Nobuhiko Okada

A type III secretion system (T3SS) is utilized by a large number of gram-negative bacteria to deliver effectors directly into the cytosol of eukaryotic host cells. One essential component of a T3SS is an ATPase that catalyzes the unfolding of proteins, which is followed by the translocation of effectors through an injectisome. Here we demonstrate a functional role of the ATPase SsaN, a component of Salmonella pathogenicity island 2 T3SS (T3SS-2) in Salmonella enterica serovar Typhimurium. SsaN hydrolyzed ATP in vitro and was essential for T3SS function and Salmonella virulence in vivo. Protein-protein interaction analyses revealed that SsaN interacted with SsaK and SsaQ to form the C ring complex. SsaN and its complex co-localized to the membrane fraction under T3SS-2 inducing conditions. In addition, SsaN bound to Salmonella pathogenicity island 2 (SPI-2) specific chaperones, including SsaE, SseA, SscA, and SscB that facilitated translocator/effector secretion. Using an in vitro chaperone release assay, we demonstrated that SsaN dissociated a chaperone-effector complex, SsaE and SseB, in an ATP-dependent manner. Effector release was dependent on a conserved arginine residue at position 192 of SsaN, and this was essential for its enzymatic activity. These results strongly suggest that the T3SS-2-associated ATPase SsaN contributes to T3SS-2 effector translocation efficiency.

Lactobacillus casei ATCC 27139 enhances host innate immunity, and the J1 phage-resistant mutants of this strain lose the activity. A transposon insertion mutant library of L. casei ATCC 27139 was constructed, and nine J1 phage-resistant mutants out of them were obtained. Cloning and sequencing analyses identified three independent genes that were disrupted by insertion of the transposon element: asnH, encoding asparagine synthetase, and dnaJ and dnaK, encoding the molecular chaperones DnaJ and DnaK, respectively. Using an in vivo mouse model of Listeria infection, only asnH mutant showed deficiency in their ability to enhance host innate immunity, and complementation of the mutation by introduction of the wild-type asnH in the mutant strain recovered the immuno-augmenting activity. AsnH protein exhibited asparagine synthetase activity when the lysozyme-treated cell wall extracts of L. casei ATCC 27139 was added as substrate. The asnH mutants lost the thick and rigid peptidoglycan features that are characteristic to the wild-type cells, indicating that AsnH of L. casei is involved in peptidoglycan biosynthesis. These results indicate that asnH is required for the construction of the peptidoglycan composition involved in the immune-activating capacity of L. casei ATCC 27139.

Background: The global ppGpp-mediated stringent response in pathogenic bacteria plays an important role in the pathogenesis of bacterial infections. In Salmonella enterica serovar Typhimurium (S. Typhimurium), several genes, including virulence genes, are regulated by ppGpp when bacteria are under the stringent response. To understand the control of virulence genes by ppGpp in S. Typhimurium, agarose 2-dimensional electrophoresis (2-DE) combined with mass spectrometry was used and a comprehensive 2-DE reference map of amino acid-starved S. Typhimurium strain SH100, a derivative of ATCC 14028, was established. Results: Of the 366 examined spots, 269 proteins were successfully identified. The comparative analysis of the wild-type and ppGpp⁰ mutant strains revealed 55 proteins, the expression patterns of which were affected by ppGpp. Using a mouse infection model, we further identified a novel virulence-associated factor, STM3169, from the ppGpp-regulated and Salmonella-specific proteins. In addition, Salmonella strains carrying mutations in the gene encoding STM3169 showed growth defects and impaired growth within macrophage-like RAW264.7 cells. Furthermore, we found that expression of stm3169 was controlled by ppGpp and SsrB, a response regulator of the two-component system located on Salmonella pathogenicity island 2. Conclusions: A proteomic approach using a 2-DE reference map can prove a powerful tool for analyzing virulence factors and the regulatory network involved in Salmonella pathogenesis. Our results also provide evidence of a global response mediated by ppGpp in S. enterica.

