of many bacteria (**Figure 2B**). S-layers have been shown to possess strong adjuvant properties and represent excellent carrier candidates for immunotherapeutic vaccines (Raha et al., 2005; Sleytr et al., 2014). The general applicability of S-layers as vaccine carriers for treatment of type I allergy was tested using S-layer self-assembly products from Lysinibacillus sphaericus or Thermoanaerobacter thermohydrosulfuricus chemically conjugated with recombinant Bet v 1 (Jahn-Schmid et al., 1996). In a subsequent study by Jahn-Schmid et al. (1997), T cell lines derived from PBMCs of birch pollenallergic patients were induced either using recombinant Bet v 1 alone or Bet v 1/S-layer conjugates. After re-stimulation with Bet v 1, T cell lines induced with conjugates showed substantial increase in IFN-γ production compared to T cell lines induced with allergen only. The presence of IFN-γ in the induction phase of T cell lines has been described to lead to a preferential development of T cell clones with a TH1-like phenotype. Indeed, most of the T cell clones derived from the Bet v 1-induced T cell lines (55%) exhibited a TH2-like pattern of cytokine production and majority (79%) of the T cell clones established with the Bet v 1/S-layer conjugates revealed TH1 pattern. In PBMC cultures stimulation with S-layer proteins and Bet v 1/S-layer conjugates but not recombinant Bet v 1 increased production of IL-12, an essential mediator of TH1 response. This indicates an adjuvant effect of S-layer mediated by IL-12 (Jahn-Schmid et al., 1997).

In the following years, recombinant fusion of the Bet v 1 to S-layer proteins successfully replaced the procedures of chemical coupling. For example, recombinant fusion of Bet v 1 with S-layer proteins, SbpA from Bacillus sphaericus and SbsC from Bacillus stearothermophilus, yielded two S-layer/allergen recombinant constructs, which showed strongly reduced capacity to bind IgE compared to free Bet v 1 and possessed the ability to induce allergen-specific TH0/TH1 and regulatory T cell immune responses (Breitwieser et al., 2002; Ilk et al., 2002; Gerstmayr et al., 2007). Initially, the S-layer/allergen fusion proteins were expressed in Gram-negative host E. coli and the associated endotoxin was subsequently removed by purification procedure, which is costly and time-consuming. In a recent study a Gram-positive, non-pathogenic bacteria with naturally high secretory capacity, Bacillus subtilis, was tested for expression of the endotoxin-free recombinant protein. The obtained fusion protein consisting of Bet v 1 and S-layer surface protein SbpA from Lysinibacillus sphaericus showed excellent recrystallization properties and immune reactivity (Ilk et al., 2011). Bacterial S-layers also proved to be applicable as carriers for the development of a peanut allergen-derived peptides. In a study by Anzengruber et al. (2017), a fusion protein of the S-layer protein SlpB from Lb. buchneri and the peptide AH3a42, containing immunodominant B-cell epitopes and one T cell epitope of major peanut allergen Ara h 2, was generated. The fusion protein SlpB-AH3a42 was recognized by IgE from 69% of the allergic patients and did not induce β-hexosaminidase release from sensitized rat basophil leukemia cells. However, IgG antibodies induced by immunization of rabbits with the SlpB-AH3a42 molecule weakly inhibited IgE-binding to the natural Ara h 2 (no more than 30% reduction observed with 20 patient sera) in comparison with the inhibition by anti-Ara h 2 rabbit IgG antibodies (48% reduction). These results indicate that more than one peptide, derived from allergen, would probably be needed to promote wider patient protection.

#### Albumin Binding Protein

In the study by Ganglberger et al. (2001), the Bet v 1 mimotopes were expressed as fusion proteins with streptococcal albumin binding protein as a monovalent carrier and their antigenicity and allergenicity were examined. The fusion proteins were shown to selectively bind to anti-Bet v 1 human IgE thus demonstrating that the mimotopes fused to albumin binding protein resemble the genuine IgE epitopes. Even though they possess IgE binding structures, the recombinant mimotopealbumin binding protein constructs did not cause skin test reactivity in Bet v 1-allergic mice, indicating that mimotopes of IgE epitopes are safe for immunotherapy when presented in a monovalent form. Furthermore, upon vaccination of BALB/c mice, the constructs were able to induce Bet v 1-specific IgGs that inhibited recognition of Bet v 1 by patients' IgE.

### CONCLUSION

Microbial delivery vehicles have been applied in allergy immunotherapy to enhance its efficacy, reduce side effects, and shorten the treatment. Several promising viral and bacterial carriers have been developed and tested. Regarding phage carriers, apart from regulatory constraints, the preexisting immunity and possible transfer of antibiotic resistance prevent their broader application in spite of the exceptional stability, cost-effectiveness, and ease-of-production. In general, delivery vehicles that are not genetically modified and are not problematic from the regulatory point of view are gaining momentum in today's research. From this perspective, non-recombinant alternatives to GRAS probiotic carriers displaying particularly good performance in mucosal and gastrointestinal delivery are receiving attention. However, none of the proposed whole bacterial or whole viral delivery vehicles have reached the clinical phase of investigation thus far.

Despite numerous applications which have been proposed and their proof-of-principle demonstrated on animal models, only two virus-derived carriers have entered human trials. VLPs (in combination with CpGs) showed suitable clinical tolerance and beneficial immunological and clinical effects. Based on their viral immunomodulatory properties, VLPs in general and CpGs as adjuvants were successfully used in the treatment of allergic rhinitis. However, large controlled studies are needed to collect more extensive clinical experience with this new technology. Hepatitis B virus PreS surface protein is another viral carrier that successfully underwent phase 2b clinical trial, in which it proved

to be safe and effective as a vehicle for subcutaneous delivery of B cell epitope peptides in grass pollen allergy.

In the future, the expression of multiple allergens or allergenderived peptides on a single carrier will probably be necessary to cover a larger repertoire of the epitopes and to elicit optimal anti-allergic immune responses. This strategy also opens possibilities for patient-specific immunotherapy. Knowledge of the epitopes characteristic for the individual allergic patient, together with technology for development of the appropriate carriers, would allow targeted, personalized therapy. Moreover, the combination of several functional molecules (e.g., delivery vehicle, adjuvant, and allergen; such as VLP/CpG/house dust mite allergens) might be required to maximize vaccine efficiency. Finally, a deeper understanding of cellular and humoral factors involved in immune responses will contribute to optimization of these delivery systems and their faster translation to clinical practice.

#### REFERENCES

#### AUTHOR CONTRIBUTIONS

ML conceived of the presented idea. AZ reviewed the literature and collected the data. Both ML and AZ contributed to the final version of the manuscript.

### FUNDING

The authors acknowledge financial support of Slovenian Research Agency through Research Program P4-0127.

#### ACKNOWLEDGMENTS

The authors thank Assoc. Prof. Dr. Tomaž Bratkovic for critical ˇ reading of the manuscript.

the aeroallergen Bet v 1 biases towards Th1 and T-regulatory responses upon systemic sensitization. Allergy 66, 368–375. doi: 10.1111/j.1398-9995.2010. 02488.x

guidelines for allergic disease prevention (GLAD-P): vitamin D. World Allergy Organ. J. 9:17. doi: 10.1186/s40413-016-0108-1

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Zahirovi´c and Lunder. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Lactic Acid Bacteria for Delivery of Endogenous or Engineered Therapeutic Molecules

#### Peter A. Bron1,2 and Michiel Kleerebezem2,3 \*

1 NIZO Food Research BV, Ede, Netherlands, 2 BE-Basic Foundation, Delft, Netherlands, 3 Host-Microbe Interactomics Group, Department of Animal Sciences, Wageningen University & Research, Wageningen, Netherlands

Food-grade lactic acid bacteria (LAB) are considered suitable vehicles for the production and/or delivery of health promoting or therapeutic, bioactive molecules. The molecules considered for health-beneficial use include the endogenous effector molecules produced by probiotics (mostly lactobacilli), as well as heterologous bioactives that can be produced in LAB by genetic engineering (mostly using lactococci). Both strategies aim to deliver appropriate dosages of specific bioactive molecules to the site of action. This review uses specific examples of both strategies to illustrate the different avenues of research involved in these applications as well as their translation to human health-promoting applications. These examples pinpoint that despite the promising perspectives of these approaches, the evidence for their effective applications in human populations is lagging behind.

Keywords: lactic acid bacteria, probiotics, Lactococcus lactis, Lactobacillus, genetic engineering, therapeutic molecules

### LAB FOR FOOD-GRADE BIOACTIVE DELIVERY

Lactic acid bacteria (LAB) have a long history of safe consumption in the form of fermented food products and as a consequence are considered food-grade. LAB encompass various bacterial genera, including Lactococcus, Streptococcus, Lactobacillus, Leuconostoc, and Pediococcus. The safety status of these bacteria has inspired their use for the production and delivery of bioactive molecules that intend to improve consumers' health. An important concept in this domain is the use of probiotics that are live microorganisms that when administered in adequate amounts confer a health benefit on the host (Hill et al., 2014), a domain dominated by species of the Lactobacillus genus. An alternative approach employs LAB for the engineered production and/or delivery of heterologous bioactive molecules, which mostly employs Lactococcus lactis as a production host, because of its extensive genetic toolbox. Both conceptual approaches are illustrated in this review through specific examples that in our opinion are closest to actual application as health promoting concepts. This highlights the impressive potential of using these bacteria for bioactive delivery, but also exemplifies the complexity of translation toward predictable effects in human applications.

#### Edited by:

Aleš Berlec, Jožef Stefan Institute (IJS), Slovenia

#### Reviewed by:

Nikhil Unni Nair, Tufts University, United States Matthew Wook Chang, National University of Singapore, Singapore

> \*Correspondence: Michiel Kleerebezem<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Infectious Diseases, a section of the journal Frontiers in Microbiology

Received: 20 May 2018 Accepted: 20 July 2018 Published: 03 August 2018

#### Citation:

Bron PA and Kleerebezem M (2018) Lactic Acid Bacteria for Delivery of Endogenous or Engineered Therapeutic Molecules. Front. Microbiol. 9:1821. doi: 10.3389/fmicb.2018.01821

## THE PROBIOTIC LACTOBACILLI

Lactobacillus species encompass a group of LAB that are encountered in a diverse range of nutrition-rich environments, including the human and animal gastrointestinal tract, plants, as well as food and feed (Duar et al., 2017). Specific Lactobacillus strains are marketed as probiotics.

An intrinsic characteristic of these probiotics lactobacilli is that they produce and deliver (specific) health promoting effector molecules in situ that modulate host physiology in a health-promoting direction. For many probiotic lactobacilli the molecular mechanism by which they impact host health remains largely unknown. However, in depth molecular research on specific probiotic strains has generated an impressive knowledgebase of the effector molecules of these strains that play a role in the modulation of specific host pathways. Many of the probiotic effector molecules that have been described appear to reside in the bacterial cell-envelope (Bron et al., 2011, 2013; Lebeer et al., 2018). Since the research related to probiotic effector molecule discovery has recently been reviewed, we do not expand this section beyond the exemplary case discussed below. The chosen example is in our opinion among the best-described in this field, and includes technological improvements by encapsulation for in situ delivery of the probiotic bacteria and/or their effector molecules (Li et al., 2016).

The major secreted proteins P40 and P75 produced by the extensively studied probiotic strain Lactobacillus rhamnosus GG were demonstrated to modulate epidermal growth factor receptor (EGF-R) activity in epithelial cells, thereby modulating Akt activation. This interaction was shown to inhibit cytokineinduced apoptosis in epithelial cells in vitro, and to promote epithelial cell growth ex vivo in human and mouse colon-tissue explants (Yan et al., 2011; Yan and Polk, 2012). Importantly, EGF-R modulation by P40 protected mice against chemically induced colitis (Yan et al., 2011; Yan and Polk, 2012). Notably, homologues of these two secreted proteins are present in other L. rhamnosus strains as well as in the related species Lactobacillus casei, implying that the effects reported for L. rhamnosus GG could potentially also be achieved with related strains (Bauerl et al., 2010). The same effector molecules probably underlie the observation that L. rhamnosus GG is able to stimulate epithelial wound healing in in vitro scratch assays using both skin- and gingival- human epithelial-cell lines (Mohammedsaeed et al., 2015; Fernandez-Gutierrez et al., 2017). Interestingly, in vivo transcriptome responses in human duodenal tissues following L. rhamnosus GG consumption revealed significant modulation of epithelial wound healing pathways in the human intestinal mucosa (van Baarlen et al., 2011), indicating the congruency of these molecular interactions in different model systems. However, the connection of these conserved responses to reliable health benefits in human populations remains highly challenging. This may be due to the extensive degree of human individuality and the corresponding individualized physiological relevance of such cellular modulations, which could predominantly depend on the baseline molecular state of an individual. This conceptual view was previously coined as the "band-width of health" and may represent a principal hurdle in the reliable application of probiotics and/or their effector molecules in human (Bron et al., 2011; van Baarlen et al., 2011; Klaenhammer et al., 2012).

### ENGINEERED LACTOCOCCI

For specific diseases it is well established which human proteins are underrepresented in the diseased population and/or have an established role in therapy. LAB engineered to produce such heterologous bioactive and/or therapeutic molecules provide an approach to delivering such molecules in situ as live bacterial therapeutics, or may serve as a production host to obtain these molecules in purified form. General genetic engineering capacities and in particular the controlled gene expression toolboxes available for the lactobacilli are lagging behind those that have been developed for the paradigm LAB L. lactis (Kok et al., 2017). As a consequence, the majority of these engineering approaches for the production (and delivery) have employed L. lactis as a production host. A lot of effort has been invested in the production and delivery of vaccineantigens and allergens by engineered lactococci, and since this area has recently been reviewed (Wyszynska et al., 2015; Wang et al., 2016; Szatraj et al., 2017) we focus on some specific examples of alternative categories of bioactive molecules, including bacterial enzymes and human factors known to modulate mucosal physiology and/or immune function. The examples presented were chosen on basis of available scaleup and commercialization strategies and employ L. lactis as a production host.

