Lipopolysaccharide (LPS) is a major virulence factor of Salmonella enterica serovar Typhimurium and is composed of lipid A, core oligosaccharide (C-OS), and O-antigen polysaccharide (O-PS). While the functions of the gene products involved in synthesis of core and O-antigen have been elucidated, the effect of removing O-antigen and core sugars on the virulence and immunogenicity of Salmonella enterica serovar Typhimurium has not been systematically studied. We introduced nonpolar, defined deletion mutations in waaG (rfaG), waaI (rfaI), rfaH, waaJ (rfaJ), wbaP (rfbP), waaL (rfaL), or wzy (rfc) into wild-type S. Typhimurium. The LPS structure was confirmed, and a number of in vitro and in vivo properties of each mutant were analyzed. All mutants were significantly attenuated compared to the wild-type parent when administered orally to BALB/c mice and were less invasive in host tissues. Strains with ⌬waaG and ⌬waaI mutations, in particular, were deficient in colonization of Peyer's patches and liver. This deficiency could be partially overcome in the ⌬waaI mutant when it was administered intranasally. In the context of an attenuated vaccine strain delivering the pneumococcal antigen PspA, all of the mutations tested resulted in reduced immune responses against PspA and Salmonella antigens. Our results indicate that nonreversible truncation of the outer core is not a viable option for developing a live oral Salmonella vaccine, while a wzy mutant that retains one O-antigen unit is adequate for stimulating the optimal protective immunity to homologous or heterologous antigens by oral, intranasal, or intraperitoneal routes of administration.

We have devised and constructed a biological containment system designed to cause programmed bacterial cell lysis with no survivors. We have validated this system, using Salmonella enterica serovar Typhimurium vaccines for antigen delivery after colonization of host lymphoid tissues. The system is composed of two parts. The first component is Salmonella typhimurium strain 8937, with deletions of asdA and arabinose-regulated expression of murA, two genes required for peptidoglycan synthesis and additional mutations to enhance complete lysis and antigen delivery. The second component is plasmid pYA3681, which encodes arabinoseregulated murA and asdA expression and C2-regulated synthesis of antisense asdA and murA mRNA transcribed from the P22 P R promoter. An arabinose-regulated c2 gene is present in the chromosome. 8937(pYA3681) exhibits arabinose-dependent growth. Upon invasion of host tissues, an arabinose-free environment, transcription of asdA, murA, and c2 ceases, and concentrations of their gene products decrease because of cell division. The drop in C2 concentration results in activation of P R, driving synthesis of antisense mRNA to block translation of any residual asdA and murA mRNA. A highly antigenic ␣-helical domain of Streptococcus pneumoniae Rx1 PspA was cloned into pYA3681, resulting in pYA3685 to test antigen delivery. Mice orally immunized with 8937(pYA3685) developed antibody responses to PspA and Salmonella outer membrane proteins. No viable vaccine strain cells were detected in host tissues after 21 days. This system has potential applications with other Gram-negative bacteria in which biological containment would be desirable.programmed cell lysis ͉ rPspA ͉ Rx1

The outermost protective structure found in endospores of Bacillus subtilis is a thick protein shell known as the coat, which makes a key contribution to the resistance properties of the mature spore and also plays a role in its interaction with compounds able to trigger germination. The coat is organized as a lamellar inner layer and an electron-dense outer layer and has a complex polypeptide composition. Here we report the cloning and characterization of an operon, cotJ, located at about 62؇ on the B. subtilis genetic map, whose inactivation results in the production of spores with an altered pattern of coat polypeptides. The cotJ operon was identified by screening a random library of lacZ transcriptional fusions for a conditional (inducer-dependent) Lac . Studies with double-reporter strains bearing a cotJ-gusA fusion and lacZ fusions to other cot genes confirmed that expression of cotJ is initiated during sporulation prior to activation of genes known to encode coat structural proteins (with the sole exception of cotE). An in vitro-constructed insertion-deletion mutation in cotJ resulted in the formation of spores with no detectable morphological or resistance deficiency. However, examination of the profile of electrophoretically separated spore coat proteins from the null mutant revealed a pattern that was essentially identical to that of a wild-type strain in the range of 12 to 65 kDa, except for polypeptides of 17 and 24 kDa, the putative products of the second (cotJB) and third (cotJC) cistrons of the operon, that were missing or reduced in amount in the coat of the mutant. Polypeptides of the same apparent sizes are detected in spores of a cotE null mutant, on which basis we infer that the products of the cotJ operon are required for the normal formation of the inner layers of the coat or are themselves structural components of the coat. Because the onset of cotJ transcription is temporally coincident with the appearance of active E

We isolated spontaneous mutations (pmrA)

Vesicle shedding from bacteria is a universal process in most Gram-negative bacteria and a few Gram-positive bacteria. In this report, we isolate extracellular membrane vesicles (MVs) from the supernatants of Gram-positive pathogen Clostridium perfringens (C. perfringens). We demonstrated vesicle production in a variety of virulent and nonvirulent type A strains. MVs did not contain alpha-toxin and NetB toxin demonstrated by negative reaction to specific antibody and absence of specific proteins identified by LC-MS/MS. C. perfringens MVs contained DNA components such as 16S ribosomal RNA gene (16S rRNA), alpha-toxin gene (plc) and the perfringolysin O gene (pfoA) demonstrated by PCR. We also identified a total of 431 proteins in vesicles by 1-D gel separation and LC-MS/MS analysis. In vitro studies demonstrated that vesicles could be internalized into murine macrophage RAW264.7 cells without direct cytotoxicity effects, causing release of inflammation cytokines including granulocyte colony stimulating factor (G-CSF), tumor necrosis factor-alpha (TNF-α) and interleukin-1 (IL-1), which could also be detected in mice injected with MVs through intraperitoneal (i.p.) route. Mice immunized with C. perfringens MVs produced high titer IgG, especially IgG1, antibodies against C. perfringens membrane proteins. However, this kind of antibody could not provide protection in mice following challenge, though it could slightly postpone the time of death. Our results indicate that release of MVs from C. perfringens could provide a previously unknown mechanism to induce release of inflammatory cytokines, especially TNF-α, these findings may contribute to a better understanding of the pathogenesis of C. perfringens infection.