Many bacteria use quorum sensing (QS) to regulate virulence factor production in response to changes in population density. QS is mediated through the production, secretion, and detection of ...signaling molecules called autoinducers (AIs) to modulate population-wide behavioral changes. Four histidine kinases, LuxPQ, CqsS, CqsR and VpsS, have been identified in Vibrio cholerae as QS receptors to activate virulence gene expression at low cell density. Detection of AIs by these receptors leads to virulence gene repression at high cell density. The redundancy among these receptors is puzzling since any one of the four receptors is sufficient to support colonization of V. cholerae in the host small intestine. It is believed that one of the functions of such circuit architecture is to prevent interference on any single QS receptor. However, it is unclear what natural molecules can interfere V. cholerae QS and in what environment interference is detrimental. We show here mutants expressing only CqsR without the other three QS receptors are defective in colonizing the host large intestine. We identified ethanolamine, a common intestinal metabolite that can function as a chemical source of QS interference. Ethanolamine specifically interacts with the ligand-binding CACHE domain of CqsR and induces a premature QS response in V. cholerae mutants expressing only CqsR without the other three QS receptors. The effect of ethanolamine on QS gene expression and host colonization is abolished by mutations that disrupt CqsR signal sensing. V. cholerae defective in producing ethanolamine is still proficient in QS, therefore, ethanolamine functions only as an external cue for CqsR. Our findings suggest the inhibitory effect of ethanolamine on CqsR could be a possible source of QS interference but is masked by the presence of the other parallel QS pathways, allowing V. cholerae to robustly colonize the host.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Kelsey Barrasso, Samit Watve, Chelsea A. Simpson Affiliation: Department of Biology, Indiana University, Bloomington, Indiana, United States of America Logan J. Geyman Affiliation: Department of ...Biology, Indiana University, Bloomington, Indiana, United States of America ORCID logo https://orcid.org/0000-0001-6697-6725 Julia C. van Kessel * E-mail: jcvk@indiana.edu (JCVK); wai-leung.ng@tufts.edu (WLN) Affiliation: Department of Biology, Indiana University, Bloomington, Indiana, United States of America Wai-Leung Ng * E-mail: jcvk@indiana.edu (JCVK); wai-leung.ng@tufts.edu (WLN) Affiliations Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, United States of America, Program in Molecular Microbiology, Graduate School of Biomedical Sciences, Tufts University, Boston, Massachusetts, United States of America ORCID logo https://orcid.org/0000-0002-8966-6604 Introduction Quorum-sensing (QS) systems, which rely on the production and detection of chemical signals called autoinducers (AIs) made by the bacteria themselves, are classically thought to be employed as a means to sense “self,” ensuring that bacteria cooperate and share resources to benefit their kin. ...most QS receptors are found to be specific for their cognate AIs. Vibrio QS systems that detect host-generated signals Many Vibrio species including Vibrio cholerae, Vibrio harveyi, and Vibrio fischeri spend part of their life cycle inside animal hosts either as a pathogen or as a symbiont. Phosphorylated LuxO promotes and inhibits the production of master transcriptional regulators AphA and HapR, respectively, resulting in the activation of virulence and biofilm gene expression at LCD, which is critical for V. cholerae host colonization 18. The VqmA/DPO pathway inhibits biofilm formation in V.c. AI-2, autoinducer-2; CAI-1, cholera autoinducer-1; CP, cytoplasm; DPO, 3,5-dimethyl-pyrazin-2-ol; HAI-1, harveyi autoinducer-1; HK, histidine kinase; H-NOX, heme nitric oxide/oxygen binding; NO, nitric oxide; PP, periplasm; QS, quorum sensing.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Recent studies indicate that the human intestinal microbiota could impact the outcome of infection by
the etiological agent of the diarrheal disease cholera. A commensal bacterium,
was previously ...identified in high abundance in stool collected from individuals infected with
when compared to stool from uninfected persons. However, if and how
interacts with
has not been experimentally determined; moreover, whether any association between this bacterium alters the behaviors of
to affect the disease outcome is unclear. Here, we show that
and
together form dual-species biofilm structure at the air-liquid interface, with previously uncharacterized novel features. Importantly, the presence of
within the murine small intestine enhances
colonization in the same niche that is dependent on the
exopolysaccharide and other major components of mature
biofilm. These studies illustrate that multispecies biofilm formation is a plausible mechanism used by a gut microbe to increase the virulence of the pathogen, and this interaction may alter outcomes in enteric infections.
