Background & Aims The Helicobacter pylori toxin vacuolating cytotoxin (VacA) promotes gastric colonization and its presence (VacA+) is associated with more-severe disease. The exact mechanisms by which VacA contributes to infection are unclear. We previously found that limited exposure to VacA induces autophagy of gastric cells, which eliminates the toxin; we investigated whether autophagy serves as a defense mechanism against H pylori infection. Methods We investigated the effect of VacA on autophagy in human gastric epithelial cells (AGS) and primary gastric cells from mice. Expression of p62, a marker of autophagy, was also assessed in gastric tissues from patients infected with toxigenic (VacA+) or nontoxigenic strains. We analyzed the effect of VacA on autophagy in peripheral blood monocytes obtained from subjects with different genotypes of ATG16L1, which regulates autophagy. We performed genotyping for ATG16L1 in two cohorts of infected and uninfected subjects. Results Prolonged exposure of AGS and mouse gastric cells to VacA disrupted induction of autophagy in response to the toxin, because the cells lacked cathepsin-D in autophagosomes. Loss of autophagy resulted in the accumulation of p62 and reactive oxygen species. Gastric biopsies samples from patients infected with VacA+, but not nontoxigenic strains of H pylori, had increased levels of p62. Peripheral blood monocytes isolated from individuals with polymorphisms in ATG16L1 that increase susceptibility to Crohn's disease had reduced induction of autophagy in response to VacA+ compared to cells from individuals that did not have these polymorphisms. The presence of the ATG16L1 Crohn’s disease risk variant increased susceptibility to H pylori infection in 2 separate cohorts. Conclusions Autophagy protects against infection with H pylori; the toxin VacA disrupts autophagy to promote infection, which could contribute to inflammation and eventual carcinogenesis.

Host cell responses to

Pseudomonas aeruginosa is an opportunistic, nosocomial bacterial pathogen that forms persistent infections due to the formation of protective communities, known as biofilms. Once the biofilm is formed, the bacteria embedded within it are recalcitrant to antimicrobial treatment and host immune defenses. Moreover, the presence of biofilms in wounds is correlated with chronic infection and delayed healing. The current standard of care for chronic wound infections typically involves physical disruption of the biofilm via debridement and subsequent antimicrobial treatment. The glycoside hydrolases PelAh and PslGh have been demonstrated in vitro to disrupt biofilm integrity through degradation of the key biofilm matrix exopolysaccharides Pel and Psl, respectively. Herein, we demonstrate that PslGh hydrolase therapy is a promising strategy for controlling P. aeruginosa wound infections. Hydrolase treatment of P. aeruginosa biofilms resulted in increased antibiotic efficacy and penetration into the biofilm. PslGh treatment of P. aeruginosa biofilms also improved innate immune activity leading to greater complement deposition, neutrophil phagocytosis, and neutrophil reactive oxygen species production. Furthermore, when P. aeruginosa-infected wounds were treated with a combination of PslGh and tobramycin, we observed an additive effect leading to greater bacterial clearance than treatments of tobramycin or PslGh alone. This study demonstrates that PelAh and PslGh have promising therapeutic potential and that PslGh may aid in the treatment of P. aeruginosa wound infections.

Previous work has suggested that a group of ␣/␤-type small, acid-soluble spore proteins (SASP) is involved in the resistance of Clostridium perfringens spores to moist heat. However, this suggestion is based on the analysis of C. perfringens spores lacking only one of the three genes encoding ␣/␤-type SASP in this organism. We have now used antisense RNA to decrease levels of ␣/␤-type SASP in C. perfringens spores by ϳ90%. These spores had significantly reduced resistance to both moist heat and UV radiation but not to dry heat. These results clearly demonstrate the important role of ␣/␤-type SASP in the resistance of C. perfringens spores.Clostridium perfringens is a gram-positive, spore-forming, anaerobic bacterium that causes both histotoxic and gastrointestinal (GI) diseases in humans and animals (8, 10). C. perfringens isolates are classified into one of five types, types A through E (10, 11), based upon their ability to produce the four major lethal toxins, the alpha-, beta-, epsilon-and iota-toxins. The major lethal toxins, however, are not the only medically important toxins; some C. perfringens isolates (mostly those belonging to type A) produce C. perfringens enterotoxin (CPE). The CPE-producing type A isolates are important human GI pathogens, causing C. perfringens type A food poisoning as well as non-food-borne GI diseases (10,19). In addition to producing CPE, C. perfringens food poisoning isolates have the ability to form spores that are extremely resistant to heat and other environmental stress factors (20), facilitating spore survival in primary food vehicles (e.g., meat and poultry products) for C. perfringens type A food poisoning (10).The molecular basis for the resistance of C. perfringens spores to heat and other environmental stress factors remains unknown. However, the ␣/␤-type small, acid-soluble spore proteins (SASP) play a major role in the resistance of Bacillus subtilis spores to heat, UV radiation, and peroxides (24). Previous studies (1, 2) have shown that the genomes of Clostridium species contain multiple genes (termed ssp) encoding ␣/␤-type SASP, with three genes (ssp1, ssp2, and ssp3) in C. perfringens (13,25). However, in contrast to Bacillus species, Clostridium species do not contain genes encoding the ␥-type SASP (1). Evidence obtained recently has suggested that ␣/␤-type SASP plays a role in the resistance of C. perfringens spores to moist heat, as spores of a strain with an ssp3 deletion had slightly lower moist heat resistance than wild-type spores, and this defect was eliminated by complementing the ssp3 mutant with wild-type ssp3 (18). However, the link between ␣/␤-type SASP levels and spore heat resistance was tentative because only one of the three ssp genes in C. perfringens was deleted.In order to fully explore the relationship between levels of ␣/␤-type SASP and C. perfringens spore resistance, it would be necessary to generate a C. perfringens strain lacking all three ssp genes. However, since no technique is currently available to introduce multiple knockout muta...

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