Summary: Chromobacterium violaceum is a Gram‐negative bacterium that causes fatal septicaemia in humans and animals. C. violaceum ATCC 12472 possesses genes associated with two distinct type III secretion systems (T3SSs). One of these systems is encoded by Chromobacterium pathogenicity islands 1 and 1a (Cpi‐1/‐1a), another is encoded by Chromobacterium pathogenicity island 2 (Cpi‐2). Here we show that C. violaceum causes fulminant hepatitis in a mouse infection model, and Cpi‐1/‐1a‐encoded T3SS is required for its virulence. In addition, using C. violaceum strains with defined mutations in the genes that encode the Cpi‐1/‐1a or Cpi‐2 locus in combination with cultured mammalian cell lines, we found that C. violaceum is able to induce cytotoxicity in a Cpi‐1/‐1a‐dependent manner. Characterization of Chromobacterium‐induced cytotoxicity revealed that cell lysis by C. violaceum infection involves the formation of pore structures on the host cell membrane, as demonstrated by protection by cytotoxicity in the presence of osmoprotectants. Finally, we demonstrated that CipB, a Cpi‐1/‐1a effector, is implicated in translocator‐mediated pore formation and the ability of CipB to form a pore is essential for Chromobacterium‐induced cytotoxicity. These results strongly suggest that Cpi‐1/‐1a‐encoded T3SS is a virulence determinant that causes fatal infection by the induction of cell death in hepatocytes.

The type III secretion system (T3SS) encoded by Salmonella pathogenicity island 2 (SPI-2) is involved in systemic infection and intracellular replication of Salmonella enterica serovar Typhimurium. In this study, we investigated the function of SsaE, a small cytoplasmic protein encoded within the SPI-2 locus, which shows structural similarity to the T3SS class V chaperones. An S. enterica serovar Typhimurium ssaE mutant failed to secrete SPI-2 translocator SseB and SPI-2-dependent effector PipB proteins. Coimmunoprecipitation and mass spectrometry analyses using an SsaE-FLAG fusion protein indicated that SsaE interacts with SseB and a putative T3SS-associated ATPase, SsaN. A series of deleted and point-mutated SsaE-FLAG fusion proteins revealed that the C-terminal coiled-coil domain of SsaE is critical for protein-protein interactions. Although SseA was reported to be a chaperone for SseB and to be required for its secretion and stability in the bacterial cytoplasm, an sseA deletion mutant was able to secrete the SseB in vitro when plasmid-derived SseB was overexpressed. In contrast, ssaE mutant strains could not transport SseB extracellularly under the same assay conditions. In addition, an ssaE ( I55G ) point-mutated strain that expresses the SsaE derivative lacking the ability to form a C-terminal coiled-coil structure showed attenuated virulence comparable to that of an SPI-2 T3SS null mutant, suggesting that the coiled-coil interaction of SsaE is absolutely essential for the functional SPI-2 T3SS and for Salmonella virulence. Based on these findings, we propose that SsaE recognizes translocator SseB and controls its secretion via SPI-2 type III secretion machinery.

Salmonella pathogenicity islands are inserted into the genome by horizontal gene transfer and are required for expression of full virulence. Here, we performed tRNA scanning of the genome of Salmonella enterica serovar Typhimurium and compared it with that of nonpathogenic Escherichia coli in order to identify genomic islands that contribute to Salmonella virulence. Using deletion analysis, we identified four genomic islands that are required for virulence in the mouse infection model. One of the newly identified pathogenicity islands was the pheV-tRNA-located genomic island, which is comprised of 26 126 bp, and encodes 22 putative genes, including STM3117-STM3138. We also showed that the pheV tRNA-located genomic island is widely distributed among different nontyphoid Salmonella serovars. Furthermore, genes including STM3118-STM3121 were identified as novel virulence-associated genes within the pheV-tRNA-located genomic island. These results indicate that a Salmonella-specific pheV-tRNA genomic island is involved in Salmonella pathogenesis among the nontyphoid Salmonella serovars.

