The afc antifungal activity cluster, which is under tight regulatory control of ShvR, is essential for transition from intracellular persistence of Burkholderia cenocepacia to acute pro-inflammatory infection
[['B . cenocepacia belongs to the Burkholderia cepacia complex ( Bcc ) , currently encompassing 21 officially named species [1 , 2] .', 'These opportunistic bacteria are notorious pathogens of cystic fibrosis ( CF ) patients [3 , 4] and are emerging as the culprit of serious infections in non-CF conditions , both in and outside the hospital [5–8] .', 'Infection of CF airways by bacteria belonging to the Bcc can be asymptomatic but often result in chronic infection with intermittent acute exacerbations resulting in progressive worsening of lung function [9 , 10] .', 'B . cenocepacia is particularly associated with reduced survival and a high risk for development of unpredictable acute fatal necrotizing pneumonia and sepsis , termed “cepacia syndrome” .', 'Bcc bacteria are well-known for their intrinsic resistance to stress conditions and antibiotics [11–13] , complicating disease management and treatment strategies .', 'Bcc bacteria are ubiquitously present in the environment and in industrial settings; they have evolved intricate signalling networks to rapidly adapt to changing or stressful environments , for instance in competition for nutrients with other bacteria or fungi , or as a defence mechanism against nematodes and protozoan hosts [14–18] .', 'These signalling networks could also play important roles in pathogenicity and adaptation during human opportunistic infections .', 'As an example , the GacA/GacS signalling cascade in Pseudomonas spp .', 'has been shown to be involved in adaptation to natural conditions , while playing a crucial role during infection of CF patients by P . aeruginosa by providing a switch from acute ( Type 3 secretion system-dependent ) to chronic ( biofilm life stage ) infections [19 , 20] .', 'Several regulators , including the quorum sensing systems CepI/R and CciI/R , and the global regulator AtsR , have been shown to be involved in virulence of B . cenocepacia using different model hosts [21–23] .', 'ShvR is a LysR-type transcriptional regulator ( LTTR ) that has been shown to be involved in lung inflammation caused by B . cenocepacia K56-2 in a rat agar bead model of chronic lung infection [24] .', 'The shvR mutant , however , was highly persistent in the infected rat lungs .', 'In addition , the absence of ShvR resulted in loss of antifungal activity , reduced biofilm formation on abiotic surfaces [24] and a loss of rough colony morphotype , hence the name “Regulator of SHiny Variant” .', 'The absence of shvR has been shown to result in moderate changes in the expression of over 1000 genes [25] .', 'Interestingly , the expression of the adjacent afc operon , encoding 24 proteins specifying the synthesis of a lipopeptide with antifungal activity against Rhizoctonia solani [26] was reduced 100-fold , showing it is under tight positive regulatory control of ShvR [25] .', 'Mutation of the afcE gene ( BCAS0208 ) , encoding an acyl-CoA dehydrogenase , resulted in the same phenotypes as those seen for the shvR mutant , including loss of antifungal activity , reduced biofilm formation , and shiny colony morphology [25 , 27 , 28] .', 'Furthermore , mutation of afcE resulted in significantly reduced chronic lung inflammation in experimentally infected rats , similar to that found for a shvR mutant [28] .', 'The high persistence of the shvR mutant in a rat infection model , alongside its reduced ability to form biofilms prompted us to investigate a role for ShvR and AfcE in virulence of B . cenocepacia K56-2 using zebrafish embryos , particularly their contribution during intramacrophage stages .', 'B . cenocepacia is an intracellular pathogen and we have recently shown that macrophages play a crucial role in bacterial proliferation and acute fatal infection by B . cenocepacia in vivo using zebrafish larvae [29] .', 'Importantly , other Bcc strains were able to persist in macrophages , but could not efficiently disseminate and induce robust host pro-inflammatory responses [29 , 30] .', 'The reason for these different disease outcomes , which may have important clinical impact , is not clear .', 'In this work , we find that the expression of shvR and afcE is critical for B . cenocepacia K56-2 to progress from an intracellular persistent stage with low pro-inflammatory responses , to disseminated acute fatal infection .', 'Bioinformatic analysis indicates that the shvR/afc genes are present in the genomes of 50% of the 19 Bcc species