# MOLECULAR PATHOGENESIS OF PNEUMOCOCCUS: 2nd Edition

EDITED BY : Guangchun Bai and Jorge Eugenio Vidal PUBLISHED IN : Frontiers in Cellular and Infection Microbiology

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ISSN 1664-8714 ISBN 978-2-88974-939-3 DOI 10.3389/978-2-88974-939-3

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# MOLECULAR PATHOGENESIS OF PNEUMOCOCCUS: 2nd Edition

Topic Editors: Guangchun Bai, Albany Medical College, United States Jorge Eugenio Vidal, University of Mississippi Medical Center, United States

Publisher's note: In this 2nd edition, the following article has been added: Simmons SR, Tchalla EYI, Bhalla M and Bou Ghanem EN (2022) The Age-Driven Decline in Neutrophil Function Contributes to the Reduced Efficacy of the Pneumococcal Conjugate Vaccine in Old Hosts. Front. Cell. Infect. Microbiol. 12:849224. doi: 10.3389/fcimb.2022.849224

Citation: Bai, G., Vidal, J. E., eds. (2023). Molecular Pathogenesis of Pneumococcus: 2nd Edition. Lausanne: Frontiers Media SA. doi: 10.3389/978-2-88974-939-3

# Table of Contents

Irfan Manzoor, Sulman Shafeeq, Muhammad Afzal and Oscar P. Kuipers

*17 Induction of Central Host Signaling Kinases During Pneumococcal Infection of Human THP-1 Cells*

Thomas P. Kohler, Annemarie Scholz, Delia Kiachludis and Sven Hammerschmidt

*29 Characterization of Spbhp-37, a Hemoglobin-Binding Protein of*  Streptococcus pneumoniae

María E. Romero-Espejel, Mario A. Rodríguez, Bibiana Chávez-Munguía, Emmanuel Ríos-Castro and José de Jesús Olivares-Trejo

*38 Surface Proteins and Pneumolysin of Encapsulated and Nonencapsulated*  Streptococcus pneumoniae *Mediate Virulence in a Chinchilla Model of Otitis Media*

Lance E. Keller, Jessica L. Bradshaw, Haley Pipkins and Larry S. McDaniel

Jiaqi J. Zheng, Dhriti Sinha, Kyle J. Wayne and Malcolm E. Winkler

Muhammad Afzal, Sulman Shafeeq, Irfan Manzoor, Birgitta Henriques-Normark and Oscar P. Kuipers

*111 The Age-Driven Decline in Neutrophil Function Contributes to the Reduced Efficacy of the Pneumococcal Conjugate Vaccine in Old Hosts* Shaunna R. Simmons, Essi Y. I. Tchalla, Manmeet Bhalla and Elsa N. Bou Ghanem

# Editorial: Molecular Pathogenesis of Pneumococcus

#### Guangchun Bai 1 \* and Jorge E. Vidal 2, 3

1 Department of Immunology and Microbial Disease, Albany Medical College, Albany, NY, United States, 2 Hubert Department of Global Health, Rollins School of Public Health, Emory University, Atlanta, GA, United States, 3 Emory Antibiotic Resistance Center, School of Medicine, Emory University, Atlanta, GA, United States

#### Keywords: Streptococcus pneumoniae, pathogenesis, gene regulation, biofilm, co-infection, drug resistance, microbial, macrophages

### **Editorial on the Research Topic**

#### **Molecular Pathogenesis of Pneumococcus**

Streptococcus pneumoniae has been for decades the number one bacterial killer of children worldwide, but it is also a commensal of the human nasopharynx during childhood. Although vaccination with pneumococcal conjugate vaccines has helped decrease the burden of pneumococcal disease (PD), mortality caused by this pathogen remains high. The introduction of pneumococcal vaccines has also created a niche for vaccine-escape serotypes. Moreover, the rise of multidrug-resistant clones around the world has posed a serious threat in recent years. A better understanding of pneumococcal biology and pathogenesis is needed to further reduce the burden of PD. In the current research topic, nine papers were published for this perspective.

Complex molecules of human epithelia, including the human upper and lower airways, contain N-acetylglucosamine (NAG) that can be metabolized by pneumococcus to survive and persist during colonization, and succeed during pathogenesis. Afzal et al. investigated the transcriptome of the pneumococcal strain D39 growing in media with NAG as a sole source of carbohydrates, and demonstrated upregulated transcription of operons involved in NAG uptake. They confirmed that nagA and nagB are essential for the growth of pneumococcus in the presence of NAG and found that NagR regulates genes involved in the metabolism of NAG (Afzal et al.).

Phosphorus and metal ions are essential nutrients for all living organisms (Porcheron et al., 2013). Most bacteria encode a single Pst transporter for the uptake of inorganic phosphate (Pi). However, Zheng et al. demonstrated that S. pneumoniae possesses two distinct Pst transporters, Pst1 and Pst2. Interestingly, Pi uptake by these two Pst proteins can be compensated by a Na+/Pi co-transporter, NptA, in non-encapsulated S. pneumoniae (NESp) strains. In contrast, either Pst1 or Pst2 is needed for the growth of encapsulated strains. The Pi uptake transporters in S. pneumoniae are tightly controlled by two transcription factors, PhoU1 and PhoU2, to maintain high Pi transport in different Pi availability, which is required for optimal capsule biosynthesis (Zheng et al.).

It is known that AdcR-regulated genes play a role in pneumococcal pathogenesis. Manzoor et al. found that a number of genes belonging to the AdcR and the PsaR regulons are highly upregulated in the presence of Ni2+, suggesting the importance of Ni2+ in pneumococcal virulence. Additionally, most bacteria have developed strategies for iron acquisition from the host hemoglobulin (Hb) or heme (Andrews et al., 2003). S. pneumoniae Spbhp-37 is a cell surface protein and has an high affinity with Hb. The expression of Spbhp-37 is upregulated when S. pneumoniae is grown in media where Hb is provided as the sole iron source (Romero-Espejel et al.). Therefore, Spbhp-37 might be essential for S. pneumoniae to establish an infection in the host.

Another surface protein of S. pneumoniae, pneumococcal surface protein K (PspK), is known to increase colonization and virulence by NESp during otitis media (OM) episodes

#### Edited by:

Lorenza Putignani, Bambino Gesù Ospedale Pediatrico (IRCCS), Italy

### Reviewed by:

Stephen Peter Kidd, University of Adelaide, Australia

> \*Correspondence: Guangchun Bai<EMAIL_ADDRESS>
Received: 29 January 2017 Accepted: 23 June 2017 Published: 11 July 2017

#### Citation:

Bai G and Vidal JE (2017) Editorial: Molecular Pathogenesis of Pneumococcus. Front. Cell. Infect. Microbiol. 7:310. doi: 10.3389/fcimb.2017.00310 (Keller et al., 2014). Therefore, PspK is considered as a NESpspecific virulence factor. Interestingly, it was demonstrated that expression of pspK in an encapsulated pneumococcal strain significantly increased adhesion and invasion of human epithelial cells. Additionally, Murine colonization was increased when a capsule mutant expressed pspK (Keller et al.). These findings indicate that PspK plays distinct roles in pathogenesis between encapsulated S. pneumoniae and NESp.

It is known that macrophages play an essential role in the recognition and clearance of bacterial infections by either innate immunity or present antigen to establish acquired immunity. Phosphoinositide 3-kinase (PI3K) is an essential regulator for Fcγ and CR-mediated phagocytosis (Cox et al., 1999). Protein kinase B (Akt or PKB) is one of the major signal transducers and is a downstream target of PI3K (Alessi et al., 1997). By using immuno-fluorescence microscopy, antibiotic protection assays, and addition of inhibitors of central host cell kinase, new evidence showed that PI3K and Akt play an important role in uptake and killing of S. pneumoniae by human THP-1 macrophages, which is independent of Fcγ or CR-mediated phagocytosis pathways (Kohler et al.).

Through investigating interactions between pneumococcal biofilms and biofilms produced by other human pathogens, Khan et al. discovered that pneumococcal strains produce a factor(s) that efficiently eradicates, i.e., within hours, Staphylococcus aureus biofilms including those produced by methicillin-resistant S. aureus (MRSA) strains. Eradication was more efficient when S. pneumoniae and S. aureus had direct contact, which mimicked the condition that the strains would naturally encounter on the human upper airways. Physical-mediated killing did not require production of hydrogen peroxide since a S. pneumoniae mutant in spxB gene, encoding for an enzyme involved in production of H2O2, was able to eradicate S. aureus biofilms. Authors hypothesized that research on factors allowing eradication of S. aureus will provide alternative therapeutics for S. aureus infections (Khan et al.).

Influenza pandemics and seasonal outbreaks have enhanced susceptibility to secondary infection with S. pneumoniae.

## REFERENCES

Boianelli et al. developed a computational approach to evaluate the efficacy of treatment with Oseltamivir for flu infection and secondary pneumococcal pneumonia. Their mathematical algorithms revealed that increasing the recommended dose (i.e., 75 mg) to 150 mg, or 300 mg, almost doubles the effect of Oseltamivir against secondary bacterial infection. Other aspects of the treatment were also evaluated using their computational approach. Overall, their study warrants clinical trials to determine the efficacy of treatment regimens with Oseltamivir against lethality of secondary pneumococcal infection post influenza (Boianelli et al.).

Macrolides inhibit bacterial protein synthesis by binding to the large 50S ribosomal subunit (Schroeder and Stephens). These antibiotics have been extensively used for treatment of pneumococcal infection, which raises a strong selective pressure contributing to the expansion of macrolide-resistant S. pneumoniae. Macrolide resistance in S. pneumoniae is predominantly due to ribosomal methylation by the gene product encoded by erm(B) and macrolide efflux by a twocomponent efflux pump encoded by mef (E)/mel (Schroeder and Stephens). Current pneumococcal vaccines are effective in reducing macrolide resistance caused by vaccine serotypes. On the other hand, "serotype replacement" post vaccination might lead to macrolide resistance in new serotypes, which remains a public health concern.

## AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

## FUNDING

GB is partly supported by grants of R56AI122763 and R35HL135756 from the National Institutes of Health (NIH). JEV is in part supported by an NIH grant 5R21AI112768-02. The content is solely the responsibility of the authors and does not necessarily represent the official view of the NIH.

Porcheron, G., Garenaux, A., Proulx, J., Sabri, M., and Dozois, C. M. (2013). Iron, copper, zinc, and manganese transport and regulation in pathogenic Enterobacteria: correlations between strains, site of infection and the relative importance of the different metal transport systems for virulence. Front. Cell. Infect. Microbiol. 3:90. doi: 10.3389/fcimb.2013.00090

**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 © 2017 Bai and Vidal. 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) or licensor 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.

# The Regulation of the AdcR Regulon in *Streptococcus pneumoniae* Depends Both on Zn2+- and Ni2+-Availability

Irfan Manzoor 1, 2 † , Sulman Shafeeq1, 3 †, Muhammad Afzal 1, 2 and Oscar P. Kuipers 1 \*

*1 Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, Netherlands, 2 Department of Bioinformatics and Biotechnology, Government College University Faisalabad, Faisalabad, Pakistan, 3 Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden*

By using a transcriptomic approach, we have elucidated the effect of Ni2+ on the global gene expression of *S. pneumoniae* D39 by identifying several differentially expressed genes/operons in the presence of a high extracellular concentration of Ni2+. The genes belonging to the AdcR regulon (*adcRCBA*, *adcAII*-*phtD, phtA*, *phtB*, and *phtE*) and the PsaR regulon (*pcpA*, *prtA*, and *psaBCA*) were highly upregulated in the presence of Ni2+. We have further studied the role of Ni2+ in the regulation of the AdcR regulon by using ICP-MS analysis, electrophoretic mobility shift assays and transcriptional *lacZ*-reporter studies, and demonstrate that Ni2+ is directly involved in the derepression of the AdcR regulon *via* the Zn2+-dependent repressor AdcR, and has an opposite effect on the expression of the AdcR regulon compared to Zn2+.

#### *Edited by:*

*Jorge Eugenio Vidal, Emory University, USA*

*Reviewed by: Hilde De Reuse, Institut Pasteur, France Stephen Peter Kidd, University of Adelaide, Australia*

### *\*Correspondence:*

*Oscar P. Kuipers<EMAIL_ADDRESS>
*† These authors have contributed equally to this work.*

*Received: 27 August 2015 Accepted: 17 November 2015 Published: 08 December 2015*

#### *Citation:*

*Manzoor I, Shafeeq S, Afzal M and Kuipers OP (2015) The Regulation of the AdcR Regulon in Streptococcus pneumoniae Depends Both on Zn*2+ *and Ni*2+*-Availability. Front. Cell. Infect. Microbiol. 5:91. doi: 10.3389/fcimb.2015.00091* Keywords: metal homeostasis, pneumococcus, nickel, zinc, AdcR, Pht family proteins, AdcR regulon, PsaR regulon

## INTRODUCTION

In bacteria, the transition metal ions play an important role in the proper functioning of many enzymes, transporters, and transcriptional regulators. Transition metal ions are the prerequisite for the proper bacterial growth at low concentrations, but metal ions can be lethal at higher concentrations (Blencowe and Morby, 2003; Finney and O'Halloran, 2003; Moore and Helmann, 2005; Ge et al., 2012). Therefore, proper homeostasis of metal ions is very important for the survival of bacteria, which is maintained by the dedicated metal transport- and efflux-systems (Tottey et al., 2008; Waldron and Robinson, 2009; Lisher et al., 2013). These systems are tightly regulated by metal-responsive transcriptional regulators to ensure the proper functioning of the cell by maintaining the minimum levels of metal ions inside the cell.

Streptococcus pneumoniae is one of the most common human pathogens that reside asymptomatically in the human nasopharynx (Mitchell, 2003). However, it may occasionally translocate to the lungs, the eustachian tube, the blood, and the nervous system, causing pneumoniae, otitis media, bacteremia, and meningitis, respectively (Obaro and Adegbola, 2002; Bogaert et al., 2004). During translocation from the nasopharynx to other infection sites, S. pneumoniae may encounter different environmental conditions including varying metal ions concentrations, which might affect the expression of different genes including virulence genes (Gupta et al., 2009; Shafeeq et al., 2011b, 2013; Plumptre et al., 2014a). However, the exact conditions that S. pneumoniae might face during infections, are poorly understood.