The type III secretion system encoded by Salmonella pathogenicity island 2 (SPI-2) is essential for the intracellular survival and replication of Salmonella enterica. The expression of SPI-2 genes is dependent on a two-component regulatory system, SsrA (SpiR)/SsrB, encoded in the SPI-2 region. This paper shows that SlyA regulates transcription of the sensor kinase SsrA by binding to the ssrA promoter, indicating that SlyA is directly involved in the regulation of SPI-2 gene expression. A structure model of the SlyA dimer in complex with DNA was constructed. The model of SlyA indicated that its structure is very similar to that of other MarR family proteins. Based on this model, site-directed mutagenesis of residues located in the winged-helix region required for DNA binding and in the α-helices of the N-terminal and C-terminal regions required for dimerization of the SlyA protein was performed to identify the residues that are critical for SlyA function. Nine mutants of SlyA with single substitutions were unable to activate ssrA transcription in vivo. These mutant SlyA proteins revealed that the residues Leu-63, Val-64, Arg-65, Leu-67, Leu-70, Arg-86 and Lys-88 within the winged-helix region are required for DNA binding, and residues Leu-12 and Leu-126 within the α-helices of the N-terminal and C-terminal regions are required for efficient dimer formation. A Salmonella slyA mutant strain carrying a plasmid expressing SlyA derivatives containing mutations at these amino acid positions did not exhibit restored SlyA function in infected HeLa cells, thereby confirming the structural and functional relationships of the SlyA protein.

Serum resistance is a crucial virulence factor for the development of systemic infections, including bacteraemia, by many pathogenic bacteria.Salmonella entericaserovar Choleraesuis is an important enteric pathogen that causes serious systemic infections in swine and humans. Here, it was found that, when introduced intoEscherichia coli, a recombinant plasmid carrying thepagCgene from a plasmid-based genomic library ofS. entericaserovar Choleraesuis conferred a high-level resistance to the bactericidal activity of pooled normal swine serum. The resistance was equal to the level conferred byrck, a gene encoding a 17 kDa outer-membrane protein which promotes the serum resistance phenotype inS. entericaserovar Typhimurium. Insertional mutagenesis of the clonedpagCgene generated a mutation that resulted in the loss of the serum resistance phenotype inE. coli. When this mutation was introduced into the chromosome ofS. entericaserovar Choleraesuis by homology recombination with the wild-type allele, the resulting strain could not produce PagC, and it showed a decreased level of resistance to complement-mediated killing. The mutation could be restored by introduction of the intactpagCgene on a plasmid, but not by introduction of the point-mutatedpagCgene. In addition, PagC was able to promote serum resistance in theS. entericaserovar Choleraesuis LPS mutant strain, which is highly sensitive to serum killing. Although PagC is not thought to confer serum resistance directly, these results strongly suggest that PagC is an important outer-membrane protein that plays an important role in the serum resistance ofS. entericaserovar Choleraesuis.

We studied the effect of fosfomycin (FOM) on the intracellular growth of two different facultative intracellular bacteria, Salmonella enterica serovar Typhimurium and Listeria monocytogenes, in an enterocyte-like cell line, Caco-2. These bacteria replicate in different compartments within the host cells; Salmonella serovar Typhimurium grow inside phagosomes, whereas L. monocytogenes escape the phagosomal environment and multiply in the cytosol of the host cells. At concentrations equal to 0.25 of the MIC and 4 times the MIC, respectively, FOM effectively decreased the number of intracellular Salmonella serovar Typhimurium. Although FOM was ineffective at inhibiting the extracellular growth of L. monocytogenes in vitro, this antibiotic induced a marked reduction in intracellular L. monocytogenes, indicating that, comparatively, FOM may be more effectively taken up by L. monocytogenes in the intracellular environment.