BackgroundTuberculosis (TB) chemotherapy clears bacterial burden in the lungs of patients and allows the tuberculous lesions to heal through a fibrotic process. The healing process leaves pulmonary scar tissue that can impair lung function. The goal of this study was to identify fibrotic mediators as a stepping-stone to begin exploring mechanisms of tissue repair in TB.MethodsHematoxylin and eosin staining and Masson’s trichrome stain were utilized to determine levels of collagenization in tuberculous granulomas from non-human primates. Immunohistochemistry was then employed to further interrogate these granulomas for markers associated with fibrogenesis, including transforming growth factor-β (TGFβ), α-smooth muscle actin (αSMA), phosphorylated SMAD-2/3, and CD163. These markers were compared across states of drug treatment using one-way ANOVA, and Pearson’s test was used to determine the association of these markers with one another.ResultsTGFβ and αSMA were present in granulomas from primates with active TB disease. These molecules were reduced in abundance after TB chemotherapy. Phosphorylated SMAD-2/3, a signaling intermediate of TGFβ, was observed in greater amounts after 1 month of drug treatment than in active disease, suggesting that this particular pathway is blocked in active disease. Collagen production during tissue repair is strongly associated with TGFβ in this model, but not with CD163+ macrophages.ConclusionsTissue repair and fibrosis in TB that occurs during drug treatment is associated with active TGFβ that is produced during active disease. Further work will identify mechanisms of fibrosis and work towards mitigating lung impairment with treatments that target those mechanisms.Electronic supplementary materialThe online version of this article (doi:10.1186/s13069-016-0043-3) contains supplementary material, which is available to authorized users.

Vibrio cholerae is an aquatic organism and facultative human pathogen that colonizes the small intestine. In the small intestine, V. cholerae is exposed to a variety of antimicrobial compounds, including bile. V. cholerae resistance to bile is multifactorial and includes alterations in the membrane permeability barrier that are mediated by ToxR, a membrane-associated transcription factor. ToxR has also been shown to be required for activation of the LysR family transcription factor leuO in response to cyclic dipeptides. LeuO has been implicated in the regulation of multiple V. cholerae phenotypes, including biofilm production and virulence. In this study, we investigated the effects of bile on leuO expression. We show that leuO transcription increased in response to bile and bile salts but not in response to other detergents. The bile-dependent increase in leuO expression was dependent on ToxR, which was found to bind directly to the leuO promoter. The periplasmic domain of ToxR was required for basal leuO expression and for the bile-dependent induction of both leuO and ompU transcription. V. cholerae mutants that did not express leuO exhibited increased bile susceptibility, suggesting that LeuO contributes to bile resistance. Our collective results demonstrate that ToxR activates leuO expression in response to bile and that LeuO is a component of the ToxR-dependent responses that contribute to bile resistance. IMPORTANCEThe success of Vibrio cholerae as a human pathogen is dependent upon its ability to rapidly adapt to changes in its growth environment. Growth in the human gastrointestinal tract requires the expression of genes that provide resistance to host antimicrobial compounds, including bile. In this work, we show for the first time that the LysR family regulator LeuO mediates responses in V. cholerae that contribute to bile resistance. Vibrio cholerae is a Gram-negative bacterial pathogen and the causal agent of the severe diarrheal disease cholera. V. cholerae exists naturally in aquatic reservoirs and is capable of colonizing the human small intestine. The transition of V. cholerae from the aquatic ecosystem to growth in the human gastrointestinal tract is mediated by transcriptional responses that are required for colonization and disease development. Many of the genes that contribute to intestinal colonization are under the control of the membrane-associated regulatory protein ToxR, which functions as one of the primary regulators in the ToxR regulon (reviewed in reference 1). The ToxR regulon is divided into two branches, a ToxTdependent branch, which controls the expression of virulence factors, and a ToxT-independent branch, which reciprocally regulates the production of the outer membrane porins OmpU and OmpT. The ToxT-dependent branch of the ToxR regulon is a hierarchical regulatory cascade that regulates the expression of genes encoding the production of cholera toxin (CT) and the toxin-coregulated pilus (TCP) in response to environmental cues in the host.ToxR is a membrane-associated ...