The role of manganese (Mn2+), zinc (Zn2+), copper (Cu2+), iron (Fe2+), cobalt (Co2+), and cadmium (Cd2+) on the gene regulation of S. pneumoniae have already been established and several metal-specific acquisition- and efflux-systems have been characterized. These systems include AdcRCBA (the Zn2+ uptake system), CzcD (the Zn2+-efflux system), PsaBCA (the Mn2+-uptake system), MntE (the Mn2+-efflux system), the cop operon (the Cu2+-efflux system), and PiaABCD, PiuBCDA, and PitADBC (the Fe2+- and Fe3+-uptake systems) (Kloosterman et al., 2007, 2008; Hendriksen et al., 2009; Rosch et al., 2009; Bayle et al., 2011; Shafeeq et al., 2011a, 2013; Manzoor et al., 2015c). These systems have further been shown to be regulated by metal-specific transcriptional regulators in S. pneumoniae. The Zn2+-uptake system (AdcRCBA) is repressed by transcriptional regulator AdcR in the presence of Zn2+ (Shafeeq et al., 2011a). Similarly, the psaBCA operon encoding Mn2+-uptake system are repressed by transcriptional regulator PsaR in the presence of Mn2+ (Johnston et al., 2006; Kloosterman et al., 2008), whereas, this PsaR-mediated repression is relieved by the addition of Zn2+, Co2+, Cd2+, or Ni2+ (Kloosterman et al., 2008; Jacobsen et al., 2011; Begg et al., 2015; Manzoor et al., 2015a,b,c).

Ni2+ is an essential micronutrient for certain bacteria, due to its role in various cellular processes like methane formation, hydrolysis of urea, and consumption of molecular hydrogen (Chen and Burne, 2003; Mulrooney and Hausinger, 2003; Rodionov et al., 2006; Anwar et al., 2007). In Escherichia coli, the nik operon (nikABCDE) involved in the transport of Ni2+ is shown to regulate by transcriptional regulator NikR (De Pina et al., 1999). Moreover, the expression of NmtA, an ATPdependent transporter involved in the efflux of Ni2+ and Co2+, is tightly regulated by Ni2+-responsive transcriptional regulator NmtR in Mycobacterium tuberculosis (Cavet et al., 2002). Ni2+ is also shown to regulate the expression of urease activity in Streptococcus salivarius and Helicobacter pylori (van Vliet et al., 2001; Chen and Burne, 2003). The amount of Ni2+ in the human blood is estimated to be 0.83 ng ml−1 (Alimonti et al., 2005) and it is likely that S. pneumoniae may encounter Ni2+ during infection in blood. So far, very little is known about the impact of Ni2+ on the global gene expression of S. pneumoniae. Previously, the role of Ni2+ in the regulation of the Zn2+-efflux system czcD was reported (Kloosterman et al., 2007). It was shown that the SczA-mediated expression of czcD was highly increased in the presence of Zn2+, Co2+, or Ni2+ (Kloosterman et al., 2007). Moreover, a number of proteins and motif with Co2+- and Ni2+-binding capacity has been identified by Immobilized metal affinity column (IMAC) and LTQ-Orbitrap mass spectrometry (MS) that have diverse functions in the S. pneumoniae (Sun et al., 2013). In a recent study, we demonstrated the role of Ni2+ in regulation of the PsaR regulon and showed that Ni2+ not only alleviates the Mn2+-dependent binding of PsaR to the promoter regions of the PsaR regulon genes, but also cause Mn2+ deficiency possibly by blocking Mn2+-uptake via PsaA, hence leading to the high expression of the PsaR regulon in the presence of Ni2+ (Manzoor et al., 2015b).

In this current study, we used a transcriptomic analysis approach for the identification of differentially expressed genes/operons in response to high extracellular Ni2+ in S. pneumoniae. The expression of genes belonging to the AdcR regulon and the PsaR regulon was highly upregulated in the presence of Ni2+. We further studied the role of Ni2+ in the AdcR-mediated regulation of the adcRCBA, adcAII-phtD, phtA, phtB, and phtE by using transcriptional lacZ-reporter studies, inductively coupled plasma-mass spectrometry (ICP-MS) analysis and electrophoretic mobility shift assays (EMSAs), and showed that Ni2+ and Zn2+ play an opposite role in the regulation of the adcRCBA, adcAII-phtD, phtA, phtB, and phtE.

## MATERIALS AND METHODS

### Bacterial Strains and Media

Bacterial strains used in this study are listed in **Table 1**. Growth of bacteria and DNA manipulation were performed as described (Shafeeq et al., 2011a; Manzoor et al., 2015a). All experiments in thisstudy were performed in chemically defined medium (CDM).

#### TABLE 1 | List of strains and plasmids used in this study.

TABLE 2 | List of primers used in this study.

Salts of metal ion ZnSO4.7H2O and NiSO4.6H2O were used as specified in the Results section. Primers used in this study are based on the genome sequence of S. pneumoniae D39 and are listed in **Table 2**.

## DNA Microarray and Data Analysis

For microarray analysis in response to Ni2+, S. pneumoniae D39 wild-type was grown in two biological replicates in CDM with and without the addition of 0.5 mM NiSO4.6H2O. To analyze the impact of adcR deletion on the transcirptome of S. pneumoniae in the presence of Ni2+, D39 wild-type and 1adcR (SS200) (Shafeeq et al., 2011a) were grown in two biological replicates in CDM with 0.3 mM of NiSO4.6H2O. All other procedures regarding microarray experiments and data analysis were done as described before (Shafeeq et al., 2011b; Afzal et al., 2015). For the identification of differentially expressed genes a Bayesian p < 0.001 and a fold change cut-off of 2 was applied. The DNA microarray data have been submitted to gene expression omnibus (GEO) database under the accession number GSE73852.

## Construction of Transcriptional *lacZ*-fusions and β-galactosidase Assays

Chromosomal transcriptional lacZ-fusions to the promoter regions of adcR, adcAII, phtA, phtB, and phtE were constructed in plasmid pPP2 (Halfmann et al., 2007) with the primer pairs listed in **Table 2**, resulting in pIM501-505. These plasmids were introduced into D39 wild-type and 1adcR (SS200) (Shafeeq et al., 2011a) resulting in strains IM501-505 and IM551-554, respectively. All plasmids were checked for the presence of correct insert by means of PCR and DNA sequencing. For βgalactosidase activity, the derivatives of S. pneumoniae were grown in triplicate in CDM supplemented with different metal ion concentrations (w/v) mentioned in the Results and harvested at the mid-exponential growth phase. The β-galactosidase activity was measured as described before (Kloosterman et al., 2006). Standard deviations were calculated from three independent replicates of each sample.

## Inductively Coupled Plasma-mass Spectrometry (ICP-MS) Analysis

To determine the cell-associated concentration of metal ions, an ICP-MS analysis was performed on the cells grown in triplicates in CDM with and without the addition of 0.5 mM Ni2+ till the mid-exponential growth phase. Cell cultures were centrifuged at 4 ◦C and washed twice with overnight Chelex (Sigma) treated phosphate-buffered saline (PBS) with 1 mM nitrilotriacetic acid. Cells were dried overnight in a Speedvac at room temperature. The dried cells were dissolved in 2.5% nitric acid (Ultrapure, Sigma Aldrich) and lysed at 95oC for 10 min by vigorous vortexing after each 30 s. The lysed cell samples were used for ICP-MS analysis as described (Jacobsen et al., 2011). Metal ion concentrations were expressed as µ g g−1 dry weight of cells.

## Overexpression and Purification of Strep-tagged AdcR

The nisin-inducible (NICE) expression system (Kuipers et al., 1998) in Lactococcus lactis strain NZ9000 was used for the overexpression of C-terminally Strep-tagged AdcR (Shafeeq et al., 2011a). Cells were grown until an OD600 of 0.4 in 1 L culture followed by the induction with 10 ng ml−1 nisin. The purification of AdcR-Strep tag was performed using the Streptactin column from IBA according to the supplier's instructions (www.iba-go.com). The purified protein was eluted in buffers without EDTA and stored at a concentration of 0.5 mg/ml in the elution buffer (100 mM Tris-HCl [pH 8], 150 mM NaCl, 2.5 mM desthiobiotin, and 1 mM β-mercaptoethanol) with 10% glycerol at −80◦C.

## Electrophoretic Mobility Shift Assays

Electrophoretic mobility shift assays (EMSAs) were performed as described (Kloosterman et al., 2008). In short, PCR products of the promoter regions of adcR, adcAII, phtA, phtB, and pcpA were labeled with [γ-33P] ATP. All the EMSAs were performed with 5000 cpm of [γ-33P] ATP-labeled PCR products in buffer containing 20 mM Tris-HCL (pH 8.0), 5 mM MgCl2, 8.7% (w/v) glycerol, 62.5 mM KCl, 25µg/ml bovine serum albumin and 25µg/ml poly (dI-dC). Various metal ions were added in concentrations as described in the Results section. Reactions were incubated at 30◦C for 30 min before loading on gels. Gels were run in 1 M Tris-borate buffer (pH 8.3) at 95 V for 90 min.

## RESULTS

## Identification of Ni2+-dependent Genes in *S. pneumoniae*

To investigate the impact of Ni2+ on the transcriptome of S. pneumoniae, a DNA microarray-based comparison of D39 wildtype grown in CDM with 0.5 mM Ni2+ to the same strain grown in CDM with 0 mM Ni2+ was performed. **Table 3** summarizes the list of differentially expressed genes in the presence of 0.5 mM Ni2+. The PsaR regulon consisting of the operon psaBCA (encoding Mn2+-dependent ABC transporters, PsaBCA), pcpA (encoding a choline binding protein, PcpA), and prtA (encoding a serine protease PrtA) were highly upregulated in the presence of

*aGene numbers refer to D39 locus tags.*

*bD39 annotation/TIGR4 annotation (Hoskins et al., 2001; Lanie et al., 2007).*

*cRatios* >*2.0 or* <*2.0 (wild-type* + *0.5 mM Ni*2+*/wild-type* + *0 mM Ni*2+*).*

Ni2+. The Ni2+-dependent upregulation of the PsaR regulon in the presence of Ni2+ is consistent with our recent study, where we have explored the Ni2+-dependent regulation of the PsaR regulon in more details (Manzoor et al., 2015b). Expression of a gene cluster including the cation efflux system gene czcD, the MerR family transcriptional regulator, and the Zn2+-containing alcohol dehydrogenase adhB was increased more than 35-fold in

the presence of Ni2+. The cation efflux system CzcD was shown to protect S. pneumoniae against the intracellular Zn2+-stress (Kloosterman et al., 2007). A novel TetR family transcriptional regulator SczA has been shown to activate the expression of czcD in the presence of Zn2+, Co2+, or Ni2+ (Kloosterman et al., 2007). Therefore, the upregulation of czcD in our transcriptomic analysis is consistent with the finding presented in previous study (Kloosterman et al., 2007). Furthermore, genes encoding a heat shock protein (HtpX) and a Dpr homolog (spd\_1402) were also differentially expressed. The Dpr protein has been shown to protect bacterial cells from oxidative stress (Pulliainen et al., 2003).

The genes belonging to the AdcR regulon were also upregulated in the presence of Ni2+. The expression of the adc operon was 4-fold upregulated. The expression of adcAIIphtD operon was upregulated 2-fold. The expression of other genes encoding for Pht family proteins (PhtA and PhtE), was upregulated more then 7-fold. Previously, it was shown that the expression of the AdcR regulon is repressed by the transcriptional regulator AdcR in the presence of Zn2+ (Shafeeq et al., 2011a). Transcriptome data was further validated by qRT-PCR analysis (Supplementary data: Table S1). Upregulation of the AdcR regulon in the presence of Ni2+ might also indicate the putative role of Ni2+ in the regulation of the AdcR regulon by the transcriptional regulator AdcR. Therefore, we decided to further explore the role of Ni2+ in the regulation of the AdcR regulon and to determine the intracellular concentrations of metal ions in S. pneumoniae D39 grown in the presence of either 0.5 mM Ni2+ or 0 mM Ni2+ in CDM.

## *S. pneumoniae* Accumulates More Ni2+ When Grown in the Presence of 0.5 mM Ni2+

To investigate whether the observed transcriptomic responses correlated with high cell-associated concentration of Ni2+, we performed an ICP-MS analysis on the same conditions used for performing the transcriptome analysis, i.e., cells grown either in the presence of 0.5 mM Ni2+ or 0 mM Ni2+ in CDM. Our ICP-MS data revealed that the cells grown in the presence of 0.5 mM Ni2+ accumulate 30-fold more cell-associated Ni2+ compared to the cells grown in 0 mM Ni2+ (30µg g−l dry mass of cells vs. <1µg g−l dry mass of cells) (**Figure 1**). Moreover, 2.6 fold decrease in the cell-associated concentration of Mn2+ was observed. The cell-associated concentration of other metal ions was not changed in the presence of 0.5 mM Ni2+ compared to 0 mM Ni2+. Therefore, it is likely that the transcriptomic changes observed in the presence of 0.5 M Ni2+ are due to the high intracellular concentration of Ni2+.

## Ni2+-dependent Expression of the AdcR Regulon

To explore the transcriptional regulation of the genes/operons belonging to the AdcR regulon (adcRCBA, adcAII-phtD, phtA, phtB, and phtE) found in our microarray analysis, transcriptional lacZ-fusions were constructed to the promoter regions of adcR, adcAII, phtA, phtB, and phtE in plasmid pPP2 (Halfmann et al., 2007) and transferred to S. pneumoniae D39 wild-type. The expression of PadcR-lacZ, PadcAII-lacZ, PphtA-lacZ, PphtB-lacZ, and PphtE-lacZ was measured in CDM and CDM-Zn2+ (Zn2+ depleted medium) with the addition of 0, 0.1, 0.3, or 0.5 mM Ni2+. As AdcR represses the expression of the AdcR regulon in the presence of Zn2+, we also used Zn2+-depleted medium (CDM-Zn2+). β-galactosidase activity (Miller Units) showed that the elevated concentration of Ni2+ led to the high expression of all these promoters in CDM and CDM-Zn2+ (**Figures 2A,B**). However, the expression of these promoters was much higher in CDM-Zn2+ compared to CDM. The full CDM contains minor amounts of Zn2+ (around 883µg l−1 ) (Manzoor et al., 2015a), which could explain the lower expression of these promoters in CDM compared to CDM-Zn2+. This data not only suggests the role of Ni2+ in the regulation of the adcRCBA, adcAII-phtD, phtA, phtB, and phtE, but also indicate the ability of Ni2+ to derepress the Zn2+-dependent repression of these genes.

