Carbapenem-Resistant Enterobacter hormaechei Uses Mucus Metabolism to Facilitate Gastrointestinal Colonization.

The emergence and global spread of carbapenem-resistant Enterobacter cloacae complex species presents a pressing public health challenge. Carbapenem-resistant Enterobacter species cause a wide variety of infections, including septic shock fatalities in newborns and immunocompromised adults. The intestine may be a major reservoir for these resistant strains, either by facilitating contamination of fomites and transfer to susceptible individuals, or through translocation from the gut to the bloodstream. For this reason, we sought to establish a neonatal mouse model to investigate the mechanisms underpinning gut colonization by carbapenem-resistant Enterobacter hormaechei . We describe a new mouse model to study gut colonization by Enterobacter species, leading to vital insights into the adaptation of carbapenem-resistant E. hormaechei to the gut environment during the early stages of intestinal colonization. We observed successful colonization and proliferation of E. hormaechei in the five-day old infant mouse gut, with primary localization to the colon following oral inoculation. We also uncovered evidence that E. hormaechei uses mucus as a carbon source during colonization of the colon. Our findings underscore the importance of oxygen-dependent metabolic pathways, including the pyruvate dehydrogenase complex, and N- acetyl-D-glucosamine metabolism, in gut colonization and proliferation, which aligns with previous human studies. These insights are essential for developing novel therapeutic strategies that can serve as decolonization therapies in at-risk populations. Importance: Bloodstream infections caused by Enterobacter species pose a significant clinical threat. The intestine acts as the primary site for colonization and serves as a reservoir for infection. To combat this pathogen, it is crucial to understand how carbapenem-resistant Enterobacter species colonize the gut, as such knowledge can pave the way for alternative therapeutic targets. In this study, we developed a novel neonatal mouse model for gastrointestinal colonization by Enterobacter species and discovered that mucus plays a key role as a carbon source during colonization. Additionally, we identified two mucus catabolism pathways that contribute to intestinal colonization by carbapenem-resistant E. hormaechei . This new mouse model offers valuable insights into host-pathogen interactions and helps identify critical gastrointestinal fitness factors of Enterobacter , potentially guiding the development of vaccines and alternative therapeutic strategies to minimize intestinal carriage in patient populations at risk for infection with Enterobacter species.

Fitness factor genes conserved within the multi-species core genome of Gram-negative Enterobacterales species contribute to bacteremia pathogenesis.

There is a critical gap in knowledge about how Gram-negative bacterial pathogens, using survival strategies developed for other niches, cause lethal bacteremia. Facultative anaerobic species of the Enterobacterales order are the most common cause of Gram-negative bacteremia, including Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, Citrobacter freundii, and Enterobacter hormaechei. Bacteremia often leads to sepsis, a life-threatening organ dysfunction resulting from unregulated immune responses to infection. Despite a lack of specialization for this host environment, Gram-negative pathogens cause nearly half of bacteremia cases annually. Based on our existing Tn-Seq fitness factor data from a murine model of bacteremia combined with comparative genomics of the five Enterobacterales species above, we prioritized 18 conserved fitness genes or operons for further characterization. Mutants were constructed for all genes in all five species. Each mutant was used to cochallenge C57BL/6 mice via tail vein injection along with each respective wild-type strain to determine competitive indices for each fitness gene. Five fitness factor genes, when mutated, attenuated mutants in four or five species in the spleen and liver (tatC, ruvA, gmhB, wzxE, arcA). Five additional fitness factor genes or operons were validated as outcompeted by wild-type in three, four, or five bacterial species in the spleen (xerC, prc, apaGH, atpG, aroC). Overall, 17 of 18 fitness factor mutants were attenuated in at least one species in the spleen or liver. Together, these findings allow for the development of a model of bacteremia pathogenesis that may include future targets of therapy against bloodstream infections.

An essential host dietary fatty acid promotes TcpH inhibition of TcpP proteolysis promoting virulence gene expression in Vibrio cholerae.

