SummaryThe type III secretion system of Salmonella pathogenicity island 2 (SPI-2) is required for systemic infection of this pathogen in mice. Cloning and sequencing of a central region of SPI-2 revealed the presence of genes encoding putative chaperones and effector proteins of the secretion system. The predicted products of the sseB, sseC and sseD genes display weak but significant similarity to amino acid sequences of EspA, EspD and EspB, which are secreted by the type III secretion system encoded by the locus of enterocyte effacement of enteropathogenic Escherichia coli. The transcriptional activity of an sseA::luc fusion gene was shown to be dependent on ssrA, which is required for the expression of genes encoding components of the secretion system apparatus. Strains carrying nonpolar mutations in sseA, sseB or sseC were severely attenuated in virulence, strains carrying mutations in sseF or sseG were weakly attenuated, and a strain with a mutation in sseE had no detectable virulence defect. These phenotypes were reflected in the ability of mutant strains to grow within a variety of macrophage cell types: strains carrying mutations in sseA, sseB or sseC failed to accumulate, whereas the growth rates of strains carrying mutations in sseE, sseF or sseG were only modestly reduced. These data suggest that, in vivo, one of the functions of the SPI-2 secretion system is to enable intracellular bacterial proliferation.

SummaryThe enteric pathogen Salmonella typhimurium co-ordinates the expression of virulence determinants in response to environmental cues from the host organism. S. typhimurium possesses Salmonella pathogenicity island 2 (SPI2), a large virulence locus encoding a type III secretion system for virulence determinants required for systemic infections and accumulation inside host cells. We have generated transcriptional fusions to SPI2 genes to analyse expression and used antibodies against recombinant SPI2 proteins to monitor levels of SPI2 proteins under various conditions. Here, we demonstrate that SPI2 gene expression is induced by Mg 2þ deprivation and phosphate starvation. These conditions are likely to represent the environmental cues encountered by S. typhimurium inside the phagosome of infected host cells. The induction of SPI2 gene expression is modulated by the global regulatory system PhoPQ and is dependent on SsrAB, a two-component regulatory system encoded by SPI2.

SummaryA range of bacteria are able to use tetrathionate as a terminal respiratory electron acceptor. Here we report the identification and characterization of the ttrRSBCA locus required for tetrathionate respiration in Salmonella typhimurium LT2a. The ttr genes are located within Salmonella pathogenicity island 2 at centisome 30.5. ttrA, ttrB and ttrC are the tetrathionate reductase structural genes. Sequence analysis suggests that TtrA contains a molybdopterin guanine dinucleotide cofactor and a [4Fe-4S] cluster, that TtrB binds four [4Fe-4S] clusters, and that TtrC is an integral membrane protein containing a quinol oxidation site. TtrA and TtrB are predicted to be anchored by TtrC to the periplasmic face of the cytoplasmic membrane implying a periplasmic site for tetrathionate reduction. It is inferred that the tetrathionate reductase, together with thiosulphate and polysulphide reductases, make up a previously unrecognized class of molybdopterindependent enzymes that carry out the reductive cleavage of sulphur-sulphur bonds. Cys-256 in TtrA is proposed to be the amino acid ligand to the molybdopterin cofactor. TtrS and TtrR are the sensor and response regulator components of a two-component regulatory system that is absolutely required for transcription of the ttrBCA operon. Expression of an active tetrathionate reduction system also requires the anoxia-responsive global transcriptional regulator Fnr. The ttrRSBCA gene cluster confers on Escherichia coli the ability to respire with tetrathionate as electron acceptor.

SummaryAspergillus fumigatius is a ubiquitous saprophytic fungus that has become the most prevalent airborne fungal pathogen for immunocompromised patients during the last two decades. In this report we have analysed how macrophages recognize this microorganism. Using transfected human HEK 293 cells we demonstrate that NF-k k k k B-dependent promoter activation triggered by A. fumigatus is mediated by Toll-like receptors TLR2 and TLR4, whereas no activation was observed in cells overexpressing other distinct TLR proteins (TLR1, TLR3, TLR5-10). Using macrophages derived from mice lacking TLR2 expression, expressing defective TLR4 or both we found that A. fumigatus conidia and hyphae induce NF-k k k k B translocation, release of pro-inflammatory molecules, like TNF a a a a , and the chemoattractant MIP-2 in a TLR2-and TLR4-dependent manner. Recognition of A. niger and A. fumigatus , was similar in terms of the parameters analysed, suggesting that pathogenic and nonpathogenic aspergilli are sensed by macrophages in a similar fashion. Finally, we found that recruitment of neutrophils is severely impaired in mice lacking both functional TLR2 and TLR4, but is less impaired in single TLR2-or TLR4-deficient mice, providing evidence that both receptors are required for an optimal immune response to Aspergillus in vivo.