sequenced to date .', 'B . cenocepacia H111 is closely related to K56-2 , but shows reduced virulence in different animal models , including zebrafish .', 'We show that acute virulence of H111 also depends on the presence of the afc cluster .', 'Strikingly , constitutive expression of shvR from a lac-promoter increased virulence of H111 to almost K56-2 levels in an afc-dependent manner .', 'Altogether , our results demonstrate an important role for the afc gene cluster in pathogenicity , the severity of which may depend on strain-specific upstream regulatory control mechanisms .'], ['We first analysed the role of B . cenocepacia K56-2 ShvR in virulence in the zebrafish infection model .', 'Zebrafish embryos were micro-injected at 30 hours post-fertilization ( hpf ) with B . cenocepacia K56-2 ( mCherrypCR11 ) , ΔshvR ( mCherrypCR11 ) or the complemented ΔshvR ( pshvR:shvR;mCherrypCR11 ) .', 'Analysis of embryo survival showed that ΔshvR was unable to cause fatal infection over the time course of the experiment , in contrast to its wildtype parent which caused 100% embryo mortality in 3 days ( Fig 1A and [30] ) .', 'In agreement with the observed reduced host mortality there was a significantly lower bacterial burden of ΔshvR compared to K56-2 at 24 and 48 hours post infection ( hpi ) ( Fig 1B ) .', 'Although all embryos survived infection with ΔshvR , some contained significantly more bacteria at 24 and 48 hpi compared to T = 0 ( Fig 1B ) , which suggests the mutant bacteria are able to replicate .', 'Expression of shvR from its endogenous promoter introduced on a single copy plasmid restored virulence to the ΔshvR mutant ( Fig 1 ) .', 'These results show that ShvR is required for development of acute fatal infection .', 'Our previous work showed that macrophages efficiently phagocytose intravenously injected B . cenocepacia K56-2 , and that they are critical for bacterial replication and ensuing fatal pro-inflammatory infection in zebrafish embryos [29] .', 'Phagocytosis of bacteria from the blood results in infected macrophages distributed over the yolk and the tail region [30] .', 'To analyse the interaction of ΔshvR with macrophages in the host , we quantified the percentage of ΔshvR bacteria that were internalised by macrophages using confocal imaging of infected Tg ( mpeg:mCherry-F ) transgenic embryos , which express a membrane localised mCherry protein ( Fig 2A and S1 Movie ) .', 'One hour after injection , ΔshvR bacteria were as efficiently phagocytosed by macrophages as wildtype bacteria ( Fig 2B ) .', 'Real time experiments showed that the ΔshvR bacteria still localised in macrophages at later time points , where they sometimes reached high numbers ( Fig 2C ) in agreement with the observed increase in CFU counts ( Fig 1B ) .', 'In contrast , at these later time points wildtype K56-2 bacteria have replicated to very high intracellular numbers , formed local infection sites ( Fig 2D ) , re-entered the blood circulation and spread systemically as shown earlier [29] .', 'Quantification of the number of intracellular ΔshvR bacteria in infected macrophages using Tg ( mpeg:mCherry-F ) embryos at 1 hpi and 24 hpi ( Fig 2E ) showed a significant decrease in the number of macrophages containing 1 or 2 bacteria and an increase in the number of macrophages containing more than 4 bacteria , respectively , demonstrating that the ΔshvR mutant is able to survive and replicate inside macrophages .', 'The total number of infected macrophages that were observed per embryo decreased in time from 12 . 3 ± 2 . 1 ( SEM; n = 15 embryos ) at 1 hpi to 7 . 6 ± 0 . 9 ( SEM; n = 10 embryos ) at 24 hpi , suggesting that a proportion of ΔshvR bacteria can establish a replication niche , while others get degraded .', 'In contrast to wildtype K56-2 , which disseminates from initially infected macrophages at around 10–12 hpi culminating in acute fatal infection [29 , 30] , ΔshvR did not spread throughout the embryos .', 'Instead , the infection was characterised by low overall bacterial burden and the absence of tissue inflammation as seen for K56-2 and the complemented mutant ( Fig 3A ) .', 'However , real time observations showed that small infection sites with multiple infected cells sometimes developed from single infected macrophages .', 'Although we have been unable to quantify this event due to the cell dynamics of the in vivo infection with immune cells moving around , and the mechanism behind the local spread is not known , it suggests the ΔshvR mutant can maintain low levels of chronic infection .', 'Together , the results demonstrate that ShvR is not essential for intracellular