## Opposite Effect of Zn2+ and Ni2+ on the Expression of the AdcR Regulon

β-galactosidase activities shown above indicate that Ni2+ might compete with Zn2+ and that both metal ions have opposite effects

replications is indicated with error bars. Statistical significance of the differences in the expression levels was determined by One-way ANOVA (NS, not significant, \**P* < 0.05, \*\**P* < 0.001, and \*\*\**P* < 0.0001).

deviation of three independent replications is indicated with error bars. Statistical significance of the differences in the expression levels was determined by One-way ANOVA (\**P* < 0.05, \*\**P* < 0.001, and \*\*\**P* < 0.0001). on the expression of the adcRCBA, adcAII-phtD, phtA, phtB, and phtE. In order to study the interplay of Ni2+ and Zn2+ in the regulation of adcRCBA, adcAII-phtD, phtA, phtB, and phtE in more details, we performed β-galactosidase assays with PadcR-lacZ, PadcAII-lacZ, PphtA-lacZ, PphtB-lacZ, and PphtElacZ in CDM with the addition of varying concentrations of Ni2+ and Zn2+ together. β-galactosidase data (Miller Units) showed that addition of Zn2+ in the medium leads to the repression of PadcR-lacZ, PadcAII-lacZ, PphtA-lacZ, PphtB-lacZ, and PphtElacZ, even in the presence of Ni2+. However, repression caused by Zn2+ was much weaker at higher concentrations of Ni2+ (**Figures 3A–E**). This data confirm that Ni2+ and Zn2+ have an opposite effects on the expression of adcRCBA, adcAII-phtD, phtA, phtB, and phtE, where Zn2+ represses and Ni2+ derepresses the expression of these genes.

## Role of the Transcriptional Regulator AdcR in the Ni2+-dependent Expression of the AdcR Regulon

Previously, it has been shown that the transcriptional regulator AdcR represses the expression of adcRCBA, adcAII-phtD, phtA, phtB, and phtE in the presence of Zn2+ (Shafeeq et al., 2011a). In this study, our transcriptomic analysis and transcriptional lacZreporter data indicate that Ni2+ derepresses the expression of these genes. To identify whether the transcriptional regulator AdcR is also responsible for the Ni2+-dependent expression of adcRCBA, adcAII-phtD, phtA, phtB, and phtE, we have transformed PadcR-lacZ, PadcAII-lacZ, PphtA-lacZ, PphtB-lacZ, and PphtE-lacZ into the adcR mutant (SS200) and performed β-galactosidase assays. β-galactosidase data revealed that the deletion of adcR leads to increase expression of PadcR-lacZ, PadcAII-lacZ, PphtA-lacZ, PphtB-lacZ, and PphtE-lacZ even in the absence of Ni2+ (**Figure 4**). Upregulation of these transcriptional lacZ-fusions in the adcR mutant indicates that Ni2+-dependent expression of adcRCBA, adcAII-phtD, phtA, phtB, and phtE is mediated by transcriptional regulator AdcR.

To elucidate the Ni2+-dependent role of AdcR in more details and find more targets of AdcR in the presence of Ni2+, microarray comparison of the adcR mutant with D39 wildtype was performed in CDM with 0.3 mM Ni2+. As expected, the expression of genes belonging to the AdcR regulon was highly upregulated (**Table 4**), except for the adc operon, which was downregulated in our transcriptome analysis (**Table 4**). For creating an adcR mutant in previous study, an erythromycinresistance gene cassette was used to replace the adcR gene (Shafeeq et al., 2011a). Therefore, downregulation of the adc operon might be due to the polar effect of adcR deletion on the downstream genes of adcR (Shafeeq et al., 2011a). We further validated our DNA microarray data by qRT-PCR. qRT-PCR data is also in agreement with our transcriptome data (Supplementary data: Table S2).

## Binding of AdcR to Its Target Is Zn2+-and Ni2+-dependent

To study the direct interaction of AdcR with the promoter regions of the genes belonging to the AdcR regulon in the

#### TABLE 4 | Summary of transcriptome comparison of *S. pneumoniae* D39 wild-type with 1*adcR* (SS200) grown in CDM with 0.3 mM Ni2+.

*aGene numbers refer to D39 locus tags.*

*bD39 annotation/TIGR4 annotation (Hoskins et al., 2001; Lanie et al., 2007).*

*cRatios* >*2.0 or* <*2.0 (SS*200 + 0*.3 mM Ni*2+*/wild-type* + *0.3 mM Ni*2+*).*

presence of Ni2+, we performed EMSAs with purified Streptagged AdcR (Ad-Strep tag) and 33P-labeled promoters of adcR, adcAII, phtA, phtB, and pcpA. To prevent the interference of metal ions with Ad-Strep tag, all the experiments were performed in EDTA free gels and buffers. The pcpA promoter region was taken as a negative control. Ad-Strep tag was unable to shift the promoter regions of adcR, adcAII, phtA, and phtB in the absence of metal ions (Lane 2 in **Figure 5**). However, the addition of 0.2 mM Zn2+ led to the binding of Ad-Strep tag to the promoter regions of adcR, adcAII, phtA, and phtB (Lane 3 in **Figures 5A–D**), which is consistent with our previous study (Shafeeq et al., 2011a). Interestingly, 0.2 and 0.4 mM Ni2+ were unable to stimulate the binding of Ad-Strep tag with the promoter regions of adcR, adcAII, phtA, and phtB (Lane 4 and 5 in **Figures 5A–D**). In our transcriptome data mentioned above, Ni2+ showed a derepressive effect on the expression of the AdcR regulon. Therefore, we also decided to check the interaction of Ad-Strep tag with the promoter regions of adcR, adcAII, phtA, and phtB in the presence of both Zn2+ and Ni2+ together. The Zn2+-dependent interaction of AdcR with these promoters in the presence of 0.2 mM Zn2+ was alleviated with the addition of 0.2 mM or 0.4 mM Ni2+ (Lane 6 and 7 in **Figures 5A–D**). Under the same conditions, we did not see any band shift with the promoter region of pcpA as a negative control (**Figure 5E**). Thus, this data indicates that Zn2+ and Ni2+ have an opposite effects on the interaction of AdcR with the promoter regions of adcR, adcAII, phtA, and phtB.

## Effect of Ni2+on SczA-mediated Expression of the Zn2+-efflux system *czcD*

To investigate the regulation of czcD in the presence of Ni2+, we studied the transcriptional response of PczcD-lacZ grown in complete CDM with the addition of different concentrations of Ni2+. β-galactosidase assays showed that PczcD-lacZ responded to Ni2+ and its expression was highly increased with an increasing concentration of Ni2+ (**Figure 6**). This data is in agreement with our transcriptomic data mentioned above and suggests the putative role of CzcD in Ni2+ homeostasis.

## DISCUSSION

Transition metal ions such as Mn2+, Zn2+, Cu2+, Fe2+, Co2+, and Cd2+ have been shown to play a pivotal role in the

metabolism and virulence of S. pneumoniae (Brown et al., 2001; Kloosterman et al., 2008; Shafeeq et al., 2011b; Begg et al., 2015). However, the role of Ni2+ on the global gene expression of S. pneumoniae has not been studied before. In this study, we analyze the transcriptome changes in S. pneumoniae D39 wildtype in response to high Ni2+ concentration. The expression of a number of important genes and operons with diverse functions, including the AdcR regulon (adcRCBA, adcAII-phtD, phtA, phtB, and phtE), the PsaR regulon (pcpA, prtA, and psaBCA) regulon, and the Zn2+-efflux system czcD were significantly altered in the

presence of Ni2+. We further studied the role of Ni2+ in the regulation of the AdcR regulon and demonstrated that Ni2+ plays an opposite role compared to Zn2+ in the regulation of the AdcR regulon.

The AdcR regulon consists of adcRCBA, adcAII-phtD, phtA, phtB, phtE, and adhC in S. pneumoniae. The adc operon (adcRCBA) is involved in Zn2+ acquisition, and encodes for a Zn2+-responsive MarR family transcriptional regulator, AdcR, two ABC transporter proteins AdcC and AdcB, and an extracellular Zn2+-binding protein AdcA (Dintilhac et al., 1997; Dintilhac and Claverys, 1997; Bayle et al., 2011). The adcAII gene encodes an adhesion lipoprotein which has an overlapping specificity with AdcA for Zn2+ (Bayle et al., 2011). AdcAII belongs to the LraI-lipoprotein family and is organized in an operon with a phtD gene encoding pneumococcal histidine triade protein precursor D (PhtD). phtA, phtB, and phtE encodes for pneumococcal histidine triade protein A, B, and E, respectively. Recent studies have demonstrated the role of the PhT family proteins (PhtA, PhtB, PhtE, and PhtD) in intracellular Zn2+ acquisition and pathogenesis in S. pneumoniae (Hava and Camilli, 2002; Ogunniyi et al., 2009; Plumptre et al., 2014b). The adhC gene encodes for a Zn2+-containing alcohol dehydrogenase. Previously, it was demonstrated that the expression of adcRCBA, adcAII-phtD, phtA, phtB, and phtE is repressed, while the expression of adhC is activated by the transcriptional regulator AdcR in the presence of Zn2+ (Shafeeq et al., 2011a). Here, we show that Ni2+ also plays a role in the regulation of adcRCBA, adcAII-phtD, phtA, phtB, and phtE. Our β-galactosidase assays showed that the expression of adcRCBA, adcAII-phtD, phtA, phtB, and phtE was increased with increasing concentrations of Ni2+. However, we did not find any significant change in the expression of adhC in our both transcriptome analysis performed in this study. This might exclude the role of Ni2+ in the AdcR mediated regulation of adhC.

High concentrations of Ni2+ can be very toxic for bacteria (Macomber and Hausinger, 2011). Therefore, bacteria must limit the toxic amount of Ni2+ to perform normal cellular functions. In many bacteria, CDF-family efflux pumps help to maintain proper concentrations of heavy metals in the cell. For example, in Bacillus subtilis, the CzcD heavy metal efflux pump is involved in the homeostasis of Zn2+, Co2+, Cu2+, and Ni2+, and is regulated by CzrA (Moore et al., 2005). It is also important to note that the expression of czcD is highly upregulated in our transcriptome analysis in response to Ni2+. Expression of czcD is regulated by the TetR family transcriptional regulator SczA in the presence of Zn2+, Co2+, or Ni2+ (Kloosterman et al., 2007). Moreover, Zn2+, Co2+, or Ni2+ has been shown to stimulate the binding of SczA to the promoter region of czcD (Kloosterman et al., 2007). In this study, we further confirmed the expression of czcD in the presence of Ni2+ by transcriptional lacZ-reporter study with PczcD-lacZ and our results are consistent with a previous study (Kloosterman et al., 2007).

The PsaR regulon consists of psaBCA, pcpA, and prtA that encodes for the Mn2+ uptake system (PsaBCA), a choline binding protein (PcpA), and a serine protease (PrtA), respectively. The expression of the PsaR regulon is shown to be repressed by the DtxR family transcriptional regulator PsaR in the presence of Mn2+ (Johnston et al., 2006). Notably, Zn2+ and Co2+ can bind with PsaR to relieve the Mn2+-dependent repression of the PsaR regulon (Kloosterman et al., 2008; Manzoor et al., 2015a). Recently, we have studied the regulation of the PsaR regulon in the presence of Ni2+ and demonstrated that like Zn2+ and Co2+, Ni2+ also has the ability to derepress the Mn2+-dependent repression of the PsaR regulon, and that high concentrations of Ni2+ leads to cell-associated Mn2+ deficiency (Manzoor et al., 2015b). In this study, we have also observed the significant upregulation of the PsaR regulon in our transcriptome analysis performed in the presence of Ni2+ (**Table 3**). Upregulation of the PsaR regulon in our transcriptome further verifies our previous

## REFERENCES

results (Manzoor et al., 2015b). Moreover, we have also observed the cell-associated deficiency of Mn2+ in our ICP-MS analysis performed in this study (**Figure 1**), which is also in consistent with our previous results (Manzoor et al., 2015b).

The interplay, or competition, of metal ions plays an important role in the regulation of metal responsive genes. In S. pneumoniae, competition of Mn2+ with Zn2+, Co2+, or Ni2+ in the regulation of the PsaR regulon by transcriptional regulator PsaR has already extensively been studied (Kloosterman et al., 2008; Manzoor et al., 2015a,b). Similarly, the interplay of Cu2+ and Zn2+ in the regulation of cop operon by transcriptional regulator CopY was studied before, where Cu2+ induces and Zn2+ represses the CopY-mediated expression of cop operon (Shafeeq et al., 2011b). Here, we elaborated for the first time the interplay of Ni2+ and Zn2+ in the regulation of genes belonging to the AdcR regulon. Our lacZ-reporter studies determined the ability of Ni2+, in derepressing the Zn2+-dependent repression of adcRCBA, adcAII-phtD, phtA, phtB, and phtE. Our in vitro data showed that the Zn2+-dependent binding of AdcR to the promoter regions of the genes belonging to the AdcR regulon was alleviated by the addition of Ni2+. Recently, it has been shown that Cd2+-uptake reduces the accumulation of cell-associated Mn2+ and Zn2+ (Begg et al., 2015). Our ICP-MS comparison of cells grown in CDM with 0.5 mM to 0 mM Ni2+ has not shown any difference in the concentration of Zn2+ or other metal ions, which also indicates the direct role of Ni2+ in the regulation of adcRCBA, adcAII-phtD, phtA, phtB, and phtE. Moreover, the role of genes belonging to the AdcR regulon in the pathogenesis of S. pneumoniae has already been demonstrated, which also suggests the important role of Ni2+ in pneumococcal virulence.

## SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fcimb. 2015.00091

basis for cadmium toxicity in Streptococcus pneumoniae. Nat. Commun. 6:6418. doi: 10.1038/ncomms7418

**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 © 2015 Manzoor, Shafeeq, Afzal and Kuipers. 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) or licensor 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.