#### LACTOCOCCAL PRODUCTION OF AN ANTIMICROBIAL ENZYME

Staphylococcus simulans harbors lss which encodes a 25 kDa antibacterial protein termed lysostaphin that hydrolyzes the Gly–Gly bond present in the cell wall of Staphylococcus aureus, thereby lysing this important pathogen (Thumm and Gotz, 1997). Lysostaphin administration in a nasal spray effectively reduces S. aureus carriage, supporting the prevention of spreading of this bacterium in hospital environments (Quickel et al., 1971). Biosynexus Inc. is involved in development of protein-based pharmaceutical products aiming to ameliorate staphylococcal infections in infants, including lysostaphin. To provide a suitable production system for this selective antimicrobial enzyme, the encoding lss gene was cloned under control of the PnisA promoter and introduced in the expression host-strain NZ3900, allowing food-grade, lactosebased plasmid selection and induction of lysostaphin production through the NICE system (Mierau and Kleerebezem, 2005; Mierau et al., 2005). Following optimization of lysostaphin production at 1 L scale it was shown to be readily scalable to 300 and 3000 L production facilities. In addition, the protein could be purified from the lactococcal culture supernatant via straightforward and industrially scalable chromatography procedures (Mierau et al., 2005). This example

illustrates that L. lactis is a suitable host for the production and purification of relevant bioactive enzymes, in this case an anti-Staphylococcus agent. Purification of the compound of interest. avoids the consequences of releasing genetically modified L. lactis strains and is suitable for bioactive molecules that do not depend on the presence or co-modulatory effects of the production host in the application.

### LIVE-BIOTHERAPEUTICS USING ENGINEERED L. lactis

Intrexon develops so-called Actobiotics, a class of orally delivered biopharmaceuticals employing engineered and biologically contained L. lactis production hosts for the in situ production and delivery of various proteins and peptides in the oral and gastrointestinal tract of humans. The selection of the bioactives to be produced within this delivery system aim to provide therapies against oral, gastrointestinal, metabolic, allergic, and autoimmune diseases, focusing on bioactives with known pharmacological activity and that are supported by documented efficacy and safety information. The company's ambition is strongly based on the landmark study that demonstrated that chemically induced colitis in mice could be effectively treated with L. lactis strains that produce interleukin-10 (IL-10) (Steidler et al., 2000). Later work illustrated that the L. lactis based intestinal IL-10 delivery could also contribute to reduction of food-induced anaphylaxis in mice models, which may support the prevention of IgE-type sensitization to common food allergens (Frossard et al., 2007). Likewise, IL-10 delivery combined with secretion of the type-1 diabetes (T1D) auto-antigen GAD65370-575 by the same L. lactis strain was effective in suppressing T1D in a preclinical NOD-mouse model (Robert and Steidler, 2014; Robert et al., 2014). Besides these IL-10 producing L. lactis strains and their derivatives, the same platform for production and delivery of bioactives is employed for a variety of other molecules intending to provide live lactococcal therapeutics for a range of human and animal diseases (Vandenbroucke et al., 2004; McLean et al., 2017). Moreover, some comparative work using similar IL-10 producing Lactobacillus strains has been reported and may expand or enhance the application potential of these concepts (Steidler, 2003). Besides the bioactive production and delivery technology, the Actobiotics platform is supported by effective strategies for the biological containment of genetically engineered bacteria as well as scalable production and processing procedures for the live biotherapeutic bacteria (Steidler, 2003). However, initial human trials in patients suffering from inflammatory bowel disease established the safety and biological containment of the IL-10 producing lactococcal delivery vehicle (Braat et al., 2006), but failed to deliver convincing clinical efficacy evidence for disease symptom reduction to date, illustrating that translation of the mousemodel outcomes to human applications still requires further investigation.

Aurealis Pharma is a pharmaceutical company that employs genetically modified L. lactis strains for the delivery of combinations of therapeutic proteins to diseased tissues. For example, L. lactis MG1363 was engineered to allow plasmidbased expression of three genes encoding human fibroblast growth factor 2 (FGF-2), interleukin 4 (IL-4), and colony stimulating growth factor 1 (CSF-1) that were selected based on their functional properties (Yun et al., 2010; Hume and MacDonald, 2012; Sica and Mantovani, 2012) and availability of clinical safety data. An animal study demonstrated the efficacy of the combination therapy provided by the three proteins and the L. lactis strain in a delayed wound healing model in diabetic mice1 . Aurealis Pharma is currently producing a clinical grade product for a clinical phase 1 trial to build a safety dossier for the use of this live bacterial therapeutic in diabetic foot ulcer patients.

### PERSPECTIVES

This short review illustrates that significant advances have been made in understanding the mechanism of action of endogenous probiotic Lactobacillus effector molecules, and that heterologous production of therapeutic bioactives in engineered lactococci has reached technological maturity. The examples chosen employed in this review are actively pursued by commercial initiatives and involve either purified bioactives or the use of the engineered LAB as live biotherapeutic.

However, in both strategies, the translation of the healthbeneficial effects toward reliable human application is lagging behind despite the consistent findings across the different layers of the translational pipeline progressing from molecular insight in molecular interactions involved toward their conservation in in situ human response. Lagging efficacy evidence may be largely due to human individuality in health and disease, which implies that careful consideration should be given to selecting the appropriate target population. This may require novel strategies toward subject stratification and responsiveness prediction to better select and treat human subjects that are likely to have a perceivable benefit from the response elicited by the bioactive supplied.

### AUTHOR CONTRIBUTIONS

Both authors conceived the review, wrote it together and decided to submit it to this Research Topic.

## FUNDING

Part of this work was carried out within the BE-Basic R&D program (Grant F10.002.01), which was granted an FES subsidy from the Dutch Ministry of Economic Affairs.

1 http://ewma.conference2web.com/#users/153991

### REFERENCES

**Conflict of Interest Statement:** PB was employed by the company NIZO Food Research BV. Part of the work by PB and MK was funded by the BE-Basic Program. Neither employer, nor funding agency had a role in the preparation of the manuscript.

Copyright © 2018 Bron and Kleerebezem. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

Instituto de Ciências Biológicas,

# Anti-tumoral Effects of Recombinant Lactococcus lactis Strain Secreting IL-17A Cytokine

Elsa Jacouton1† , Edgar Torres Maravilla1† , Anne-Sophie Boucard1 , Nicolas Pouderous1 , Ana Paula Pessoa Vilela1,2, Isabelle Naas1 , Florian Chain1 , Vasco Azevedo2 , Philippe Langella1 and Luis G. Bermúdez-Humarán1 \*

1 Micalis Institute, AgroParisTech, INRA, Université Paris-Saclay, Jouy-en-Josas, France, 2

Federal University of Minas Gerais, Belo Horizonte, Brazil

#### Edited by:

Aleš Berlec, Jožef Stefan Institute (IJS), Slovenia

#### Reviewed by:

Scott Kenneth Durum, National Cancer Institute (NCI), United States Apon Zaenal Mustopa, Indonesia Institute of Sciences (LIPI), Indonesia

#### \*Correspondence:

Luis G. Bermúdez-Humarán<EMAIL_ADDRESS>†These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Infectious Diseases, a section of the journal Frontiers in Microbiology

Received: 24 May 2018 Accepted: 31 December 2018 Published: 23 January 2019

#### Citation:

Jacouton E, Torres Maravilla E, Boucard A-S, Pouderous N, Pessoa Vilela AP, Naas I, Chain F, Azevedo V, Langella P and Bermúdez-Humarán LG (2019) Anti-tumoral Effects of Recombinant Lactococcus lactis Strain Secreting IL-17A Cytokine. Front. Microbiol. 9:3355. doi: 10.3389/fmicb.2018.03355 Interleukin-17A (IL-17A) is a pro-inflammatory cytokine produced by TH17 cells that participates and contributes in host defense and autoimmune disease. We have recently reported antitumor properties of the probiotic strain of Lactobacillus casei BL23 in mice and TH17 cells was shown to play an important role in this beneficial effect. In order to better understand the role of IL-17A in cancer, we constructed a recombinant strain of Lactococcus lactis producing this cytokine and we determined its biological activity in: (i) a bioassay test for the induction of IL-6 production by murine fibroblasts 3T3 L1 cells line and (ii) in a mouse allograft model of human papilloma virus (HPV)-induced cancer. Our data show that recombinant L. lactis produces and efficiently secretes biologically active IL-17A cytokine. Interestingly, ∼26% of mice intranasally treated with L. lactis-IL-17A and challenged with TC-1 cells remained tumor free over the experiment, in contrast to control mice treated with the wild type strain of L. lactis which developed 100% of aggressive tumors. In addition, the median size of the ∼74% tumor-bearing mice treated with recombinant L. lactis-IL-17A, was significantly lower than mice treated with L. lactis-wt. Altogether, our results demonstrate that intranasal administration with L. lactis secreting IL-17A results in a partial protection against TC-1-induced tumors in mice, confirming antitumor effects of this cytokine in our cancer model.

Keywords: Lactococcus lactis, lactic acid bacteria, IL-17A, cancer, HPV

## INTRODUCTION

Cancer remains a serious health concern in human society worldwide and colorectal cancer (CRC), prostrate, lung, stomach, liver and breast cancers are among the major types associated with significant mortality every year (Ferlay et al., 2012). Cancer is generally considered to be a disease involving both host genetics and environmental factors; however microorganisms (such as viruses and bacteria) are associated in ∼20% of human cancers (de Martel et al., 2012). Recent studies suggest that probiotics can help to fight cancer. Probiotics are live

microorganism which, when administered in adequate amounts confer a health benefit on the host (Food and Agriculture Organization, 2002). For instance, probiotics can induce dendritic cells (DC) maturation (Delcenserie et al., 2008), enhance natural killer (NK) cells cytotoxicity (Takagi et al., 2001), and upregulate cytokine secretion (Delcenserie et al., 2008; Azcarate-Peril et al., 2011). It has also been reported that some strains of Lactobacillus can induce DC maturation and TH1 (antiviral and bacterial immunity) and TH17 (inflammation and auto-immunity) differentiation (Kemgang et al., 2014; Cai et al., 2016; Lee et al., 2016). However, despite the great number of studies that have demonstrated anticancer effects of different strains of Lactobacillus (Khazaie et al., 2012; Konishi et al., 2016; Lenoir et al., 2016), the precise host molecular mechanisms of these antitumor properties remain unclear. Next generation probiotics, such as Akkermansia muciniphila and Faecalibacterium genus as well as genetically modified microorganisms (GMOs) (O'Toole et al., 2017) have demonstrated beneficial effects in the context of cancer, promoting the immune checkpoints inhibitors therapy targeting the programmed cell death protein 1 (PD-1) and cytotoxic lymphocyte-associated antigen (CTLA-4). In addition, other studies support the role of Bifidobacterium, Bacteroides, Faecalibacterium and Akkermansia species in cancer therapy targeting the immune checkpoint blockade (CTLA-4, PD-1), showing a T cellspecific anti-tumor-induced response (Sivan et al., 2015; Vetizou et al., 2015; Gopalakrishnan et al., 2018; Routy et al., 2018).

We previously demonstrated that mucosal administration of the probiotic strain of Lactobacillus casei BL23 displays antitumor properties in three different murine models of cancer (Lenoir et al., 2016; Jacouton et al., 2017). Interestingly, we showed that this strain was able to modulate a T-cell immune response toward a TH17-biased immune response, accompanied by the expression of regulatory cytokines (e.g., IL-6, IL-17, IL-10, and TGF-β), in a murine model of CRC (Lenoir et al., 2016). In particular we were intrigued by IL-17 induction, since IL-17 seems to be essential for both metastasis and elimination of tumor cells (Murugaiyan and Saha, 2009). Thus, IL-17-producing TH17 cells have recently gained considerable importance in cancer (Maniati et al., 2010). Therefore, we hypothesized that IL-17 induced by L. casei BL23 could play an important role in the anti-tumor effect of this probiotic strain. We thus decided to use a genetically modified strain of Lactococcus lactis, the model lactic acid bacterium (LAB), to produce and deliver exogenous murine IL-17 and to determine its anti-tumor effect in a mouse allograft model of human papilloma virus (HPV)-induced cancer.

#### MATERIALS AND METHODS

#### Bacterial Strains and Growth Conditions

L. lactis MG1363 (Gasson, 1983) was grown in M17 medium (Difco Laboratories, England) supplemented with 0.5% glucose (GM17) and 15 µg/ml of chloramphenicol at 30◦C without agitation.

### Construction of a Recombinant Strain of L. lactis Secreting Murine IL-17

To construct a vector which will allow stress-inducible IL-17 expression in L. lactis a DNA fragment encoding IL-17 mature sequence was obtained from a recombinant plasmid containing murine il-17 gene (synthesized by Geneart, Invitrogen) with NsiI/EcoRI enzymes. As previously described (Benbouziane et al., 2013), we used pLB333 vector containing nucB gene under the control of the stress inducible groESL promotor. pLB333 was digested with the same enzymes to replace nuc gene by il-17 gene. The resulting vector, pSICE:IL-17 (**Figure 1A**), was established into L. lactis MG1363 strain to obtain L. lactis-IL-17. For detection of IL-17, L. lactis-IL-17 strain (L. lactis-wt was used as negative control) was grown overnight (ON, optical density (OD)600nm = 2.0–2.5) as described above. Plasmid DNA isolation and general procedures for DNA manipulation were essentially performed as described previously (Sambrook et al., 1989). PCR amplification was performed using High Fidelity PCR Enzyme Mix (Fermentas) with a thermal cycler (Applied Biosystem). DNA sequences were confirmed by sequencing (MWG-Genomic Company, Germany).

#### In vitro Validation of IL-17 Production and Secretion by Recombinant L. lactis

Over-night cultures were washed twice using PBS and culture pursued (after a 1:10 dilution in GM17 medium) until OD600nm ∼0.6. Then, cultures were induced with 2.5% NaCl for 30 min and protein samples prepared from 2 ml of the induced cultures. After centrifugation (10 min, 17500g), the cellular pellet (C) and supernatant (S) were treated separately. The S samples were precipitated with 200 µl of trichloroacetic acid (TCA) 100% for 1 h on ice to recover proteins (centrifugation at 17500g at 4 ◦C for 30 min) and resuspended in 200 µl of 50mM NaOH. The C fraction was resuspended in 200 µl of PBS plus protease inhibitors (Roche) and sonicated 30 s with alternated pulses on ice (on: 5 s, off: 30 s). Protein samples were diluted 1:1 in Laemmli sample buffer containing 355 mM β-mercaptoethanol and denaturated 5 min at 95◦C. Equal amounts of proteins were loaded and separated on a Mini-PROTEAN TGX stain free 4–20% SDS gel at 200 V and further transferred to a PVDF membrane using a Trans-Blot Turbo transfer system (Biorad). Membrane was probed with primary antibody antimouse IL-17A (R&D Systems) and secondary anti-rat IgG HRP-conjugated antibody (Abliance) at 1:1000 dilutions. Bound secondary antibody was visualized by the Clarity ECL Western Substrate (Bio-Rad) and Chemidoc imaging system (Biorad). The concentration of IL-17A secreted in the medium was assessed by ELISA (mouse IL-17 ELISA Development Kit, Mabtech).

