Clostridium perfringens type F (formerly enterotoxigenic C. perfringens type A) strains produce an enterotoxin (CPE) to cause acute cases of food poisoning and chronic nonfoodborne human gastrointestinal diseases (NFD), e.g., antibiotic-associated diarrhea (AAD). NFD strains also produce NanI sialidase, an extracellular enzyme that releases sialic acids from sialyated host macromolecules. Recent in vitro studies suggested that NanI may contribute to NFD strain intestinal colonization by enhancing the adherence of such strains to intestinal cells and promoting their bacterial growth using generated sialic acid as an energy source. The current study tested this hypothesis by developing a mouse intestinal colonization model involving clindamycin pretreatment to produce conditions mimicking those during AAD. In this model, the type F NFD strain F4969 persisted for at least 4 days in the small intestine, cecum, and colon. When clindamycin-pretreated mice were challenged by oral gavage with equivalent numbers of F4969 bacteria or its isogenic nanI null mutant, significantly lower numbers of the nanI mutant were recovered from all intestinal segments, and it was completely cleared from the small intestine by day 4. Complementation of the mutant to restore NanI production also promoted colonization. When the same nanI null mutant strain was coinoculated into the mouse model together with a nanI-producing strain, the numbers of this mutant were restored to wild-type F4969 levels in all intestinal segments. This result suggests that sialidases produced by other bacteria might also provide some support for C. perfringens intestinal colonization. Collectively, these in vivo findings identify NanI to be the first known significant contributor to chronic intestinal colonization by NFD strains.

Clostridium perfringens enterotoxin (CPE) is a pore-forming toxin that causes the symptoms of common bacterial food poisoning and several non-foodborne human gastrointestinal diseases, including antibiotic-associated diarrhea and sporadic diarrhea. In some cases, CPE-mediated disease can be very severe or fatal due to the involvement of enterotoxemia. Therefore, the development of potential therapeutics against CPE action during enterotoxemia is warranted. Mepacrine, an acridine derivative drug with broad-spectrum effects on pores and channels in mammalian membranes, has been used to treat protozoal intestinal infections in human patients. A previous study showed that the presence of mepacrine inhibits CPE-induced pore formation and activity in enterocyte-like Caco-2 cells, reducing the cytotoxicity caused by this toxin in vitro Whether mepacrine is similarly protective against CPE action in vivo has not been tested. When the current study evaluated whether mepacrine protects against CPE-induced death and intestinal damage using a murine ligated intestinal loop model, mepacrine protected mice from the enterotoxemic lethality caused by CPE. This protection was accompanied by a reduction in the severity of intestinal lesions induced by the toxin. Mepacrine did not reduce CPE pore formation in the intestine but inhibited absorption of the toxin into the blood of some mice. Protection from enterotoxemic death correlated with the ability of this drug to reduce CPE-induced hyperpotassemia. These in vivo findings, coupled with previous in vitro studies, support mepacrine as a potential therapeutic against CPE-mediated enterotoxemic disease.

In light of the demonstrated antagonism of Wnt5A signaling toward the growth of several bacterial pathogens, it was important to study the influence of Wnt5A on gut-resident bacteria and its outcome. Here, we demonstrate that in contrast to inhibiting the survival of the established gut pathogen Salmonella enterica, Wnt5A clearly promotes the survival of the common gut commensals Enterococcus faecalis and Lactobacillus rhamnosus within macrophages through a self-perpetuating Wnt5A-actin axis. A Wnt5A-actin axis furthermore regulates the subsistence of the natural bacterial population of the Peyer's patches, as is evident from the diminution in the countable bacterial CFU therein through the application of Wnt5A signaling and actin assembly inhibitors. Wnt5A dependency of the gut-resident bacterial population is also manifested in the notable difference between the bacterial diversities associated with the feces and Peyer's patches of Wnt5A heterozygous mice, which lack a functional copy of the Wnt5A gene, and their wild-type counterparts. Alterations in the gut commensal bacterial population resulting from either the lack of a copy of the Wnt5A gene or inhibitor-mediated attenuation of Wnt5A signaling are linked with significant differences in cell surface major histocompatibility complex (MHC) II levels and regulatory versus activated CD4 T cells associated with the Peyer's patches. Taken together, our findings reveal the significance of steady state Wnt5A signaling in shaping the gut commensal bacterial population and the T cell repertoire linked to it, thus unveiling a crucial control device for the maintenance of gut bacterial diversity and T cell homeostasis. IMPORTANCE Gut commensal bacterial diversity and T cell homeostasis are crucial entities of the host innate immune network, yet the molecular details of host-directed signaling pathways that sustain the steady state of gut bacterial colonization and T cell activation remain unclear. Here, we describe the protective role of a Wnt5A-actin axis in the survival of several gut bacterial commensals and its necessity in shaping gut bacterial colonization and the associated T cell repertoire. This study opens up new avenues of investigation into the role of the Wnt5A-actin axis in protection of the gut from dysbiosis-related inflammatory disorders.