persistence and replication , but that it regulates factors that are critical for the bacteria to disseminate efficiently and cause robust pro-inflammatory infection .', 'We have shown earlier that acute infection caused by B . cenocepacia K56-2 correlates with systemic phagocyte death , whereas persistent infection , caused by for instance B . stabilis LMG14294 , triggers neutrophilic inflammation [29] .', 'Neutrophil numbers were quantified during infection with wildtype K56-2 , ΔshvR and the complemented mutant at 0 , 24 , 43 , and 72 hpi using Tg ( mpx:eGFP ) transgenic embryos , which express GFP specifically in neutrophils ( S1 Fig and Fig 3B ) .', 'Embryos infected with B . cenocepacia K56-2 showed a strong decline in the number of neutrophils , with only few cells remaining at the end stage of infection ( Fig 3B and [29] ) .', 'In contrast , infection with ΔshvR resulted in neutrophilic inflammation with a significant increase in the number of neutrophils compared to non-infected control embryos from 43 hpi ( Fig 3B ) .', 'The complemented mutant caused neutropenia , as seen for the wildtype strain , confirming restoration of virulence by expression of shvR in trans .', 'B . cenocepacia K56-2 induces a robust increase in host pro-inflammatory cytokine expression ( [29]; Fig 3C and 3D ) .', 'The expression of il1b and cxcl8/il8 upon infection with ΔshvR was , however , only moderately increased compared to PBS-injected embryos ( Fig 3C and 3D ) , as shown earlier for B . stabilis [29] .', 'Moreover , at 24 hpi , no significant difference in gene expression was observed between embryos injected with PBS or ΔshvR .', 'These results confirm that ShvR is required for the induction of robust pro-inflammatory responses .', 'Based on the requirement for macrophages for intracellular bacterial replication of K56-2 and subsequent pro-inflammatory fatal infection [29] , and the restriction of the ΔshvR mutant to macrophages , we hypothesised that ShvR could be an important switch between intracellular persistence and acute pro-inflammatory infection .', 'To address this hypothesis , we used a rhamnose inducible expression system [31] to temporally control the induction of shvR expression in the ΔshvR mutant during infection in zebrafish embryos .', 'This allowed us to study the effect of experimental induction of shvR gene expression during intramacrophage stages of B . cenocepacia on disease outcome .', 'The shvR coding sequence was cloned under control of the rhamnose-inducible promoter PrhaB , resulting in PrhaB:shvR and introduced in ΔshvR .', 'The PrhaB- control plasmid , lacking the shvR coding sequence , served as a negative control .', 'Zebrafish embryos were injected at 30 hours post fertilisation ( hpf ) with ΔshvR ( PrhaB- ) and ΔshvR ( PrhaB:shvR ) .', 'At 24 h post injection of bacteria , at the time ΔshvR resided in macrophages ( Fig 2 ) , a 2% rhamnose solution , or PBS as a control , was injected into the circulation of the infected embryos ( see Fig 4A for schematic representation ) .', 'In order to maintain shvR expression , the embryos were further incubated in E3 medium with 2% rhamnose .', 'The presence of PrhaB- in ΔshvR had no effect on embryo survival or bacterial burden , even after injection of rhamnose ( Fig 4B and 4C ) .', 'Strikingly , application of rhamnose , but not PBS , restored virulence to ΔshvR harbouring PrhaB:shvR , measured as host mortality and increase in bacterial burden ( Fig 4B and 4C ) .', 'These results confirm a critical role for ShvR in acute infection .', 'To visualise the effect of temporal induction of shvR expression on intracellular B . cenocepacia , fluorescence microscopy was performed using Tg ( mpx:eGFP ) transgenic embryos infected with ΔshvR ( PrhaB:shvR ) expressing mCherry .', 'While injection with PBS in embryos infected prior with ΔshvR ( PrhaB:shvR ) did not alter the infection progression of the ΔshvR mutant , injection of rhamnose resulted in changes that are characteristic of acute infection ( Fig 5A ) .', 'These changes include neutrophil recruitment towards infection sites , indicative of increased pro-inflammatory signalling , and as described for acute infection with B . cenocepacia K56-2 [29] .', 'The infection rapidly worsened and at 24 h post rhamnose injection the embryos were neutropenic ( Fig 5A ) , as seen for infection with wildtype ( Fig 3A ) .', 'The 2-log increase in bacterial burden within 48 hours post rhamnose injection ( Fig 4C ) was in agreement with real time observations using fluorescence microscopy and time lapse confocal imaging ( Fig 5A and S2 Movie ) .', 'At later time