#### Determination of the Biological Activity of IL-17 Produced by Recombinant L. lactis

Murine fibroblasts 3T3 L1 cells line, grown in DMEM medium (Lonza, Switzerland) supplemented with 10% heat-inactivated fetal calf serum (FCS), 50 U/ml penicillin and 50 U/ml streptomycin (Lonza, Levallois-Perret, France) were cultivated

at 1 × 105 cells per well during 24 h at 37◦C, 5% CO2. Then, medium was changed and bacterial preparations added at 10% for supernatants, pellet and control medium or MOI 100 for bacteria suspensions during 24 h. Supernatants of co-incubations were collected and stored at −80◦C before ELISA analyses (mouse IL-6 DuoSet ELISA, R&D).

markers is indicated on the left. The position of mature murine IL-17 is given by an arrow. Values are mean ± SEM.

### Mice and TC-1 Cell Line

Specific pathogen-free C57BL/6 mice (females, 6–8 weeks old; Janvier SAS, St. Berthevin, France) were housed in a pathogenfree isolator (n = 4 mice per cage) under sterile conditions in 12-h light cycles in the animal facilities of the French National Institute for Agricultural Research (INRA, IERP, Jouy-en-Josas, France). Animals were supplied with water and fed ad libitum (normal chow: R 03-40, SAFE). Temperature and moisture were carefully controlled. Mice were observed once a day to ensure their welfare. All protocols were carried out in accordance with the institutional ethical guidelines of the ethics committee COMETHEA (Comité d'Ethique en Expérimentation Animale of the Centre INRA of Jouy-en-Josas and AgroParisTech), which approved this study.

The mouse (C57BL/6) lung tumor cell line TC-1 (generated by transduction with a retroviral vector harboring HPV-16 E6/E7 genes plus a retrovirus expressing activated human oncogene c-Ha-ras (Lin et al., 1996)) was grown in RPMI medium 1640 (Lonza, Switzerland) supplemented with 10% heat-inactivated FCS, 50 U/ml penicillin, 50 U/ml streptomycin (Lonza, Levallois-Perret, France), 0.4 mg/ml G418 and 0.2 mg/ml hygromycin in 5% CO2 atmosphere.

## TC-1 Cell Line Challenge and Bacteria Administration

Groups of mice (n = 22 from 3 independent in vivo experiments) were intranasally (i.n.) administered using a micropipette with 1 × 109 colony-forming units (CFU) of either L. lactis-wt or L. lactis-IL-17 strain (suspended in 10 µl of PBS). ON cultures were washed two times and finally suspended in PBS at 1 × 1011 CFU/ml. Each mouse received 5 µl of the solution in each nostril on days −35, −21, and −7. Control mice received identical quantities of PBS (i.e., 10 µl). Mice were challenged 7 days after the final bacterial administration (D0) by subcutaneous (s.c.) injection in the right rear flank with 5 × 104 TC-1 cells in 100 µl of sterile PBS. The dimensions of the tumor at the site of injection were measured every week in two perpendicular directions with a caliper, and tumor volume was estimated as (length × width2 )/2 (Bermudez-Humaran et al., 2005). Mice were sacrificed by vertebral dislocation at D28.

### Analysis of the Immune Response in Mice Treated With Recombinant L. lactis and Challenged With TC-1 Cells

Mice were euthanatized at D28 and spleens collected and isolated via gentle extrusion of the tissue through a 50-µm-mesh nylon cell strainer (BD). Cells were resuspended in DMEM medium supplemented with 10% FCS, 2 mM L-glutamine, 50 U/mg penicillin and 50 U/mg streptomycin. Erythrocytes

were lysed with red-blood-cell lysing buffer (Sigma-Aldrich). For stimulation experiments, 1 × 106 cells per well were stimulated for 48 h (37◦C, 10% CO2) in DMEM medium in P24 plates in presence of PMA (phorbol 12-myristate 13-acetate) ionomycin cocktail 1× (eBioscience). Culture supernatant was frozen at −80◦C until processing. Levels of the cytokines IL-6 (mouse IL-6 DuoSet ELISA, R&D), IL-17A, and IFN-γ (ELISA Development Kit, Mabtech) were determined using ELISA according manufacturer's instructions.

#### Statistical Analysis

All data are expressed as mean values and standard deviations. Data analysis was performed using the GraphPad Prism Software V.5.00. Experiments were analyzed using an unpaired t-test. The two-tailed unpaired Mann–Whitney test was used to evaluate differences between two groups. In all experiments, a value of P < 0.05 was considered significant (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001).

#### RESULTS

#### Characterization of IL-17 Production by Recombinant Lactococcus lactis

Before to test the biological effect of the genetically modified strain of L. lactis harboring pSICE:IL-17 plasmid (**Figure 1A**), we first analyzed IL-17 production and secretion from both nonstressed and stressed L. lactis-wt and L. lactis-IL-17 cultures by Western blot (**Figure 1B**). A band of approximately 15 kDa was detected in the supernatant (S) fraction from induced cultures of L. lactis-IL-17 strain, which corresponds to secreted mature murine IL-17. IL-17 secretion and quantification was then determined by ELISA in C and S samples. As shown in **Figure 1C**, a better production of IL-17 was observed (∼3-fold) under stress conditions (i.e., NaCl 2.5%): ∼15,000 pg/ml versus ∼5000 pg/ml. As expected no IL-17 signal was detected in the negative control L. lactis-wt.

#### Recombinant Lactococcus lactis Secretes a Biologically Active IL-17 Cytokine

Besides IL-17 detection in S samples of bacterial cultures, we determined the biological activity of this cytokine secreted by recombinant L. lactis. IL-17A is known to stimulate several cytokines (including IL-6) in different cell lines (such as fibroblast, epithelial cells and immune cells). Thus, we selected murine fibroblasts 3T3 L1 cells to assess specific IL-6-induction by recombinant L. lactis. Our results showed that S samples of L. lactis-IL-17 strain and stressed with NaCl 2.5% induced a significant IL-6 secretion in 3T3 L1 cells (**Figure 2A**) in comparison with their respective negative control. No significant IL-6 production was observed in bacterial cultures without stress induction. In parallel we confirmed by ELISA the presence of IL-17 cytokine in S samples of recombinant bacteria (**Figure 2B**).

### L. lactis IL-17 Has a Protective Effect Against Tumors in TC-1 Allograft Model of HPV-Induced Cancer

To further evaluate in vivo the biological activity of IL-17 produced by recombinant L. lactis, and in particular the impact of this cytokine in the TC-1 mouse allograft model of HPVinduced cancer, we analyzed the effect after i.n. administration of this strain in the TC-1 tumor mice. Mice were immunized as described in Material and Methods and tumor absence/presence monitored every week. As shown in **Figure 3A**, L. lactis IL-17 displayed a protective effect against tumor development at D28 (the end of the experiment) since 77% (5/22) of mice that had been administered L. lactis IL-17 developed tumors with a mean tumor size of ∼0.80 cm3 (**Figure 3B**) compared to 100% (22/22) of mice receiving L. lactis-wt control strain (mean tumor size of ∼1.2 cm3 ) (**Figures 3A,B**). These results confirm

that IL-17 cytokine display anti-tumor effects in our cancer model.

### L. lactis IL-17 Induces IL-6 and IL-17 Secretion in Reactivated Splenocytes From Mice Challenged With the Tumoral Cell Line

In order to further explore the impact on the immune response of mice treated with recombinant L. lactis IL-17 we analyzed cytokines release by reactivated splenocytes from mice 28 days after i.n administration of recombinant bacteria and challenged with TC-1 cells. Interestingly, L. lactis IL-17 induced a significant IL-6 secretion in splenocytes compared to L. lactis-wt (**Figure 4**). This modulation was correlated with a slight IL-17 induction but without reach statistical significance (**Figure 4**). No effect was observed on IFN-γ production by recombinant bacteria (**Figure 4**).

### DISCUSSION AND CONCLUSION

A better understanding of the interactions between cancer cells and stromal components in the tumor associated pro-inflammatory microenvironment would be important for the management of this disease (Ferlay et al., 2012). Anti-tumor response involves different components of the immune system, such as NK cells, DC, macrophages and T cells. A growing body of evidence suggests that probiotics can help to combat cancer by either protecting against gastrointestinal infections or enhancing immune response. Indeed, it has been shown that probiotics can induce DC maturation (Delcenserie et al., 2008), enhance NK cell cytotoxicity (Takagi et al., 2001), and upregulate cytokine secretion (Delcenserie et al., 2008; Azcarate-Peril et al., 2011). In addition, recent studies described the role of specific members of microbiota in cancer therapy by targeting the immune checkpoint blockade (CTLA-4, PD-1) (Sivan et al., 2015; Vetizou et al., 2015; Gopalakrishnan et al., 2018; Routy et al., 2018).

FIGURE 3 | Effect of recombinant L. lactis expressing IL-17 against tumors in TC-1 allograft model of HPV-induced cancer. Mice were i.n. treated with 1 × 109 CFU resuspended in 10 µl of PBS (5 µl were administered with a micropipette into each nostril) on D-35, -21, and -7. Seven days after the last administration (D0), a challenge with the tumoral cell line TC-1 was performed, and the presence and size of the tumor was monitored once a week. (A) Tumor incidence with proportions of tumor-free animals (B) Individual tumor volume at the end of week 10. Data represented mean ± SEM from 3 independent in vivo experiments.

FIGURE 4 | Cytokines production by reactivated splenocytes from mice treated with recombinant bacteria and challenged with TC-1 cells. Splenocytes were stimulated for 48 h with PMA ionomycin before measuring cytokines levels. Data represented mean ± SEM from 4 (L. lactis wt) to 8 mice (L. lactis IL-17). Data are analyzed with unpaired t-test followed by Mann–Withney post-test.

Among the potential anti-tumoral mechanisms of probiotics, two of the most known are the modulation of the immune response and the induction of cellular apoptosis. For instance, two strains of L. casei are able to decrease tumor cell proliferation and enhance apoptosis in allograft models of CRC (Lee et al., 2004; Baldwin et al., 2010; Konishi et al., 2016). Similarly, oral administration of a L. casei strain reduces the onset of chemically induced tumors via the stimulation of IL-12 or NK-cell cytotoxicity mechanisms (Takagi et al., 2001, 2008). Furthermore, our team recently demonstrated protective effects of the probiotic strain L. casei BL23 in three different mouse models of cancer, including CRC (Lenoir et al., 2016; Jacouton et al., 2017) and the TC-1 allograft model (Lenoir et al., 2016). In one of our two CRC models (Lenoir et al., 2016), the anti-tumor effects of L. casei BL23 were associated with the reduction of pro-inflammatory cytokines, but the precise molecular and cellular mechanisms involved in tumor prevention of this bacterium remain unclear. Since cancer therapy includes chemotherapy, drug, vaccines, and cytokines, and for instance, current therapies are toward to enhance the immune system as use of pro-inflammatory cytokines (such as IL-2, one of the first cytokines used in cancer therapy) and immune check points inhibitors (CTLA-4, PD-1). In this work, we constructed a recombinant strain of L. lactis expressing IL-17. Strikingly, we showed that i.n. administration of this strain results in a lower tumor incidence and that tumor size was reduced in comparison to the control L. lactis-wt, a LAB strain for which no positive effect has been reported in the HPVinduced cancer model. Our results not only suggest a positive effect of IL-17 but also reinforce the idea that some of the molecular mechanisms of L. casei BL23 against cancer could be related to activation of TH and NK via TH17. IL-17 is a pro-inflammatory cytokine, although its role is controversially, some studies report that IL-17 deficiency state may have a protective role or a harmful role in tumorigenesis (Welch et al., 2015; Qian et al., 2017). For example, in IL-17 deficient mice, enhanced lung and subcutaneous tumor growth and metastasis is correlated to a decrease in the number of IFNγ producing NK cells (Kryczek et al., 2009). Recent research provided substantial insights into the mode of action of IL-17 cytokines in a variety of tumors, suggesting an anti-tumor activity of IL-17 could be achieved by means of a T celldependent mechanism increasing generation of specific cytotoxic

#### REFERENCES

T lymphocytes (Alshaker and Matalka, 2011). Paradigms are changing, and IL-17 cytokines are double-edged agents acting in a cancer-type depending manner as anti- and pro-tumor cytokines (Fabre et al., 2016). IL-6 is a pro-inflammatory cytokine involved, in part, in a TH17-related immune response with a feedback loop. Thus, we assessed the in vivo ability of L. lactis secreting IL-17 to stimulate the TH17 pathway. Here, we demonstrated an in vivo induction of IL-6 resulting in an increase of IL-17. We hypothesized that the anti-tumoral effect of recombinant lactococci secreting biologically active IL-17 could due to a TH17 immune response even if more experiments are needed to further decipher the precise molecular mechanisms.

In conclusion, our results demonstrate that i.n. administration with a genetically modified strain of L. lactis secreting IL-17 results in a partial protection against TC-1-induced tumors in mice, confirming antitumor effects of this cytokine in this model.

#### AUTHOR CONTRIBUTIONS

EJ and LB-H conceived and designed the study. EJ, ETM, and A-SB conducted all experiments. NP, APV, IN, FC, and VA contributed analytic tools. EJ, ETM, PL and LB-H performed the data analysis. ETM and LB-H wrote the manuscript.

#### FUNDING

This work was partially funded by the Association pour la Recherche sur le Cancer (ARC, France): action no PGA12014020851. ETM received a post-doctoral fellowship mobility from CONACyT (Consejo Nacional de Ciencia y Tecnología, Mexico) and APV from CAPES-COFECUB (action no 849/15).