The type III secretion system encoded by Salmonella pathogenicity island 2 (SPI2) is required for systemic infections and intracellular accumulation of Salmonella enterica. This system is induced by intracellular Salmonella and subsequently transfers effector proteins into the host cell. Growth conditions either inducing expression of the type III secretion system or the secretion of substrate proteins were defined. Here we report the identification of a set of substrate proteins consisting of SseB, SseC, and SseD that are secreted by the SPI2 system in vitro. Secretion was observed if bacterial cells were exposed to acidic pH after growth in minimal medium with limitation of Mg 2؉ or phosphate. SseB, -C, and -D were isolated in a fraction detached from the bacterial cell surface by mechanical shearing, indicating that these proteins are predominantly assembled into complexes on the bacterial cell surface. The three proteins were required for the translocation of SPI2 effector proteins SspH1 and SspH2 into infected host cells. Thus, SseB, SseC, and SseD function as the translocon for effector proteins by intracellular Salmonella.

SummaryTwo large virulence loci encoding type III secretion systems are present on the chromosome of Salmonella typhimurium. Salmonella pathogenicity island 2 (SPI2) is important for the survival of S. typhimurium in host organs and forms an insertion of about 40 kb at the tRNA Val gene. However, several indications suggested that SPI2 was not the result of a single event of horizontal gene transfer. We characterized the portion of SPI2 towards the 30 cs boundary and performed mutational analysis to investigate the contribution of this region to S. enterica virulence. This analysis indicates that SPI2 may be composed of at least two different genetic elements. About 15 kb of the 40 kb of SPI2 contain genes without a significant contribution to systemic infections in the model of murine salmonellosis. Our study allowed us to define genes in SPI2 important for virulence further and indicated that this locus has a complex mosaic structure.

The Salmonella typhimurium genome contains two pathogenicity islands (SPI) with genes encoding type III secretion systems for virulence proteins. SPI1 is required for the penetration of the epithelial layer of the intestine. SPI2 is important for the subsequent proliferation of bacteria in the spleens of infected hosts. Although most mutations in SPI2 lead to a strong reduction of virulence, they have different effects in vitro, with some mutants having significantly increased sensitivity to gentamicin and the antibacterial peptide polymyxin B. Previously we showed that certain mutations in SPI2 affect the ability of S. typhimurium to secrete SPI1 effector proteins and to invade cultured eukaryotic cells. In this study, we show that these SPI2 mutations affect the expression of the SPI1 invasion genes. Analysis of reporter fusions to various SPI1 genes reveals highly reduced expression of sipC,prgK, and hilA, the transcriptional activator of SPI1 genes. These observations indicate that the expression of one type III secretion system can be influenced dramatically by mutations in genes encoding a second type III secretion system in the same cell.

The potential use as vaccine delivery system of Salmonella typhimurium strains harboring defined mutations in thesseC (HH104) and sseD (MvP101) genes, which encode putative effector proteins of the type III secretion system ofSalmonella pathogenicity island 2, was evaluated and compared with that of the well-characterized aroA mutant strain SL7207 by using β-galactosidase (β-Gal) as a model antigen. When orally administered to immune-competent or gamma interferon-deficient (IFN-γ−/−) BALB/c mice, both mutants were found to be highly attenuated (50% lethal dose, >109 bacteria). Both strains were also able to efficiently colonize and persist in Peyer’s patches. Immunization with HH104 and MvP101 triggered β-Gal-specific serum and mucosal antibody responses equivalent to or stronger than those observed in SL7207-immunized mice. Although immunoglobulin G2 (IgG2) serum antibodies were dominant in all groups, IgG1 was also significantly increased in mice vaccinated with MvP101 and SL7207. Comparable β-Gal-specific IgA and IgG antibodies were detected in intestinal lavages from mice immunized with the different strains. Antigen-specific CD4+ T-helper cells were generated after vaccination with all vaccine prototypes; however, responses were significantly more efficient when HH104 and MvP101 were used (P < 0.05). Significantly higher levels of IFN-γ were produced by restimulated spleen cells from mice immunized with HH104 than from those vaccinated with the MvP101 or SL7207 derivatives (P ≤ 0.05). Interestingly, the three strains induced major histocompatibility complex class I-restricted CD8+ cytotoxic T cells against β-Gal; however, cytotoxic T-lymphocyte responses were significantly stronger after immunization with HH104 (P < 0.05). These novel S. typhimurium attenuated strains constitute promising delivery systems for vaccine antigens. The qualitative differences observed in the obtained responses with different carriers may be useful for those applications in which a targeted immunomodulation is required.