Toxigenic Clostridium perfringens isolated from at-risk pediatric inflammatory bowel disease patients.

BACKGROUND AND AIMS: The goal was to identify microbial drivers of IBD, by investigating mucosal-associated bacteria and their detrimental products in IBD patients. METHODS: We directly cultured bacterial communities from mucosal biopsies from pediatric gastrointestinal patients and examined for pathogenicity-associated traits. Upon identifying C. perfringens as toxigenic bacteria present in mucosal biopsies, we isolated strains and further characterized toxicity and prevalence. RESULTS: Mucosal biopsy microbial composition differed from corresponding stool samples. C. perfringens was present in 8 of 9 patients' mucosal biopsies, correlating with hemolytic activity, while not in all corresponding stool samples. Large IBD datasets showed higher C. perfringens prevalence in stool samples of IBD adults (18.7-27.1%) versus healthy (5.1%). In vitro, C. perfringens supernatants were toxic to cell types beneath the intestinal epithelial barrier, including endothelial, neuroblasts, and neutrophils, while impact on epithelial cells was less pronounced, suggesting C. perfringens may be damaging particularly when barrier integrity is compromised. Further characterization using purified toxins and genetic insertion mutants confirmed PFO toxin was sufficient for toxicity. Toxin RNA signatures were found in the original patient biopsies by PCR, suggesting intestinal production. C. perfringens supernatants also induced activation of neuroblast and dorsal root ganglion neurons in vitro, suggesting C. perfringens in inflamed mucosal tissue may directly contribute to abdominal pain, a frequent IBD symptom. CONCLUSIONS: Gastrointestinal carriage of certain toxigenic C. perfringens may have an important pathogenic impact on IBD patients. These findings support routine monitoring of C. perfringens and PFO toxins and potential treatment in patients.

Gastric Metabolomics Detects Helicobacter pylori Correlated Loss of Numerous Metabolites in Both the Corpus and Antrum.

Helicobacter pylori is a chronic bacterial pathogen that thrives in several regions of the stomach, causing inflammation that can vary by site and result in distinct disease outcomes. Whether the regions differ in terms of host-derived metabolites is not known. We thus characterized the regional variation of the metabolomes of mouse gastric corpus and antrum organoids and tissue. The uninfected secreted organoid metabolites differed between the corpus and antrum in only seven metabolites as follows: lactic acid, malic acid, phosphoethanolamine, alanine, uridine, glycerol, and isoleucine. Several of the secreted chemicals were depleted upon H. pylori infection in both regions, including urea, cholesterol, glutamine, fumaric acid, lactic acid, citric acid, malic acid, and multiple nonessential amino acids. These results suggest a model in which H. pylori preferentially uses carboxylic acids and amino acids in complex environments, and these are found in both the corpus and antrum. When organoid metabolites were compared to mouse tissue, there was little overlap. The tissue corpus and antrum metabolomes were distinct, including antrum-elevated 5-methoxytryptamine, lactic acid, and caprylic acid, and corpus-elevated phospholipid products. The corpus and antrum remained distinct over an 8-month infection time course. The antrum displayed no significant changes between the time points in contrast to the corpus, which exhibited metabolite changes that were consistent with stress, tissue damage, and depletion of key nutrients, such as glutamine and fructose-6-phosphate. Overall, our results suggest that the corpus and antrum have largely but not completely overlapping metabolomes that change moderately upon H. pylori infection.

Spatial control of the GTPase MglA by localized RomR-RomX GEF and MglB GAP activities enables Myxococcus xanthus motility.

The rod-shaped Myxococcus xanthus cells move with defined front-rear polarity using polarized motility systems. A polarity module consisting of the small GTPase MglA, its cognate GTPase activating protein (GAP) MglB and RomR establishes this polarity. Agl-Glt gliding motility complexes assemble and disassemble at the leading and lagging pole, respectively. These processes are stimulated by MglA-GTP at the leading and MglB at the lagging pole. Here, we identify RomX as an integral component of the polarity module. RomX and RomR form a complex that has MglA guanine nucleotide exchange factor (GEF) activity and also binds MglA-GTP. In vivo RomR recruits RomX to the leading pole forming the RomR-RomX complex that stimulates MglA-GTP formation and binding, resulting in a high local concentration of MglA-GTP. The spatially separated and opposing activities of the RomR-RomX GEF at the leading and the MglB GAP at the lagging cell pole establish front-rear polarity by allowing the spatially separated assembly and disassembly of Agl-Glt motility complexes. Our findings uncover a regulatory system for bacterial cell polarity that incorporates a nucleotide exchange factor as well as an NTPase activating protein for regulation of a nucleotide-dependent molecular switch and demonstrate a spatial organization that is conserved in eukaryotes.