#### ACKNOWLEDGMENTS

We are grateful to conventional animal facility' staff from INRA Jouy-en-Josas. We also thank Dr. Naima Cortes-Perez for her precious advices.

vaccine that enhances major histocompatibility class II presentation of tumor antigen. Cancer Res. 56, 21–26.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Jacouton, Torres Maravilla, Boucard, Pouderous, Pessoa Vilela, Naas, Chain, Azevedo, Langella and Bermúdez-Humarán. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# A New Bifidobacteria Expression SysTem (BEST) to Produce and Deliver Interleukin-10 in Bifidobacterium bifidum

Aurélie Mauras1 , Florian Chain2 , Aurélie Faucheux1 , Pauline Ruffié2 , Sophie Gontier2 , Bernhard Ryffel3 , Marie-José Butel1 , Philippe Langella2 , Luis G. Bermúdez-Humarán2 \* † and Anne-Judith Waligora-Dupriet1 \* †

1 EA 4065, Ecosystème Intestinal, Probiotiques, Antibiotiques, Faculté de Pharmacie de Paris, DHU Risques et Grossesse, Université Paris Descartes, Paris, France, 2 INRA, Commensals and Probiotics-Host Interactions Laboratory, Micalis Institute, AgroParisTech, Université Paris-Saclay, Jouy-en-Josas, France, 3 UMR 7355 CNRS, Laboratory of Experimental and Molecular Immunology and Neurogenetics, University of Orleans, Orleans, France

#### Edited by:

Aleš Berlec, Jožef Stefan Institute (IJS), Slovenia

#### Reviewed by:

Andrey Shkoporov, University College Cork, Ireland Gi-Seong Moon, Korea National University of Transportation, South Korea

#### \*Correspondence:

Luis G. Bermúdez-Humarán<EMAIL_ADDRESS>Anne-Judith Waligora-Dupriet<EMAIL_ADDRESS>†These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Infectious Diseases, a section of the journal Frontiers in Microbiology

Received: 07 May 2018 Accepted: 29 November 2018 Published: 21 December 2018

#### Citation:

Mauras A, Chain F, Faucheux A, Ruffié P, Gontier S, Ryffel B, Butel M-J, Langella P, Bermúdez-Humarán LG and Waligora-Dupriet A-J (2018) A New Bifidobacteria Expression SysTem (BEST) to Produce and Deliver Interleukin-10 in Bifidobacterium bifidum. Front. Microbiol. 9:3075. doi: 10.3389/fmicb.2018.03075 In the last years there has been a growing interest in the use of genetically modified bacteria to deliver molecules of therapeutic interest at mucosal surfaces. Due to the well-recognized probiotic properties of some strains, bifidobacteria represent excellent candidates for the development of live vehicles to produce and deliver heterologous proteins at mucosal surfaces. However, very few studies have considered this genus because of its complexity to be genetically manipulated. In this work, we report the development of a new Bifidobacteria Expression SysTem (BEST) allowing the production of heterologous proteins in Bifidobacterium bifidum. This system is based on: i) the broad host range plasmid pWV01, ii) a stress-inducible promoter, and iii) two different signal peptides (SPs) one issued from Lactococcus lactis (SPExp4) and issued from Bifidobacterium longum (SPBL1181). The functionality of BEST system was validated by cloning murine interleukin-10 (IL-10) and establishing the resulting plasmids (i.e., pBESTExp4:IL-10 and pBESTBL1181:IL-10) in the strain of B. bifidum BS42. We then demonstrated in vitro that recombinant B. bifidum BS42 harboring pBESTBL1181:IL-10 plasmid efficiently secreted IL-10 and that this secretion was significantly higher (sevenfold) than its counterpart B. bifidum BS42 harboring pBESTExp4:IL-10 plasmid. Finally, we validated in vivo that recombinant B. bifidum strains producing IL-10 using BEST system efficiently delivered this cytokine at mucosal surfaces and exhibit beneficial effects in a murine model of low-grade intestinal inflammation.

Keywords: Bifidobacterium bifidum, microbiota, recombinant bacteria, heterologous expression system, IL-10, low-grade intestinal inflammation

### INTRODUCTION

Bifidobacteria are strict anaerobic Gram-positive bacteria with irregular forms, belonging to the dominant microbiota in the human gut (especially in young children). Some species of bifidobacteria display beneficial properties for human health and are recognized as GRAS (Generally Regarded As Safe) microorganisms. In addition, some strains are already marketed as

food supplements (with probiotic claims) such as Bifidobacterium breve M16-V, Bifidobacterium longum BB536, and Bifidobacterium bifidum BGN4. Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host, as defined by the World Health Organization (WHO) and the Food and Agriculture Organization of the United Nations (FAO) (Hill et al., 2014).

While recombinant bacteria are used in industry for the production of insulin or vaccines, researchers are increasingly interested by the direct administration of these recombinant bacteria to humans for either the development of mucosal vaccines or in situ delivery of proteins of therapeutic interest such as antioxidants, cytokines and protease inhibitors (Bermúdez-Humarán et al., 2013; Kumar et al., 2016). One of the main benefits in the use of probiotics as live vectors to deliver biologically active cytokines is to combine: (i) the intrinsic beneficial properties of some strains and (ii) the potential effect of the delivered protein. Moreover, several probiotics strains belong to some bacteria already present in our organism (i.e., intestinal microbiota) such as certain lactobacilli and bifidobacteria strains. This feature allows these strains to withstand gut environment wherein they can release biologically active molecules. Most bacteria used to deliver heterologous protein so far are lactobacilli and lactococci. Indeed, several trials have been carried out in animal models using either recombinant lactococci or lactobacilli to treat different human pathologies such as irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD) (Motta et al., 2012; Martín et al., 2014; Allain et al., 2016). Strikingly, some human clinical trials (phase I and II) have been conducted with L. lactis producing either IL-10 or trefoil factor (TTF) to treat, respectively IBD (Braat et al., 2006) and oral mucositis (Limaye et al., 2013), confirming both the feasibility and the potential in the use of recombinant probiotics for human health.

In contrast to lactobacilli and lactococci, Bifidobacterium sp. could be a more attractive live vector to deliver proteins. Indeed, B. breve has been shown to persist for up to 32 days into mouse digestive tract after 3 days of gavage (Sheehan et al., 2007), in contrast to Lactobacillus plantarum which persists only 10 days (Pavan et al., 2003) and Lactococcus lactis that did not colonize with a transit time of 24–48 h (Kimoto et al., 2003). Finally, bifidobacteria have a low intrinsic and acquired resistance to antibiotics, showing therefore a safe profile for use in humans (Moubareck et al., 2005). Today, only few strains of bifidobacteria have been proposed as live delivery vehicles. For instance, some strains of B. longum have been used as a delivery vector for the development of: (i) oral vaccine against Hepatitis C virus (HVC) and Enterovirus 71 (Yu et al., 2013; Takei et al., 2014), (ii) anti-tumoral treatment (Shirakawa and Kitagawa, 2017), and (iii) to treat metabolic disorders in mice (Zhang et al., 2018). Other reports have described the use of genetically modified bifidobacteria to express diverse heterologous proteins (Khokhlova et al., 2010; Losurdo et al., 2013).

Currently, several expression systems are available for both lactobacilli and lactococci, including constitutive and inducible promoters and the possibility to express the desired protein into different cellular locations (i.e., intracellular, extracellular, or cell wall-attached) (Bermúdez-Humarán et al., 2013). However, as some proteins (such as cytokines) can accumulate in the cytoplasm and consequently be deleterious to bacteria growth, there is a need to develop new controlled-expression systems. These systems could avoid the negative outcomes of the overproduction of heterologous proteins by controlling expression through an inductor such as changes in either the pH, temperature or even the addition of bile salts or antimicrobial peptides. One of the best-described expression systems in L. lactis is the NICE system (for NIsin Controlled Expression) (Kuipers et al., 1995), where gene expression is turned on by the addition of nisin to the culture medium. However, NICE system requires the presence of some regulatory genes (i.e., nisRK) that hampers its further applications in both fed-batch production and in vivo models. One alternative is the use of an expression system that does not require either the presence of regulatory genes or induction of bacterial cultures before use. Thus, we developed in our laboratory two in vivo stress-inducible expression systems for both lactococci [i.e., the SICE system (Benbouziane et al., 2013)] and lactobacilli [i.e., the LIVE system (Allain et al., 2016)], which allow the direct delivery of heterologous proteins in situ by recombinant bacteria. In the current work, we seek to develop a comparable system to deliver proteins of health interest by recombinant bifidobacteria. This system, named BEST for Bifidobacteria Expression SysTem, is based in the use of the broad-host range plasmid pWV01 (Morello et al., 2008), a dnaK promoter from B. longum dnaK operon, and two different signal peptides (SPs): one issued from the lactic acid bacterium (LAB) model L. lactis (SPExp4) (Morello et al., 2008), and one from B. longum: SPBL1181. Furthermore, the functionality of BEST system was validated in vivo. For this, we cloned murine interleukin-10 (IL-10) an anti-inflammatory cytokine successfully expressed in different LAB (Braat et al., 2006), under BEST system and we determine its beneficial effects in a model of murine colitis (Martín et al., 2014). Our results show that genetically modified B. bifidum BS42 harboring IL-10 under BEST system successfully delivered the recombinant cytokine at mucosal surfaces and display beneficial properties in the murine model of intestinal low-grade inflammation.

### MATERIALS AND METHODS

### Bacterial Strains, Plasmids, and DNA Manipulation

Strains and plasmids used in this work are listed in **Table 1**. Escherichia coli was grown in Luria-Bertani medium (Oxoid, Dardilly, France) at 37◦C, under aerobic conditions with shaking. L. lactis was grown in M17 medium (Difco Laboratories, Detroit, MI, United States) supplemented with 1% glucose at 30◦C without agitation. Bifidobacteria were cultured in de Man Rogosa Sharpe medium (MRS, Oxoid), supplemented prior to inoculation with cysteine (500 mg/L, Oxoid), at 37◦C under anaerobic conditions (N2:CO2:H2; 80:10:10) in an anaerobic chamber (MACS, Chemunex AES Laboratoire, Bruz, France). Recombinant bacteria were selected by the addition of chloramphenicol (10 mg/L) except for E. coli harboring TOPO plasmid which was selected with kanamycin (50 mg/L).

TABLE 1 | Bacterial strains and plasmids used in this study.

fmicb-09-03075 December 20, 2018 Time: 17:25 # 3

aFor strains origin are given; for plasmids and cloned cassette characteristic are given. Ap, ampicillin; K, kanamycin; Cm, chloramphenicol.

General procedures for DNA manipulation were essentially performed as described previously (Sambrook et al., 1989). Plasmid DNA isolation was performed using the Qiagen Midiprep kit (Qiagen, Courtaboeuf, France). DNA sequences were confirmed by sequencing (Genewiz, Paris, France). Plasmid constructions were first established in either E. coli or L. lactis by electrotransformation (Sambrook et al., 1989) and then transferred into bifidobacteria. Electrocompetent B. bifidum protocols were adapted from Alvarez (Álvarez-Martín et al., 2008). Briefly, bifidobacteria were grown up to the optical density 0.6 at 600 nm (OD600nm) in MRS supplemented with 0.5 M of sucrose. Cultures were then put on ice for 20 min. After centrifugation for 15 min at 4700 rpm, 4◦C, pellets were washed three times with cold 0.5 M sucrose + 10% glycerol. This step allowed a 200-fold bifidobacteria concentration. Aliquots were incubated 1 h on ice, and then frozen at −80◦C. Cells (5 × 1010 colony-forming units, CFU/mL) were electroporated at 200 , 2.5 kV/cm, 25 µF (Bio-Rad, Marnes-la-Coquette, France) with 0.1–0.3 µg of DNA and resuspended in MRS for 3 h at 37◦C in anaerobic condition before plating on MRS agar plus chloramphenicol (10 mg/L).

#### Construction of the Bifidobacteria Expression SysTem (BEST)

For the construction of a vector allowing heterologous protein production in Bifidobacterium sp., we cloned dnaK promoter from a strain of B. longum into pLB141 vector (**Table 1**; Morello et al., 2008), a derivative of the broad-host range plasmid pWV01 (GenBank Accession Number: NC\_002192). Briefly, a DNA fragment (241 bp) containing dnaK promoter was amplified by PCR from genomic DNA of B. longum (GenBank Accession Number: NC\_004307.2) with primers: BglII-PdnaK (50 -CCA**AGATCT**CAAAAAACBTGAGCCWAMMWYRCTCA ACTT-30 ) for the coding strand and NheI-PdnaK (50 -**AGCT AGC**GTTAATAAAGCAAGGTTTATTTTTTTCATAACGTGTG CTCCTTAATTAYTCGTTTGTTCTTACGTTSTTYG-30 ) for the complementary strand (with B = C or G or T; W = A or T; M = A or C; Y = C or T; R = A or G). The PCR product was then subcloned into a pCR-TOPO plasmid (Invitrogen, Carlsbad, CA, United States) resulting in pCR-TOPO:dnaK (**Table 1**). Then, the DNA fragment containing dnaK promoter was recovered from pCR-TOPO:dnaK plasmid with BglII/NheI enzymes (New England Biolabs, Evry, France) and cloned into purified backbone isolated from BglII/NheI-cut pLB141 vector, resulting in pLB141:dnaK:Nuc. Nucleotide sequence analysis of this construction revealed a deletion in the SPExp4 (data not shown). To repair this, we performed a directed mutagenesis step by PCR. Briefly, we established pLB141:dnaK:Nuc vector in E. coli TG1 strain in order to purify large quantities of the plasmid, we then performed a first PCR reaction with forward primer: 50 -GTTATGAAA AAAATAAACCTTGCTTTATTAACGCTA-30 using Pfu turbo DNA polymerase (Promega). Parameters of PCR were a first cycle at 95◦C 1 min and 5 cycles at 95◦C 30 s, 55◦C 1 min, and 68◦C 4 min. Afterward, we performed a second PCR reaction on the first PCR product with forward and reverse primers: 50 - TAGCGTTAATAAAGCAAGGTTTATTTTTTTCATAAC-30 ). PCR parameters were as follow: first cycle 95◦C, 1 min; 20 cycles

at 95◦C 30 s/55◦C 1 min/68◦C 4 min; 4◦C. Then we performed one digestion with DpnI enzyme (New England Biolabs, Evry, France) before establishment of the plasmid into L. lactis. The final construction pBESTExp4:Nuc (**Figure 1** and **Table 1**) was confirmed by sequencing.