points after rhamnose injection , tissue damage as a sign of strong pro-inflammatory responses could be observed ( Fig 5A ) .', 'Next , we analysed whether global cytokine gene expression levels were induced after restoration of shvR expression in ΔshvR ( PrhaB:shvR ) bacteria .', 'While no difference in relative expression levels was observed between rhamnose and PBS conditions in embryos infected with ΔshvR ( PrhaB- ) , a significant increase in the expression of cxcl8 and il1b was observed in embryos infected with ΔshvR ( PrhaB:shvR ) after injection with rhamnose ( Fig 5B and 5C ) .', 'Thus , ShvR strongly controls the expression of genes that are required for the transition from intracellular persistence to acute pro-inflammatory infection .', 'ShvR has been described as a global regulator of virulence factors in B . cenocepacia , moderately ( 2 to 4 fold ) changing the expression of a large set of genes .', 'The antifungal activity cluster afc , comprising 24 genes organised in two divergently expressed operons localised adjacent to shvR , has been described to show strongly decreased expression levels ( 100 fold ) in the absence of shvR , demonstrating afc expression is under tight positive control of ShvR [25] .', 'In addition , mutation of afcE mimics the phenotypes found for ΔshvR , including shiny colony morphology , altered membrane properties , and reduced virulence in a rat infection model [25 , 27 , 28] , suggesting the afc cluster is a major target of ShvR .', 'In analogy to the experiments described above for ΔshvR , we therefore analysed a role for AfcE in persistent/acute transition .', 'Infection experiments showed that ΔafcE ( DSRedpBBR ) was severely attenuated in virulence in zebrafish embryos ( Fig 6A and 6B ) .', 'In agreement with the inability of the mutant to cause fatal infection , ΔafcE showed significantly lower bacterial burden compared to K56-2 at 24 hpi , similar to that observed for ΔshvR ( Fig 1 ) .', 'Expression of afcE in trans ( ΔafcE ( Plac:afcE;DSRedpBBR ) complemented virulence to the ΔafcE mutant ( Fig 6A and 6B ) .', 'ΔafcE was taken up by macrophages as efficiently as the wildtype and ΔshvR mutant from the blood circulation ( Fig 2B and S2A Fig ) .', 'After phagocytosis by macrophages , ΔafcE bacteria were observed in macrophages also at later time points , sometimes , like ΔshvR , at high numbers ( Fig 6C ) .', 'Quantification of intracellular ΔafcE bacteria at 1 hpi and 24 hpi ( S2B Fig ) confirmed that the ΔafcE mutant is able to survive and replicate inside macrophages , as shown for ΔshvR ( Fig 2E ) .', 'Using non-invasive real time imaging , we observed that the ΔafcE mutant bacteria did not cause neutropenia nor disseminated infection , while expression of the afcE gene restored these wildtype features ( Fig 6D ) .', 'Further , analysis of the global pro-inflammatory response of the host showed that the expression of the cytokine genes cxcl8 and il1b is induced to lower levels during infection with ΔafcE compared to wildtype K56-2 ( Fig 6E and 6F ) , similar to that shown for ΔshvR ( Fig 3C and 3D ) .', 'These results clearly demonstrate that AfcE is required for acute , disseminated pro-inflammatory infection .', 'Experiments using rhamnose-mediated induction of afcE during intramacrophage stages , as shown for shvR ( Fig 4 ) , were performed .', 'Whereas infection with ΔafcE ( PrhaB- ) after injection of rhamnose or PBS at 24 hpi remained persistent , embryos infected with ΔafcE ( PrhaB:afcE ) and injected with rhamnose succumbed to the infection ( Fig 7A ) , which correlated with an increase in bacterial CFU at 72 hpi ( Fig 7B ) .', 'Not all embryos showed increased bacterial numbers , however , and further experiments are needed to determine the reasons for the apparently larger biological variation in this assay for the ΔafcE mutant compared to the ΔshvR mutant ( Fig 4C ) .', 'In addition , global cytokine expression of embryos infected with ΔafcE ( PrhaB:afcE ) was significantly induced at 24 h post injection of rhamnose , but not PBS ( Fig 7C and 7D ) .', 'Together , these results demonstrate that AfcE is essential for bacterial dissemination and the robust pro-inflammatory fatal response seen for B . cenocepacia K56-2 , in agreement with afc being the direct target of ShvR required for acute infection caused by B . cenocepacia K56-2 .', 'To better understand a role for ShvR/Afc in virulence within the Bcc , we performed bio-informatics analysis .', 'The Burkholderia cepacia complex has seen a rapid increase in the number of bacterial species it contains due to improved characterization and reclassification [32] .', 'The current methods for discrimination between species are based on recA gene sequences , multilocus sequencing and whole genome studies ( reviewed in [33] ) .', 'At present , the complex includes 21 formally named species [1 , 2] .', 'For the construction of a phylogenetic tree , representative genomes of each species were included ( in some cases more than one strain ) with either a complete genome and annotation status or whole genome sequence ( WGS , see S2 Table ) .', 'A phylogenetic inference tree of these species was constructed using the nucleotide sequences of the Multilocus Sequence Typing ( MLST ) housekeeping genes ( atpD , gyrB , gltB , lepA , recA , phaC and trpD ) [34] .', 'Fig 8 shows a maximum-likelihood phylogeny generated from concatenation of these 7 genes .', 'B . pseudomallei K96243 , B . thailandensis E264 and Ralstonia pickettii 12J were used as out-groups .', 'To better understand the evolution of the LTTR regulator ShvR within the complex , BLAST-analysis was performed to identify orthologs of the gene BCAS0225 from B . cenocepacia J2315 .', 'A gene with high similarity to BCAS0225 ( identity >85% ) was identified in a subclade containing strains from 10 out of the 20 species for which complete genome sequences are available ( B . arboris has not been sequenced ) .', 'In all cases , the gene was located on the megaplasmid pC3 .', 'Similarly , orthologs were identified for afcE ( BCAS0208 ) .', 'Fig 8 shows that the presence/absence of shvR within the Bcc complex matches that of afcE .', 'Further investigation of the adjacent afc cluster showed that the 24 afc genes and their organization , including the presence and location of the shvR gene are well-conserved among shvR-containing Bcc species ( S3 Fig ) .', 'In addition to the 19 analysed Bcc strains ( covering 10 species ) that carry the shvR-afc cluster , B . pseudomallei and B . mallei have a subset of the afc genes , including afcE .', 'The operon in B . pseudomallei , located on chromosome 1 , shares similarity to 15 of the 22 genes in the Bcc afc cluster and is likely regulated by an LTTR regulator unrelated to ShvR , encoded by an ORF adjacent to the cluster ( BPSL0494 , S3 Fig ) .', 'This regulator shares amino acid similarities with only the DNA binding domain of the ShvR protein ( 27% amino acid identity ) .', 'BPSL0494 shares 77% nucleotide identity with BCAS0283 , another LTTR identified in B . cenocepacia J2315 encoded on pC3 , with homology mainly in the 5’ 200 bp and the 3’ 150 bp .', 'A maximum-likelihood phylogeny of afcE ( S4 Fig ) confirmed that the acyl-CoA dehydrogenase from B . pseudomallei is the most distant in the Burkholderia group , although they share 80% of nucleotide similarity .', 'Analysis of the region upstream of the shvR coding sequence amongst the shvR-positive strains of the Bcc complex showed nucleotide variations ( SNPs , indels ) , not only between species but also between strains belonging to the same species ( S5 Fig ) .', 'As expected , strains belonging to the same species contain fewer variations , compared to , for example , those observed between the B . cenocepacia and B . lata strains .', 'Within the B . cenocepacia species , the recA lineage IIIA and IIIB ( Fig 8 ) each appear to have their own consensus sequence for the shvR upstream region .', 'Interestingly , a 2-nucleotide gap , at position -238 from the translation start site ( S5 Fig ) , followed by a short highly non-homologous region compared to the conserved sequence found in the other cluster , is observed in the strains of B . cepacia , B . pyrrocinia , B . stabilis , B . lata , B . contaminans , B . metallica and B . vietnamiensis .', 'This is in agreement with the phylogenetic relationship shown in Fig 8 , and suggests that this deletion and the following non-homologous region occurred before speciation .', 'B . cenocepacia , B . cepacia and B . contaminans , Bcc species which are generally correlated with more severe disease in humans , all carry genes encoding ShvR and Afc proteins .', 'Several B . cenocepacia strains , including the epidemic CF isolates K56-2 and J2315 , are highly virulent in different model systems , including zebrafish [29] .', 'However , B . cenocepacia H111 , which is very closely related to K56-2 , was shown to be less virulent in G . mellonella and rats [35 , 36] .', 'In zebrafish , we previously described that this strain can show substantial variation between experiments [37] , with mortality rates between 20 and 60% at 4 days post fertilisation ( dpf ) , but always less virulent than K56-2 ( Fig 9 ) .', 'We also found that H111 