Resistance-nodulation-division (RND) efflux systems are ubiquitous transporters in Gram-negative bacteria that are essential for antibiotic resistance. The RND efflux systems also contribute to diverse phenotypes independent of antimicrobial resistance, but the mechanism by which they affect most of these phenotypes is unclear. This is the case in Vibrio cholerae where the RND systems function in antimicrobial resistance and virulence factor production. Herein, we investigated the linkage between RND efflux and V. cholerae virulence. RNA sequencing revealed that the loss of RND efflux affected the activation state of periplasmic sensing systems including the virulence regulator ToxR. Activation of ToxR in an RND null mutant resulted in ToxR-dependent transcription of the LysR-family regulator leuO. Increased leuO transcription resulted in the repression of the ToxR virulence regulon and attenuated virulence factor production. Consistent with this, leuO deletion restored virulence factor production in an RND-null mutant, but not its ability to colonize infant mice; suggesting that RND efflux was epistatic to virulence factor production for colonization. The periplasmic sensing domain of ToxR was required for the induction of leuO transcription in the RND null mutant, suggesting that ToxR responded to metabolites that accumulated in the periplasm. Our results suggest that ToxR represses virulence factor production in response to metabolites that are normally effluxed from the cell by the RND transporters. We propose that impaired RND efflux results in periplasmic metabolite accumulation, which then activates periplasmic sensors including ToxR and two-component regulatory systems to initiate the expression of adaptive responses.

Indole is a degradation product of tryptophan that functions as a signaling molecule in many bacteria. This includes Vibrio cholerae, where indole was shown to regulate biofilm and type VI secretion in nontoxigenic environmental isolates. Indole is also produced by toxigenic V. cholerae strains in the human intestine, but its significance in the host is unknown. We investigated the effects of indole on toxigenic V. cholerae O1 El Tor during growth under virulence inducing conditions. The indole transcriptome was defined by RNA sequencing and showed widespread changes in the expression of genes involved in metabolism, biofilm production, and virulence factor production. In contrast, genes involved in type VI secretion were not affected by indole. We subsequently found that indole repressed genes involved in V. cholerae pathogenesis, including the ToxR virulence regulon. Consistent with this, indole inhibited cholera toxin and toxin-coregulated pilus production in a dose-dependent manner. The effects of indole on virulence factor production and biofilm were linked to ToxR and the ToxR-dependent regulator LeuO. The expression of leuO was increased by exogenous indole and linked to repression of the ToxR virulence regulon. This process was dependent on the ToxR periplasmic domain, suggesting that indole was a ToxR agonist. This conclusion was further supported by results showing that the ToxR periplasmic domain contributed to indole-mediated increased biofilm production. Collectively, our results suggest that indole may be a niche-specific cue that can function as a ToxR agonist to modulate virulence gene expression and biofilm production in V. cholerae.

Vibrio cholerae encodes six resistance-nodulation-division (RND) efflux systems which function in antimicrobial resistance, virulence factor production, and intestinal colonization. Among the six RND efflux systems, VexAB exhibited broad substrate specificity and played a predominant role in intrinsic antimicrobial resistance. The VexAB system was encoded in an apparent three gene operon that included vexR; which encodes an uncharacterized TetR family regulator. In this work we examined the role of vexR in vexRAB expression. We found that VexR bound to the vexRAB promoter and vexR deletion resulted in decreased vexRAB expression and increased susceptibility to VexAB antimicrobial substrates. Substrate-dependent induction of vexRAB was dependent on vexR and episomal vexR expression provided a growth advantage in the presence of the VexAB substrate deoxycholate. The expression of vexRAB increased, in a vexR-dependent manner, in response to the loss of RND efflux activity. This suggested that VexAB may function to export intracellular metabolites. Support for this hypothesis was provided by data showing that vexRAB was upregulated in several metabolic mutants including tryptophan biosynthetic mutants that were predicted to accumulate indole. In addition, vexRAB was found to be upregulated in response to exogenous indole and to contribute to indole resistance. The collective results indicate that vexR is required for vexRAB expression in response to VexAB substrates and that the VexAB RND efflux system modulates the intracellular levels of metabolites that could otherwise accumulate to toxic levels.