#### Expression of IL-10 Cytokine Using BEST System

For the expression of IL-10 using BEST system, a DNA fragment encoding for IL-10 mature sequence was obtained from a plasmid harboring murine il-10 gene (cassette SPUsp45:IL-10) under the control of PnisA promoter, also known as pLB270

plasmid (Motta et al., 2012; Benbouziane et al., 2013), with NsiI/SpeI enzymes (Thermo Scientific, Courtaboeuf, France) and cloned into pBESTExp4:Nuc vector digested with the same enzymes and replacing the corresponding nuc DNA fragment. The resulting vector pBESTExp4:IL-10 was established into B. bifidum.

In parallel and in order to determine the impact of SPExp4 (issued from L. lactis) for heterologous protein secretion in bifidobacteria, we replaced this SP with a new one issued from B. longum: SP of the hypothetical protein BL1181 from B. longum NCC2705 (Schell et al., 2002). Indeed, after in silico analysis (using SignalP software1 ) of homologous proteins efficiently secreted by bifidobacteria we put a particular interest in this SP since it was one with the highest score of "cleavage site." For the cloning: a DNA fragment containing the SP of BL1181 (SPBL1181) was PCR amplified from genomic DNA of B. longum strain (GenBank Accession Number: NC\_004307.2) with the following primers: EcoRV-SPBL1181 (50 -G**GATATC**AAGGAGATATAAAA ATGACGAACGTACGTGTGATCAAGCCCGC-30 ) for the coding strand and NsiI-SPBL1181 (50 -TG**ATGCAT**CTGCCTGGG CAGGCTGTGCCGAGCTGAACG-30 ) for the complementary strand. The PCR product (132 pb) was subcloned into a pCR-TOPO kit (Invitrogen, Carlsbad, CA, United States) resulting in pCR-TOPO:SPBL1181 (**Table 1**). Afterward, the DNA fragment containing SPBL1181 was obtained from this plasmid with EcoRV/NsiI enzymes (Thermo Scientific) and cloned into purified backbone isolated from bluntended-AflIII/NsiI-cut pBESTExp4:IL-10 vector. The resulting plasmid pBESTBL1181:IL-10 (**Figure 1**) was established into B. bifidum.

For IL-10 detection, bacteria were grown until exponential phase (OD600nm = 0.4–0.8) and the centrifuged culture resuspended (after one wash step with PBS) into the culture medium: MRS plus cysteine (500 mg/L) in order to apply different stresses: pH4, pH8, 42◦C, NaCl (25 g/L) or bile salts (100 or 500 mg/L, Sigma-Aldrich, Saint-Quentin-Fallavier, France). Induction was performed in anaerobic conditions during 30 min. Culture extraction was performed as followings. Protein samples were prepared from 2 ml of induced cultures. After centrifugation (5 min, 5000 rpm), the cell pellet (C) and supernatant (S) were treated separately. The S samples were treated by filtration on 0.25 µm filter then frozen at −20◦C. The C fraction was obtained after treatment in a PBS complemented with antiprotease 1× and lysozyme (100 mg/mL, Roche), at 37◦C during 1 h and then sonicated (6 × 10 sec with a 30 s of timeout on ice). IL-10 concentration in S and C was assessed by ELISA (eBioscience, Paris, France) and normalized by the final OD600nm.

For western blot experiments, the C fraction was obtained as described for ELISA detection. The S samples were also treated by filtration on 0.25 µm filter plus a 50-fold concentration by the addition and precipitation of 10% trichloroacetic acid (TCA) during 15 min on ice and then centrifuged. Pellets were resuspended in NaOH. Protein concentration was determined by Bradford assay. Ten micrograms of protein were mixed with the blue gel-loading buffer and loaded in pre-casted gel (Bio-Rad). Proteins were then transferred on PVDF membrane and then blocked overnight in 5% milk in TBST. Membranes were stained with a primary rabbit polyclonal antibody against murine IL-10 (Abcam, ab9735) and a second goat HRP-conjugated against Rabbit IgG (BI2407, Abliance). Blots were reveled using Bio-Rad Kit.

#### Detection of IL-10 in vivo

The two strains BS42:BESTExp4:IL-10 and BS42:BESTBL1181:IL-10 were administered by oral gavage to 6 weeks old IL-10-KO mice during 11 days. To mimic the context of our standard experiment in WT mice (**Figure 2**), animals received one intrarectal injection of a low dose of 2,4-DiNitroBenzene Sulfonic acid (DNBS, 2 mg/mouse), 7 days after the beginning of the gavage with bacteria. After sacrifice, colonic content and caecal content were collected. After being weighted, contents were diluted at 1/4 in PBS 150 mM pH7.2 with 1× anti-protease cocktail (Sigma-Aldrich, Saint-Quentin-Fallavier, France), and then placed 30 min at 4◦C under mechanic agitation. Supernatants were collected after centrifugation and IL-10 was measured using ELISA (BioLegend, San Diego, CA, United States).

#### DNBS-Induced Colitis Model

Male C57BL/6 mice (6 to 8-week-old) (Janvier, Le-Genest-Saint-Isle, France) were maintained at the animal care facilities of the National Institute of Agricultural Research (IERP, INRA, Jouy-en-Josas, France). Mice were housed under standard conditions for a minimum of 1 week before experimentation. All experiments were performed in accordance with European Community rules for animal care and were approved by COMETHEA C2EA-45 with authorization n ◦ 3445-2016010615159974.

For the induction of colitis, we used an adapted protocol from Martín (Martín et al., 2014; **Figure 2**) based on the intrarectal administration of DNBS. Briefly, mice (∼20 g) were anesthetized by intraperitoneal injection of 150 µL of 0.1% ketamine (Imalgene 1000, Merial) and 0.06% xylazine (Rompun). Then, a 4 cm-long catheter (Ecimed, Boissy-Saint-Léger, France) was attached to a tuberculin syringe, inserted into the colon and a dose of 100 mg/Kg of DNBS solution (Sigma-Aldrich, Saint-Quentin Fallavier, France) in 30% ethanol (EtOH) was injected intra-rectally to induce colitis in all group except control mice. Control mice (without colitis) received only 30% EtOH. Ten days after the so-called DNBS period, 1X109 CFU of bacteria in PBS or PBS alone were intragastrically administered daily for 10 days (gavage period). The inflammation was reactivated 21 days after the first DNBS injection (recovery period) with a second injection of 100 mg/Kg of DNBS solution. Weight loss was monitored daily to assess the severity of colitis. Experiment was performed twice and mice were treated as follow: with ETOH-PBS (control mice without colitis, n = 16), with DNBS-PBS (colitis control group, n = 20), with B. bifidum BS42 wild-type strain (WT-BS42, n = 16), with B. bifidum BS42 harboring pBESTExp4:IL-10 plasmid (BS42:BESTExp4:IL-10, n = 16), and with B. bifidum BS42 harboring pBESTBL1181:IL-10 plasmid (BS42:BESTBL1181:IL-10, n = 16).

1http://www.cbs.dtu.dk/services/SignalP/

### Macroscopic Damage Scores

Macroscopic scores were recorded in the abdominal cavity using previously described system (Wallace and Keenan, 1990). Briefly, the macroscopic criteria (assessed on a scale from 0 to 9) include macroscopic mucosal damages such as ulcers, thickening of the colon wall, the presence of adhesions between the colon and other intra-abdominal organs, the consistency of fecal material (as an indicator of diarrhea), and the presence of hyperemia in colon (increase of blood flow).

### Histology on Colonic Samples

Fragment of colon were fixed in Formalin then dehydrated with ethanol 70%, and paraffin-embedded. Section of 3 µmthick sections were stained with hematoxylin and eosin (H&E). As previously described (Cooper et al., 1993), colonic damage was scored according to architectural derangement, goblet cell depletion, edema/ulceration, and degree of inflammatory cell infiltration.

## Lipocalin-2 (LCN-2) Levels

Colonic contents were homogenized in FastPrep instrument with cold PBS. Supernatants were collected and stored at −80◦C until analysis. LCN-2 was measured using R&D ELISA kit (R&D System, Minneapolis, MN, United States) and following manufacturer instruction.

### Cytokine Production by CD3/CD28-Stimulated Splenocytes

The day of sacrifice, spleens were removed and placed in RPMI medium for culture (Breyner et al., 2017). Briefly, spleens were crushed and filtered through a 70 µm nylon filter (BD, Le Pont-de-Claix, France) and were resuspended in RPMI (Lonza, Levallois-Perret, France) completed with 100 Unit of Streptomycin, Penicillin and 10% Fetal Calf Serum (FCS). Red blood cells were removed with buffered Blood Cell Lysis Buffer (Sigma-Aldrich, France). Plates were pre-coated with anti-CD3 and anti-CD28 antibodies, 4 µg/ml of each antibody (eBioscience) in PBS with 0.5% FCS. Splenocytes cells were adjusted to 2 × 106 cells/ml per well and were cultured in 24-well plates at 37◦C in a 5% CO2 and 95% air atmosphere. Supernatants were collected after 48 h of culture and were stored at −80◦C until further analysis. Splenocytes culture supernatant levels of IL-4, IL-5 (Mabtech, Stockholm, Sweden), IL-6, IFN-γ (eBioscience), and IL-10 (BioLegend, San Diego, CA, United States) were quantified by ELISA, following manufacturer instructions.

### Statistical Analyses

Data were analyzed using GraphPad Prism (GraphPad Software Inc, San Diego, CA, United States) by non-parametric Kruskal– Wallis test followed by a Mann–Whitney test. A P-value of less than 0.05 was considered significant.

## RESULTS

### Development of a New Expression System to Produce and Deliver IL-10 Cytokine in Bifidobacterium bifidum

To obtain a functional expression plasmid allowing in vivo stressinducible heterologous protein expression in bifidobacteria, we first cloned the promoter from B. longum dnaK operon (Sanchez et al., 2005) into pLB270 plasmid (**Table 1**) resulting in pBESTExp4:Nuc vector, replacing PnisA promoter by PdnaK promoter (**Figure 1**). We chose this promoter, because dnaK operon encodes heat-shock proteins partially involved in the adaptation and survival of some bacteria (such as Bifidobacteria) in the gut (Sanchez et al., 2005). In addition, in this genus, transcription of heat-shock proteins (and more particularly DnaK), are greatly enhanced by exposure to both bile salts and different pH.

In order to validate the functionality of BEST system in vivo, nuc gene was replaced from this vector for murine IL-10 to obtain plasmid pBESTExp4:IL-10. The resulting vector, pBESTExp4:IL-10 (**Figure 1**), was established into B. bifidum BS42. As expected, no IL-10 was detected with the BS42 wild-type strain. As shown in **Figure 3**, B. bifidum BS42 harboring pBESTExp4:IL-10 plasmid

without any stress revealed IL-10 both in C and S samples reaching a maximal concentration of 2000 ng/L/OD. This phenomenon suggests a basal activity of the BEST system. To further explore if we can improve IL-10 secretion we replaced the SPExp4 which is issued from L. lactis by a new one issued from a B. longum (SPBL1181). Our results show that there was no significant difference in C samples between the two recombinant strains; however, the constitutive production of IL-10 was sevenfold higher in S samples (9283 ± 2981 ng/L/OD) prepared from recombinant BS42:BESTBL1181:IL-10 compared to BS42:BESTExp4:IL-10 strain: S (1377 ± 840 ng/L/OD) (p < 0.05) (**Figure 3**). Due to protein concentration and the sensitivity of western blot together with the fact that this cytokine is acid sensitive, only IL-10 produced by BS42:BESTBL1181:IL-10 in S samples was detected (**Supplementary Figure S1**). The in vitro production of IL-10 from recombinant B. bifidum BS42 harboring either pBESTExp4:IL-10 or pBESTBL1181:IL-10 plasmid was studied under 6 different stress conditions during 30 min and compared to non-induced cultures in order to validate the inducibility of the BEST system (**Figures 4A,B**).

Regardless of the stress conditions, IL-10 concentration was higher in S fraction from BS42:BESTBL1181:IL-10 than BS42:BESTExp4:IL-10 (p < 0.05, **Figure 4C**). Heat (42◦C), NaCl, and bile salts stresses did not induce a significant difference in IL-10 concentrations neither in C nor S samples in BS42:BESTBL1181:IL-10 strain compared to non-induced condition (**Figure 4A**). In contrast, compared to non-induced conditions, IL-10 concentrations were significantly lower in S fraction at pH4 (p < 0.05) or higher at pH8 (p = 0.05). IL-10 amount was much higher in S than in C under pH8 and bile salt stress conditions (p < 0.05). Concerning the analysis in BS42:BESTExp4:IL-10 strain (**Figure 4B**), nonetheless the stress condition, IL-10 levels were similar in both C and S samples even if IL-10 amount decreased in C at pH4 (p = 0.05). Finally, the two recombinant strains displayed a different IL-10 secretion profile.

### Detection of IL-10 Delivered in vivo in IL-10 KO Mice

Our IL-10 KO mice did not develop colitis: percentage of weight loss and macroscopic score were measured but no differences were observed (data not shown) between control and DNBS mice. Our results revealed that IL-10 could be detected in (i) ceacal samples of either 62.5% of mice (5/8) treated with BS42:BESTBL1181:IL-10 strain or 50% of mice (4/8) treated with BS42:BESTExp4:IL-10, with a mean level of 0.759 and 0.554 pg/g, respectively (**Supplementary Figure S2A**); and (ii) colonic content samples of either 50% of mice (4/8) treated with BS42:BESTBL1181:IL-10 strain or 25% of mice (2/8) treated with BS42:BESTExp4:IL-10, with a mean level of 0.280 and 0.097 pg/g, respectively (**Supplementary Figure S2B**). The presence of IL-10 in caecal and colonic content in IL-10 KO mice shows that the recombinant BS42:BESTBL1181:IL-10 and BS42:BESTExp4:IL-10 are able to bring IL-10 in vivo within the digestive tract.