# Induction of Central Host Signaling Kinases during Pneumococcal Infection of Human THP-1 Cells

Thomas P. Kohler\*, Annemarie Scholz, Delia Kiachludis and Sven Hammerschmidt

Department Genetics of Microorganisms, Interfaculty Institute for Genetics and Functional Genomics, Ernst-Moritz-Arndt Universität Greifswald, Greifswald, Germany

Streptococcus pneumoniae is a widespread colonizer of the mucosal epithelia of the upper respiratory tract of human. However, pneumococci are also responsible for numerous local as well as severe systemic infections, especially in children under the age of five and the elderly. Under certain conditions, pneumococci are able to conquer the epithelial barrier, which can lead to a dissemination of the bacteria into underlying tissues and the bloodstream. Here, specialized macrophages represent an essential part of the innate immune system against bacterial intruders. Recognition of the bacteria through different receptors on the surface of macrophages leads thereby to an uptake and elimination of bacteria. Accompanied cytokine release triggers the migration of leukocytes from peripheral blood to the site of infection, where monocytes differentiate into mature macrophages. The rearrangement of the actin cytoskeleton during phagocytosis, resulting in the engulfment of bacteria, is thereby tightly regulated by receptor-mediated phosphorylation cascades of different protein kinases. The molecular cellular processes including the modulation of central protein kinases are only partially solved. In this study, the human monocytic THP-1 cell line was used as a model system to examine the activation of Fcγ and complement receptor-independent signal cascades during infection with S. pneumoniae. Pneumococci cultured either in chemically defined or complex medium showed no significant differences in pneumococcal phagocytosis by phorbol 12-myristate 13-acetate (PMA) differentiated THP-1 cells. Double immuno-fluorescence microscopy and antibiotic protection assays demonstrated a time-dependent uptake and killing of S. pneumoniae 35A inside of macrophages. Infections of THP-1 cells in the presence of specific pharmacological inhibitors revealed a crucial role of actin polymerization and importance of the phosphoinositide 3-kinase (PI3K) and Protein kinase B (Akt) as well during bacterial uptake. The participation of essential host cell signaling kinases in pneumococcal phagocytosis was deciphered for the kinase Akt, ERK1/2, and p38 and phosphoimmunoblots showed an increased phosphorylation and thus activation upon infection with pneumococci. Taken together, this study deciphers host cell kinases in innate immune cells that are induced upon infection with pneumococci and interfere with bacterial clearance after phagocytosis.

#### *Edited by:*

Guangchun Bai, Albany Medical College, USA

#### *Reviewed by:*

Kishore Alugupalli, Thomas Jefferson University, USA Glen C. Ulett, Griffith University, Australia Jose Yuste, Instituto de Salud Carlos III, Spain

> *\*Correspondence:* Thomas P. Kohler<EMAIL_ADDRESS>
*Received:* 25 January 2016 *Accepted:* 13 April 2016 *Published:* 26 April 2016

#### *Citation:*

Kohler TP, Scholz A, Kiachludis D and Hammerschmidt S (2016) Induction of Central Host Signaling Kinases during Pneumococcal Infection of Human THP-1 Cells. Front. Cell. Infect. Microbiol. 6:48. doi: 10.3389/fcimb.2016.00048

Keywords: *Streptococcus pneumoniae*, phagocytosis, THP-1 cells, cell signaling, p38, Akt, PI3K, ERK1/2

## INTRODUCTION

Streptococcus pneumoniae is a common colonizer of the upper respiratory tract of human, with increased colonization rates in children and the elderly (Garenne et al., 1992; Bogaert et al., 2004b; Hussain et al., 2005). Beside its role as a harmless colonizer, pneumococci are also a common cause of otitis media, pneumonia, meningitis and sepsis, especially in children under the age of 5 years (Bogaert et al., 2004a; Sleeman et al., 2006). S. pneumoniae possesses a wide variety of virulence factors to colonize the host, invade into tissues and to evade the human immune system (Jonsson et al., 1985; Gamez and Hammerschmidt, 2012; Voss et al., 2012). The epithelia of the upper respiratory tract of human represent thereby a physical barrier which needs to be conquered in the process of invasive pneumococcal diseases. Pneumococci therefore release amongst others pneumolysin, neuraminidase, and hyaluronidase to the environment, leading to disruption of connective tissues and extracellular matrices, promoting dissemination of the bacteria into underlying tissues, and the blood system (Kelly and Jedrzejas, 2000; Feldman et al., 2007; Trappetti et al., 2009; Mitchell and Mitchell, 2010). In this scenario, phagocytic cells play an essential role in the recognition and clearance of bacterial infections (van Furth and Cohn, 1968; van Furth et al., 1972). Macrophages represent an important link between the innate and the acquired immune system due to the possibility to phagocytose and digest bacteria and to present part fragments of processed bacteria in association with major histocompatibility complex (MHC) class I or II to Tcells (Greenberg and Grinstein, 2002). Bacterial recognition and uptake by macrophages can be initiated by the activation of different surface receptors. Fc receptor-mediated phagocytosis is initiated by recognition of immunoglobulin G (IgG) opsonized microorganisms. Here, members of the Fcγ receptor family are able to recognize and bind the constant Fc region of IgG molecules that opsonize pathogenic microorganisms (Gomez et al., 1994; Ravetch, 1997). Microorganisms can also be opsonized for complement receptor-mediated phagocytosis by proteins of the complement system, like C3b or C4b, resulting from cleavage of complement factors (Ghiran et al., 2000).

Besides recognition of opsonized microorganisms, cells of the innate immune system have the capability to sense bacteria directly via their target specific molecular structures, the so called pathogen-associated molecular patterns (PAMPs) via pattern recognition receptors (PRRs). These receptors are located on the surface of host cells, intracellularly and are also be secreted (Janeway and Medzhitov, 2002; Iwasaki and Medzhitov, 2015). For the recognition of pneumococci various PRRs have been described, including the C-reactive protein (CRP), members of the toll-like receptor family (TLRs), Nod proteins, the LPS binding protein (LBP), and CD14 (Mold et al., 2002; Weber et al., 2003; Currie et al., 2004; Echchannaoui et al., 2005; Malley et al., 2005). Furthermore, the C-type lectin SIGN-R1 and the scavenger receptor MARCO, both expressed by macrophages, have been described as PRRs important for the recognition of pneumococci (Arredouani et al., 2004; Kang et al., 2004).

However, recognition of bacteria via PAMPs and sensing of opsonized bacteria leads to the initiation of signal transduction cascades catalyzed by different protein kinases ending up in the activation of proteins involved in actin remodeling and therefore phagocytosis (Freeman and Grinstein, 2014). Nevertheless, the molecular process of actin remodeling during phagocytosis is only partially understood and best studied in Fcγ and complement-3 receptor-(CR3) mediated phagocytosis.

The signaling network of protein kinases involved in phagocytosis is rather complex and dependent from the activated surface receptors and cross-talk between different signaling pathways.

Members of the phosphoinositide-3-kinase (PI3K) family are essential for many cellular processes by transducing outsideinside signaling. Amongst others, this signaling leads to the activation of downstream effector pathways, including the reorganization of the cytoskeleton via exchange factors that regulate the small GTPase Rac and activation of the protein kinase C (PKC) as well as the serine/threonine protein kinase B (PKB/Akt) (Hawkins et al., 1995; Katso et al., 2001; Engelman et al., 2006). Moreover, experiments with pharmacological inhibitors of the PI3K (Wortmannin and LY294002) revealed also an essential role in FCγ and complement receptor-mediated phagocytosis (Cox et al., 1999; Aderem, 2003). Blocking of the PI3K leads not to an inhibition of opsonized particle binding or initial actin polymerization but seems to be required for membrane extension and fusion during engulfment (Araki et al., 1996; Cox et al., 1999).

Another important component of intracellular signaling processes is the protein kinase B (Akt), a serine/threonine (Ser/Thr) protein kinase involved in a wide variety of signaling pathways concerning such as cell growth, survival, or cellular metabolism (del Peso et al., 1997; Wullschleger et al., 2006; Manning and Cantley, 2007). Akt is an important downstream target of the PI3K, and represents therefore a mediator of the PI3K activity, as shown for example by blocking Akt activation using the PI3K inhibitor Wortmannin (James et al., 1996). Also, Akt was further shown to be activated during the process of Fcγ receptor-mediated phagocytosis (Ganesan et al., 2004).

A third protein family widely involved in cellular signal transduction pathways is the mitogen-activated protein kinase (MAPK) family. These kinases are also Ser/Thr protein kinases converting extracellular stimuli into a cellular response and they are involved in many physiological processes (Widmann et al., 1999). Examples for conventional MAPKs are the two MAPK isoforms ERK1 and ERK2 which can be activated by a number of different growth factors such as platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) as well as in response to insulin, ligand binding on heteromeric G-protein coupled receptors (GPCR), cytokines, osmotic stress, and microtubule disorganization (Boulton et al., 1990; Raman et al., 2007).

ERK1/2 was shown to be activated during the process of Fcγmediated phagocytosis (Karimi and Lennartz, 1998; Fitzer-Attas et al., 2000). Interestingly, ERK1/2 was shown to be inhibited in the early phase of CR3-dependent bacterial phagocytosis as shown by infection of human macrophages with Francisella tullarensis (Dai et al., 2013).

The p38 kinases are a further sub-group of the MAPK family, which includes the p38 α, β, γ, and δ kinases. The p38 kinases are highly activated by cytokines and environmental stress and were shown to be critical for the regulation of immune and inflammatory processes (Cuenda and Rousseau, 2007). Bacterial binding to various TLRs initiates bacterial phagocytosis via signaling cascades involving amongst others p38 (Doyle et al., 2004).

A widely-used model cell line for the analysis of macrophage/pathogen interactions are the human monocytic THP-1 cells. The addition of phorbol 12-myristate 13-acetate (PMA) to the growth medium leads to the differentiation of monocytes into macrophage-like phagocytic cells. Due to their availability of Fc receptors, C3b receptors and various pattern recognition receptors, as well as the lack of surface and cytoplasmatic immunoglobulins, THP-1 cells can be used for immunocytochemical studies (Tsuchiya et al., 1980; Matsumoto et al., 1990).

In this study, the human pathogen S. pneumoniae and human THP-1cells were used to evaluate the role of the bacterial growth medium on phagocytosis, to study the time-dependent uptake of pneumococci and to visualize the intracellular fate over time within the macrophages. On the other hand Fcγ- and CR-independent signaling mechanisms during phagocytosis of S. pneumoniae in differentiated THP-1 cells were analyzed. Therefore, infection experiments in the presence of pharmacological inhibitors of actin polymerization, PI3K and Akt were carried out. Furthermore, cell lysates from different time points of infected THP-1 cells were analyzed by immunoblot analysis to identify the participation of important cellular protein kinases involved in cell signaling during pneumococcal phagocytosis.

## MATERIALS AND METHODS

## Bacterial Strains, Media, and Culture Conditions

Streptococcus pneumoniae NCTC 10319 (serotype 35A, low encapsulated) (Pracht et al., 2005) was grown in complex medium Todd-Hewitt broth (Oxoid) supplemented with 0.5% yeast extract (THY), defined chemical medium RPMI modified (RPMImod) (Schulz et al., 2014), or on blood agar plates (Oxoid) at 37◦C and 5% CO2.

## Bacterial Growth Curves

Bacteria were plated from cryo cultures on blood agar (Columbia agar with sheep blood, Oxoid) and incubated at 37◦C and 5% CO2. THY medium or RPMImod (40 ml in polypropylene tubes) was inoculated with freshly grown bacteria to an initial OD600 = 0.1–0.15 and incubated without agitation in a water bath at 37◦C. Growth was monitored at appropriate time points by measuring absorbance at OD600 (BioPhotometer, Eppendorf).

## Phagocytosis Experiments

Monocytic THP-1 cells were seeded in 24-well plates (2 × 105 cells per well in RPMI-1640 supplemented with 10% heat inactivated FCS in a volume of 1 ml) and differentiation was stimulated by the addition of 200 nmol/ml phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich). Prior to infection THP-1 cells were incubated for 72 h at 37◦C and 5% CO2. Pneumococci cultured in RPMImod to mid-log phase (OD600 = 0.35–0.45), were centrifuged and washed with infection medium (RPMI-1640, PAA) containing 1% heat inactivated fetal bovine serum (Gibco).

THP-1 cells were infected with S. pneumoniae using a multiplicity of infection (MOI) of 50 bacteria per phagocyte at 37◦C and 5% CO2 in infection medium. Bacteria were slightly centrifuged (2 min, 200 × g) onto the cells to initiate a simultaneous contact with phagocytes. Post infection, phagocytes were washed with infection medium and subsequently incubated with Penicillin G (100 units/ml, Sigma-Aldrich) and Gentamicin (0.1 mg/ml, Sigma-Aldrich) for 1 h at 37◦C and 5% CO2. After washing, the phagocytes were lysed using a 1% saponin solution. The colony forming units (cfu) of released intracellular pneumococci was determined by plating the bacteria in appropriate dilutions on blood agar plates (Hermans et al., 2006; Noske et al., 2009; Hartel et al., 2011).

Phagocytosis was also analyzed in the presence of the pharmacological inhibitors of PI3-kinase (Wortmannin, 50 nM and LY294002 50 µM, ENZO Life Sciences), Akt kinase (Akt-inhibitor VIII, 2.5 and 5 µM, Calbiochem), and actin polymerization (Cytochalasin D, 0.125 and 0.25 µM, ENZO Life Sciences).

## Double Immunofluorescence Staining

Pneumococci attached to or phagocytosed by PMA-differentiated THP-1 cells were visualized by double immunofluorescence microscopy (DIF). Therefore, THP-1 cells (2 × 105 ) were seeded on sterile glass cover slips (12 mm, Hartenstein) and cultured at 37◦C and 5% CO2, 72 h prior to the infection (in the presence of 200 nmol/ml PMA) and infected as described above. Post-infection, THP-1 cells were washed with infection medium to remove unbound bacteria and then fixed with 1% paraformaldehyde (Roth). After blocking with 5% bovine serum albumin (BSA, Roth), extracellular bacteria were stained using a polyclonal anti-pneumococcal serum (1:200) and secondary goat anti-rabbit IgG coupled to Alexa-Fluor-488 (1:500, Abcam). Intracellular pneumococci were stained with Alexa-Fluor-568 goat anti-rabbit IgG (1:500, Abcam) after permeabilization of the THP-1 cells with 0.1% Triton-X-100 (Sigma) (10 min, room temperature) and pneumococcal antiserum as primary antibody (1:200). For the statistical analysis 100 cells per experiment and time point were analyzed for the number of intracellular bacteria.