The Helicobacter pylori Autotransporter ImaA Tempers the Bacterium's Interaction with α5ß1 Integrin.

The human pathogen Helicobacter pylori uses the host receptor α5ß1 integrin to trigger inflammation in host cells via its cag pathogenicity island (cag PAI) type IV secretion system (T4SS). Here, we report that the H. pylori ImaA protein (HP0289) decreases the action of the cag PAI T4SS via tempering the bacterium's interaction with α5ß1 integrin. Previously, imaA-null mutants were found to induce an elevated inflammatory response that was dependent on the cag PAI T4SS; here we extend those findings to show that the elevated response is independent of the CagA effector protein. To understand how ImaA could be affecting cag PAI T4SS activity at the host cell interface, we utilized the Phyre structural threading program and found that ImaA has a region with remote homology to bacterial integrin-binding proteins. This region was required for ImaA function. Unexpectedly, we observed that imaA mutants bound higher levels of α5ß1 integrin than wild-type H. pylori, an outcome that required the predicted integrin-binding homology region of ImaA. Lastly, we report that ImaA directly affected the amount of host cell ß1 integrin but not other cellular integrins. Our results thus suggest a model in which H. pylori employs ImaA to regulate interactions between integrin and the T4SS and thus alter the host inflammatory strength.

Spatial and Temporal Shifts in Bacterial Biogeography and Gland Occupation during the Development of a Chronic Infection.

Gland colonization may be one crucial route for bacteria to maintain chronic gastrointestinal infection. We developed a quantitative gland isolation method to allow robust bacterial population analysis and applied it to the gastric pathobiont Helicobacter pylori After infections in the murine model system, H. pylori populations multiply both inside and outside glands in a manner that requires the bacteria to be motile and chemotactic. H. pylori is able to achieve gland densities averaging 25 to 40 bacteria/gland after 2 to 4 weeks of infection. After 2 to 4 weeks of infection, a primary infection leads to colonization resistance for a secondary infection. Nonetheless, about ~50% of the glands remained unoccupied, suggesting there are as-yet unappreciated parameters that prevent gastric gland colonization. During chronic infections, H. pylori populations collapsed to nearly exclusive gland localization, to an average of <8 bacteria/gland, and only 10% of glands occupied. We analyzed an H. pylori chemotaxis mutant (Che-) to gain mechanistic insight into gland colonization. Che- strains had a severe inability to spread to new glands and did not protect from a secondary infection but nonetheless achieved a chronic gland colonization state numerically similar to that of the wild type. Overall, our analysis shows that bacteria undergo substantial population dynamics on the route to chronic colonization, that bacterial gland populations are maintained at a low level during chronic infection, and that established gland populations inhibit subsequent colonization. Understanding the parameters that promote chronic colonization will allow the future successful design of beneficial microbial therapeutics that are able to maintain long-term mammalian colonization. IMPORTANCE: Many bacteria have an impressive ability to stay in the gastrointestinal tract for decades despite ongoing flow and antimicrobial attacks. How this staying power is achieved is not fully understood, but it is important to understand as scientists plan so-called designer microbiomes. The gastrointestinal tract is lined with repeated invaginations called glands, which may provide one niche for chronic colonization. We developed a quantitative gland isolation method to allow robust and efficient bacterial population analysis and applied it to the gastric pathogen Helicobacter pylori Bacterial populations increased inside and outside glands at early time points but were found exclusively within glands during late time points in the chronic state. H. pylori required the ability to swim to move to new glands. Last, a fit gland bacterial population leads to colonization resistance of a second one. Our approach identified previously unappreciated aspects of gland occupation, supporting the idea that glands are the desired niche for stable, chronic colonization.

How Helicobacter pylori senses, targets and interacts with the gastric epithelium.