Vibrio cholerae is the causative agent of the disease cholera, which remains a global threat to public health and serves as an indicator of inequity and lack of socio-economic development. The ...research presented in this thesis investigates the interplay between V. cholerae and the components inside the host that affect the pathogenicity of this pathogen. Understanding how V. cholerae interacts with various components of the host gastrointestinal environment has important implications to develop new strategies to combat this disease. The first study evaluates how V. cholerae maintains the robustness of the quorum sensing (QS) network within the host. Four histidine kinases comprise the QS circuit in V. cholerae. I hypothesized that one major function of such circuit architecture is to prevent signal interference by external chemical molecules. Indeed, mutants expressing only one of these QS receptors, CqsR, is defective in inhabiting the host large intestine, a niche that may be important in asymptomatic carriage of V. cholerae. Ethanolamine, a common intestinal metabolite, was identified to bind to CqsR and inhibit its kinase activity. These findings suggest the inhibitory effect of ethanolamine on CqsR could be a possible source of QS interference within the host but is masked by the presence of the other parallel QS pathways, allowing V. cholerae to robustly colonize the host.The second study presented in this thesis is concerned with understanding how the human gut microbiota impacts V. cholerae infection. Ample evidence suggests that the presence of specific gut microbes correlates with the susceptibility to various enteric infections, yet studies linking these associations to the disease causality remain limited. An individual microbe has never been shown to increase V. cholerae virulence. Here I identify that a gut microbe, Paracoccus aminovorans, which blooms during active cholera, forms dual-species biofilm structures with V. cholerae and increases host colonization of the pathogen. Together, my study has identified a novel mechanism by which a gut microbe increases V. cholerae virulence and establishes a plausible explanation of why certain microbiota species are more readily to be found in individuals with symptomatic cholera.
Abstract
Background
In chronic liver disease (CLD), gut microbiome disruption worsens with disease severity and growing evidence suggests it is a key driver of infection risk through a combination of ...intestinal pathogen expansion from a loss of colonization resistance and dysfunction of the gastrointestinal (GI) barrier. For the SER-147 preclinical program, we evaluated rationally designed cultivated consortia of human commensal bacteria in vitro and in vivo for the ability to reduce the abundance of pathogens commonly observed in patients with CLD, such as Enterococcus and Enterobacteriaceae.
Methods
Candidate consortia for SER-147 were designed by leveraging genomic data from public observational CLD patient studies and Seres interventional human trial data to include taxa associated with reduced risk of infection and select strains from Seres’ strain library to maximize features associated with decolonizing key pathogens. We developed a novel in vitro gut-ecology model (iGEM) system that propagates human stool-derived microbial communities to evaluate candidate consortia for their ability to reduce titers of vancomycin-resistant Enterococcus faecium (VRE), and both carbapenem-resistant Klebsiella pneumoniae (CR-Kpn) and Escherichia coli (CR-Ec). In vivo, candidate consortia were evaluated for their ability to reduce fecal titers of VRE and CR-Kpn in mouse models of gut pathogen colonization.
Results
In the iGEM system, following introduction and expansion of select pathogens in the microbial community, intervention with a subset of five unique candidate consortia from the SER-147 program were able to significantly reduce titers of VRE and Cr-Ec compared to controls and in the case of Cr-Kpn, titers were reduced to the limit of detection of the assay (Figure 1). In the in vivo colonization models, oral administration of the same five candidate consortia led to a 1-3 Log10 reduction in VRE and Cr-Kpn fecal titers compared to vehicle-only treated mice (Figure 2).Figure 1.Reduction in abundance of Cr-Kpn, Cr-Ec, and VRE after microbiome intervention in the iGEM system.
