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Reducing food intake is a common host response to infection, yet it remains unclear whether fasting is detrimental or beneficial to an infected host. Despite the gastrointestinal tract being the primary site of nutrient uptake and a common route for infection, studies have yet to examine how fasting alters the host’s response to an enteric infection. To test this, mice were fasted before and during oral infection with the invasive bacterium Salmonella enterica serovar Typhimurium. Fasting dramatically interrupted infection and subsequent gastroenteritis by suppressing Salmonella’s SPI-1 virulence program, preventing invasion of the gut epithelium. Virulence suppression depended on the gut microbiota, as Salmonella’s invasion of the epithelium proceeded in fasting gnotobiotic mice. Despite Salmonella’s restored virulence within the intestines of gnotobiotic mice, fasting downregulated pro-inflammatory signaling, greatly reducing intestinal pathology. Our study highlights how food intake controls the complex relationship between host, pathogen and gut microbiota during an enteric infection., Author summary Most animals, including humans, lose their appetites when sick. Whether this sickness behavior has evolved as a protective mechanism is unclear. In addition, fasting therapies have become popular in recent years and show promise for treating chronic inflammatory diseases, but it is uncertain whether fasting-induced immunosuppression could leave an already fasted host more vulnerable to infection than a fed host. To test this, we fasted mice and orally infected them with the invasive bacterium Salmonella Typhimurium. This pathogen causes gastroenteritis (food poisoning) in humans and in antibiotic-pretreated mice. Notably, the fasted mice were protected from infection. While Salmonella rapidly expanded in the intestines of fed mice, their expansion was reduced in fasted mice. Moreover, Salmonella in the fasted mice did not cause any intestinal tissue damage as the bacteria were unable to invade the intestinal wall. This protection was found to be partially due to the gut microbiome, since fasting was unable to prevent Salmonella infection in mice lacking a microbiome, although the mice suffered less gastroenteritis. We therefore conclude that fasting can protect hosts from intestinal bacterial infections, in part through the actions of the gut microbiome.

Attaching/Effacing (A/E) bacteria include human pathogens enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC), and their murine equivalent Citrobacter rodentium (CR), of which EPEC and EHEC are important causative agents of foodborne diseases worldwide. While A/E pathogen infections cause mild symptoms in the immunocompetent hosts, an increasing number of studies show that they produce more severe morbidity and mortality in immunocompromised and/or immunodeficient hosts. However, the pathogenic mechanisms and crucial host-pathogen interactions during A/E pathogen infections under immunocompromised conditions remain elusive. We performed a functional screening by infecting interleukin-22 (IL-22) knockout (Il22-/-) mice with a library of randomly mutated CR strains. Our screen reveals that interruption of the espF gene, which encodes the Type III Secretion System effector EspF (E. coli secreted protein F) conserved among A/E pathogens, completely abolishes the high mortality rates in CR-infected Il22-/- mice. Chromosomal deletion of espF in CR recapitulates the avirulent phenotype without impacting colonization and proliferation of CR, and EspF complement in ΔespF strain fully restores the virulence in mice. Moreover, the expression levels of the espF gene are elevated during CR infection and CR induces disruption of the tight junction (TJ) strands in colonic epithelium in an EspF-dependent manner. Distinct from EspF, chromosomal deletion of other known TJ-damaging effector genes espG and map failed to impede CR virulence in Il22-/- mice. Hence our findings unveil a critical pathophysiological function for EspF during CR infection in the immunocompromised host and provide new insights into the complex pathogenic mechanisms of A/E pathogens., Author summary Emerging evidence demonstrates that attaching/effacing (A/E) pathogens, the important causative agents of foodborne illnesses, cause more severe morbidity and mortality in immunocompromised hosts. However, the molecular and cellular mechanisms during A/E pathogen infections under immunocompromised conditions have remained obscure. More specifically, the critical host-pathogen interactions that account for the severe symptoms are not fully understood. Utilizing an immunodeficient mouse model for a functional screening, we have identified E. coli secreted protein F (EspF) as a critical virulence protein that plays a pivotal role in A/E pathogen Citrobacter rodentium (CR) infection-induced severe morbidity and mortality in immunocompromised animals. We have also revealed that the EspF-dependent disruption of tight junctions is crucial for the loss of colonic epithelial barrier function and the systemic spread of pathogens during infection. Our findings highlight the previously underappreciated pathophysiological relevance of EspF during A/E pathogen infections and increase our understanding of the sophisticated pathogenic mechanisms of foodborne diseases.

Attaching/Effacing (A/E) pathogens including enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC) and the rodent equivalent Citrobacter rodentium are important causative agents of foodborne diseases. Upon infection, a myriad of virulence proteins (effectors) encoded by A/E pathogens are injected through their conserved type III secretion systems (T3SS) into host cells where they interfere with cell signaling cascades, in particular the nuclear factor kappaB (NF-κB) signaling pathway that orchestrates both innate and adaptive immune responses for host defense. Among the T3SS-secreted non-LEE-encoded (Nle) effectors, NleC, a metalloprotease, has been recently elucidated to modulate host NF-κB signaling by cleaving NF-κB Rel subunits. However, it remains elusive how NleC recognizes NF-κB Rel subunits and how the NleC-mediated cleavage impacts on host immune responses in infected cells and animals. In this study, we show that NleC specifically targets p65/RelA through an interaction with a unique N-terminal sequence in p65. NleC cleaves p65 in intestinal epithelial cells, albeit a small percentage of the molecule, to generate the p651–38 fragment during C. rodentium infection in cultured cells. Moreover, the NleC-mediated p65 cleavage substantially affects the expression of a subset of NF-κB target genes encoding proinflammatory cytokines/chemokines, immune cell infiltration in the colon, and tissue injury in C. rodentium-infected mice. Mechanistically, the NleC cleavage-generated p651–38 fragment interferes with the interaction between p65 and ribosomal protein S3 (RPS3), a ‘specifier’ subunit of NF-κB that confers a subset of proinflammatory gene transcription, which amplifies the effect of cleaving only a small percentage of p65 to modulate NF-κB-mediated gene expression. Thus, our results reveal a novel mechanism for A/E pathogens to specifically block NF-κB signaling and inflammatory responses by cleaving a small percentage of p65 and targeting the p65/RPS3 interaction in host cells, thus providing novel insights into the pathogenic mechanisms of foodborne diseases., Author Summary The nuclear factor kappaB (NF-κB) signaling pathway is crucial for host defense, as it orchestrates both innate and adaptive immune responses. Beyond the best-studied Rel proteins (p65, RelB, c-Rel, p50 and p52), RPS3 has been recently identified as a “specifier” component of NF-κB, modulating the promoter selectivity and transcriptional specificity of NF-κB. In particular, the RPS3/p65-conferred signaling pathway was recently shown to play a critical role in host proinflammatory transcription and immune responses. Attaching and effacing (A/E) pathogens and others have acquired sophisticated mechanisms to modulate host NF-κB signaling pathways. We have found that NleC, a metalloprotease effector secreted by A/E pathogens, modulates host NF-κB signaling and inflammatory responses through a novel mechanism. NleC specifically recognizes and cleaves a small percentage of p65 and the generated N-terminal fragment of p65 interferes with the p65/RPS3 interaction, thereby amplifying the effect of cleaving only a small percentage of p65 molecules to selectively inhibit NF-κB gene expression. Our findings highlight a previously unappreciated mechanism through which pathogen-encoded proteases interfere with signaling cascades and inflammatory responses in host cells.