Abstract Background The aberrant inflammation that is the hallmark of the inflammatory bowel diseases (IBD) is associated with several factors, including changes in the intestinal microbiota. Here, we confirmed that an intestinal microbiota is needed for development of typhlocolitis in Helicobacter hepaticus infected IL-10-/- C57BL/6 mice, and investigated the role of the microbiota in modulating disease. Results We altered the murine microbiota by treatment with the antibiotics vancomycin or cefoperazone prior to H. hepaticus infection. Through surveys of the 16S rRNA encoding-gene, analyses of histology and changes in expression of host mediators, we correlated alterations in the microbiota with host responses. We found that resident microbes are essential for initiation of disease, as animals mono-associated with H. hepaticus did not develop colitis. Despite the requirement for an indigenous microbiota for the initiation of disease, the severity of disease was independent of antibiotic-induced changes in the microbial community structure. Despite differences in the expression of host inflammatory mediators associated with shifts in the microbiota, H. hepaticus infection led to similar histopathologic lesions in microbial communities exposed to either cefoperazone or vancomycin. Conclusion In conclusion, we demonstrate that colitis due to H. hepaticus infection can be initiated and progress in the presence of several different microbial communities. Furthermore, H. hepaticus is the main driver of inflammation in this model, while the specific structure of the microbiota may modulate the host pathways that lead to chronic inflammation.

Clostridium perfringens enterotoxin (CPE) is thought to cause lethal enterotoxemia when absorbed from the intestinal lumen into the circulation. CPE action sequentially involves receptor-binding, oligomerization into a prepore, and pore formation. To explore the mechanistic basis by which CPE alters permeability, this study tested the permeability effects of several recombinant CPE (rCPE) species: rCPE and rCPE_C186A (which form pores), rC-CPE and rCPE_D48A (which bind to receptors but cannot oligomerize), rCPE_C186A/F91C (which binds and oligomerizes without pore formation), and rCPE_Y306A/L315A (which has poor receptor-binding ability). On Caco-2 cells, i) only rCPE and rCPE_C186A were cytotoxic; ii) rCPE and rCPE_C186A affected transepithelial resistance (TEER) and 4 kDa fluorescent dextran (FD4) transit more quickly than binding-capable, but noncytotoxic, rCPE variants; whereas iii) rCPE_Y306A/L315A did not affect TEER or FD4 transit. Using mouse intestinal loops, rCPE (but not noncytotoxic rC-CPE, rCPE_D48A or rCPE_Y306A/L315A) was lethal and caused intestinal histologic damage within 4 h. After 2 h of treatment, rCPE was more strongly absorbed into the serum than those noncytotoxic rCPE species but by 4 h rC-CPE and rCPE_D48A became absorbed similarly as rCPE, while rCPE_Y306A/L315A absorption remained low. This increased rC-CPE and rCPE_D48A absorption from 2 to 4 h did not involve a general intestinal permeability increase because Evans Blue absorption from the intestines did not increase between 2 and 4 h of treatment with rC-CPE or rCPE_D48A. Collectively, these results indicate that CPE receptor binding is sufficient to slowly affect permeability, but CPE-induced cytotoxicity is necessary for rapid permeability changes and lethality. IMPORTANCE Clostridium perfringens enterotoxin (CPE) causes lethal enterotoxemia when absorbed from the intestines into the bloodstream. Testing recombinant CPE (rCPE) or rCPE variants impaired for various specific steps in CPE action showed that full CPE-induced cytotoxicity causes rapid Caco-2 monolayer permeability alterations, as well as enterotoxemic lethality and rapid CPE absorption in mouse small intestinal loops. However, receptor binding-capable, but noncytotoxic, rCPE variants did cause slow-developing in vitro and in vivo permeability effects. Absorption of binding-capable, noncytotoxic rCPE variants from the intestines did not correlate with general intestinal permeability alterations, suggesting that CPE binding can induce its own uptake. These findings highlight the importance of binding and, especially, cytotoxicity for CPE absorption during enterotoxemia and may assist development of permeability-altering rCPE variants for translational purposes.