#### Impact of IL-10 Delivery by Recombinant B. bifidum Using BEST System in a Low-Grade Inflammation Murine Model

In vivo delivery of IL-10 through administration of BS42:BESTBL1181:IL-10 and BS42:BESTExp4:IL-10 strains

of NaCl; SB100 = 100 mg/L of bile salts; SB500 500 mg/L of bile salts. IL-10 was measured with ELISA in Cell-pellet and filtered Supernatant. Data (means plus SEMs) are the result of four separate experiments. Comparison involved the non-parametric Mann–Whitney test. ∗p < 0.05.

were then studied in a murine model of low-grade intestinal inflammation in wild type mice (**Figure 2**). As shown in **Figure 5A**, compared to ethanol, DNBS-treatment induced a weight loss (90.6 ± 0.94% at day 31, p < 0.001) after the second rectal injection and the next 3 days. Mice treated with IL-10-producing bifidobacterial strains tended to lose less weight, 93.06 ± 1.02% and 92.5 ± 1.18% for BS42:BESTBL1181:IL-10 and BS42:BESTExp4:IL-10 groups, respectively, compared to the DNBS-PBS control group, (i.e., colitis control). Nevertheless, only the Ethanol-PBS group is significantly different compared to this colitis control group. The macroscopic score was significantly higher in the DNBS-PBS group compared to Ethanol-PBS group (p < 0.0001, **Figure 5B**). Mice treated with BS42 strain showed almost the same score as DNBS-PBS group. In contrast, BS42:BESTBL1181:IL-10 treatment significantly decreased this score (p < 0.05) and BS42:BESTExp4:IL-10 induced a non-significant slight decrease in macroscopic score.

LCN-2, an early marker of inflammation, was measured in colonic content at day 33 (**Figure 5C**). The level of LCN-2 was significantly higher in the DNBS-PBS group compared to negative control (p < 0.0001). LCN-2 level was significantly lower with BS42:BESTBL1181:IL-10 group (p < 0.05), and, although not significant, tended to be decreased following treatment with BS42 and BS42:BESTExp4:IL-10.

The histological score was significantly lower in BS42:BESTBL1181:IL-10 and BS42:BESTExp4:IL-10 groups (respectively p < 0.05 and p < 0.0001) than in the DNBS-PBS group (**Figures 5D,E**).

The impact on systemic T-helper cells balance was determined by measuring IL-4, IL-5, IL-6, IFN-γ, and IL-10 cytokines in supernatant from mice splenocytes after 48H of CD3/CD28 restimulation (**Figure 6**). For all cytokines, there were significant differences between the colitis control group and the negative control group, confirming the inflammatory status of DNBS-PBS group. The intake of BS42:BESTBL1181:IL-10 slightly decreased IL-5, IL-6, and IFN-γ (not significant). However, there was no effect on IL-4 and IL-10. The intake of BS42:BESTExp4:IL-10 as well as the BS42 tended to decrease the level of IL-5 and IFN- γ, with no effect on other cytokines.

#### DISCUSSION

In this work, we sought to construct an inducible expression system for bifidobacteria allowing in vivo delivery of proteins of health interest. For this, we developed a Bifidobacterium expression system (BEST) regulated by stress that we validated in B. bifidum. The functionality of BEST system was validated by cloning murine IL-10 cytokine and confirming both its correct expression and biological activity in vivo in a mouse model of low-grade intestinal inflammation. For the expression of IL-10 we confirm that the two recombinant B. bifidum strains were able to produce and secrete (even without any stress) the same IL-10 levels than those previously reported in recombinant bifidobacteria (Escogido et al., 2007; Khokhlova et al., 2010). For instance, a recombinant strain of B. longum was reported to produce 22 pg/ml of IL-10 (Escogido et al., 2007) whereas a recombinant strain of B. breve produced 1900 pg/ml of IL-10 (Khokhlova et al., 2010). In our study, we observed a constitutive production of IL-10 comparable or even greater (up to sevenfold) than those described with another plasmid, with an average yield of 1000 pg/ml with B. bifidum harboring pBESTExp4:IL-10 plasmid and reaching 7000 pg/ml with B. bifidum harboring pBESTSP1181:IL-10. Moreover, at pH 8 (a stress condition under basic pH), we observed an IL-10

overproduction reaching 24000 pg/ml. This pH, which could reflect the pH of pancreatic juice when it is spilled into the duodenum, models a condition that Bifidobacterium can shortly encounter following oral administration. It shows the inductivity of our system as, in the large intestine where they are mainly located, Bifidobacterium will encounter a combination of factors and stresses which is difficult to reproduce in vitro. It is also interesting to note that pH8 can also stabilize IL-10. Indeed,

it has been shown that at pH8.5, human IL-10 is under a highly stable dimer form (Syto et al., 1998). In the same study, the authors showed that IL-10 retained high activity between pH6 and 10. Below pH6.0, its activity decreased significantly and at pH2.5 only a small percent of IL-10 activity remained. A comparison analysis with commercial murine recombinant IL-10 allowed us to confirm that IL-10 produced by recombinant Bifidobacteria was stable in all the tested stresses conditions except at pH4 and lower (data not shown). In addition, pH4 can impact bifidobacteria growth. Except for some strains of B. thermacidophilus, bifidobacteria can survive (but not grow) at pH4.5–5 (Biavati and Mattarelli, 2015), this can explain that after 30 min at pH4, no IL-10 was detected in the culture medium.

To improve secretion, we included in the BEST system a new signal peptide issued from B. longum: SPBL1181. At pH8 (pancreatic juice), IL-10 secretion was 2.7-fold increased, confirming the interest of both SPBL1181 and PDnaK promoter. Concentrations of IL-10 in the C fraction were similar for our two recombinant BS42; however, a sevenfold higher concentration was observed in the S of BS42:BESTBL1181:IL-10 compared to that of BS42:BESTExp4:IL-10. SPExp4 allows only a small amount of IL-10 to be secreted compared to SPBL1181. This can be easily understandable by the bifidobacterial origin of this SP compared to SPExp4 which is issued from L. lactis. Thus, BEST system using SPBL1181 seems to be an efficient system to secrete heterologous protein in B. bifidum. In addition, this SP (issued from B. longum) could also work in other Bifidobacterium strains, including B. longum itself; and although preliminary data suggest that SP1181 is functional in B. longum and B. breve, this remains to be confirmed.

To assess IL-10 in vivo delivery by recombinant bifidobacteria, we used IL-10 KO mice model. In these mice, following oral administration of the recombinant strains, we detected IL-10 in ceacal and colonic content confirming the production of IL-10 in vivo. Of note, these young IL-10 KO mice do not develop colitis despite the administration of DNBS, so we assess the biological efficacy of these recombinant bacteria on a well characterized low-grade intestinal inflammation model (Martín et al., 2014; Allain et al., 2016).

Several studies have reported beneficial effects of different strains of B. bifidum in a murine model of acute colitis (Preising et al., 2010; Alard et al., 2018). Our B. bifidum BS42 seems to have no beneficial impact as demonstrated on the murine model of low-grade intestinal inflammation. In our experiments, BS42:BESTBL1181:IL-10 had a greater beneficial effect on gut inflammation with a significant

decrease in LCN-2 level than BS42. Despite not significant, BS42:BESTBL1181:IL-10 improved systemic inflammation as observed by the decrease in IL-6 and IFN-γ cytokines. Impact of BS42:BESTExp4:IL-10 was less obvious confirming that IL-10 secretion is an advantage. In a previous work Martín et al. (2014) tested a recombinant strain of L. lactis producing murine IL-10 in the same model of low-grade intestinal inflammation and using the same dose of bacteria (i.e., 1 × 109 CFU/mouse) for the same administration period (11 days). If we compare the efficacy between the two recombinant strains, the effects of our BS42:BESTBL1181:IL-10 were more efficient regarding the weight evolution, the macroscopic and histologic scores and LCN-2 levels. In contrast, IL-10-producing L. lactis had a better effect on gut permeability and in cytokines levels. Despite each strain display its intrinsic properties, in the two cases, the presence of IL-10 in the intestine has beneficial effect on the low-grade intestinal inflammation model. It will be interesting to test in a near future a combination of these two recombinant strains producing IL-10 in different murine models of either IBS or IBD. Indeed, the ecological niche of L. lactis is the small intestine whereas for Bifidobacterium it is the colon and we can thus expect a synergic beneficial effect.

#### CONCLUSION

In conclusion, we succeeded in the construction of a new Bifidobacteria Expression SysTem (BEST) that allowed in vivo IL-10 delivery by B. bifidum. Furthermore, the use of recombinant B. bifidum BS42 secreting IL-10 cytokine in a model of lowgrade intestinal inflammation showed its ability to decrease local inflammation and confirmed therefore its potential for delivery of therapeutic molecules in the colon. Despite we consider our expression system is promising, more studies are needed, such as evaluate either daily or weekly bacteria administration which will allow us to determine the advantages of the higher persistency of

#### REFERENCES

this genus versus other recombinant bacteria (e.g., lactococci and lactobacilli).

#### AUTHOR CONTRIBUTIONS

LB-H and A-JW designed the study. AF performed the cloning. AM performed the in vitro characterization of IL-10 production. AM, FC, PR, and A-JW performed the animal experiments. AM performed all dosages. BR read the histological score. SG provided the analytical tools. AM, LB-H, and A-JW interpreted the data and wrote the manuscript. PL and M-JB provided the critical feedback, and helped to shape the research, analysis, and manuscript.

#### ACKNOWLEDGMENTS

The authors would like to thank Laurie Watterlot, Thomas Larcher, and Jasmine Sridar for their involvement in the recombinant bacteria construction. The authors also thank the members of the IERP animal facilities from INRA, Jouy-en-Josas, for their assistance in mouse care and experiments.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2018.03075/full#supplementary-material

FIGURE S1 | Western blot analyses of IL-10 in the C and S sample following or not 42◦C induction.

FIGURE S2 | In vivo production of IL-10. IL-10 KO-mice received daily BS42:BESTExp4:IL-10 or BS42:BESTBL1181:IL-10, during 11 days by oral gavage. IL-10 were then measured in (A) caecal content and (B) colonic content by ELISA.

use of the term probiotic. Nat. Rev. Gastroenterol. Hepatol. 11, 506–514. doi: 10.1038/nrgastro.2014.66

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Mauras, Chain, Faucheux, Ruffié, Gontier, Ryffel, Butel, Langella, Bermúdez-Humarán and Waligora-Dupriet. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Engineering Components of the Lactobacillus S-Layer for Biotherapeutic Applications

Courtney Klotz1,2 and Rodolphe Barrangou1,2 \*

1 Genomic Sciences Graduate Program, North Carolina State University, Raleigh, NC, United States, 2 Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC, United States

Lactic acid bacteria (LAB) are frequently harnessed for the delivery of biomolecules to mucosal tissues. Several species of Lactobacillus are commonly employed for this task, of which a subset are known to possess surface-layers (S-layers). S-layers are two-dimensional crystalline arrays of repeating proteinaceous subunits that form the outermost coating of many prokaryotic cell envelopes. Their periodicity and abundance have made them a target for numerous biotechnological applications. In the following review, we examine the multi-faceted S-layer protein (Slp), and its use in both heterologous protein expression systems and mucosal vaccine delivery frameworks, through its diverse genetic components: the strong native promoter, capable of synthesizing as many as 500 Slp subunits per second; the signal peptide that stimulates robust secretion of recombinant proteins; and the structural domains, which can be harnessed for both cell surface display of foreign peptides or adhesion enhancement of a host bacterium. Although numerous studies have established vaccine platforms based on one or more components of the Lactobacillus S-layer, this area of research still remains largely in its infancy, thus this review is meant to not only highlight past works, but also advocate for the future usage of Slps in biotherapeutic research.

#### Edited by:

Aleš Berlec, Jožef Stefan Institute, Slovenia

#### Reviewed by:

Akinobu Kajikawa, Tokyo University of Agriculture, Japan Olivia Cano Garrido, Centre for Research in Agricultural Genomics (CRAG), Spain

> \*Correspondence: Rodolphe Barrangou<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Infectious Diseases, a section of the journal Frontiers in Microbiology

Received: 05 June 2018 Accepted: 05 September 2018 Published: 02 October 2018

#### Citation:

Klotz C and Barrangou R (2018) Engineering Components of the Lactobacillus S-Layer for Biotherapeutic Applications. Front. Microbiol. 9:2264. doi: 10.3389/fmicb.2018.02264 Keywords: Lactobacillus, probiotic, S-layer, biotherapeutic, mucosal vaccine, CRISPR

## INTRODUCTION

Lactic acid bacteria (LAB) are Gram-positive, anaerobic or microaerophilic, non-sporulating microorganisms that inhabit diverse environments including milk and plant surfaces, as well as the mouth, gastrointestinal tract, and vaginal tract of humans and animals (Liu et al., 2014). Traditionally, they have been employed for food and dairy fermentations, but have more recently garnered attention for their health-promoting properties with many species used widely as probiotics (Klein et al., 1998; Klaenhammer et al., 2005). Recombinant LABs are frequently harnessed for mucosal delivery of biomolecules such as therapeutic proteins or vaccine antigens (Berlec et al., 2012). In comparison to traditional intravenous or intramuscular vaccine administration, the mucosal route enables immunizations to be performed orally, reducing potential side effects while increasing specificity for chronic illnesses and infections associated with mucosal tissues (Bermudez-Humaran et al., 2011;

Wyszynska et al., 2015). Furthermore, many LABs are bile and acid tolerant, act as natural adjuvants, and interact with cells of the immune system (Wells and Mercenier, 2008), making them ideal candidates for antigen carriage.

The LABs most frequently chosen for vaccine delivery are Lactococcus lactis and select species of the Lactobacillus genus (Wells and Mercenier, 2008; Bermudez-Humaran et al., 2011, 2013; Wyszynska et al., 2015). However, unlike L. lactis, several species Lactobacillus have been shown to possess surface-layers (S-layers) (Hynonen and Palva, 2013). S-layers have been detected on both Gram-positive and Gram-negative bacteria and are nearly ubiquitous in archaea (Fagan and Fairweather, 2014). They are defined as two-dimensional crystalline arrays composed of repeating proteinaceous subunits that constitute the outermost layer of a cell envelope (Fagan and Fairweather, 2014). The S-layer proteins (Slps) attach to the underlying peptidoglycan via electrostatic interactions and possess inherent, entropy-driven affinities to self-assemble with each other (Hynonen and Palva, 2013). Thus far, S-layers have been characterized for their role in maintaining cell shape; acting as molecular sieves; serving as binding sites for large molecules, ions, or phages; and mediating surface adhesion (Sleytr et al., 2014). Additionally, Slps are some of the most abundant proteins synthesized by the cell, making them metabolically expensive but also underscoring their importance to the organism (Sara and Sleytr, 2000; Fagan and Fairweather, 2014). Their high expression, periodicity, and self-assembling properties have made them a target for numerous applications in biotechnology and nanotechnology (Avall-Jaaskelainen and Palva, 2005; Hynonen and Palva, 2013; Sleytr et al., 2014).