Helicobacter pylori is a human-specific pathogen that chronically infects about 50% of the world's population. After travelling through the harsh environment of the stomach lumen, H. pylori colonizes the mucosal surface and within the glands of the human stomach. During colonization, H. pylori uses motility and its chemotaxis signalling system to sense the environment to reach the gastric epithelium for colonization, where it is able to attach to the epithelial surface. The H. pylori population inside the stomach contains a subgroup of bacteria that are attached to the gastric epithelium and a larger subgroup of non-attached bacteria that are freely swimming. To establish a tight interaction between H. pylori and epithelial cells, the bacterium produces a variety of adhesins and delivers virulence factors. These lead to alterations in the host signalling pathways, inducing pro-inflammatory responses, apoptosis, uncontrolled cell proliferation, and eventually peptic ulcers and gastric cancer. To prevent disease and find a vaccine or better treatments, it is crucial to understand how H. pylori is able to sense its niche for chronic infection inside the stomach and how its virulence factors interact with the epithelial target cells.

MglC, a Paralog of Myxococcus xanthus GTPase-Activating Protein MglB, Plays a Divergent Role in Motility Regulation.

UNLABELLED: In order to optimize interactions with their environment and one another, bacteria regulate their motility. In the case of the rod-shaped cells of Myxococcus xanthus, regulated motility is essential for social behaviors. M. xanthus moves over surfaces using type IV pilus-dependent motility and gliding motility. These two motility systems are coordinated by a protein module that controls cell polarity and consists of three polarly localized proteins, the small G protein MglA, the cognate MglA GTPase-activating protein MglB, and the response regulator RomR. Cellular reversals are induced by the Frz chemosensory system, and the output response regulator of this system, FrzZ, interfaces with the MglA/MglB/RomR module to invert cell polarity. Using a computational approach, we identify a paralog of MglB, MXAN_5770 (MglC). Genetic epistasis experiments demonstrate that MglC functions in the same pathway as MglA, MglB, RomR, and FrzZ and is important for regulating cellular reversals. Like MglB, MglC localizes to the cell poles asymmetrically and with a large cluster at the lagging pole. Correct polar localization of MglC depends on RomR and MglB. Consistently, MglC interacts directly with MglB and the C-terminal output domain of RomR, and we identified a surface of MglC that is necessary for the interaction with MglB and for MglC function. Together, our findings identify an additional member of the M. xanthus polarity module involved in regulating motility and demonstrate how gene duplication followed by functional divergence can add a layer of control to the complex cellular processes of motility and motility regulation. IMPORTANCE: Gene duplication and the subsequent divergence of the duplicated genes are important evolutionary mechanisms for increasing both biological complexity and regulation of biological processes. The bacterium Myxococcus xanthus is a soil bacterium with an unusually large genome that carries out several social processes, including predation of other bacterial species and formation of multicellular, spore-filled fruiting bodies. One feature of the large M. xanthus genome is that it contains many gene duplications. Here, we compare the products of one example of gene duplication and divergence, in which a paralog of the cognate MglA GTPase-activating protein MglB has acquired a different and opposing role in the regulation of cellular polarity and motility, processes critical to the bacterium's social behaviors.

H. pylori GPS: Modulating Host Metabolites for Location Sensing.

Almost 20 years ago, urea was described as a chemotaxis attractant for Helicobacter pylori. In this issue of Cell Host & Microbe, Huang et al. (2015) report that H. pylori employs its urease enzyme to destroy urea to bring the concentration into a range that provokes an attractant response.

Contact- and Protein Transfer-Dependent Stimulation of Assembly of the Gliding Motility Machinery in Myxococcus xanthus.

Bacteria engage in contact-dependent activities to coordinate cellular activities that aid their survival. Cells of Myxococcus xanthus move over surfaces by means of type IV pili and gliding motility. Upon direct contact, cells physically exchange outer membrane (OM) lipoproteins, and this transfer can rescue motility in mutants lacking lipoproteins required for motility. The mechanism of gliding motility and its stimulation by transferred OM lipoproteins remain poorly characterized. We investigated the function of CglC, GltB, GltA and GltC, all of which are required for gliding. We demonstrate that CglC is an OM lipoprotein, GltB and GltA are integral OM ß-barrel proteins, and GltC is a soluble periplasmic protein. GltB and GltA are mutually stabilizing, and both are required to stabilize GltC, whereas CglC accumulate independently of GltB, GltA and GltC. Consistently, purified GltB, GltA and GltC proteins interact in all pair-wise combinations. Using active fluorescently-tagged fusion proteins, we demonstrate that GltB, GltA and GltC are integral components of the gliding motility complex. Incorporation of GltB and GltA into this complex depends on CglC and GltC as well as on the cytoplasmic AglZ protein and the inner membrane protein AglQ, both of which are components of the gliding motility complex. Conversely, incorporation of AglZ and AglQ into the gliding motility complex depends on CglC, GltB, GltA and GltC. Remarkably, physical transfer of the OM lipoprotein CglC to a ΔcglC recipient stimulates assembly of the gliding motility complex in the recipient likely by facilitating the OM integration of GltB and GltA. These data provide evidence that the gliding motility complex in M. xanthus includes OM proteins and suggest that this complex extends from the cytoplasm across the cell envelope to the OM. These data add assembly of gliding motility complexes in M. xanthus to the growing list of contact-dependent activities in bacteria.