does not show afc-dependent antifungal activity as seen for K56-2 [37] .', 'Comparison of the shvR coding sequences showed 3 silent SNPs between H111 and our K56-2 strain .', 'Of note , the sequenced K56-2Valvano strain has a 4th SNP giving rise to a variant Tyr78 to Cys78 .', 'We set out to analyse whether differences in shvR expression could account for the differences in virulence observed between K56-2 and H111 , and thus confirm the role of shvR/afc in virulence of B . cenocepacia .', 'To be able to study the role of shvR and afc in virulence of H111 in the absence of upstream regulatory signals , we cloned the shvR gene of H111 under control of the lac-promoter on pBBR1MCS and constructed an H111 mutant lacking the complete afc cluster .', 'K56-2 , H111 and H111Δafc were transformed with Plac-shvRpBBR or the pBBR1MCS control plasmid .', 'Expression of shvR from the lac promoter led to a change in colony morphotype of H111 from shiny to rough , in a manner that depended on afc ( S6 Fig ) .', 'This suggests that the endogenous shvR gene is not expressed in H111 on agar plates , but that expression of shvR in trans induces the expression of a functional AFC lipoprotein .', 'H111 caused fatal disease in on average 40% of injected embryos over a 4-day time period , compared to 100% mortality in 2 to 3 days for K56-2 ( Fig 9 ) .', 'Zebrafish embryos injected with the H111Δafc mutant survived and remained without any clinical signs of infection during the experimental time , in agreement with the experiments performed with K56-2ΔafcE ( Fig 6B ) .', 'The finding that afc is essential for acute fatal infection also in H111 suggests that shvR and afc expression is induced in wildtype H111 upon injection in zebrafish larvae , but probably does not reach the level of that in K56-2 .', 'Strikingly , overexpression of shvR from the lac promoter increased virulence in H111 , but not the H111Δafc mutant , almost to that seen with B . cenocepacia K56-2 ( Fig 9 ) .', 'These data validate the results obtained with K56-2 that shvR/afc are critically important for acute virulence in zebrafish larvae .', 'They also unambiguously demonstrate that shvR/afc are functional in H111 , and suggest that differences in inducing signals or upstream elements render H111 less virulent in zebrafish compared to K56-2 .'], ['The LysR-type transcriptional regulator ShvR has been shown to tightly control the expression of an adjacent operon , called afc , involved in antifungal activity [25] .', 'The zebrafish embryo model , in which persistent and acute infection caused by different Bcc strains can be studied in detail in the context of an innate immune response [29 , 30] was exploited to gain better insight into a role for ShvR and AfcE in virulence of B . cenocepacia K56-2 .', 'We found that while shvR and afcE are not required for bacterial persistence in the host , they are both essential for the induction of a robust fatal pro-inflammatory infection .', 'Our study shows that ShvR and AfcE are key elements in the transition from an intracellular bacterial stage to disseminated pro-inflammatory infection in zebrafish larvae .', 'A previous microarray study has shown that the expression of the afc cluster is under tight positive regulatory control of ShvR [25] .', 'The afc cluster consists of 24 genes arranged in two divergently expressed operons that specify the synthesis of a lipopeptide located in the membrane of the bacterial cells , previously identified as AFC BC11 [26] .', 'The role of several genes in the afc cluster has been analysed in more detail .', 'While afcA , afcB , afcC , afcD , afcE and afcF have been shown to be important for antifungal activity against Fusarium solani , BCAS0204 ( ABC transporter ) and BCAS0207 ( citrate synthase ) are dispensable [26 , 27] .', 'In addition , AfcE and AfcF , the latter being a putative FAD-dependent oxidoreductase , but not BCAS0204 and BCAS0207 were involved in virulence in alfalfa seedlings .', 'Previous studies have further shown that ΔafcE and ΔafcF mutants have altered membrane lipid profiles , although additional membrane properties , including membrane permeability and membrane morphology , were affected only in the ΔafcE mutant [27] .', 'Additionally , B . cenocepacia lacking afcE produces less biofilms and displays a shiny colony morphology [27] , similar to the phenotypes observed for ΔshvR .', 'The similarity in observed phenotypes between the ΔshvR and ΔafcE mutants suggests that the afc operon is an important regulatory target of ShvR , with a significant role for the afcE gene product .', 'Recently , we showed the essential role