Sharon Okamoto

Identification of Salmonella SPI-2 secretion system components required for SpvB-mediated cytotoxicity in macrophages and virulence in mice

The Salmonella SpvB protein possesses ADP-ribosyl transferase activity. SpvB, acting as an intracellular toxin, covalently modifies monomeric actin, leading to loss of F-actin filaments in Salmonella-infected human macrophages. Using defined Salmonella mutants, different functional components of the SPI-2 type three secretion system (TTSS), ssaV, spiC, sseB, sseC, and sseD, were found to be required for SpvB-mediated actin depolymerization in human macrophages. Expression of SpvB protein in Salmonella was not affected by any of the SPI-2 mutants and the effects of these loci were not due to reduced numbers of intracellular bacteria. Interestingly, the major SPI-2 virulence effector, SifA, is not required for SpvB action. Further, caspase-3 activation is an additional marker of cytotoxicity in Salmonella-infected human macrophages. Caspase-3 activity depended on SpvB and SPI-2 TTSS function, but not on SifA. These human macrophage cell culture results were corroborated by virulence studies in mice. Using competitive infection of mice with mixed inocula of single and double mutants, spvBmut1 mutation did not have an effect independent of ssaJ mutation, essential for SPI-2 TTSS function. In contrast, competitive infection studies in mice confirmed that SpvB and SifA have independent virulence effects, as predicted by the macrophage studies.

A Model of Salmonella Colitis with Features of Diarrhea in SLC11A1 Wild-Type Mice

Background Mice do not get diarrhea when orally infected with S. enterica, but pre-treatment with oral aminoglycosides makes them susceptible to Salmonella colitis. However, genetically susceptible ItyS mice (Nramp1G169D allele) die from systemic infection before they develop diarrhea, so a new model is needed to study the pathogenesis of diarrhea. We pretreated ItyR mice (Nramp1G169) with oral kanamycin prior to infecting them with virulent S. Typhimurium strain 14028s in order to study Salmonella-induced diarrhea. We used both a visual scoring system and the measurement of fecal water content to measure diarrhea. BALB/c.D2Nramp1 congenic started losing weight 5 days post-infection and they began to die from colitis 10–14 days after infection. A SPI-1 (invA) mutant caused cecal, but not colonic inflammation and did not cause diarrhea. A phoP- mutant did not cause manifestations of diarrhea in either normal or NADPH-deficient (gp91phox) mice. However, strain 14028s caused severe colitis and diarrhea in gp91phox-deficient mice on an ItyR background. pmr A and F mutants, which are less virulent in orally infected BALB/c mice, were fully virulent in this model of colitis. Conclusions S. enterica must be able to invade the colonic epithelium and to persist in the colon in order to cause colitis with manifestations of diarrhea. The NADPH oxidase is not required for diarrhea in Salmonella colitis. Furthermore, a Salmonella phoP mutant can be cleared from the colon by non-oxidative host defenses.

Diarrhea and colitis in mice require the Salmonella pathogenicity island 2-encoded secretion function but not SifA or Spv effectors.

ABSTRACT We investigated the roles of Salmonella pathogenicity island 2 (SPI-2) and two SPI-2 effectors in Salmonella colitis and diarrhea in genetically resistant BALB/c.D2 Slc11a1 congenic mice with the wild-type Nramp1 locus. Wild-type Salmonella enterica serovar Typhimurium 14028s caused a pan-colitis, and the infected mice developed frank diarrhea with a doubling of the fecal water content. An ssaV mutant caused only a 26% increase in fecal water content, without producing the pathological changes of colitis, and it did not cause weight loss over a 1-week period of observation. However, two SPI-2 effector mutants, the spvB and sifA mutants, and a double spvB sifA mutant caused diarrhea and colitis, even though the sifA mutant was sensitive to killing by bone marrow-derived macrophages from BALB/c.D2 mice and was severely impaired in extraintestinal growth but not in growth in the cecum. These results demonstrate that systemic S. enterica infection and diarrhea/colitis are distinct pathogenic processes and that only the former requires spvB and sifA.

Salmonella-induced diarrhea occurs in the absence of IL-8 receptor (CXCR2)-dependent neutrophilic inflammation

BACKGROUND Gastroenteritis is the most common manifestation of nontyphoidal Salmonella enterica infections, but little is known about the pathogenesis of diarrhea in this infection METHODS To determine whether polymorphonuclear neutrophils (PMNs) are required for diarrhea for Salmonella colitis, we infected kanamycin-pretreated interleukin 8R (IL-8R) mutant mice and controls, both with nonmutant Slc11a1 (Nramp1, ItyR). We compared the 2 mouse strains for increases in fecal water content (diarrhea) 3 days after infection, changes in expression of ion transporters in colonic epithelial cells, proliferation of epithelial cells, and severity of infection as measured by colony-forming units (CFUs). RESULTS The IL-8R knockout mice had fewer PMNs in the colon but the other variables we measured were unaffected except for an increase in CFUs in the colon. The pathologic changes in the cecum were similar in both groups except for the lack of PMNs in the IL-8R knockout mice. There was minimal damage to the colon more distally. CONCLUSIONS In the early stage of Salmonella colitis, PMNs are not required for diarrhea or for the decrease in expression of colonic epithelial cell apical ion transporters. They contribute to defense against infection in the cecum but not extracolonically at this stage of Salmonella colitis.

Myeloid Differentiation Factor 88 and Interleukin-1R1 Signaling Contribute to Resistance to Coccidioides immitis

ABSTRACT Rodents are a natural host for the dimorphic pathogenic fungi Coccidioides immitis and Coccidioides posadasii, and mice are a good model for human infection. Humans and rodents both express Dectin-1 and Toll-like receptor 2 (TLR2) on myeloid cells, and those receptors collaborate to maximize the cytokine/chemokine responses to spherules (the tissue form of the fungi) and to formalin-killed spherules (FKS). We showed that Dectin-1 is necessary for resistance to pulmonary coccidioidomycosis, but the importance of TLR2 in vivo is uncertain. Myeloid differentiation factor 88 (MyD88) is the adapter protein for TLR2 and -4, interleukin-1R1 (IL-1R1), and IL-18R1. MyD88/TRIF−/− and MyD88−/− mice were equally susceptible to C. immitis infection, in contrast to C57BL/6 (B6) controls. Of the four surface receptors, only IL-1R1 was required for resistance to C. immitis, partially explaining the susceptibility of MyD88−/− mice. We also found that FKS stimulated production of IL-1Ra by bone marrow-derived dendritic cells (BMDCs), independent of MyD88 and Dectin-1. There also was a very high concentration of IL-1Ra in the lungs of infected B6 mice, supporting the potential importance of this regulatory IL-1 family protein in the largely ineffective response of B6 mice to coccidioidomycosis. These results suggest that IL-1R1 signaling is important for defense against C. immitis infection.