## SDS-Page and Immunoblotting

To analyze the phosphorylation status of selected host kinases during pneumococcal infection, cell lysates were prepared as followed. THP-1 cells (1 × 106 ) cells were seeded in 6-well plates in a volume of 2 ml/well. Cells were infected as described above and infection was stopped at different time-points by washing cells with ice-cold infection medium, following cell disruption by the addition of Triton-X-100 lysis buffer (10 mM TRIS, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 0.1% SDS, 1% Triton-X-100, 10% glycerin, 0.5% sodium deoxycholate) supplemented with a protease inhibitor (Complete R , Roche). Afterwards, plates were incubated on ice for 10 min followed by sonication (2 × 30 s). After centrifugation (18,234 × g, 10 min, 4◦C) protein concentration of the supernatant was determined using the Bradford assay (Sigma) and stored at −20◦C. Cell lysates (50 µg/sample) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 12% gels. Proteins were transferred on a nitrocellulose membrane (GE Healthcare) by semi-dry blotting. After transfer, the membrane was blocked overnight at 4◦C in TBS + 5% skim milk (Roth). Immunodetection was carried out using specific primary and horse radish peroxidase-(HRP)-conjugated secondary antibodies (listed in **Table 1**). A primary antibody against glycerinaldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Luminol was used as substrate for HRP. Detection was carried out using a chemiluminescence-detecting camera (ChemoCam, INTAS).

## Statistics and Quantitative Analysis of Immunoblots

All data are reported as the mean ± SD. Statistical analysis was performed with unpaired, two-tailed Student's t-test using GraphPad Prism R software v5.01. In all analyzes a p < 0.05 was considered statistically significant.

The image processing program ImageJ was used for quantificational analysis of immunoblots by densitometry (Girish and Vijayalakshmi, 2004; Schneider et al., 2012).

## RESULTS

## Influence of Bacterial Growth Medium on Pneumococcal Phagocytosis by THP-1 Cells

To assess the impact of the bacterial growth medium on pneumococcal phagocytosis by PMA-differentiated THP-1 cells, S. pneumoniae were grown in complex medium (THY) or chemically defined medium (RPMImod)prior to infection. The growth curves of S. pneumoniae in the different growth media (**Figure 1A**) showed a delayed growth in the chemically defined medium in comparison to the complex medium, which is probably due to the high adaptation of pneumococci to the overall nutrient availability in RPMImod. In consequence, pneumococci cultured in THY showed an earlier achievement of the stationary phase and a higher optical density.

Pneumococci used in infection assays were harvested from the exponential growth phase (OD600 = 0.35–0.45). PMAdifferentiated THP-1 cells were infected for 1 h. The number of recovered intracellular surviving bacteria was determined by the antibiotic protection assay. The results revealed no significant influence of the bacterial growth medium on pneumococcal uptake by PMA-differentiated THP-1 (**Figures 1B,C**).

## Kinetics of *S. pneumoniae* Phagocytosis by THP-1 Cells

In the results of the antibiotic protection assay a time-dependent increase of phagocytized pneumococci could be observed over time without saturation 90 min post infection (**Figure 2A**). In the complementary infection assay, adherent extracellular, and ingested intracellular pneumococci were visualized and illustrated using DIF-staining. Similar to the antibiotic protection assay, an increase of intracellular bacteria was monitored over time as shown by representative microscopic images (**Figure 2B**). The enumeration of intracellular bacteria in 100 infected THP-1 cells using immunofluorescence microscopy confirmed the results of the antibiotic protection assay. This approach also demonstrated a time-dependent increase of intracellular bacteria upon increasing infection times, without reaching saturation 90 min post-infection (**Figure 2C**).

## Intracellular Fate of Phagocytized Pneumococci

The intracellular fate of S. pneumoniae after phagocytosis was analyzed in a time-dependent manner post-infection of THP-1 cells. Extracellular bacteria were killed after infection by the addition of antibiotics. The intracellular number of recovered, viable bacteria was determined after lysis of the THP-1 cells and plating of the bacteria on blood agar plates. The THP-1 cells

showed a continuous killing of the engulfed pneumococci during the evaluation period as determined by a constant decrease of bacterial cfu over time (**Figure 3A**). Similar, the DIF-staining and fluorescence microscopy confirmed the results of the antibiotic protection assay (**Figures 3B,C**). Representative images and numbers of intracellular bacteria within infected THP-1 cells confirmed the decrease of intracellular pneumococci over the observed time period. Taken together, these results revealed an effective time-dependent intracellular killing of phagocytized pneumococci by THP-1 cells.

## Inhibition of Pneumococcal Phagocytosis by Inhibitors of Central Host Cell Kinases

Phagocytosis and hence, the rearrangement of the host cell cytoskeleton requires most likely bacterial binding to cell surface receptors, followed by cell signaling through different protein kinases and finally activation of proteins involved in the reorganization of the cytoskeleton. To demonstrate the

Values represent means ± SD of at least three independent experiments.

influence of actin polymerization during phagocytosis, infection experiments were carried out in the presence of Cytochalasin D (CytoD), a potent inhibitor of actin polymerization. PMAdifferentiated THP-1 cells were infected with S. pneumoniae 35A in the presence of different concentrations of CytoD. DIFstaining and immunofluorescence microscopy were applied to visualize the effect of CytoD on the phagocytosis rate. The

addition of increasing concentrations of CytoD resulted in a dose-dependent decline of intracellular pneumococci within the infected THP-1 cells (**Figure 4A**). To quantify ingested bacteria the number of intracellular bacteria was enumerated in infected cells by DIF-staining and subsequent immunofluorescence microscopy (**Figure 4C**). A significant, dose-dependent effect of CytoD on the phagocytosis rate (reduction of intracellular bacteria to 40 and 16%) was visible. These results were confirmed with an antibiotic protection assay where the amount of living

extracellular bacteria significantly declined dose-dependently (45 and 25%) with increasing concentrations of CytoD (**Figure 4B**).

This experimental approach was further chosen to analyze the role of the protein kinases PI3K and Akt in the process of pneumococcal phagocytosis by THP-1 cells. The presence of the PI3K inhibitors Wortmannin and LY294002 resulted in a significant inhibition of pneumococcal uptake after 1 h of infection. The microscopic images of the DIF-stained infections (**Figure 5A**) and counted bacteria within the THP-1 cells (**Figure 5C**) showed a significant decrease of intracellular bacteria (33 and 30%) compared to the control cells treated only with DMSO. These results were comparable to the results obtained in the complementary antibiotic protection assay (**Figure 5B**). To analyze the role of the protein kinase Akt, different concentrations of the Akt inhibitor VIII were used during pneumococcal infection of THP-1 cells. The results of the antibiotic protection assay (**Figure 6A**) in the presence of Akt inhibitor VIII revealed a dose-dependent decrease of viable intracellular pneumococci (66 and 39%) with increasing concentrations of the inhibitor. Taken together, these results reflect the essential role of actin polymerization and the involvement of the protein kinases PI3K and Akt in the Fcγ

and CR-independent phagocytosis of S. pneumoniae through PMA-differentiated THP-1 cells.

## Time-Dependent Phosphorylation of Selected Protein Kinases during Bacterial Phagocytosis

To evaluate the involvement and time-dependent phosphorylation of various protein kinases during pneumococcal infection, PMA-differentiated THP-1 cells were infected with S. pneumoniae for different time periods. Cell lysates were used for SDS-PAGE and immunoblot analysis with specific antibodies against phosphorylated and non-phosphorylated forms of different protein kinases (**Table 1**). The inhibition of Akt in the presence of Akt inhibitor VIII was already demonstrated to be involved in pneumococcal uptake as shown by an antibiotic protection assay (**Figure 6A**). The immunoblots of Akt/pAkt indicated an increase in Akt phosphorylation 10 min post infection with the highest amount after 20 min (**Figure 6B**). A densitometric approach was chosen to quantify the obtained results from the immunoblot. The analysis reflects thereby the observed increase of the phosphorylated form of Akt as shown in **Figures 6 C,D**. Moreover, the phosphorylation of the MAPKs ERK1/2 and p38 during pneumococcal uptake was analyzed (**Figure 7**). Here the amount of phosphorylated ERK1/2 (pERK1/2) starts to increase 20 min post-infection, whereas the amount of phosphorylated p38 (pp38) increases already after 5 min post-infection.

## DISCUSSION

The innate immune system comprises an enormous arsenal of defense strategies to recognize and eliminate foreign microbes, colonizing or invading the human body. These include amongst others physical barriers like epithelia and mucus, secreted enzymes, antimicrobial peptides, and phagocytes (Someya et al., 2013; Sperandio et al., 2015). A critical function of the immune system is the detection and elimination of invading microbes in normally sterile compartments of the human body. In such a case, professional phagocytes recognize microbes amongst others via PRRs, triggering intracellular signal cascades leading in the end to engulfment and elimination of foreign intruders (Silva, 2010).

In this study, we investigated the interaction of pneumococci with macrophage-like, PMA-differentiated THP-1 cells in the absence of human antibodies or complement. This approach allowed us to analyze the host cell response independently of Fcγ or CR-mediated phagocytosis pathways.

Pneumococci have to adapt to various environmental conditions, including nutrient availability, during colonization of mucosal surfaces in humans and invasive infections. Changes in the nutrient composition or availability results in an alteration of intracellular metabolites, influencing regulatory networks and as a consequence gene expression and protein production (Orihuela et al., 2004; Tang, 2011; Schulz and Hammerschmidt, 2013). Pneumococcal growth in a chemically defined medium reflects much more the in vivo situation, in which certain nutrients and/or carbon sources are limited. As shown in the pneumococcal growth curves (**Figure 1A**), growth was indeed affected due to the limitation of several nutrients. Interestingly, no difference in bacterial uptake through THP-1 cells could be observed using chemically defined (RPMImod) or complex medium (THY). This indicates that the composition of surfaceexposed bacterial structures necessary for the recognition and uptake of S. pneumonia by THP-1 cells are not substantially changed or reduced due to the composition of the chosen growth media.

Kinetics of pneumococcal phagocytosis by THP-1 cells were analyzed by DIF-staining and subsequent fluorescence microscopy. With this approach, all intracellular bacteria can be visualized, while no differentiation between living and nonviable bacteria is possible. First intracellular pneumococci within THP-1 cells were detected after 15–30 min of infection. The monitored pneumococcal internalization happened in the same time-frame as carried out with other bacteria like S. aureus and THP-1 cells (Miller et al., 2011). A saturation in pneumococcal uptake was not observed within the analyzed time period (15– 90 min), suggesting a higher capacity of THP-1 cells to take up bacteria.

A major virulence factor of S. pneumoniae is the polysaccharide capsule protecting the bacteria from phagocytosis (Wood et al., 1946). In our experimental approach, the low encapsulated strain 35A was chosen to facilitate recognition of surface exposed PAMPs by macrophage PRRs. Intracellular killing of bacteria in macrophages takes place in the phagolysosome via proteases, antimicrobial peptides and reactive oxygen and nitrogen species (Garin et al., 2001). Whereas, several bacterial species evolved strategies to survive or escape from phagolysosomes, such mechanisms are unknown for pneumococci. However, it was shown that during pneumococcal phagocytosis by dendritic cells, a minor proportion of the pneumococci escape from the intracellular vacuoles and resides in the cytosol (Noske et al., 2009). The reason or mechanisms for the pneumococcal escape from phagosomes is unknown. For experiments regarding the intracellular fate of the phagocytized pneumococci during infection, one time point was chosen. Pneumococci phagocytized after 1 h of infection were nearly completely killed within 5 h post-infection in a time-dependent manner, demonstrating the inability of S. pneumoniae 35A to survive within the macrophages. However, a minor amount of remaining intracellular pneumococcal cfu 5 h post-infection

could be explained with the outbreak of some of the pneumococci from macrophage phagosomes into the cytoplasm.

The participation of the actin machinery and central cellular protein kinases involved in intracellular signaling pathways was analyzed by pneumococcal infection assays in the presence of specific pharmacological inhibitors and immunoblot analysis of THP-1 cell lysates from different time points of infection. The engulfment of pneumococci into phagosomes requires the recognition of the bacteria via surface receptors on THP1 cells, activation of signal cascades and in the end remodeling of the cytoskeleton. The inhibition of actin polymerization by Cytochalasin D during phagocytosis leads in the conducted experiments to a dose-dependent reduction of pneumococcal uptake up to 75%. These results underline the essential function of cytoskeleton rearrangement in pneumococcal phagocytosis by PMA-differentiated THP-1 cells.

The PI3K is an essential regulator of phagocytosis as shown for Fcγ and CR-mediated phagocytosis (Araki et al., 1996; Cox et al., 1999). The enzyme catalyzes after activation the phosphorylation of phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-trisphosphate (PIP3) (Domin and Waterfield, 1997). PIP3 was shown to be a key player in signaling

pathways controlling phagocytosis (Gerisch et al., 2009). The activation of the PI3K can, besides others, also occur via PRRs as shown for various TLRs (Arbibe et al., 2000; Monick et al., 2001). Also in our experiments, using non-opsonized pneumococci, inhibition of the PI3K by the pharmacological inhibitors Wortmannin or LY294002 leads to strong decrease in pneumococcal phagocytosis. The protein kinase B (Akt) is one of the major signal transducers, activated by PIP3 of the PI3K (Chan et al., 1999). After 10 min of pneumococcal infection, the amount of phosphorylated Akt started to increase, with a maximum 20 min post-infection (**Figure 4B**). Inhibition of Akt by Akt inhibitor VIII led to a dose-dependent decrease of phagocytized pneumococci within the THP-1 cells. First intracellular bacteria where observed after 15–30 min after infection, which is in concert with the activation of Akt after 10 min, which in turn leads to activation of proteins involved in cytoskeleton rearrangement and therefore engulfment of the pneumococci.

Besides the activation of the PI3K and Akt, we were interested in the activation of the mitogen activated kinases ERK1/2 and p38. The infection of THP-1 cells with S. pneumoniae resulted in an increase of pERK1/2 20 min post-infection, without saturation after 60 min. The downstream substrates of ERK1/2 includes amongst others transcription factors, kinases, and cytoskeletal proteins (Yoon and Seger, 2006) involved in proliferation, differentiation, and activation of macrophages (Rao, 2001). Further investigations are needed here, especially with a focus on the role of ERK1/2 in the induction of cytokines during phagocytosis. The second MAPK we focused on is p38.  Doyle et al. proposed a model in which the activation of TLRs in macrophages leads amongst others to the activation of p38 resulting in the upregulation of scavenger receptors like MARCO and therefore enhanced phagocytosis (Doyle et al., 2004). In our experiments, the phosphorylation of p38 started within the first 5 min post-infection indicating an important role in the early phase of pneumococcal phagocytosis.