In the following review, we examine Slp applications in recombinant protein expression and biotherapeutic delivery via their distinct genetic building blocks: the strong native promoter, which can synthesize as many as 500 Slp subunits per second; the signal peptide, that can trigger robust secretion of target molecules; and the structural domains, which can be harnessed for both cell surface display of heterologous proteins or enhancement of host adhesion (**Figure 1A**). Despite the existence of several recombinant protein expression systems based on one or more components of the Lactobacillus S-layer, this area of research still remains largely underexploited. Thus, the purpose of this review is to not only shed light on past S-layer studies, but also to advocate for future utilization of Slps in mucosal vaccine and biotherapeutic delivery research.

#### HARNESSING THE STRONG NATIVE PROMOTER

The bacterial S-layer array is composed of an estimated 5 × 105 subunits, representing 10–15% of the total protein content of the bacterium (Sleytr and Messner, 1983; Avall-Jaaskelainen and Palva, 2005). During exponential growth phase, approximately 500 subunits per second must be synthesized, translocated to the cell surface, and incorporated into the existing lattice structure (Sleytr et al., 2014). In order to obtain these high levels of transcription, S-layer genes possess strong, efficient promoters, which can be harnessed for protein production systems (**Figure 1B**). Within the Lactobacillus genus, this research has predominately been limited to slp promoters of Lactobacillus acidophilus and Lactobacillus brevis.

Several studies have investigated the versatility of Lactobacillus S-layer promoters for driving heterologous protein expression in various LAB hosts. Lactobacilli regularly possess multiple slp genes within the same strain that are not all concurrently active (Hynonen and Palva, 2013). Although SlpA is the major constituent of the L. acidophilus S-layer, it can be moved to an inactive position triggering expression of the once silent SlpB (Boot et al., 1996a; Konstantinov et al., 2008). When the L. acidophilus ATCC 4356 slpA and slpB promoters were evaluated in Lactobacillus casei ATCC 393, only slpA remained active under all tested growth phases (Boot et al., 1996b). However, the same slpA promoter, although highly efficient in L. lactis, was nearly inactive in isolates of Lactobacillus reuteri (Lizier et al., 2010). The L. acidophilus NCFM slpA promoter was used to drive expression of a green fluorescent protein (GFP) in shuttle vectors based on oriV1 and oriV2 replicons (Chen et al., 2014). Similarly, the plasmids exhibited distinct properties based on which strain they were ported into as well as growth-phase-dependent effects. Both plasmids were capable of replicating in strains of L. casei and Lactobacillus delbrueckii, but only the oriV1 plasmid, pEL5.6, could replicate in Lactobacillus paracasei (Chen et al., 2014). The functionality of the L. brevis ATCC 8287 slpA promoter was evaluated in three LAB hosts: L. lactis MG1614, Lactobacillus plantarum NCDO1193 and Lactobacillus gasseri NCK334, via the expression of various reporter genes (Kahala and Palva, 1999). The S-layer promoter was recognized in each strain, but was particularly active in L. lactis and L. plantarum. In fact, aminopeptidase N (PepN) reporter activity within L. plantarum was 30-fold higher compared to the Lactobacillus helveticus PepN native host and composed a staggering 28% of the total cellular protein during late exponential growth phase. In summary, Lactobacillus slp promoters are effective tools for driving recombinant protein expression, but optimization based on host and growth conditions is essential.

The S-layer promoter has also been harnessed for reporter expression in situ. Plasmid instability and antibiotic markers can complicate the use of these constructs when moving into human clinical trials or to market, thus chromosomal insertions have gained immense popularity. For L. acidophilus NCFM, a pORI-based upp counterselective gene replacement system (Goh et al., 2009) has considerably aided this effort (see "Engineering Platforms"). Originally intended for knockout characterizations, it was first employed for a knock-in by cloning a β-glucuronidase (gusA) reporter downstream of the slpA gene (Douglas and Klaenhammer, 2011). The resulting mutant exhibited a three log increase in GusA activity in comparison to the gusA-negative parent, and the study established a framework for the exploitation of highly expressed genomic regions for heterologous protein production (Douglas and Klaenhammer, 2011). Although this particular system was never evaluated within the context of

vaccine delivery, the technique was shown effective for expressing antigens using an alternative highly expressed region within the L. acidophilus genome (O'Flaherty and Klaenhammer, 2016).

Moving beyond reporter genes and targeting more specific therapeutics, the L. brevis JCM 1559 slpA promoter was evaluated in an L. casei IGM393 host via incorporation into a mouse interleukin 10 (IL-10) secretion system (Kajikawa et al., 2010). Administration of IL-10 was previously shown to be an effective treatment of murine colitis when delivered via recombinant L. lactis (Steidler et al., 2000). The authors sought to improve upon this design by substituting in a strain hypothesized to be better adapted to mammalian body temperature. The delivery system was successful in that it yielded high levels of IL-10 secretion when cloned into L. casei, but accumulation of the protein varied widely based on pH (Kajikawa et al., 2010). Maximum efficiency occurred at pH 8.0 and dropped drastically as the pH became more acidic. Although the authors attributed low levels of the protein to its physical characteristics (Kajikawa et al., 2010), the selection of a promoter from a free-living species (e.g., L. brevis) over a low pH tolerating, vertebrate-adapted organism (e.g., L. acidophilus), may have also contributed (Duar et al., 2017). Nonetheless, the S-layer promoter has repeatedly demonstrated its utility for driving protein production systems, and thus merits future research with a focus on therapeutic molecule delivery.

#### EXPLOITATION OF THE SECRETION SIGNAL PEPTIDE

All Lactobacillus S-layer proteins characterized thus far are preceded by a 25–32 amino acid signal peptide sequence indicative of secretion through the general secretory pathway (Hynonen and Palva, 2013). By cloning this short sequence upstream of an antigen or biotherapeutic molecule, one can obtain robust secretion of a target protein (**Figure 1C**), thus this peptide is frequently harnessed for biotechnological applications.

The L. brevis ATCC 8287 slpA signal sequence, along with the promoter and transcription terminator, were used to drive expression and secretion of the Escherichia coli β-lactamase (bla) reporter gene in L. lactis, L. brevis, L. plantarum, L. gasseri, and L. casei, using a low-copy-number plasmid derived from pGK12 (Savijoki et al., 1997). In all hosts

tested, Bla was expressed and released into the culture medium, though highest yields were obtained by recombinant L. lactis and the strain of L. brevis from which the SlpA components were derived. Production of Bla was mainly restricted to exponential growth phase (Savijoki et al., 1997). Since the system was under control of an slpA promoter, it was unsurprising that efficiency was host-specific and growth-phase-dependent (see "Harnessing the strong native promoter").

The Slp secretion signal has frequently been harnessed for targeted therapeutic applications. In a study seeking to develop a recombinant L. lactis mucosal vaccine against porcine postweaning diarrhea and edema disease, signal peptides from the L. lactis major extracellular protein, Usp45, and L. brevis SlpA, were used to stimulate the secretion of an E. coli F18 fimbrial adhesion protein (FedF) fused to a proteinase PrtP cell wall anchor (Lindholm et al., 2004). Both expression systems induced secretion of all tested FedF-PrtP fusions, however, the quantity of fusion proteins found in the culture medium was four to sixfold higher in those containing the SlpA signal peptide. This was a compelling find considering that the Usp45 signal sequence, previously shown to be one of the most effective L. lactis secretion signals (Nouaille et al., 2003), was outperformed by the heterologous SlpA signal sequence. A similar result was obtained with the human interferon alpha 2b (hIFNα-2b) gene (Zhang et al., 2010), used worldwide for the treatment of diseases such as hepatitis B and C (Chelbi-Alix and Wietzerbin, 2007). The addition of the L. brevis SlpA signal peptide increased the secretion efficiency threefold in comparison to the lactococcal Usp45 signal sequence (Zhang et al., 2010).

The SlpA signal sequence has also been employed for secretion of chromosomally inserted heterologous proteins. The Bacillus anthracis protective antigen (PA), fused to a dendritic cell (DC)-targeting peptide, was previously shown to induce protective immunity when delivered on a plasmid via L. acidophilus NCFM (Mohamadzadeh et al., 2009) and L. gasseri (Mohamadzadeh et al., 2010). Since a major advantage of using recombinant microbes for vaccine delivery is their ability to express multiple antigens (Wells and Mercenier, 2008), the B. anthracis PA was co-expressed with the Clostridium botulinum Serotype A neurotoxin heavy-chain antigen (O'Flaherty and Klaenhammer, 2016). The C. botulinum vaccine cassette, which utilizes an L. acidophilus SlpA secretion signal, was chromosomally inserted downstream of the highly expressed enolase gene. Western blot analysis and RNA sequencing confirmed expression of the two antigens (O'Flaherty and Klaenhammer, 2016). Although this strain was never evaluated in vivo, a similarly, constructed strain, also utilizing the SlpA secretion signal, but carrying only the C. botulinum antigen (O'Flaherty and Klaenhammer, 2016), was used to vaccinate mice but unable to confer complete protection (Sahay et al., 2018). Rather, vaccine efficacy was enhanced by intradermal injection of the purified immunogenic subunit, which elicited robust memory B cell responses and rendered mice resistant to lethal doses of botulinum neurotoxin serotype A (Sahay et al., 2018).

The Slp signal sequence is capable of stimulating ample secretion of target molecules, prompting its continued use in vaccine research platforms. Although antigen secretion by recombinant LABs has been shown effective for treating disease (Mohamadzadeh et al., 2005, 2009, 2010), exposure to proteolytic enzymes, low pH, and bile salts may encourage protein degradation and therefore decreased functionality (Wells and Mercenier, 2008); consequently cell wall anchoring has become a popular alternative. However, surface display is a balancing act. High exposure of a protein implies optimal host interaction, but also increased susceptibly to degradation. Alternatively, low exposure, via compact protein folding or embedment within the cell wall, confers protection in exchange for diminished efficacy (Michon et al., 2016). Several S-layer-mediated surface display approaches have attempted to resolve this relationship.

### THE SELF-ASSEMBLING AND ANCHORING DOMAINS OF THE Slp

Cell wall anchoring via fusion to Slp structural domains (**Figure 1D**) enables direct interaction of target peptides with host mucosal tissues while simultaneously protecting them from degradation (Bermudez-Humaran et al., 2011; Wang et al., 2016). The first S-layer-mediated display of a foreign epitope was generated by fusing the S-layer homology (SLH) domain of B. anthracis to the normally secreted levansucrase of Bacillus subtilis (Mesnage et al., 1999). The surface-exposed levansucrase retained its enzymatic and antigenic properties, prompting a new area of research which exploits the anchoring abilities of the Slp for cell surface presentation. Lactobacillus Slps do not possess SLH domains and are instead composed of two structural domains: a variable terminal for monomer self-assembly (N- or C- depending on the species), and a highly conserved peptidoglycan anchor (Smit et al., 2001; Hynonen and Palva, 2013).

Several studies have used S-layer-mediated anchoring for recombinant protein display on S-layer-deficient bacteria. The cell wall binding domain of the L. crispatus F5.7 Slp (LbsA) in conjunction with the Slp promoter and secretion signal, were used for surface display of a GFP on several Lactobacillus chicken isolates (Mota et al., 2006). The intention was to generate a vaccine delivery framework for the immunization of broilers against infectious diseases, but never evolved past proof of concept. Vaccination via recombinant bacteria is a particularly attractive option for livestock and poultry operations since the lyophilized microorganisms can be blended into feed; a process that is easily scaled up (Wang et al., 2016). For human applications, the complete SlpA from L. acidophilus ATCC 4356 was fused to a GFP reporter for external presentation on a plasmid-cured, lactose-deficient derivative of L. casei ATCC 334 (Qin et al., 2014). The authors were able to develop a food-grade cell surface display vector by substituting lactose metabolism genes in place of antibiotic selection markers and verified gastrointestinal stability via in vitro modeling (Qin et al., 2014). The L. crispatus K2-4-3 SlpB C-terminal domain,

defense (Cascade) and the signature Cas3 nuclease (Barrangou, 2015).

LcsB, was employed for the display of not only a GFP reporter (Hu et al., 2011), but also a carcinoembryonic antigen (CEA) (Zhang et al., 2016). Previous studies have shown that CEA is capable of eliciting strong T-cell and humoral immune responses, which can hinder tumor growth (Greiner et al., 2002). Oral administration of recombinant CEA-presenting L. lactis to mice yielded significantly higher levels of CEA-specific secretory IgA and a higher spleen index in comparison to CEA antigen alone or the negative control, demonstrating the potential of L. lactis CEA as a cancer vaccine (Greiner et al., 2002).

Since the S-layer of L. crispatus is capable of binding intestinal extracellular matrices such as collagen and laminin, heterologous expression of Slps has also been used to improve the adhesive capacity of host organisms. For example, the L. crispatus JCM5810 collagen-binding S-layer protein, CbsA (Martinez et al., 2000), as well as its individual domains (Antikainen et al., 2002),

were expressed on the surface of L. casei enabling recombinant organisms to bind various extracellular matrices. Similarly, a surface display cassette consisting of the L. brevis ATCC 8287 SlpA receptor-binding domain fused to a PrtP spacer enhanced L. lactis adherence to Intestine 407 cells (Avall-Jaaskelainen et al., 2003). Both approaches were able to significantly increase the adhesive capacity of engineered organisms, but yet to be evaluated is the probable synergistic effect of combining improved adhesion with an S-layer anchored antigen in what could potentially be a potent vaccine design platform.