(A) Human stool-derived microbial communities were passaged anaerobically in 96-well deep well plates, with cultures stamped into fresh deep well plates every 24 hours with different wells representing different treatments and conditions. Individual pathogens were introduced on day 4 of the experiment, followed by a vancomycin pre-treatment from days 5-8. On days 10-16, five unique candidate consortia (DE061, DE456, DE240, DE714, and DE492) were dosed daily as the microbiome intervention. (B, C, D) The titers of Cr-Kpn, Cr-Ec, or VRE were quantified at specific timepoints during the study by plating culture aliquots on selective and differential media. The mean CFU per milliliter of culture was calculated for each arm of the study and plotted on the line graph (N=4 per arm). Error bars represent the standard deviation from the mean. L.O.D., limit of detection.Figure 2:Reduction in abundance of Cr-Kpn and VRE after microbiome intervention in mouse models of gut pathogen colonization.
(A) Wild-type C57BL/6 mice underwent antibiotic conditioning prior to pathogen challenge. Following the introduction of pathogen (Day 0) mice were dosed daily vial oral gavage with five unique candidate consortia (DE061, DE456, DE240, DE714, and DE492) as the microbiome intervention or vehicle only for 6 days and fecal titers are monitored for a 3-week period to evaluate pathogen abundance (B,C) The titers of VRE or CR-Kpn were quantified in fecal pellets by plating on selective agar at the indicated time-points. The median (B) CR-Kpn and (C) VRE CFU per gram of feces was calculated for each group and plotted on the line graph (n=9 per group). L.O.D., limit of detection.
Conclusion
Preclinical assessments in vitro and in vivo support the ability of SER-147 candidate consortia to reduce VRE, CR-Kpn, and CR-Ec abundance in the gut. These results support the potential ability of microbiome therapeutics to reduce the risk of infection in patients with CLD.
Disclosures
Elizabeth Halvorsen, PhD, Seres Therapeutics: Employee|Seres Therapeutics: Stocks/Bonds Marin Vulić, PhD, Seres Therapeutics: Employee|Seres Therapeutics: Stocks/Bonds Kelsey Barrasso, PhD, Seres Therapeutics: Stocks/Bonds Nicholas Beauchemin, n/a, Seres Therapeutics: Employee|Seres Therapeutics: Stocks/Bonds Jessica Brown, n/a, Seres Therapeutics: Employee|Seres Therapeutics: Stocks/Bonds Nathaniel J. Ennis, n/a, Seres Therapeutics: Employee|Seres Therapeutics: Stocks/Bonds Melissa Mayol, n/a, Seres Therapeutics: Employee|Seres Therapeutics: Stocks/Bonds Jenna Wurster, PhD, Seres Therapeutics: Paid Employee|Seres Therapeutics: Stocks/Bonds Edward J. O'Brien, PhD, Seres Therapeutics: Employee|Seres Therapeutics: Stocks/Bonds Christopher Ford, PhD, Seres Therapeutics: Employee|Seres Therapeutics: Stocks/Bonds Matthew Henn, PhD, Seres Therapeutics: Employee|Seres Therapeutics: Stocks/Bonds
Summary
Sporulation in Bacillus subtilis is governed by a cascade of alternative RNA polymerase sigma factors. We previously identified a small protein Fin that is produced under the control of the ...sporulation sigma factor σF to create a negative feedback loop that inhibits σF‐directed gene transcription. Cells deleted for fin are defective for spore formation and exhibit increased levels of σF‐directed gene transcription. Based on pull‐down experiments, chemical crosslinking, bacterial two‐hybrid experiments and nuclear magnetic resonance chemical shift analysis, we now report that Fin binds to RNA polymerase and specifically to the coiled‐coil region of the β′ subunit. The coiled‐coil is a docking site for sigma factors on RNA polymerase, and evidence is presented that the binding of Fin and σF to RNA polymerase is mutually exclusive. We propose that Fin functions by a mechanism distinct from that of classic sigma factor antagonists (anti‐σ factors), which bind directly to a target sigma factor to prevent its association with RNA polymerase, and instead functions to inhibit σF by competing for binding to the β′ coiled‐coil.
During the developmental process of sporulation in Bacillus subtilis a cascade of alternative sigma factors associate with RNA polymerase to direct gene expression. We report that Fin, a novel RNAP‐binding protein produced during sporulation, inhibits the function of the sporulation sigma factor σF by competing with the binding of region 2 (Rg2) of σF to the coiled‐coil region of the β′ subunit of RNA polymerase (β′ CC).
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