The activation of the aforementioned kinases during pneumococcal infection seems not to be restricted to professional phagocytes. Pneumococci were shown to interact with the cellular polymeric immunoglobulin receptor (pIgR) of respiratory epithelial cells via the pneumococcal surface protein C (PspC). The modulated signaling cascades resulting in uptake of the bacteria involves amongst others the PI3K, Akt, and the MAPKs ERK1/2 (Agarwal and Hammerschmidt, 2009; Agarwal et al., 2010). Furthermore, pneumococci were shown to be taken

## REFERENCES

up by human epithelial pharyngeal cells (Detroit 562) exploiting vitronectin as a bridging molecule to interact with αvβ3 integrins. In addition to the integrin linked kinase (ILK) the PI3K plays an essential role in pneumococcal endocytosis via the vitronectin mechanism (Bergmann et al., 2009).

The analysis of further signaling cascades like the JNK, Src, and focal adhesion kinases is necessary to gain deeper insights into signaling events triggered by pneumococci during phagocytosis.

Moreover, pneumococcal infection of macrophages was shown to contribute to apoptosis (Ulett and Adderson, 2006). Therefore, it could be of great interest to analyze the participation of pneumococcal triggered cell signaling pathways on the induction of apoptosis.

Taken together, we used the model of PMA-differentiated THP-1 cells to characterize the interaction of S. pneumoniae with professional phagocytes. The relevance of the bacterial growth medium on phagocytosis as well as the time-dependent uptake and killing was demonstrated. Furthermore, insights into cell signaling processes during bacterial uptake were deciphered by using pharmacological inhibitors and performing immunoblot analysis.

## AUTHOR CONTRIBUTIONS

TK was writing the paper and performed experiments. TK was the supervisor of AS. AS executed experiments, DK executed experiments, SH was writing the paper, supervisor of DK, project leader.

## ACKNOWLEDGMENTS

We thank Peggy Stremlow for excellent technical assistance. This work was supported by Deutsche Forschungsgemeinschaft Grant DFG HA 3125/4-2.

pneumococcal pneumonia and inhaled particles. J. Exp. Med. 200, 267–272. doi: 10.1084/jem.20040731

complement but not Fc gamma receptors. J. Immunol. 168, 6375–6381. doi: 10.4049/jimmunol.168.12.6375

**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 © 2016 Kohler, Scholz, Kiachludis and Hammerschmidt. This is an openaccess 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) or licensor 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.

# Characterization of Spbhp-37, a Hemoglobin-Binding Protein of *Streptococcus pneumoniae*

María E. Romero-Espejel 1 , Mario A. Rodríguez 1 , Bibiana Chávez-Munguía1 , Emmanuel Ríos-Castro2 and José de Jesús Olivares-Trejo3 \*

1 Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del IPN, México, México, 2 Unidad de Genómica, Proteómica y Metabolómica. LaNSE-CINVESTAV, Centro de Investigación y de Estudios Avanzados del IPN, México, México, 3 Laboratorio de Bacteriología y Nanomedicina, Posgrado en Ciencias Genómicas, Universidad Autónoma de la Ciudad de México, México, México

Streptococcus pneumoniae is a Gram-positive microorganism that is the cause of bacterial pneumonia, sinusitis and otitis media. This human pathogen also can cause invasive diseases such as meningitis, bacteremia and septicemia. Hemoglobin (Hb) and haem can support the growth and viability of S. pneumoniae as sole iron sources. Unfortunately, the acquisition mechanism of Hb and haem in this bacterium has been poorly studied. Previously we identified two proteins of 37 and 22 kDa as putative Hb- and haem-binding proteins (Spbhp-37 and Spbhp-22, respectively). The sequence of Spbhp-37 protein was database annotated as lipoprotein without any function or localization. Here it was immunolocalized in the surface cell by transmission electron microscopy using specific antibodies produced against the recombinant protein. The expression of Spbhp-37 was increased when bacteria were grown in media culture supplied with Hb. In addition, the affinity of Sphbp-37 for Hb was determined. Thus, in this work we are presenting new findings that attempt to explain the mechanism involved in iron acquisition of this pathogen. In the future these results could help to develop new therapy targets in order to avoid the secondary effects caused by the traditional therapies.

#### *Edited by:*

Jorge Eugenio Vidal, Emory University, USA

*Reviewed by:* Gregory T. Robertson, Colorado State University, USA Zehava Eichenbaum,

Georgia State University, USA

### *\*Correspondence:*

José de Jesús Olivares-Trejo<EMAIL_ADDRESS>
*Received:* 26 January 2016 *Accepted:* 12 April 2016 *Published:* 04 May 2016

#### *Citation:*

Romero-Espejel ME, Rodríguez MA, Chávez-Munguía B, Ríos-Castro E and Olivares-Trejo JJ (2016) Characterization of Spbhp-37, a Hemoglobin-Binding Protein of Streptococcus pneumoniae. Front. Cell. Infect. Microbiol. 6:47. doi: 10.3389/fcimb.2016.00047

Keywords: *Streptococcus pneumoniae*, haem, iron, iron starvation, haem-binding protein

## INTRODUCTION

Streptococcus pneumoniae is the most important cause of bacterial pneumonia and moreover this pathogen can cause infections as septicemia, bacteremia, and meningitis (Yaro et al., 2006; Thornton et al., 2010). This bacterium causes considerable human morbidity and mortality throughout the world, especially among children, the elderly and immunocompromised individuals (Gray et al., 1979; Austrian, 1989; Musher, 1992; Butler and Schuchat, 1999). However, the mechanisms for pneumococcal disease are not fully understood. There is a necessity for the discovering of novel therapeutic strategies focused on bacterial iron acquisition systems, because many bacteria pathogens require iron as an essential nutrient to infect the human (Klebba et al., 1982; Ratledge and Dover, 2000; Simpson et al., 2000; Crosa and Walsh, 2002; Andrews et al., 2003). Due to that the iron is required in several cellular processes, most bacteria have developed strategies for iron scavenging from host proteins (Wooldridge and Williams, 1993; Raymond et al., 2003; Ge and Sun, 2012; Andrews et al., 2013). One of the best studied bacterial iron acquisition systems is based on siderophores, which are secreted from the bacterial cell to scavenge free iron (Wooldridge and Williams, 1993; Guerinot, 1994; Wandersman and Delepelaire, 2004). Even though many pathogens secrete siderophores for iron acquisition during infection (Wandersman and Stojiljkovic, 2000; Genco and Dixon, 2001; Wandersman and Delepelaire, 2004), there are not biochemical or genetic evidences that S. pneumoniae produces siderophores (Tai et al., 1993; Brown et al., 2001; Romero-Espejel et al., 2013). As a result of the powerful reactivity of haem, it is generally sequestered within human cells by hemoproteins such as hemoglobin (Hb; Wandersman and Stojiljkovic, 2000; Wandersman and Delepelaire, 2004). In accordance, many bacteria have developed systems involved in iron acquisition from host hemoproteins (Tai et al., 1993; Brown et al., 2001; Genco and Dixon, 2001; Romero-Espejel et al., 2013). There are several studies on bacterial haem acquisition systems based mostly on Gram-negative bacteria (Stojiljkovic et al., 1996; Lewis et al., 1998; Wandersman and Stojiljkovic, 2000; Genco and Dixon, 2001; Olczak et al., 2001). Comparatively, less is known about how Gram-positive pathogens utilize host hemoproteins as an iron source. Recently, some surface proteins of Streptococcus pyogenes have been shown that bind haem (Shr and Shp, and haem-specific ATP-binding cassette transporter HtsABC). Shp has been shown to rapidly transfer its haem to the HtsA lipoprotein of HtsABC (Lei et al., 2002, 2003; Bates et al., 2003). In addition, it has been proposed that Shr is a source of haem for Shp and that the Shr-to-Shp haem transfer is a step of the haem acquisition process in S. pyogenes (Zhu et al., 2008).

Staphylococcus aureus acquires iron from haem by the Isd (iron-regulated surface determinant) system, which is formed by cell wall-anchored surface proteins (IsdA, IsdB, IsdC, and IsdH), a membrane transporter (composed by IsdD, IsdE, and IsdF), a transpeptidase (SrtB), and cytoplasmic haem-degrading monooxygenases (IsdG and IsdI) (Mazmanian et al., 2000, 2002, 2003; Skaar and Schneewind, 2004; Wu et al., 2005). Unfortunately, the mechanism of Hb and haem uptake in S. pneumoniae has been poorly studied. This pathogenic bacterium can grow using Hb or haem as a sole iron source. Hb acquisition is vital to microbial survival (Tai et al., 1993; Brown et al., 2001; Romero-Espejel et al., 2013). Previously, we detected two potential S. pneumoniae Hb- and haem-binding proteins (Spbhp) of 22 and 37 kDa, termed by us as Spbhp-22 and Spbhp-37. The Spbhp-37 protein had homology with a lipoprotein (Bierne et al., 2002; Romero-Espejel et al., 2013). Interestingly, several proteins required for virulence in Gram-positive bacteria are lipoproteins; for instance, FhuD which is an iron-siderophore transporter (Schneider and Hantke, 1993). Therefore, the aim of this work was to confirm the role of Spbhp-37 as Hb-binding protein and to determinate the affinity of Sphbp-37 for Hb.

## MATERIALS AND METHODS

## *S. pneumoniae* Growth Conditions

S. pneumoniae strain R6 was grown under microaerophilic conditions in 5% CO2 for 24 h at 37◦C on agar plates supplemented with 5% sheep blood. The cellular cultures were then inoculated in plates containing Todd-Hewitt Broth (THB), supplemented with 0.5% yeast extract (THB-Y) and incubated for 16 h at 37◦C with 5% CO2. For testing alternative iron sources the bacteria were cultivated in well culture plates containing medium THB, supplemented with 0.5% yeast extract (THB-Y) and 700 µm of 2,2′dipyridyl (a chelating agent) was added to eliminate free iron from the culture medium. Then, incubation was followed for 16 h at 37◦C with 5% CO2. The cellular growth was adjusted to 0.1 (OD600) by spectrophotometry. After 3 h under iron starvation, the culture medium was supplemented with 2 µM human Hb.

## Cloning and Expression of Spbhp-37 Recombinant Protein

The coding region of the Spbhp-37 gene, excluding the signal peptide, was amplified by PCR from S. pneumoniae genomic DNA. For its directional cloning we used as sense primer an oligonucleotide containing the BamHI recognition site (5′ - GGGGGGGATCCATGAACAAGAAACAATGGCTAGGTC-3′ ), and as anti-sense primer an oligonucleotide that included the SalI recognition site (5′ -GGGGGGTCGACTTATTTT TCAGGAACTTTTACGCTTCCATC-3′ ). Then, amplicon was cloned in frame with the glutathione-S-transferase (GST), tag of the pGEX-6P-1 construction vector (GE Healthcare) using the BamHI and SalI restriction sites. The nucleotide sequence was corroborated by automated DNA sequencer.

For expression, Escherichia coli (strain BL21) competent cells were transformed with the pGEX-6P-1 empty vector, used as a negative control, or with the construction containing the Spbhp-37 gene (pGEX-spbhp-37). Induction of recombinant proteins (GST and Spbhp-37-GST) was induced with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 3 h at 37◦C.

## Purification of the Spbhp-37 Recombinant Protein

Inclusion bodies (IB), where the recombinant protein was accumulated, were purified as described (Vallejo et al., 2002). Briefly, after protein induction, cultures were centrifuged at 1500 g for 40 min and bottom was suspended in 50 ml of buffer A (100 mM Tris-HCl pH 8.0, 10 mM EDTA, 100 mM NaCl) in the presence of 1 mM PMSF. Cells were sonicated for 30 s (100 W) and 50 s off time for a total sonication time (including the off time) of 10 min. Then, an equal volume of buffer A having 8 M urea and 1 mM PMSF was added, stirred for 1 h at 4◦C and centrifuged at 10000 g for 30 min. Pellet was resuspended in 500 ml of buffer B (100 mM Tris-HCl pH 8.0, 1 mM EDTA, 1 M NaCl) and centrifuged at 10000 g for 30 min. Pellet was resuspended in 500 ml of water, centrifuged again, and frozen at −70◦C. After that, pellet was resuspended in buffer C (2 M urea, 20 mM Tris-HCl pH 8.0, 0.5 M NaCl, and 2% Triton X-100), centrifuged for 15 min at 10000 g, washed with the same volume of buffer B and centrifuged again. Then, the wet pellet of IB (2.1 g) was dissolved in 20 ml of solubilization buffer (8 M urea, Tris-HCl pH 8.0, 0.5 M NaCl and 1 mM 2-mercaptoethanol) and stirred for 2 h at room temperature. Sample was centrifuged at 12000 g for 30 min at 4◦C and supernatant was extensively dialyzed against a freshly prepared solution containing 20 mM Tris-HCl pH 8.0 and 4 mM urea. Finally, dialyzed samples were filter through a 0.45 µm membrane and stored in aliquots at −70◦C. Recombinant protein was then purified by affinity chromatography using Glutathione–agarose beads (GE Healthcare) following the manufacturer's recommendations. Induction of the recombinant protein and its purification were examined by SDS-PAGE and western blotting assays using antibodies against GST. Cleavage of the GST tag was achieved using Pre-Scission protease following the manufacturer's recommendations.

## Production of Specific Antibodies

Recombinant protein was used as antigen to produce specific antibodies against Spbhp-37. Thus, recombinant protein was mixed with a volume of TiterMax Gold Adjuvant (Sigma 145380- 33-2). Then, a New Zealand white rabbit was injected with 150 µg of protein in 1 ml of suspension. For immunization, protein suspension was divided in four doses of 250 µl each, which were injected into two subcutaneous and two intramuscular sites. Immunization was performed three times for periods of 15 days. The study was conducted in accordance with Good Laboratory Practices (GLP) and Use of Laboratory Animals (NOM-062-ZOO-1999). The study protocol was approved by the Institutional Animal Care and Use Committee (IACUC)- Cinvestav. Thereafter, antiserum was obtained and tested using total S. pneumoniae extracts or Spbhp-37 purified protein.