of macrophages in the establishment of a replicative niche for B . cenocepacia K56-2 and in the development of a robust pro-inflammatory response that becomes rapidly fatal for zebrafish larvae [29] .', 'In contrast , infection with the ΔshvR and ΔafcE mutants was not fatal and led to only minimal induction of inflammatory responses ( Figs 3 and 6 ) .', 'Importantly , after phagocytosis by macrophages , the ΔshvR and ΔafcE mutants did not disseminate and total bacterial burden only slightly increased over time , in contrast to the dissemination and rapid increase in bacterial numbers seen for the wildtype [30] .', 'However , the absence of shvR and afcE did not prevent the bacteria from replicating intracellularly ( Fig 2 and S2 Fig ) .', 'These data show that shvR and afcE , although not required for persistence and replication in macrophages , are critical for the development of fatal pro-inflammatory disease .', 'This is in agreement with results obtained in a chronic lung infection model in rats , where ΔshvR and ΔafcE mutants showed significant reduction in lung inflammation , while bacteria persisted in the lungs , sometimes replicating to higher numbers than the wildtype [24 , 28] .', 'We are now investigating in more detail the virulence profile of mutants in other genes of the afc operon , and whether general changes in membrane properties or a dysfunctional AFC lipopeptide itself , caused by the absence of AfcE , are responsible for the observed lack in mounting a robust pro-inflammatory response by B . cenocepacia K56-2 .', 'To test our hypothesis that ShvR and AfcE have an important role in the transition from intracellular persistence to acute infection we used a rhamnose-inducible expression system [31] .', 'Our data show that the temporal induction of shvR and afcE expression in intracellular ΔshvR and ΔafcE bacteria , respectively , restored properties that are essential for bacterial dissemination and induction of pro-inflammatory responses .', 'Rhamnose-mediated activation of shvR and afcE resulted in neutrophil recruitment and increased global expression of cxcl8 and il1b in the embryos ( Figs 5 and 7 ) .', 'The infection rapidly worsened and became fatal for the zebrafish embryos ( Figs 4B and 7A ) , confirming our hypothesis that ShvR and AfcE are essential for persistent to acute transition in this model .', 'Bacteria belonging to the Burkholderia cepacia complex ( Bcc ) are life threatening opportunistic pathogens of cystic fibrosis patients and immunocompromised people .', 'One of the major concerns in CF infections is the recurrent acute exacerbations during infection that severely deteriorate the health of the patients with often fatal consequences .', 'The underlying signals and mechanisms that regulate pathogenesis and lead to chronic-acute transitions are not known .', 'Studies on transcriptional regulators of Pseudomonas aeruginosa , including AmpR and RetS/LadS/GacS , have shown that these are involved in regulating the switch between acute to chronic infection [19 , 38–42] .', 'Our study describes for the first time a regulator that is absolutely required for B . cenocepacia to change a persistent intracellular lifestyle to acute pro-inflammatory infection .', 'The data strongly suggest that the downstream regulated target of ShvR that is responsible for the acute infection is the afc gene cluster , with a major role for afcE .', 'Our bioinformatics analysis shows that shvR and the afc cluster are present in the genomes of 10 of the 20 Bcc species of which the whole genome sequence has been published ( Fig 8 ) .', 'It is interesting that the shvR/afc cluster is present in most of the more virulent species in the complex including B . cenocepacia , B . cepacia and B . contaminans , while B . stabilis LMG14294 for example , which causes persistent infection in the zebrafish model [30] and is reduced virulent in a rat model [43] , lacks shvR/afc .', 'In contrast to differences in the flanking gene sequences between strains , shvR and afc have evolved as one unit with a highly conserved gene structure over time ( S3 Fig ) , suggesting an important evolutionary advantage of ShvR-dependent production of the AFC lipopeptide .', 'Interestingly , the important human pathogens B . pseudomallei , the causative agent of melioidosis or Whitmore disease , and B . mallei , causing glanders , encode a subset of the afc genes .', 'Although an afcE homolog is present , the LTTR encoded adjacent to the afc cluster is not an ortholog of shvR .', 'The presence of the afc cluster also in the B . pseudomallei cluster suggests an ancestral origin that has adapted through divergent evolution in the B . pseudomallei and Bcc clusters , with loss of the cluster several times during speciation of the Bcc .', 'Although less likely , the shvR/afc cluster may have been acquired independently during evolution in the Bcc and the B . pseudomallei cluster .', 'It would be interesting to know whether afcE has a similar role in virulence in B . pseudomallei and B . mallei .', 'O’Grady and co