Unlike much of the work presented in this review, a handful of studies achieved Slp-mediated display of foreign proteins without DNA manipulation in the host by exploiting the inherent ability of Slps to anchor and self-assemble. For instance, the L. acidophilus C-terminal anchor (SAC) was attached to a GFP reporter, then produced and purified in E. coli. The SAC-GFP fusion protein was capable of binding lithium chloridepretreated surfaces of wild-type L. acidophilus, L. helveticus, and L. crispatus (Smit et al., 2001). Similarly, L. crispatus K2-4-3 LcsB-GFP fusions were able to associate with SDS-pretreated surfaces of various S-layer-forming LABs including L. brevis, L. helveticus, L. crispatus, and Lactobacillus salivarius as well as several non-S-layer-formers, including L. lactis, L. delbrueckii, Lactobacillus johnsonii, and Streptococcus thermophilus (Hu et al., 2011). Interestingly, neither the SAC-GFP fusion nor LcsB-GFP were able to bind the surface of L. casei (Smit et al., 2001; Hu et al., 2011). This approach is unique in that it offers an alternative way to deliver foreign proteins while also circumventing the GMO (genetically modified organism) label, but is limited by its inability to generate additional heterologous protein in vivo and susceptibility to replacement by wild type Slps. However, in general, studies using heterologously expressed Slps and Slp anchors are hindered by inadequate secretion across the cell wall (Hu et al., 2011) or inability to form an array due to irregular folding and/or lack of cell surface exposure (Martinez et al., 2000). Consequently, rather than tease apart the efficient Slp secretion and display system, there is now interest in harnessing it as a whole through the direct insertion of foreign peptides within the context of the protein.

#### DELIVERY VIA DIRECT INTEGRATION WITHIN THE Slp

The extraordinarily high, stable abundance of the Slp, makes it an enticing target for antigen display and delivery via direct integration into its genome sequence (**Figure 1E**). The presentation of an exogenous protein within the context of the Slp was first achieved in Caulobacter crescentus through the random introduction of a pilin peptide from Pseudomonas aeruginosa strain K. Eleven potential sites of successful insertion were identified, demonstrating for the first time the capacity of the Slp to act as a carrier for foreign epitopes (Bingle et al., 1997).

Many subsequent studies have focused on mapping the S-layer to gain insight into ideal positioning for novel insertions, including in Lactobacillus. In L. acidophilus ATCC 4356, peptides ranging from 7 to 13 amino acids were randomly introduced into the Slp (Smit et al., 2002). Within the variable N-terminal (SAN), five of these positions maintained paracrystalline structure formation in vitro, while four others resulted in the complete abolishment of any array-forming capacity. Unsurprisingly, an insertion into the cell wall-binding domain had no effect on assembly (Smit et al., 2002). Similarly, the L. brevis SlpA was mapped via cysteine-scanning mutagenesis combined with sulfhydryl modification to identify locations of high surface accessibility and verify that the mutations did not alter selfassembly properties (Vilen et al., 2009). Combined, these works established several stable, surface-accessible insertion sites within the Lactobacillus Slp, yet few researchers have capitalized on this knowledge.

Currently, only two studies have successfully integrated antigens within the context of the Lactobacillus Slp. Through an inducible expression system, the poliovirus VP1 epitope was evaluated in four potential L. brevis ATCC 8287 slpA insertion sites (Avall-Jaaskelainen et al., 2002). The location that demonstrated the best surface expression was then targeted for chromosomal insertion of the c-Myc epitope via direct double-crossover integration (Avall-Jaaskelainen et al., 2002). A uniformly chimeric S-layer was obtained without any effect on array formation. More recently, the membrane proximal external region (MPER) epitope from human immunodeficiency virus type 1 (HIV-1) was inserted into L. acidophilus NCFM SlpA (Kajikawa et al., 2015). L. acidophilus NCFM is regularly employed for mucosal vaccine delivery due in part to its direct interactions with the dendritic cell-specific antigen DC-SIGN (Konstantinov et al., 2008) and adaptation to the harsh conditions associated with gastric transit (Sanders and Klaenhammer, 2001). Vaccination via the recombinant organism, in conjunction with an IL-1β adjuvant, successfully stimulated MPER-specific antibody production in both the serum and mucosal secretions of mice (Kajikawa et al., 2015). This study marks the first and only instance of an Slp-integrated antigen being evaluated in vivo.

The establishment of a uniformly chimeric S-layer translates to approximately 105 instances of epitope display on the surface of a single bacterium (Sleytr and Messner, 1983). Despite these considerable numbers, insert size is exceptionally limited in order to preserve S-layer array formation (Smit et al., 2002). Currently, peptides longer than 19 amino acids are unable to be inserted into SlpA without disrupting the lattice structure (Kajikawa et al., 2015). Therefore, alternative methods exploiting auxiliary proteins associated with the S-layer are now being investigated for the display and delivery of larger antigens, as seen in Slp-mediated anchoring studies (see "The self-assembling and anchoring domains of the Slp"), but at frequencies more akin to direct integration.

#### S-LAYER ASSOCIATED PROTEINS

The Lactobacillus S-layer, once thought to be solely composed of repeating monomeric Slp subunits, is actually far more

complex (Johnson et al., 2013, 2015). It is now widely accepted that S-layers can act as scaffolds for the external display of numerous auxiliary proteins, termed S-layer associated proteins (SLAPs), which can confer additional physiological functionalities (Johnson et al., 2013, 2015, 2017; Hymes et al., 2016; Johnson and Klaenhammer, 2016; Celebioglu and Svensson, 2017). Recently, the SLAP profile of L. acidophilus NCFM was quantified via multiplexing mass spectrometry (Klotz et al., 2017). Although results revealed significant growth stage-dependent alterations, they also highlighted several proteins with consistent high expression in both logarithmic and stationary growth phases (Klotz et al., 2017). The surface location and abundance of these proteins make them excellent targets for biotherapeutic delivery. Unlike Slp integrants, SLAP fusions are theoretically less limited in epitope size as they are not prone to S-layer array disruption (**Figure 1F**). Both the native SLAP promoter and secretion signal can be harnessed for this process with the intent to maintain high expression, secretion, and surface localization coupled with the display and delivery of a significantly larger and therefore more potent epitope.

#### TABLE 1 | Continued

#### Slp ENGINEERING PLATFORMS

Presently, the two most popular techniques for engineering LABs are the NICE (NIsin Controlled gene Expression) system in L. lactis and the pORI-based upp counterselective gene replacement system in L. acidophilus. There is also growing interest surrounding the use of CRISPR-Cas technology, though its application in bacterial genome editing remains relatively underrepresented (Selle and Barrangou, 2015; Hidalgo-Cantabrana et al., 2017).

#### The NICE System

The NICE system uses nisin to drive heterologous protein expression in L. lactis (Kuipers et al., 1995). Through the insertion of signal transduction genes from a nisin gene cluster into a nisin-negative L. lactis strain, NZ9000 was created (Kuipers et al., 1998). Subsequently, when a gene of interest is inserted downstream of the inducible nisA promoter, expression of that gene can be obtained by the addition of nisin to the culture medium (Mierau and Kleerebezem, 2005). Since its conception, NICE has become one of the most successful and widely used expression systems in Grampositive bacteria (Mierau and Kleerebezem, 2005). Indeed, the NICE system was even employed for the production a number of the S-layer fusion proteins mentioned above (Hu et al., 2011; Zhang et al., 2016) and to render the non-adhesive L. lactis NZ9000 adhesive via the addition of an L. brevis SlpA receptor-binding domain (Avall-Jaaskelainen et al., 2003). The availability of an easily engineered, non-S-layer-forming organism, has greatly accelerated not only our understanding of the biological role of an S-layer but also how we can exploit it.

### The pORI-Based upp System

fmicb-09-02264 September 29, 2018 Time: 16:39 # 9

The establishment of the pORI-based upp counterselective gene replacement system in S-layer-former L. acidophilus NCFM, first employed for the functional characterization of SlpX (Goh et al., 2009), has since become an invaluable tool for S-layer component engineering. The system uses a upp-encoded uracil phosphoribosyltransferase (UPRTase) as a counterselection marker to positively select for double crossover homologous recombination events. The method has been adapted for numerous Slp studies ranging from reporter integration (Douglas and Klaenhammer, 2011), anchoring/adjuvant assessment (Kajikawa et al., 2011), and targeted antigen delivery systems for disease protection (Kajikawa et al., 2015; O'Flaherty and Klaenhammer, 2016). Similar counterselective systems have also been developed in non-S-layer-formers including L. gasseri ATCC 33323 (Selle et al., 2014) and L. casei ATCC 393 (Song et al., 2014), but have yet to be harnessed for Slp analyses. In general, the technique remains a superior approach for characterizing the functional genetics of lactobacilli without the additional pressures required for plasmid maintenance.

#### CRISPR

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) together with CRISPR-associated (Cas) proteins, form the prokaryotic adaptive immune system which provides DNAencoded, RNA-guided, sequence-specific protection against viral invaders (Barrangou and Doudna, 2016). The current classification system uses two broad classes consisting of six major types (I–VI) which can be allocated into approximately 30 subtypes (Klompe and Sternberg, 2018). The simplest of these is the Type II system which relies on the activity of a singular Cas9 endonuclease and has gained immense popularity for its ability to be repurposed for genome editing (Chylinski et al., 2014; Barrangou and Doudna, 2016; Klompe and Sternberg, 2018). Depending on the organism, this can be done via harnessing of the endogenous system or supplying an exogenous Cas9:single guide RNA (sgRNA) complex. To date, no CRISPRbased genome editing has been performed in S-layer-forming lactobacilli, but it has been conducted in L. casei (Song et al., 2017) and L. lactis (Berlec et al., 2018), frequent hosts for recombinant Slp investigative research. Nevertheless, the development and delivery of a functional CRISPR-Cas9 plasmid remains a critical need, particularly in organisms such as L. acidophilus NCFM (depicted in **Figure 2A**), which does not possess an active CRISPR system (Crawley et al., 2018), but has proven to be hugely impactful in both probiotic and Slp research.

Despite the popularity of Type II engineering, Type I systems have been harnessed for genome editing in Clostridium and archaea (Pyne et al., 2016; Cheng et al., 2017) and transcriptional regulation in E. coli (Luo et al., 2015). S-layer formers, L. crispatus, and L. helveticus, frequently possess Type I systems (Crawley et al., 2018) and are also of interest for vaccine delivery. L. crispatus is a natural inhabitant of the human vaginal tract (Lepargneur, 2016), rendering it suitable for delivery of antigens targeting sexually transmitted diseases, such as HIV, while L. helveticus is predominately associated with dairy (Taverniti and Guglielmetti, 2012), which could be advantageous when considering modes of delivery and stability of the vaccine. A proposed strategy for Type I genome editing in L. crispatus is illustrated in **Figure 2B**. Harnessing of the endogenous system or delivery of a functional exogenous system both possess the potential to be powerful tools for advancing Slp-mediated biotherapeutic research.

The NICE and pORI-based upp systems have both proven effective for S-layer-mediated biotherapeutic delivery; however, next-generation genome editing tools, such as CRISPR-Cas, hold tremendous potential for bacterial engineering overall (Selle and Barrangou, 2015; Hidalgo-Cantabrana et al., 2017). Many S-layer-forming lactobacilli possess endogenous CRISPR systems, making them promising candidates for future S-layer engineering studies. Alternatively, delivery of a functional CRISPR to strains devoid of a system, will also greatly accelerate the pace at which recombinant organisms can be generated. Ironically, despite CRISPR originating as the bacterial adaptive immune system, CRISPR-based bacterial genome editing still remains relatively underexploited, though recent studies have provided valuable insights for its widespread future implementation (Selle and Barrangou, 2015).

### FUTURE DIRECTIONS

The Slp is a multi-faceted engineering target with both biotherapeutic and biotechnological applications (Sleytr et al., 2014). However, within the Lactobacillus genus, harnessing of this protein remains early in its development (Avall-Jaaskelainen and Palva, 2005; Hynonen and Palva, 2013); nonetheless, the research highlighted above (summarized in **Table 1**) advocates for its continued pursuit. The slp promoter and signal peptide are undoubtedly adept at driving robust expression and secretion of target proteins, while the structural domains have successfully displayed foreign epitopes and improved the adhesive capacity of host cells. More complex and novel display strategies, such as direct integration into the Slp or SLAP fusions, are innovative approaches for cell surface presentation that also exploit the inherent properties of S-layer-forming lactobacilli. In general, eliciting consistent immune responses via the mucosal route of administration is hindered by rapid elimination or inability to make contact with M cells and other mucosal tissues involved in antigen uptake and processing (Ogra et al., 2001). Thus the ability of recombinant LABs, and the S-layer in particular, to promote antigen uptake and stimulate the adaptive immune response is highly desirable (Konstantinov et al., 2008).

Numerous studies have established vaccine platforms based on one or more components of the Lactobacillus Slp, however, there remains a disconnect between delivery and efficacy. Despite extensive reviews touting the effectiveness of the LAB-based vaccines in vivo (Wells and Mercenier, 2008; LeCureux and Dean, 2018), few S-layer-based delivery frameworks have moved into animal models. Although successful secretion and/or surface display of the reporter/antigen is regularly achieved, only three of the recombinant organisms presented above were tested in mice (Kajikawa et al., 2015; Zhang et al., 2016; Sahay et al., 2018), whereas the remainder were more focused on establishing that antigen production/display was even possible. Thus, an important step moving forward will be connecting the delivery of these antigens with an actual vaccination event, therefore surpassing proof of concept studies and ultimately demonstrating disease protection.

#### AUTHOR CONTRIBUTIONS

fmicb-09-02264 September 29, 2018 Time: 16:39 # 10

CK and RB wrote and edited the manuscript.

#### REFERENCES

#### FUNDING

This work was supported by DuPont Nutrition and Health and the North Carolina Ag Foundation.

### ACKNOWLEDGMENTS

We would like to thank Dr. Yong Jun Goh, Dr. Sarah O'Flaherty, Dr. Claudio Hidalgo-Cantabrana, and Dr. Alexandra Crawley for their technical expertise and assistance with manuscript preparation.

Lactobacillus acidophilus NCFM. Appl. Environ. Microbiol. 82, 5687–5697. doi: 10.1128/AEM.02025-16

S-layer proteins and the Bacillus subtilis levansucrase. Mol. Microbiol. 31, 927–936.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Klotz and Barrangou. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Discovery of Bacterial Deaminases That Convert 5-Fluoroisocytosine Into 5-Fluorouracil

Agota Aucynait ˇ e˙ 1 , Rasa Rutkiene˙ 1 , Daiva Tauraite˙ 1 , Rolandas Meškys1 and Jaunius Urbonavicius ˇ 1,2 \*

1 Institute of Biochemistry, Department of Molecular Microbiology and Biotechnology, Life Sciences Center, Vilnius University, Vilnius, Lithuania, 2 Department of Chemistry and Bioengineering, Vilnius Gediminas Technical University, Vilnius, Lithuania