## Western Immunoblotting

Protein samples from different fractions during purification of the Spbhp-37 recombinant protein or total extracts of S. pneumoniae, isolated as described (Romero-Espejel et al., 2013), were loaded onto 12% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were soaked for 1 h with 5% non-fat milk in PBS in order to saturate all remaining active binding sites, and then they were incubated with anti-GST (glutathione transferase; 1:10000) or anti-Spbhp-37 (1:10000) antibodies. After that, membranes were incubated with antirabbit IgGs secondary antibodies conjugated to horseradish peroxidase (Invitrogen 65–6120; 1:10000) and the antibodies recognition was revealed by chemiluminescence (Millipore).

## Immunoelectron Microscopy

Bacteria grown in THB-Y or in the presence of Hb as only iron source were fixed in 4% paraformaldehyde and 0.5% glutaraldehyde in PBS for 1 h at room temperature. Samples were embedded in the acrylic resin (LR White) and polymerized under UV at 4◦C overnight. Thin sections (i.e., 60 nm) were obtained and mounted on Formvar-covered nickel grids. Later, sections were incubated in PBS with 10% fetal bovine serum before incubation with the anti-Spbhp-37 antibodies diluted (1:100) in 5% fetal bovine serum. Then, samples were incubated with anti-rabbit IgGs secondary antibodies conjugated to 20 nm colloidal gold spheres (Ted Pella Inc; 1:100). Finally, sections were contrasted with aqueous solutions of uranyl acetate and lead citrate before being examined in a Jeol JEM-1011 transmission electron microscope.

## *S. pneumoniae* Growth in the Presence of Hb and Anti Spbhp-37-GST Antibodies

Cells of S. pneumoniae previously cultivated and inoculated in THB (supplemented with 0.5% of yeast extract). When it was necessary to test an iron supply alternative, a chelating agent 700 µm 2,2′ -dipyridyl (Sigma D216305-25G) was used in order to eliminate iron from the medium of culture. Thereafter, the bacteria were incubated for 16 h at 37◦C with an atmosphere regulated at 5% CO2. In order to synchronize the cellular growth (OD600) was adjusted to 0.1. Thus, the cellular growth was monitored each hour. After 3 h under iron starvation the medium of culture was supplemented with: (a) only 2.5 µM of Hb (Cat. H7379, Sigma R ), (b) pre-immune serum (500 mg/ml) plus Hb or (c) anti-Spbhp-37 antibodies (500 mg/ml) plus Hb. After that, cellular growth was monitored each hour for 4 h, comparing under iron limiting condition vs. the condition when Hb was supplied as the sole iron source or when the anti-Spbhp-37 antibodies were added.

## Overlay Assays

Total extracts of S. pneumoniae were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad). Membranes were incubated for 1 h at 37◦C with 0.5% nonfat milk in PBS and 0.05% Tween 20, pH 7.4 (PBST) to block unspecific sites and then overnight at 4◦C with human Hb (2.5 µM). After that, membranes were incubated for 1 h at 37◦C with anti-hemoglobin antibodies (Santa Cruz Biotechnology SC-21005) (1:10000), and lastly 1 h at 37◦C with the horseradish peroxidase-conjugated secondary antibodies (Invitrogen 65- 6120; 1:10000). Antibodies recognition was developed by chemiluminescence (Millipore). As a control, a membrane was incubated only with PBST before the incubation with anti-globin antibodies.

## Surface Plasmon Resonance (SPR)

All SPR experiments were performed using Biacore T200 optical biosensor (GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK). SPR measurements were carried out in HBS-EP running buffer 10X (10 mM HEPES, 3 mM EDTA, 150 mM NaCl, 0.05% v/v of Tween 20 and pH 7.4) at 25◦C in a CM5 chip (coated with carboxylated dextran). For the immobilization scouting Hb was dissolved (30 µg/ml) in acetate buffer (pH 3.5, 4, 4.5, 5, and 5.5) and injected at the Biacore system at flow rate of 10 µl/min, with a contact time of 120 s and using NaOH 50 mM as a wash solution to regenerate the chip surface. Once pH was selected (pH 4), Hb was dissolved in a corresponding acetate buffer (30 µg/ml) and immobilized to the chip using amine coupling at an immobilization level of RL = 435 RU to reach a theorical RUmax = 249.53. During the coupling, the chip surface was activated using 1:1 mixture of 100 mM Nethyl-N-(dimethylaminopropyl)-carbodiimide (EDC) and 100 mM N-hydroxysuccinimide (NHS; both dissolved in water), and after Hb injection, the residual activated carboxy methyl groups on the chip surface were blocked by 1 M ethanolamine, pH 8.5. For this study, flow cell 1 was blank immobilized (without protein) for using as a reference. To analyze interactions of Spbhp-37 with immobilized Hb, Spbhp-37

was dissolved in buffer HBS-EP and injected. The same buffer was used as the running buffer. The flow rate was maintained constant throughout the kinetics experiment (30 µl/min), contact time was settled for 120 s and dissociation time was kept at 300 s. Regeneration was carried out with NaCl 1 M for 30 s. Experiments were performed with various concentrations of Sphbp-37 from 100 to 1000 nM, monitoring the refractive index changes as a function of time under constant flow conditions. The relative amount of Spbhp-37 bound to the Hb was determined by measuring the net increase of refractive index over time compared with that of running buffer alone. This change was reported in response units (RU). The data analysis was done with Biacore T200 evaluation software version 1.0 and data was fit to 1:1 binding.

markers are indicated on the left. Arrows indicate the antibodies recognition of recombinant protein Spbhp-37.

## RESULTS

## Cloning and Expression of Spbhp-37

To investigate the participation of Spbhp-37 protein on iron acquisition, its encoding gene was cloned in the plasmid pGEX-6-P-1. The construction was termed pGEX-spbhp-37 and was used to transform BL21 strain; the expression of Spbhp-37 was induced with IPTG. The overexpression of Spbhp-37 protein was confirmed by SDS-PAGE stained with Coomassie blue, we observed a band of 63 kDa when bacteria were incubated with IPTG with respect to non-induced bacteria (**Figure 1**, lane 2). This molecular weight corresponds to the expected for the recombinant protein (26 kDa from GST and 37 kDa from Spbhp-37). Unfortunately, when soluble and insoluble fractions were separated, the Spbhp-37 recombinant protein was detected in the insoluble fraction (**Figure 1**, lane 4). Therefore, a protocol to solubilize the inclusion bodies (IB) was performed (see Materials and Methods Section) (**Figure 1**, lane 5) previous to purification of the protein by affinity chromatography (**Figure 1**, lane 6). To confirm the identity of the purified protein, we carried out western blot assays using anti-GST antibodies. Results showed that antibodies recognized the purified recombinant protein (**Figure 1B**, lane 1).

## Obtaining of Anti Spbhp-37 Antibodies

In order to produce anti Spbhp-37 antibodies, the recombinant protein was used as antigen to inoculate a New Zealand rabbit. As expected, the obtained antibodies recognized the recombinant protein in western blot assays (**Figure 1B**, lane 2). After that, antibodies were characterized by western blotting on total proteins of S. pneumoniae. In these assays, antibodies recognized a single band of 37 kDa, which corresponds to the molecular weight of Spbhp-37 (**Figure 1C**, lane 3). This band was not revealed when western blotting was performed with preimmune serum or with anti-GST antibodies (**Figure 1C**, lanes 2, 4, respectively). These results allowed us to demonstrate the specificity of the antibodies raised against Spbhp-37.

## Spbhp-37 Protein is Increased Two Fold on the Surface of *S. pneumoniae* When it is Grown in the Presence of Hb As Only Iron Source

To investigate the location of Spbhp-37 in S. pneumoniae bacteria we performed immunoelectronic microscopy assays using antibodies against the recombinant protein. Our results showed the presence of Spbhp-37 protein on bacteria surface (**Figure 2B**). Signal was specific for Spbhp-37 because not signal was detected in a control incubated only with the gold-labeled secondary antibodies (**Figure 2A**). Then, to analyze the effect of Hb on the expression of Spbhp-37, we cultivated S. pneumoniae in THB in the presence of Hb as the sole iron source. In this condition we observed that the occurrence of Spbhp-37

on bacteria surface was more abundant than when cells were grown in normal medium (**Figure 2C**). To obtain a quantitative value, the positive signals were counted in each condition. Results revealed an increase of about two fold of Spbhp-37 in bacteria grown with Hb (**Figure 2D**). This result was corroborated by western blotting (data no showed). These results suggest that the presence of Hb increases two fold the abundance of Spbhp-37.

## Anti Spbhp-37-GST Antibodies Limited the Cellular Growth When Hb Was Supplied As the Sole Iron Source

To explore whether Spbhp-37 protein is related to utilization of Hb in the cellular growth of S. pneumoniae, we designed an experiment in which the anti-Spbhp-37 antibodies were supplied to block the bacteria growth under free iron limiting conditions, but using Hb as sole iron source. When S. pneumoniae was cultivated in THB under iron starvation, the cellular growth was limited, but when this media was supplemented with Hb as the sole iron source, the cellular growth was restored (**Figure 3**). Interestingly, the cellular growth was blocked when anti-Spbhp-37 antibodies were added to cellular cultures with Hb as the sole iron source (**Figure 3**). In addition, preimmune serum had a minimal effect on the cellular growth in media supplemented with Hb (**Figure 3**). Results clearly showed the cellular growth under iron limiting conditions was impaired.

FIGURE 3 | Anti Spbhp-37-GST antibodies block the cellular growth of *S. pneumoniae.* S. pneumoniae strain R6 was cultivated in Todd-Hewitt Broth under conditions of: iron sufficiency ( ); without iron ( ); without iron and supplemented with Hb ( ); without iron and supplemented with Hb in the presence of anti-Spbhp-37 antibodies ( ); and without iron and supplemented with Hb in the presence of pre-immune serum (used as unrelated antibodies) ( ). The cellular growth was monitored each hour for a period of 7 h by spectrophotometry (600 nm). Data represent mean ± SD of three independent experiments by triplicate. The cellular growth from bacteria cultivated in the medium without iron and supplemented with Hb in the presence of anti-Spbhp-37 antibodies was significantly higher compared to the cellular growth from bacteria cultivated in the medium without iron and supplemented with Hb in the presence of pre-immune serum (used as unrelated antibodies; p < 0.05, one-way ANOVA).

difference (p < 0.05).

Arrowheads indicate the location of Spbhp-37 protein in S. pneumoniae. In right is shown the magnification of cell bacteria to compare expression in both growth conditions. (D) Quantitative analysis of Spbhp-37 expression. Gold particles on bacteria growth under normal conditions (TH) and in medium with Hb as only iron source (-Fe+Hb) were counted n = 25. Data represent mean ± SD of three independent experiments. Asterisk indicates a significant

## Anti-Spbhp-37 Antibodies Block the Interaction between Spbhp-37 Protein and Hb

Inhibition of cellular growth with anti-Spbhp-37 notwithstanding the presence of Hb supports the hypothesis that Spbhp-37 is a receptor for Hb and that antibodies block the interaction between both proteins. To corroborate these

the overlay assay was incubated first with anti-Spbhp-37 antibodies. (C) Negative control. Nitrocellulose membranes were incubated only with primary and secondary antibodies (omitting the incubation with Hb). (D) Coomassie blue staining. Total proteins of S. pneumoniae were loaded onto 12% SDS-PAGE. Arrow indicates the Spbhp-37 protein.

assumptions, the interaction between Spbhp-37 protein and Hb as well the blockage of this interaction by anti Spbhp-37 antibodies were investigated by overlay assays. Thus, total proteins of S. pneumoniae (**Figure 4D**) were separated in SDS-PAGE and transferred to nitrocellulose membranes. Then, membranes were incubated with Hb and interaction was revealed with anti-Hb antibodies. At least five proteins, including a 37 kDa band Spbhp-37 protein (located in the top of gel), were recognized by Hb and anti-Hb antibodies when total proteins were used (**Figure 4A**). Interestingly, the detection of the protein was not observed when anti-Spbhp-37 antibodies were added in the overlay assays (**Figure 4B**). In addition, not bands were detected when incubation of Hb was omitted in the overlay experiments (used as a negative control) (**Figure 4C**). Results confirmed that Spbhp-37 binds to Hb and that antibodies block the interaction between both proteins.

## Spbhp-37 Showed High Affinity by Hb

To determine the affinity of Spbhp-37 for Hb, the GST tag of the Spbhp-37 recombinant protein was eliminated by digestion with the Pre-Scission protease and its affinity to Hb was analyzed by surface plasmon resonance (SPR). First, to confirm the cleavage of the GST tag, protein samples were analyzed by SDS-PAGE and western blotting. After SDS-PAGE and coomassie blue staining we observed the purified Spbhp-37-GST protein (**Figure 5A**, lane 1), the Spbhp-37 protein without GST (**Figure 5A**, lane 2), and the releasing of the GST tag (**Figure 5A**, lane 3). Identity of Spbhp-37 protein was corroborated by western blotting assays using the anti Spbhp-37 antibodies. These antibodies recognized the Spbhp-37-GST protein (**Figure 5B**, lane 1) and the Spbhp-37 protein without the GST tag (**Figure 5B**, lane 2), but GST was not revealed (**Figure 5B**, lane 3). As a control, western blotting was

performed using an anti-GST antibody, where only Spbhp-37- GST protein (**Figure 5C**, lane 1), and GST (**Figure 5C**, lane 3) were revealed.

To obtain kinetic binding data by SPR, Hb was immobilized on the sensor chip and the binding of Spbhp-37 (without GST tag) at concentrations ranged from 100 to 1000 nm was tested. In these experiments we observed a dose-dependent binding of Spbhp-37 to Hb (**Figure 6**). Curve fitting of the sensograms enabled us to determine that Kd was of 3.57 e-7 M, showing a high affinity of Spbhp-37 protein for Hb.