Vibrio cholerae is a Gram-negative gastrointestinal pathogen responsible for the diarrheal disease cholera. Expression of key virulence factors, cholera toxin and toxin-coregulated pilus, is regulated directly by ToxT and indirectly by two transmembrane transcription regulators (TTRs), ToxR and TcpP, that promote the expression of toxT. TcpP abundance and activity are controlled by TcpH, a single-pass transmembrane protein, which protects TcpP from a two-step proteolytic process known as regulated intramembrane proteolysis (RIP). The mechanism of TcpH-mediated protection of TcpP represents a major gap in our understanding of V. cholerae pathogenesis. The absence of tcpH leads to unimpeded degradation of TcpP in vitro and a colonization defect in a neonate mouse model of V. cholerae colonization. Here, we show that TcpH protects TcpP from RIP via direct interaction. We also demonstrate that α-linolenic acid, a dietary fatty acid, promotes TcpH-dependent inhibition of RIP via co-association of TcpP and TcpH molecules within detergent-resistant membranes (DRMs) in a mechanism requiring the TcpH transmembrane domain. Taken together, our data support a model where V. cholerae cells use exogenous α-linolenic acid to remodel the phospholipid bilayer in vivo, leading to co-association of TcpP and TcpH within DRMs where RIP of TcpP is inhibited by TcpH, thereby promoting V. cholerae pathogenicity. IMPORTANCE: Vibrio cholerae continues to pose a significant global burden on health and an alternative therapeutic approach is needed, due to evolving multidrug resistance strains. Transcription of toxT, stimulated by TcpP and ToxR, is essential for V. cholerae pathogenesis. Our results show that TcpP, one of the major regulators of toxT gene expression, is protected from proteolysis by TcpH, via direct interaction. Furthermore, we identified a gut metabolite, α-linolenic acid, that stimulates the co-association of TcpP and TcpH within detergent-resistant membranes (also known as lipid-ordered membrane domains), thereby supporting TcpH-dependent antagonism of TcpP proteolysis. Data presented here extend our knowledge of RIP, virulence gene regulation in V. cholerae, and, to the best of our knowledge, provides the first evidence that lipid-ordered membranes exist within V. cholerae. The model presented here also suggests that TTRs, common among bacteria and archaea, and co-component signal transduction systems present in Enterobacteria, could also be influenced similarly.

Gut metabolite L-lactate supports Campylobacter jejuni population expansion during acute infection.

How the microaerobic pathogen Campylobacter jejuni establishes its niche and expands in the gut lumen during infection is poorly understood. Using 6-wk-old ferrets as a natural disease model, we examined this aspect of C. jejuni pathogenicity. Unlike mice, which require significant genetic or physiological manipulation to become colonized with C. jejuni, ferrets are readily infected without the need to disarm the immune system or alter the gut microbiota. Disease after C. jejuni infection in ferrets reflects closely how human C. jejuni infection proceeds. Rapid growth of C. jejuni and associated intestinal inflammation was observed within 2 to 3 d of infection. We observed pathophysiological changes that were noted by cryptic hyperplasia through the induction of tissue repair systems, accumulation of undifferentiated amplifying cells on the colon surface, and instability of HIF-1α in colonocytes, which indicated increased epithelial oxygenation. Metabolomic analysis demonstrated that lactate levels in colon content were elevated in infected animals. A C. jejuni mutant lacking lctP, which encodes an L-lactate transporter, was significantly decreased for colonization during infection. Lactate also influences adhesion and invasion by C. jejuni to a colon carcinoma cell line (HCT116). The oxygenation required for expression of lactate transporter (lctP) led to identification of a putative thiol-based redox switch regulator (LctR) that may repress lctP transcription under anaerobic conditions. Our work provides better insights into the pathogenicity of C. jejuni.

Gut metabolite L-lactate supports Campylobacter jejuni population expansion during acute infection.

