Our laboratory recently molecularly characterized the type II secretion system (T2SS)-associated cytotoxic enterotoxin (Act) and the T3SS-secreted AexU effector from a diarrheal isolate SSU of Aeromonas hydrophila. The role of these toxin proteins in the pathogenesis of A. hydrophila infections was subsequently delineated in in vitro and in vivo models. In this study, we characterized the new type VI secretion system (T6SS) from isolate SSU of A. hydrophila and demonstrated its role in bacterial virulence. Study of the role of T6SS in bacterial virulence is in its infancy, and there are, accordingly, only limited, recent reports directed toward a better understanding its role in bacterial pathogenesis. We have provided evidence that the virulence-associated secretion (vas) genes vasH (Sigma 54-dependent transcriptional regulator) and vasK (encoding protein of unknown function) are essential for expression of the genes encoding the T6SS and/or they constituted important components of the T6SS. Deletion of the vasH gene prevented expression of the potential translocon hemolysin coregulated protein (Hcp) encoding gene from bacteria, while the vasK gene deletion prevented secretion but not translocation of Hcp into host cells. The secretion of Hcp was independent of the T3SS and the flagellar system. We demonstrated that secreted Hcp could bind to the murine RAW 264.7 macrophages from outside, in addition to its ability to be translocated into host cells. Further, the vasH and vasK mutants were less toxic to murine macrophages and human epithelial HeLa cells, and these mutants were more efficiently phagocytosed by macrophages. We also provided evidence that the expression of the hcp gene in the HeLa cell resulted in apoptosis of the host cells. Finally, the vasH and vasK mutants of A. hydrophila were less virulent in a septicemic mouse model of infection, and animals immunized with recombinant Hcp were protected from subsequent challenge with the wild-type (WT) bacterium. In addition, mice infected with the WT A. hydrophila had circulating antibodies to Hcp, indicating an important role of T6SS in the pathogenesis of A. hydrophila infections. Taken together, we have characterized the T6SS from Aeromonas for the first time and provided new features of this secretion system not yet known for other pathogens.

This study describes early intracellular events occurring during the establishment phase of Bacillus anthracis infections. Anthrax infections are initiated by dormant endospores gaining access to the mammalian host and becoming engulfed by regional macrophages (Mφ). During systemic anthrax, late stage events include vegetative growth in the blood to very high titres and the synthesis of the anthrax exotoxin complex, which causes disease symptoms and death. Experiments focus on the early events occurring during the first few hours of the B. anthracis infectious cycle, from endospore germination up to and including release of the vegetative cell from phagocytes. We found that newly vegetative bacilli escape from the phagocytic vesicles of cultured Mφ and replicate within the cytoplasm of these cells. Release from the Mφ occurs 4–6 h after endospore phagocytosis, timing that correlates with anthrax infection of test animals. Genetic analysis from this study indicates that the toxin plasmid pXO1 is required for release from the Mφ, whereas the capsule plasmid pXO2 is not. The transactivator atxA, located on pXO1, is also found to be essential for release, but the toxin genes themselves are not required. This suggests that Mφ release of anthrax bacilli is atxA regulated. The putative ‘escape’ genes may be located on the chromosome and/or on pXO1.

Abnormal production of inflammatory cytokines and chemokines is a key feature of bacterial endotoxin, lipopolysaccharide (LPS)-induced inflammation, and cytotoxicity; however, the mechanisms regulating production of inflammatory markers remain unclear. Herein, we show that inhibition of the aldehyde-metabolizing enzyme aldose reductase (AR; AKR1B3) modulates NF-Bdependent activation of inflammatory cytokines and chemokines in mouse serum, liver, heart, and spleen. Pharmacological inhibition or small interfering RNA ablation of AR prevented the biosynthesis of tumor necrosis factor-␣, interleukin 1␤, interleukin-6,

A total of 227 isolates of Aeromonas obtained from different geographical locations in the United States and different parts of the world, including 28 reference strains, were analyzed to determine the presence of various virulence factors. These isolates were also fingerprinted using biochemical identification and pulse-field gel electrophoresis (PFGE). Of these 227 isolates, 199 that were collected from water and clinical samples belonged to three major groups or complexes, namely, the A. hydrophila group, the A. caviae-A. media group, and the A. veronii-A. sobria group, based on biochemical profiles, and they had various pulsotypes. When virulence factor activities were examined, Aeromonas isolates obtained from clinical sources had higher cytotoxic activities than isolates obtained from water sources for all three Aeromonas species groups. Likewise, the production of quorum-sensing signaling molecules, such as N-acyl homoserine lactone, was greater in clinical isolates than in isolates from water for the A. caviae-A. media and A. hydrophila groups. Based on colony blot DNA hybridization, the heat-labile cytotonic enterotoxin gene and the DNA adenosine methyltransferase gene were more prevalent in clinical isolates than in water isolates for all three Aeromonas groups. Using colony blot DNA hybridization and PFGE, we obtained three sets of water and clinical isolates that had the same virulence signature and had indistinguishable PFGE patterns. In addition, all of these isolates belonged to the A. caviae-A. media group. The findings of the present study provide the first suggestive evidence of successful colonization and infection by particular strains of certain Aeromonas species after transmission from water to humans.

Yersinia pestis evolved from Y. pseudotuberculosis to become the causative agent of bubonic and pneumonic plague. We identified a homolog of the Salmonella enterica serovar Typhimurium lipoprotein (lpp) gene in Yersinia species and prepared lpp gene deletion mutants of Y. pseudotuberculosis YPIII, Y. pestis KIM/D27 (pigmentation locus minus), and Y. pestis CO92 with reduced virulence. Mice injected via the intraperitoneal route with 5 ؋ 10 7 CFU of the ⌬lpp KIM/D27 mutant survived a month, even though this would have constituted a lethal dose for the parental KIM/D27 strain. Subsequently, these ⌬lpp KIM/D27-injected mice were solidly protected against an intranasally administered, highly virulent Y. pestis CO92 strain when it was given as five 50% lethal doses (LD 50 ). In a parallel study with the pneumonic plague mouse model, after 72 h postinfection, the lungs of animals infected with wild-type (WT) Y. pestis CO92 and given a subinhibitory dose of levofloxacin had acute inflammation, edema, and masses of bacteria, while the lung tissue appeared essentially normal in mice inoculated with the ⌬lpp mutant of CO92 and given the same dose of levofloxacin. Importantly, while WT Y. pestis CO92 could be detected in the bloodstreams and spleens of infected mice at 72 h postinfection, the ⌬lpp mutant of CO92 could not be detected in those organs. Furthermore, the levels of cytokines/chemokines detected in the sera were significantly lower in animals infected with the ⌬lpp mutant than in those infected with WT CO92. Additionally, the ⌬lpp mutant was more rapidly killed by macrophages than was the WT CO92 strain. These data provided evidence that the ⌬lpp mutants of yersiniae were significantly attenuated and could be useful tools in the development of new vaccines.