The small G-protein MglA connects to the MreB actin cytoskeleton at bacterial focal adhesions.

In Myxococcus xanthus the gliding motility machinery is assembled at the leading cell pole to form focal adhesions, translocated rearward to propel the cell, and disassembled at the lagging pole. We show that MglA, a Ras-like small G-protein, is an integral part of this machinery. In this function, MglA stimulates the assembly of the motility complex by directly connecting it to the MreB actin cytoskeleton. Because the nucleotide state of MglA is regulated spatially and MglA only binds MreB in the guanosine triphosphate-bound form, the motility complexes are assembled at the leading pole and dispersed at the lagging pole where the guanosine triphosphatase activating protein MglB disrupts the MglA-MreB interaction. Thus, MglA acts as a nucleotide-dependent molecular switch to regulate the motility machinery spatially. The function of MreB in motility is independent of its function in peptidoglycan synthesis, representing a coopted function. Our findings highlight a new function for the MreB cytoskeleton and suggest that G-protein-cytoskeleton interactions are a universally conserved feature.

A model for spatio-temporal dynamics in a regulatory network for cell polarity.

Cell polarity in Myxococcus xanthus is crucial for the directed motility of individual cells. The polarity system is characterised by a dynamic spatio-temporal localisation of the regulatory proteins MglA and MglB at opposite cell poles. In response to signalling by the Frz chemosensory system, MglA and MglB are released from the poles and then rebind at the opposite poles. Thus, over time MglA and MglB oscillate irregularly between the poles in synchrony but out of phase. A minimal macroscopic model of the Mgl/Frz regulatory system based on a reaction-diffusion PDE system is presented. Mathematical analysis of the steady states derives conditions on the reaction terms for formation of dynamic localisation patterns of the regulatory proteins under different biologically-relevant regimes, i.e. with and without Frz signalling. Numerical simulations of the model system produce either a stationary pattern in time (fixed polarity), periodic solutions in time (oscillating polarity), or excitable behaviour (irregular switching of polarity).

Effect of the Min system on timing of cell division in Escherichia coli.

In Escherichia coli the Min protein system plays an important role in positioning the division site. We show that this system also has an effect on timing of cell division. We do this in a quantitative way by measuring the cell division waiting time (defined as time difference between appearance of a division site and the division event) and the Z-ring existence time. Both quantities are found to be different in WT and cells without functional Min system. We develop a series of theoretical models whose predictions are compared with the experimental findings. Continuous improvement leads to a final model that is able to explain all relevant experimental observations. In particular, it shows that the chromosome segregation defect caused by the absence of Min proteins has an important influence on timing of cell division. Our results indicate that the Min system affects the septum formation rate. In the absence of the Min proteins this rate is reduced, leading to the observed strongly randomized cell division events and the longer division waiting times.

Regulation of bacterial cell polarity by small GTPases.

Bacteria are polarized with many proteins localizing dynamically to specific subcellular sites. Two GTPase families have important functions in the regulation of bacterial cell polarity, FlhF homologues and small GTPases of the Ras superfamily. The latter consist of only a G domain and are widespread in bacteria. The rod-shaped Myxococcus xanthus cells have two motility systems, one for gliding and one that depends on type IV pili. The function of both systems hinges on proteins that localize asymmetrically to the cell poles. During cellular reversals, these asymmetrically localized proteins are released from their respective poles and then bind to the opposite pole, resulting in an inversion of cell polarity. Here, we review genetic, cell biological, and biochemical analyses that identified two modules containing small Ras-like GTPases that regulate the dynamic polarity of motility proteins. The GTPase SofG interacts directly with the bactofilin cytoskeletal protein BacP to ensure polar localization of type IV pili proteins. In the second module, the small GTPase MglA, its cognate GTPase activating protein (GAP) MglB, and the response regulator RomR localize asymmetrically to the poles and sort dynamically localized motility proteins to the poles. During reversals, MglA, MglB, and RomR switch poles, in that way inducing the relocation of dynamically localized motility proteins. Structural analyses have demonstrated that MglB has a Roadblock/LC7 fold, the central ß2 strand in MglA undergoes an unusual screw-type movement upon GTP binding, MglA contains an intrinsic Arg finger required for GTP hydrolysis, and MglA and MglB form an unusual G protein/GAP complex with a 1:2 stoichiometry.