How the microaerobic pathogen Campylobacter jejuni establishes its niche and expands in the gut lumen during infection is poorly understood. Using six-week-old ferrets as a natural disease model, we examined this aspect of C. jejuni pathogenicity. Unlike mice, which require significant genetic or physiological manipulation to become colonized with C. jejuni , ferrets are readily infected without the need to disarm the immune system or alter the gut microbiota. Disease after C. jejuni infection in ferrets reflects closely how human C. jejuni infection proceeds. Rapid growth of C. jejuni and associated intestinal inflammation was observed within two-three days of infection. We observed pathophysiological changes that were noted by cryptic hyperplasia through the induction of tissue repair systems, accumulation of undifferentiated amplifying cells on the colon surface, and instability of HIF-1α in colonocytes, which indicated increased epithelial oxygenation. Metabolomic analysis demonstrated that lactate levels in colon content were elevated in infected animals. A C. jejuni mutant lacking lctP , which encodes an L-lactate transporter, was significantly decreased for colonization during infection. Lactate also influences adhesion and invasion by C. jejuni to a colon carcinoma cell line (HCT116). The oxygenation required for expression of lactate transporter ( lctP ) led to discovery of a putative thiol based redox switch regulator (LctR) that may repress lctP transcription under anaerobic conditions. Our work provides new insights into the pathogenicity of C. jejuni . Significance: There is a gap in knowledge about the mechanisms by which C. jejuni populations expand during infection. Using an animal model which accurately reflects human infection without the need to alter the host microbiome or the immune system prior to infection, we explored pathophysiological alterations of the gut after C. jejuni infection. Our study identified the gut metabolite L-lactate as playing an important role as a growth substrate for C. jejuni during acute infection. We identified a DNA binding protein, LctR, that binds to the lctP promoter and may repress lctP expression, resulting in decreased lactate transport under low oxygen levels. This work provides new insights about C. jejuni pathogenicity.

Activation of a Vibrio cholerae CBASS anti-phage system by quorum sensing and folate depletion.

IMPORTANCE: To counteract infection with phage, bacteria have evolved a myriad of molecular defense systems. Some of these systems initiate a process called abortive infection, in which the infected cell kills itself to prevent phage propagation. However, such systems must be inhibited in the absence of phage infection to prevent spurious death of the host. Here, we show that the cyclic oligonucleotide based anti-phage signaling system (CBASS) accomplishes this by sensing intracellular folate molecules and only expressing this system in a group. These results enhance our understanding of the evolution of the seventh Vibrio cholerae pandemic and more broadly how bacteria defend themselves against phage infection.

Transmembrane Transcription Regulators Are Widespread in Bacteria and Archaea.

To adapt and proliferate, bacteria must sense and respond to the ever-changing environment. Transmembrane transcription regulators (TTRs) are a family of one-component transcription regulators that respond to extracellular information and influence gene expression from the cytoplasmic membrane. How TTRs function to modulate expression of their target genes while localized to the cytoplasmic membrane remains poorly understood. In part, this is due to a lack of knowledge regarding the prevalence of TTRs among prokaryotes. Here, we show that TTRs are highly diverse and prevalent throughout bacteria and archaea. Our work demonstrates that TTRs are more common than previously appreciated and are enriched within specific bacterial and archaeal phyla and that many TTRs have unique transmembrane region properties that can facilitate association with detergent-resistant membranes. IMPORTANCE One-component signal transduction systems are the major class of signal transduction systems among bacteria and are commonly cytoplasmic. TTRs are a group of unique one-component signal transduction systems that influence transcription from the cytoplasmic membrane. TTRs have been implicated in a wide array of biological pathways critical for both pathogens and human commensal organisms but were considered to be rare. Here, we demonstrate that TTRs are in fact highly diverse and broadly distributed in bacteria and archaea. Our findings suggest that transcription factors can access the chromosome and influence transcription from the membrane in both archaea and bacteria. This study challenges thus the commonly held notion that signal transduction systems require a cytoplasmic transcription factor and highlights the importance of the cytoplasmic membrane in directly influencing signal transduction.

Achieving Single-Molecule Tracking of Subcellular Regulation in Bacteria during Real-Time Environmental Perturbations.