Many Clostridium perfringens strains produce NanI as their major sialidase. Previous studies showed that NanI could potentiate C. perfringens epsilon toxin cytotoxicity by enhancing the binding of this toxin to host cells. The present study first determined that NanI exerts similar cytotoxicity-enhancing effects on C. perfringens enterotoxin and beta toxin, which are also important toxins for C. perfringens diseases (enteritis and enterotoxemia) originating in the gastrointestinal (GI) tract. Building upon previous work demonstrating that purified trypsin can activate NanI activity, this study next determined that purified chymotrypsin or mouse intestinal fluids can also activate NanI activity. Amino acid sequencing then showed that this effect involves the N-terminal processing of the NanI protein. Recombinant NanI (rNanI) species corresponding to major chymotrypsin- or small intestinal fluid-generated NanI fragments possessed more sialidase activity than did full-length rNanI, further supporting the proteolytic activation of NanI activity. rNanI species corresponding to proteolysis products also promoted the cytotoxic activity and binding of enterotoxin and beta toxin more strongly than did full-length rNanI. Since enterotoxin and beta toxin are produced in the intestines during human and animal disease, these findings suggest that intestinal proteases may enhance NanI activity, which in turn could further potentiate the activity of intestinally active toxins during disease. Coupling these new results with previous findings demonstrating that NanI is important for the adherence of C. perfringens to enterocyte-like cells, NanI sialidase is now emerging as a potential auxiliary virulence factor for C. perfringens enteritis and enterotoxemia.

Clostridium perfringens enterotoxin (CPE) is responsible for the gastrointestinal symptoms of C. perfringens type A food poisoning and some cases of nonfoodborne gastrointestinal diseases, such as antibiotic-associated diarrhea. In the presence of certain predisposing medical conditions, this toxin can also be absorbed from the intestines to cause enterotoxemic death. CPE action in vivo involves intestinal damage, which begins at the villus tips. The cause of this CPE-induced intestinal damage is unknown, but CPE can induce caspase-3-mediated apoptosis in cultured enterocyte-like Caco-2 cells. Therefore, the current study evaluated whether CPE activates caspase-3 in the intestines and, if so, whether this effect is required for the development of intestinal tissue damage or enterotoxemic lethality. Using a mouse ligated small intestinal loop model, CPE was shown to cause intestinal caspase-3 activation in a dose- and time-dependent manner. Most of this caspase-3 activation occurred in epithelial cells shed from villus tips. However, CPE-induced caspase-3 activation occurred after the onset of tissue damage. Furthermore, inhibition of intestinal caspase-3 activity did not affect the onset of intestinal tissue damage. Similarly, inhibition of intestinal caspase-3 activity did not reduce CPE-induced enterotoxemic lethality in these mice. Collectively, these results demonstrate that caspase-3 activation occurs in the CPE-treated intestine but that this effect is not necessary for the development of CPE-induced intestinal tissue damage or enterotoxemic lethality.

Helicobacter pylori is a pathogen that chronically colonizes the stomachs of approximately half of the world's population and contributes to the development of gastric inflammation. We demonstrated previously in vivo that H. pylori uses motility to preferentially colonize injury sites in the mouse stomach. However, the chemoreceptor responsible for sensing gastric injury has not yet been identified. In this study, we utilized murine gastric organoids (gastroids) and mutant H. pylori strains to investigate the components necessary for H. pylori chemotaxis. High-intensity 730-nm light (two-photon photodamage) was used to cause single-cell damage in gastroids, and repair of the damage was monitored over time; complete repair occurred within ∼10 min in uninfected gastroids. Wild-type H. pylori accumulated at the damage site after gastric damage induction. In contrast, mutants lacking motility (ΔmotB) or chemotaxis (ΔcheY) did not accumulate at the injury site. Using mutants lacking individual chemoreceptors, we found that only TlpB was required for H. pylori accumulation, while TlpA, TlpC, and TlpD were dispensable. All strains that were able to accumulate at the damage site limited repair. When urea (an identified chemoattractant sensed by TlpB) was microinjected into the gastroid lumen, it prevented the accumulation of H. pylori at damage sites. Overall, our findings demonstrate that H. pylori colonizes and limits repair at damage sites via chemotactic motility that requires the TlpB chemoreceptor to sense signals generated by gastric epithelial cells.