A strategy for identifying fluorescence intensity profiles of single rod-shaped cells.

The extraction of fluorescence intensity profiles of single cells from image data is a common challenge in cell biology. The manual segmentation of cells, the extraction of cell orientation and finally the extraction of intensity profiles are time-consuming tasks. This article proposes a routine for the segmentation of single rod-shaped cells (i.e. without neighboring cells in a distance of the cell length) from image data combined with an extraction of intensity distributions along the longitudinal cell axis under the aggravated conditions of (i) a low spatial resolution and (ii) lacking information on the imaging system i.e. the point spread function and signal-to-noise ratio. The algorithm named cipsa transfers a new approach from particle streak velocimetry to cell classification interpreting the rod-shaped as streak-like structures. An automatic reduction of systematic errors such as photobleaching and defocusing is included to guarantee robustness of the proposed approach under the described conditions and to the convenience of end-users unfamiliar with image processing. Performance of the algorithm has been tested on image sequences with high noise level produced by an overlay of different error sources. The developed algorithm provides a user-friendly, stand-alone procedure.

A response regulator interfaces between the Frz chemosensory system and the MglA/MglB GTPase/GAP module to regulate polarity in Myxococcus xanthus.

How cells establish and dynamically change polarity are general questions in cell biology. Cells of the rod-shaped bacterium Myxococcus xanthus move on surfaces with defined leading and lagging cell poles. Occasionally, cells undergo reversals, which correspond to an inversion of the leading-lagging pole polarity axis. Reversals are induced by the Frz chemosensory system and depend on relocalization of motility proteins between the poles. The Ras-like GTPase MglA localizes to and defines the leading cell pole in the GTP-bound form. MglB, the cognate MglA GTPase activating protein, localizes to and defines the lagging pole. During reversals, MglA-GTP and MglB switch poles and, therefore, dynamically localized motility proteins switch poles. We identified the RomR response regulator, which localizes in a bipolar asymmetric pattern with a large cluster at the lagging pole, as important for motility and reversals. We show that RomR interacts directly with MglA and MglB in vitro. Furthermore, RomR, MglA, and MglB affect the localization of each other in all pair-wise directions, suggesting that RomR stimulates motility by promoting correct localization of MglA and MglB in MglA/RomR and MglB/RomR complexes at opposite poles. Moreover, localization analyses suggest that the two RomR complexes mutually exclude each other from their respective poles. We further show that RomR interfaces with FrzZ, the output response regulator of the Frz chemosensory system, to regulate reversals. Thus, RomR serves at the functional interface to connect a classic bacterial signalling module (Frz) to a classic eukaryotic polarity module (MglA/MglB). This modular design is paralleled by the phylogenetic distribution of the proteins, suggesting an evolutionary scheme in which RomR was incorporated into the MglA/MglB module to regulate cell polarity followed by the addition of the Frz system to dynamically regulate cell polarity.

Structural analysis of the Ras-like G protein MglA and its cognate GAP MglB and implications for bacterial polarity.

The bacterium Myxococcus xanthus uses a G protein cycle to dynamically regulate the leading/lagging pole polarity axis. The G protein MglA is regulated by its GTPase-activating protein (GAP) MglB, thus resembling Ras family proteins. Here, we show structurally and biochemically that MglA undergoes a dramatic, GDP-GTP-dependent conformational change involving a screw-type forward movement of the central ß2-strand, never observed in any other G protein. This movement and complex formation with MglB repositions the conserved residues Arg53 and Gln82 into the active site. Residues required for catalysis are thus not provided by the GAP MglB, but by MglA itself. MglB is a Roadblock/LC7 protein and functions as a dimer to stimulate GTP hydrolysis in a 2:1 complex with MglA. In vivo analyses demonstrate that hydrolysis mutants abrogate Myxococcus' ability to regulate its polarity axis changing the reversal behaviour from stochastic to oscillatory and that both MglA GTPase activity and MglB GAP catalysis are essential for maintaining a proper polarity axis.