Bacteria rely on protein systems for regulation in response to external environmental signals. Single-molecule fluorescence imaging and tracking has elucidated the complex mechanism of these protein systems in a variety of bacteria. We recently investigated Vibrio cholerae, the Gram-negative bacterium responsible for the human cholera disease, and its regulation of the production of toxins and virulence factors through the membrane-localized transcription factors TcpP and ToxR. These experiments determined that TcpP and ToxR work cooperatively under steady-state conditions, but measurements of how these dynamical interactions change over the course of environmental perturbations were precluded by the traditional preparation of bacterial cells confined on agarose pads. Here, we address this gap in technology and access single-molecule dynamics during real-time changes by implementing two alternative sample preparations: microfluidic devices and chitosan-coated coverslips. We report the first demonstration of single-molecule tracking within live bacterial cells in a microfluidic device. Additionally, using the chitosan-coated coverslips, we show that real-time environmental changes impact TcpP-PAmCherry dynamics, activating a virulence condition in the bacteria about 45 min after dropping to pH 6 and about 20 min after inducing ToxR expression. These new technology advances open our ability for new experiments studying a variety of bacteria with single-molecule imaging and tracking during real-time environmental perturbations.

Vibrio cholerae requires oxidative respiration through the bd-I and cbb3 oxidases for intestinal proliferation.

Vibrio cholerae respires both aerobically and anaerobically and, while oxygen may be available to it during infection, other terminal electron acceptors are proposed for population expansion during infection. Unlike gastrointestinal pathogens that stimulate significant inflammation leading to elevated levels of oxygen or alternative terminal electron acceptors, V. cholerae infections are not understood to induce a notable inflammatory response. To ascertain the respiration requirements of V. cholerae during infection, we used Multiplex Genome Editing by Natural Transformation (MuGENT) to create V. cholerae strains lacking aerobic or anaerobic respiration. V. cholerae strains lacking aerobic respiration were attenuated in infant mice 105-fold relative to wild type, while strains lacking anaerobic respiration had no colonization defect, contrary to earlier work suggesting a role for anaerobic respiration during infection. Using several approaches, including one we developed for this work termed Comparative Multiplex PCR Amplicon Sequencing (CoMPAS), we determined that the bd-I and cbb3 oxidases are essential for small intestinal colonization of V. cholerae in the infant mouse. The bd-I oxidase was also determined as the primary oxidase during growth outside the host, making V. cholerae the only example of a Gram-negative bacterial pathogen in which a bd-type oxidase is the primary oxidase for energy acquisition inside and outside of a host.

SMAUG: Analyzing single-molecule tracks with nonparametric Bayesian statistics.

Single-molecule fluorescence microscopy probes nanoscale, subcellular biology in real time. Existing methods for analyzing single-particle tracking data provide dynamical information, but can suffer from supervisory biases and high uncertainties. Here, we develop a method for the case of multiple interconverting species undergoing free diffusion and introduce a new approach to analyzing single-molecule trajectories: the Single-Molecule Analysis by Unsupervised Gibbs sampling (SMAUG) algorithm, which uses nonparametric Bayesian statistics to uncover the whole range of information contained within a single-particle trajectory dataset. Even in complex systems where multiple biological states lead to a number of observed mobility states, SMAUG provides the number of mobility states, the average diffusion coefficient of single molecules in that state, the fraction of single molecules in that state, the localization noise, and the probability of transitioning between two different states. In this paper, we provide the theoretical background for the SMAUG analysis and then we validate the method using realistic simulations of single-particle trajectory datasets as well as experiments on a controlled in vitro system. Finally, we demonstrate SMAUG on real experimental systems in both prokaryotes and eukaryotes to measure the motions of the regulatory protein TcpP in Vibrio cholerae and the dynamics of the B-cell receptor antigen response pathway in lymphocytes. Overall, SMAUG provides a mathematically rigorous approach to measuring the real-time dynamics of molecular interactions in living cells.

Aerobic Metabolism in Vibrio cholerae Is Required for Population Expansion during Infection.