Alterations in diet can have significant impact on the host, with high-fat diet (HFD) leading to obesity, diabetes, and inflammation of the gut. Although membership and abundances in gut bacterial communities are strongly influenced by diet, substantially less is known about how viral communities respond to dietary changes. Examining fecal contents of mice as the mice were transitioned from normal chow to HFD, we found significant changes in the relative abundances and the diversity in the gut of bacteria and their viruses. Alpha diversity of the bacterial community was significantly diminished in response to the diet change but did not change significantly in the viral community. However, the diet shift significantly impacted the beta diversity in both the bacterial and viral communities. There was a significant shift away from the relatively abundant Siphoviridae accompanied by increases in bacteriophages from the Microviridae family. The proportion of identified bacteriophage structural genes significantly decreased after the transition to HFD, with a conserved loss of integrase genes in all four experimental groups. In total, this study provides evidence for substantial changes in the intestinal virome disproportionate to bacterial changes, and with alterations in putative viral lifestyles related to chromosomal integration as a result of shift to HFD.IMPORTANCE Prior studies have shown that high-fat diet (HFD) can have profound effects on the gastrointestinal (GI) tract microbiome and also demonstrate that bacteria in the GI tract can affect metabolism and lean/obese phenotypes. We investigated whether the composition of viral communities that also inhabit the GI tract are affected by shifts from normal to HFD. We found significant and reproducible shifts in the content of GI tract viromes after the transition to HFD. The differences observed in virome community membership and their associated gene content suggest that these altered viral communities are populated by viruses that are more virulent toward their host bacteria. Because HFD also are associated with significant shifts in GI tract bacterial communities, we believe that the shifts in the viral community may serve to drive the changes that occur in associated bacterial communities.

The functioning, health, and productivity of soil are intimately tied to a complex network of interactions, particularly in plant root-associated rhizosphere soil. We conducted a stable-isotope-informed, genome-resolved metagenomic study to trace carbon from Avena fatua grown in a ¹³CO₂ atmosphere into soil. We collected paired rhizosphere and nonrhizosphere soil at 6 and 9 weeks of plant growth and extracted DNA that was then separated by density using ultracentrifugation. Thirty-two fractions from each of five samples were grouped by density, sequenced, assembled, and binned to generate 55 unique bacterial genomes that were ≥70% complete. We also identified complete 18S rRNA sequences of several ¹³C-enriched microeukaryotic bacterivores and fungi. We generated 10 circularized bacteriophage (phage) genomes, some of which were the most labeled entities in the rhizosphere, suggesting that phage may be important agents of turnover of plant-derived C in soil. CRISPR locus targeting connected one of these phage to a Burkholderiales host predicted to be a plant pathogen. Another highly labeled phage is predicted to replicate in a Catenulispora sp., a possible plant growth-promoting bacterium. We searched the genome bins for traits known to be used in interactions involving bacteria, microeukaryotes, and plant roots and found DNA from heavily ¹³C-labeled bacterial genes thought to be involved in modulating plant signaling hormones, plant pathogenicity, and defense against microeukaryote grazing. Stable-isotope-informed, genome-resolved metagenomics indicated that phage can be important agents of turnover of plant-derived carbon in soil. IMPORTANCE Plants grow in intimate association with soil microbial communities; these microbes can facilitate the availability of essential resources to plants. Thus, plant productivity commonly depends on interactions with rhizosphere bacteria, viruses, and eukaryotes. Our work is significant because we identified the organisms that took up plant-derived organic C in rhizosphere soil and determined that many of the active bacteria are plant pathogens or can impact plant growth via hormone modulation. Further, by showing that bacteriophage accumulate CO₂-derived carbon, we demonstrated their vital roles in redistribution of plant-derived C into the soil environment through bacterial cell lysis. The use of stable-isotope probing (SIP) to identify consumption (or lack thereof) of root-derived C by key microbial community members within highly complex microbial communities opens the way for assessing manipulations of bacteria and phage with potentially beneficial and detrimental traits, ultimately providing a path to improved plant health and soil carbon storage.