['Introduction', 'Results', 'Discussion', 'Materials and methods']
['The opportunistic pathogen Burkholderia cenocepacia is particularly life-threatening for cystic fibrosis ( CF ) patients .', 'Chronic lung infections with these bacteria can rapidly develop into fatal pulmonary necrosis and septicaemia .', 'We have recently shown that macrophages are a critical site for replication of B . cenocepacia K56-2 and the induction of fatal pro-inflammatory responses using a zebrafish infection model .', 'Here , we show that ShvR , a LysR-type transcriptional regulator that is important for biofilm formation , rough colony morphotype and inflammation in a rat lung infection model , is also required for the induction of fatal pro-inflammatory responses in zebrafish larvae .', 'ShvR was not essential , however , for bacterial survival and replication in macrophages .', 'Temporal , rhamnose-induced restoration of shvR expression in the shvR mutant during intramacrophage stages unequivocally demonstrated a key role for ShvR in transition from intracellular persistence to acute fatal pro-inflammatory disease .', 'ShvR has been previously shown to tightly control the expression of the adjacent afc gene cluster , which specifies the synthesis of a lipopeptide with antifungal activity .', 'Mutation of afcE , encoding an acyl-CoA dehydrogenase , has been shown to give similar phenotypes as the shvR mutant .', 'We found that , like shvR , afcE is also critical for the switch from intracellular persistence to fatal infection in zebrafish .', 'The closely related B . cenocepacia H111 has been shown to be less virulent than K56-2 in several infection models , including Galleria mellonella and rats .', 'Interestingly , constitutive expression of shvR in H111 increased virulence in zebrafish larvae to almost K56-2 levels in a manner that absolutely required afc .', 'These data confirm a critical role for afc in acute virulence caused by B . cenocepacia that depends on strain-specific regulatory control by ShvR .', 'We propose that ShvR and AFC are important virulence factors of the more virulent Bcc species , either through pro-inflammatory effects of the lipopeptide AFC , or through AFC-dependent membrane properties .']
['Burkholderia cenocepacia is a bacterial pathogen that can infect cystic fibrosis patients , causing a chronic infection that can suddenly change to often fatal inflammatory necrotic pneumonia called cepacia syndrome .', 'The transcriptional regulator ShvR controls the expression of a lipoprotein with antifungal properties ( AFC ) .', 'Using zebrafish larvae , we earlier found that macrophages provided an important replication niche for these bacteria , and were required for the strong pro-inflammatory response .', 'Here , we contribute new data that show that ShvR and AfcE are absolutely required for the induction of pro-inflammatory disease by B . cenocepacia .', 'In the absence of ShvR and AfcE B . cenocepacia is restricted to macrophages .', 'Using a sugar-dependent expression system that allowed us to turn on shvR or afcE expression in bacteria that lack shvR or afc , respectively , we were able to mimic the transition from a restricted intracellular stage to acute disseminated infection .', 'This result supports an important role for ShvR/AFC in the acute virulence of B . cenocepacia .']
['blood cells', 'fluorescence imaging', 'medicine and health sciences', 'fish', 'immune cells', 'pathology and laboratory medicine', 'intracellular pathogens', 'pathogens', 'immunology', 'microbiology', 'vertebrates', 'opportunistic infections', 'animals', 'animal models', 'osteichthyes', 'developmental biology', 'model organisms', 'experimental organism systems', 'embryos', 'bacteria', 'bacterial pathogens', 'research and analysis methods', 'sequence analysis', 'embryology', 'infectious diseases', 'white blood cells', 'imaging techniques', 'sequence alignment', 'animal cells', 'animal studies', 'medical microbiology', 'bioinformatics', 'microbial pathogens', 'burkholderia cenocepacia', 'zebrafish', 'eukaryota', 'cell biology', 'database and informatics methods', 'biology and life sciences', 'cellular types', 'burkholderia', 'macrophages', 'organisms']