Vibrio cholerae replicates to high cell density in the human small intestine, leading to the diarrheal disease cholera. During infection, V. cholerae senses and responds to environmental signals that govern cellular responses. Spatial localization of V. cholerae within the intestine affects nutrient availability and metabolic pathways required for replicative success. Metabolic processes used by V. cholerae to reach such high cell densities are not fully known. We sought to better define the metabolic traits that contribute to high levels of V. cholerae during infection. By disrupting the pyruvate dehydrogenase (PDH) complex and pyruvate formate-lyase (PFL), we could differentiate aerobic and anaerobic metabolic pathway involvement in V. cholerae proliferation. We demonstrate that oxidative metabolism is a key contributor to the replicative success of V. choleraein vivo using an infant mouse model in which PDH mutants were attenuated 100-fold relative to the wild type for colonization. Additionally, metabolism of host substrates, including mucin, was determined to support V. cholerae growth in vitro as a sole carbon source, primarily under aerobic growth conditions. Mucin likely contributes to population expansion during human infection as it is a ubiquitous source of carbohydrates. These data highlight oxidative metabolism as important in the intestinal environment and warrant further investigation of how oxygen and other host substrates shape the intestinal landscape that ultimately influences bacterial disease. We conclude from our results that oxidative metabolism of host substrates is a key driver of V. cholerae proliferation during infection, leading to the substantial bacterial burden exhibited in cholera patients.IMPORTANCEVibrio cholerae remains a challenge in the developing world and incidence of the disease it causes, cholera, is anticipated to increase with rising global temperatures and with emergent, highly infectious strains. At present, the underlying metabolic processes that support V. cholerae growth during infection are less well understood than specific virulence traits, such as production of a toxin or pilus. In this study, we determined that oxidative metabolism of host substrates such as mucin contribute significantly to V. cholerae population expansion in vivo Identifying metabolic pathways critical for growth can provide avenues for controlling V. cholerae infection and the knowledge may be translatable to other pathogens of the gastrointestinal tract.

Plasmonic nanoparticles assemblies templated by helical bacteria and resulting optical activity.

Plasmonic nanoparticles (NPs) adsorbing onto helical bacteria can lead to formation of NP helicoids with micron scale pitch. Associated chiroptical effects can be utilized as bioanalytical tool for bacterial detection and better understanding of the spectral behavior of helical self-assembled structures with different scales. Here, we report that enantiomerically pure helices with micron scale of chirality can be assembled on Campylobacter jejuni, a helical bacterium known for severe stomach infections. These organisms have right-handed helical shapes with a pitch of 1-2 microns and can serve as versatile templates for a variety of NPs. The bacteria itself shows no observable rotatory activity in the visible, red, and near-IR ranges of electromagnetic spectrum. The bacterial dispersion acquires chiroptical activity at 500-750 nm upon plasmonic functionalization with Au NPs. Finite-difference time-domain simulations confirmed the attribution of the chiroptical activity to the helical assembly of gold nanoparticles. The position of the circular dichroism peaks observed for these chiral structures overlaps with those obtained before for Au NPs and their constructs with molecular and nanoscale chirality. This work provides an experimental and computational pathway to utilize chiroplasmonic particles assembled on bacteria for bioanalytical purposes.

Phosphate Transporter PstSCAB of Campylobacter jejuni Is a Critical Determinant of Lactate-Dependent Growth and Colonization in Chickens.