## DISCUSSION

S. pneumoniae is a human pathogen that uses Hb to cover its iron necessities. However, the iron acquisition mechanism has been poorly studied. Previously, we identified a lipoprotein of 37 kDa (Spbhp-37) as a S. pneumoniae membrane protein, which was purified by haem affinity chromatography and that could be involved in the Hb and haem acquisition. In the present work, by use of an antibody raised against Spbhp-37 recombinant protein we showed that the expression of native protein was increased on the surface of S. pneumoniae when Hb was supplemented as the sole iron source. These results clearly showed the character of receptor protein, because it was detected on the surface of the bacterium (Lei et al., 2002, 2003; Bates et al., 2003; Mazmanian et al., 2003). In addition, when these antibodies were used to analyze the bacterial growth in the presence of Hb, we noticed that they diminished about 50% the cell growth with respect to that obtained in media with Hb in the absence of this antibody or in the presence of pre-immune serum, indicating the importance of Spbhp-37 on uptake of Hb when was supplied as only iron source. This hypothesis was confirmed by overlay assays, because we found that Hb bound to Spbhp-37 protein and anti Spbhp-37 antibodies were capable to inhibit the interaction between Hb and Spbhp-37 protein. In fact, antibodies only blocked the Hb binding to one (Spbhp-37) of the all bands previously identified in S. pneumoniae total proteins (Romero-Espejel et al., 2013), which correspond to 37 kDa size, this observation clearly showed the specificity of the antibodies produced. On the other hand, the low Kd determined by SPR for the binding of Hb and Spbhp-37 protein (3.57 e−7 M) is similar to that reported for the TonB-dependent haem receptor (HasR) of Serratia marcescens, the outer membrane haem receptor (HmuR) of Porphyromonas gingivalis, and haem receptor (HasA) of S. marcescens (Ghigo et al., 1997; Olczak et al., 2001; Deniau et al., 2003), indicating that Spbhp-37 protein binds Hb with high affinity and suggesting that this protein is necessary in the mechanisms of iron acquisition to scavenge iron. Possibly other proteins are involved in this mechanism, for example those reported previously by our group, which include a stress general protein of 22 kDa, a maltosebinding protein of 45 kDa, and a glutamine synthetase type I 50 kDa (Romero-Espejel et al., 2013). These proteins could help to introduce and store the iron source to cytoplasm as it has been described for other Gram-positive bacteria (Mazmanian et al., 2002, 2003; Skaar et al., 2004; Skaar and Schneewind, 2004; Wu et al., 2005).

Our overall results attempt to explain the iron acquisition mechanism by S. pneumoniae when Hb is available. We showed that Spbhp-37 is a surface protein involved in Hb uptake, an essential mechanism of this pathogen to establish an infection process in human, because this bacterium uses Hb and haem as iron sources.

## AUTHOR CONTRIBUTIONS

ME conceived and carried out most of the experiments, analyzed data, and drafted the manuscript; BC was responsible for transmission electronic microscopy experiments; ER performed the surface plasmon resonance assays; MR and JO designed the study, analyzed data, and drafted the manuscript. All authors read and approved the final manuscript.

## FUNDING

This work was supported by Consejo Nacional de Ciencia y Tecnología (CONACyT) [grant numbers: SALUD-2012-01- 181641 and 222180].

## ACKNOWLEDGMENTS

We would like to thank Carlos Vázquez-Calzada and Mario Rodríguez-Nieves (Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del IPN, México City, México) for their help in the generation of specific antibodies and reagents preparation used in this work. We also thank Lorena Ramírez-Reyes (Unidad de Genómica, Proteómica y Metabolómica. LaNSE-CINVESTAV, Centro de Investigación y de Estudios Avanzados del IPN, México City, México) for her help in experiments of surface plasmon resonance.

## REFERENCES

of surface proteins and in the pathogenesis of animal infections. Proc. Natl. Acad. Sci. U.S.A. 97, 5510–5515. doi: 10.1073/pnas.080 520697

**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 © 2016 Romero-Espejel, Rodríguez, Chávez-Munguía, Ríos-Castro and Olivares-Trejo. 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) or licensor 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.

## Surface Proteins and Pneumolysin of Encapsulated and Nonencapsulated Streptococcus pneumoniae Mediate Virulence in a Chinchilla Model of Otitis Media

Lance E. Keller † , Jessica L. Bradshaw, Haley Pipkins and Larry S. McDaniel\*

*Department of Microbiology and Immunology, University of Mississippi Medical Center, Jackson, MS, USA*

*Streptococcus pneumoniae* infections result in a range of human diseases and are responsible for almost one million deaths annually. Pneumococcal disease is mediated in part through surface structures and an anti-phagocytic capsule. Recent studies have shown that nonencapsulated *S. pneumoniae* (NESp) make up a significant portion of the pneumococcal population and are able to cause disease. NESp lack some common surface proteins expressed by encapsulated pneumococci, but express surface proteins unique to NESp. A chinchilla model of otitis media (OM) was used to determine the effect various pneumococcal mutations have on pathogenesis in both NESp and encapsulated pneumococci. Epithelial cell adhesion and invasion assays were used to examine the effects in relation to deletion of intrinsic genes or expression of novel genes. A mouse model of colonization was also utilized for comparison of various pneumococcal mutants. It was determined that pneumococcal surface protein K (PspK) and pneumolysin (Ply) affect NESp middle ear pathogenesis, but only PspK affected epithelial cell adhesion. Experiments in an OM model were done with encapsulated strains testing the importance of native virulence factors and treatment of OM. First, a triple deletion of the common virulence factors PspA, PspC, and Ply, (1PAC), from an encapsulated background abolished virulence in an OM model while a PspC mutant had detectable, but reduced amounts of recoverable bacteria compared to wildtype. Next, treatment of OM was effective when starting antibiotic treatment within 24 h with resolution by 48 h post-treatment. Expression of NESp-specific virulence factor PspK in an encapsulated strain has not been previously studied, and we showed significantly increased adhesion and invasion of human epithelial cells by pneumococci. Murine colonization was not significantly increased when an encapsulated strain expressed PspK, but colonization was increased when a capsule mutant expressed PspK. The ability of PspK expression to increase colonization in a capsule mutant despite no increase in adhesion can be attributed to other functions of PspK, such as sIgA binding or immune modulation. OM is a substantial economic burden, thus a better understanding of both encapsulated pneumococcal pathogenesis and the emerging pathogen NESp is necessary for effective prevention and treatment.

Keywords: Streptococcus pneumoniae, pneumococcal surface proteins, PspK, nonencapsulated Streptococcus pneumoniae, NESp, pneumococcus, chinchilla, otitis media

Edited by:

*Jorge Eugenio Vidal, Emory University, USA*

#### Reviewed by:

*Kevin Mason, The Ohio State University, USA W. Edward Swords, Wake Forest University Health Sciences, USA*

> \*Correspondence: *Larry S. McDaniel<EMAIL_ADDRESS>
†Present Address:

*Lance E. Keller, Department of Molecular Genetics, University of Groningen, Groningen, Netherlands*

> Received: *25 January 2016* Accepted: *02 May 2016* Published: *18 May 2016*

#### Citation:

*Keller LE, Bradshaw JL, Pipkins H and McDaniel LS (2016) Surface Proteins and Pneumolysin of Encapsulated and Nonencapsulated Streptococcus pneumoniae Mediate Virulence in a Chinchilla Model of Otitis Media. Front. Cell. Infect. Microbiol. 6:55. doi: 10.3389/fcimb.2016.00055*

## INTRODUCTION

The pathogenesis of Streptococcus pneumoniae (the pneumococcus) is varied and complex due in part to surface structures (Gillespie and Balakrishnan, 2000; Hammerschmidt, 2006; Thornton et al., 2010). Pneumococcal diseases account for ∼1 million childhood deaths annually worldwide (O'Brien et al., 2009). However, introduction of the pneumococcal conjugate vaccine (PCV) has led to a dramatic decline in invasive pneumococcal disease (IPD) (Fitzwater et al., 2012). The currently available pneumococcal vaccines target specific pneumococcal polysaccharide serotypes, 23 in Pneumovax (Pneumovax R 23; PPSV23; Merck, Whitehouse Station, NJ, USA), and 13 in Prevnar [Prevnar 13 R ; PCV13; Pfizer (formerly Wyeth Pharmaceuticals), New York, NY, USA]. Despite extensive use of the PCV in certain parts of the world, noninvasive pneumococcal infections are still prevalent (Weinberger et al., 2011). Noninvasive pneumococcal infections include nonbacteremic pneumonia, conjunctivitis, otitis media (OM), and sinusitis. The pneumococcus is a common etiological agent of OM. In the United States, OM is responsible for most pediatrician visits (McCaig and Hughes, 1995; Gonzales et al., 2001; Lieberthal et al., 2013).

The major pneumococcal virulence factor and target of the PCV is the pneumococcal polysaccharide capsule. There are at least 97 antigenically distinct capsule types (serotypes), as well as pneumococci that do not express any capsule (Geno et al., 2015). Encapsulated strains are dependent on the presence of capsule for all stages of the life cycle, from colonization to virulence, while nonencapsulated S. pneumoniae (NESp) do not require a capsule (Kadioglu et al., 2008). Pneumococcal surface proteins are required for colonization and mediate virulence independent of the capsular status. Pneumococcal surface proteins are classified by means of surface attachment and include choline binding proteins (CBPs), LPxTG binding, lipoproteins, and nonclassical surface proteins (Bergmann and Hammerschmidt, 2006).

The surface of NESp and encapsulated pneumococci vary greatly, not only because of the presence or absence of capsule, but also based on the various surface proteins expressed. Regardless of what surface proteins are expressed in either NESp or encapsulated pneumococci, they are essential for colonization and pathogenesis (Valentino et al., 2014). The surface composition between different encapsulated strains also varies, usually as a result in variations of what genes are encoded and expressed, along with different protein isoforms. For instance, the encapsulated pneumococcal CBPs PspA and PspC have multiple variants and have been shown to be important for virulence in both invasive and noninvasive infections (Ogunniyi et al., 2007a). It is important to understand the alterations in virulence profiles as a consequence of differential surface protein expression in order to better understand which strains may be more pathogenic. Furthermore, genetic exchange between pneumococci occurs rapidly and can alter the virulence potential of a strain. Past studies examining alterations in surface proteins have shown that during OM, deletion of PspC reduced virulence while loss of PspA completely eliminated disease in a serotype 2 background (Schachern et al., 2014). NESp lack PspA and PspC but some express the LPxTG binding protein PspK, which has been shown to increase NESp colonization and virulence during OM (Park et al., 2012; Keller et al., 2013, 2014, 2015). We have previously demonstrated adherence to epithelial cells is an important function of NESp surface proteins, and increased adhesion of NESp correlated to increased bacterial burden during OM (Keller et al., 2013, 2014). Another important virulence factor that all known encapsulated and nonencapsulated pneumococci possess is pneumolysin (Ply). It has been shown that Ply has a significant function during infection (Mitchell and Mitchell, 2010). The role of Ply in NESp virulence during OM has not been previously established.

Pneumococci are known to exchange genetic material that has resulted in a high frequency of recombination within the cps locus, potentially as a consequence of selective pressure from the use of the PCV (Croucher et al., 2011). Genetic exchange at the cps locus between encapsulated pneumococci and NESp may allow encapsulated strains to persist transiently as a nonencapsulated variant despite vaccination. High rates of chromosomal recombination are also focused around regions harboring genes for antibiotic resistance (Croucher et al., 2011). Antibiotic resistance is commonly observed in pneumococcal strains, and NESp often harbor multiple drug resistances (Sulikowska et al., 2004; Chewapreecha et al., 2014; Keller et al., 2016). Due to rapid transfer of resistance genes between pneumococcal strains, prompt, and targeted treatment is necessary for pneumococcal infections. Timely treatment allows for faster resolution and may limit dissemination into other sites.

The current study demonstrates that the absence of Ply reduces the ability of NESp to cause OM. Also, epithelial cell adherence is an important function of OM virulence, but does not directly lead to pathogenesis of encapsulated strains. Additionally, we demonstrate that expression of PspK in capsule mutants partially compensates for the loss of capsule during colonization, thus affording pneumococci a way to circumvent increased use of capsule targeting vaccines.

## METHODS

## Bacterial Strains

**Table 1** contains bacterial strains, relevant mutations, and selective markers used in the current study. All pneumococcal strains were grown at 37◦C in 5% CO2 in Todd-Hewitt medium with 0.5% yeast extract (THY), or on sheep blood agar (BA) with appropriate antibiotic selection as indicated in **Table 1**. Genomic and plasmid DNA was obtained using manufacturers protocols with a DNeasy blood and tissue kit (Qiagen) or a plasmid minikit (Qiagen), respectively. Plasmids were maintained in Escherichia coli strain DH5α and grown in Luria Bertani broth (LB) with appropriate antibiotic selection.

## Genetic Manipulations

Gene deletions were made through allelic replacement. Pneumococcal transformations were performed in competence media (THY, 0.2% fresh bovine serum albumin, 0.01% CaCl2, and 0.1% glucose). Bacteria were grown in competence media to approximately OD600 0.2, diluted 1:20 in competence media, and

#### TABLE 1 | Bacterial strains used in this study.

*aTmp-trimethoprim; Tet, Tetracycline; Erm, Erythromycin; Kan, Kanamycin; Spec, Spectinomycin.*

stimulated with 2 µg/ml of competence-stimulating peptide 1 (CSP-1) for 12 min before the addition of ∼1 µg of DNA (Yother et al., 1986). Pneumococci were incubated for 4 h at 37◦C before plating on BA containing appropriate antibiotic selection.

DNA for pspK deletion in MNZ67 and cps deletion in EF3030 was obtained through polymerase chain reaction (PCR) of genomic DNA isolated from MNZ1131 (Park et al., 2012) with primers DexBF (5′ -GACTATCTAGCCAAGCTAGG-3′ ) and AliAR (5′ - CCCTGTACGAGATGTAGTTG -3′ ). DNA for ply deletion was isolated from 1Ply2 (Thornton and McDaniel, 2005) using primers UpstreamPlyF (5′ -CTAGCCTTGACAACT AGCCAATC-3′ ) and DownstreamPlyR (5′ -TGCAAATAGAAA GTTTCAGCC-3′ ). Expression of PspK was achieved through ectopic expression using plasmid pABG5. The pspK gene was isolated from genomic MNZ67 DNA using primers pABG5 PspKF (5′ -GCG**GAATTC**ATGAATAATAAGAATATCATC CCGATGAG-3′ ) and pABG5 PspKR (5′ -GCG**CTGCAG**CTA ATTTTTATGTTTAACAAATGGAAGA-3′ ). Primers contain restriction sites EcoRI and PstI, respectively, indicated by bolded and underlined section of primer. Plasmid pABG5 and pspK amplicon was digested with EcoRI (NEB) and PstI (NEB), ligated with T4 DNA ligase (Thermo Scientific) and transformed into DH5α making pABG5::pspK. Plasmid verified by sequencing and PspK levels in wildtype MNZ67 and PspK expression mutants were equivalent as determined by flow cytometry and Western blot analysis.

### Adhesion and Invasion Assays