Campylobacter jejuni causes acute gastroenteritis worldwide and is transmitted primarily through poultry, in which it is often a commensal member of the intestinal microbiota. Previous transcriptome sequencing (RNA-Seq) experiment showed that transcripts from an operon encoding a high-affinity phosphate transporter (PstSCAB) of C. jejuni were among the most abundant when the bacterium was grown in chickens. Elevated levels of the pstSCAB mRNA were also identified in an RNA-Seq experiment from human infection studies. In this study, we explore the role of PstSCAB in the biology and colonization potential of C. jejuni Our results demonstrate that cells lacking PstSCAB survive poorly in stationary phase, in nutrient-limiting media, and under osmotic conditions reflective of those in the chicken. Polyphosphate levels in the mutant cells were elevated at stationary phase, consistent with alterations in expression of polyphosphate metabolism genes. The mutant strain was highly attenuated for colonization of newly hatched chicks, with levels of bacteria at several orders of magnitude below wild-type levels. Mutant and wild type grew similarly in complex media, but the pstS::kan mutant exhibited a significant growth defect in minimal medium supplemented with l-lactate, postulated as a carbon source in vivo Poor growth in lactate correlated with diminished expression of acetogenesis pathway genes previously demonstrated as important for colonizing chickens. The phosphate transport system is thus essential for diverse aspects of C. jejuni physiology and in vivo fitness and survival.IMPORTANCECampylobacter jejuni causes millions of human gastrointestinal infections annually, with poultry a major source of infection. Due to the emergence of multidrug resistance in C. jejuni, there is need to identify alternative ways to control this pathogen. Genes encoding the high-affinity phosphate transporter PstSCAB are highly expressed by C. jejuni in chickens and humans. In this study, we address the role of PstSCAB on chicken colonization and other C. jejuni phenotypes. PstSCAB is required for colonization in chicken, metabolism and survival under different stress responses, and during growth on lactate, a potential growth substrate in chickens. Our study highlights that PstSCAB may be an effective target to develop mechanisms for controlling bacterial burden in both chicken and human.

A putative Vibrio cholerae two-component system controls a conserved periplasmic protein in response to the antimicrobial peptide polymyxin B.

The epidemic pathogen Vibrio cholerae senses and responds to different external stresses it encounters in the aquatic environment and in the human host. One stress that V. cholerae encounters in the host is exposure to antimicrobial peptides on mucosal surfaces. We used massively parallel cDNA sequencing (RNA-Seq) to quantitatively identify the transcriptome of V. cholerae grown in the presence and absence of sub-lethal concentrations of the antimicrobial peptide polymyxin B. We evaluated the transcriptome of both wild type V. cholerae and a mutant carrying a deletion of vc1639, a putative sensor kinase of an uncharacterized two-component system, under these conditions. In addition to many previously uncharacterized pathways responding with elevated transcript levels to polymyxin B exposure, we confirmed the predicted elevated transcript levels of a previously described LPS modification system in response to polymyxin B exposure. Additionally, we identified the V. cholerae homologue of visP (ygiW) as a regulatory target of VC1639. VisP is a conserved periplasmic protein implicated in lipid A modification in Salmonellae. This study provides the first systematic analysis of the transcriptional response of Vibrio cholerae to polymyxin B, raising important questions for further study regarding mechanisms used by V. cholerae to sense and respond to envelope stress.

Methylation-dependent DNA discrimination in natural transformation of Campylobacter jejuni.

Campylobacter jejuni, a leading cause of bacterial gastroenteritis, is naturally competent. Like many competent organisms, C. jejuni restricts the DNA that can be used for transformation to minimize undesirable changes in the chromosome. Although C. jejuni can be transformed by C. jejuni-derived DNA, it is poorly transformed by the same DNA propagated in Escherichia coli or produced with PCR. Our work indicates that methylation plays an important role in marking DNA for transformation. We have identified a highly conserved DNA methyltransferase, which we term Campylobacter transformation system methyltransferase (ctsM), which methylates an overrepresented 6-bp sequence in the chromosome. DNA derived from a ctsM mutant transforms C. jejuni significantly less well than DNA derived from ctsM+ (parental) cells. The ctsM mutation itself does not affect transformation efficiency when parental DNA is used, suggesting that CtsM is important for marking transforming DNA, but not for transformation itself. The mutant has no growth defect, arguing against ongoing restriction of its own DNA. We further show that E. coli plasmid and PCR-derived DNA can efficiently transform C. jejuni when only a subset of the CtsM sites are methylated in vitro. A single methylation event 1 kb upstream of the DNA involved in homologous recombination is sufficient to transform C. jejuni, whereas otherwise identical unmethylated DNA is not. Methylation influences DNA uptake, with a slight effect also seen on DNA binding. This mechanism of DNA discrimination in C. jejuni is distinct from the DNA discrimination described in other competent bacteria.