Hirofumi Danbara

Molecular analysis of spv virulence genes of the salmonella virulence plasmids

Genes on an 8 kb region common to the virulence plasmids of several serovars of Salmonella are sufficient to replace the entire plasmid in enabling systemic infection in animal models. This virulence region encompasses five genes which previously have been designated with different names from each investigating laboratory. A common nomenclature has been devised for the five genes, i.e. spv for salmonella plasmid virulence. The first gene, spvR, encodes a positive activator for the following four genes, spvABCD. DNA sequence analysis of the spv genes from Salmonella typhimurium. Salmonella dublin, and Salmonella choleraesuis demonstrated extremely high conservation of the DNA and amino acid sequences. The spv genes are induced at stationary phase and in carbon‐poor media, and optimal expression is dependent on the katF locus. The cirulence functions of the spv genes are not known, but these genes may increase the growth rate of salmonellae in host cells and affect the interaction of salmonellae with the host immune system.

Synergistic action of cholera toxin B subunit (and Escherichia coli heat-labile toxin B subunit) and a trace amount of cholera whole toxin as an adjuvant for nasal influenza vaccine

Cholera toxin B subunit (CTB) and Escherichia coli heat-labile toxin (LTB) (2 micrograms), each supplemented with a trace amount of cholera toxin (CT) (0.02-20 ng), were examined for the adjuvant effect on antibody (Ab) response against influenza inactivated HA (haemagglutinin) vaccine in Balb/c mice. Each mouse received a primary intranasal (i.n.) inoculation of the vaccine (1.5 micrograms) and the CT-containing CTB and in 4 weeks a second i.n. inoculation of the vaccine alone. The primary inoculation of the vaccine with CTB alone did not induce either anti-HA IgA or IgG Ab response, or haemagglutination-inhibition Ab responses in the serum. The vaccine with less than 2 ng of CT also failed to induce Ab response. On the other hand, the vaccine with CT-containing CTB induced a high Ab response, which increased depending on the CT dose. Moreover, the second vaccine induced a response more than ten times higher than the primary one and the response increased depending on the CT dose. Similar enhancement was found in the local anti-HA IgA Ab response in the nasal wash. Such synergistic effects were observed also between LTB and CT. The amount of Ab produced by the synergism was considered to be enough to protect against virus infection. These results suggest that CTB (or LTB) containing a trace amount of CT (about 0.1%) can be used practically as a potent adjuvant for nasal vaccination of humans against influenza.

Escherichia coli heat-labile enterotoxin B subunits supplemented with a trace amount of the holotoxin as an adjuvant for nasal influenza vaccine

Escherichia coli heat-labile enterotoxin B subunit (LTB) (2 micrograms), supplemented with a trace amount of the holotoxin (LT) (0.02-20 ng), was examined for the adjuvant effect on antibody (Ab) responses against influenza inactivated haemagglutinin (HA) vaccine in Balb/c mice. Each mouse received a primary intranasal (i.n.) inoculation with the vaccine (1.5 micrograms), prepared from PR8 (H1N1) virus, together with LT-containing LTB and in 4 weeks a second i.n. inoculation of the vaccine alone. The inoculation of the vaccine with the LT-containing LTB induced significantly high primary and secondary anti-HA IgA and IgG Ab responses in the nasal wash and the serum, while the vaccine with LTB or less than 2 ng of LT induced little response. The synergistic adjuvant effect was maximal in the concentration of LTB supplemented with 0.2-2 ng of LT. Under these conditions, the augmented IgA and IgG Ab responses, which are cross-protective to PR8 HA molecules, provided complete cross-protection against PR8 virus challenge in mice immunized with heterologous vaccine within the same subtype. These results suggest that LTB containing a trace amount of LT can be used as a potent adjuvant for nasal vaccination of humans against influenza.

Complete DNA Sequence and Comparative Analysis of the 50-Kilobase Virulence Plasmid of Salmonella enterica Serovar Choleraesuis

ABSTRACT The complete nucleotide sequence of pKDSC50, a large virulence plasmid from Salmonella enterica serovar Choleraesuis strain RF-1, has been determined. We identified 48 of the open reading frames (ORFs) encoded by the 49,503-bp molecule. pKDSC50 encodes a known virulence-associated operon, the spv operon, which is composed of genes essential for systemic infection by nontyphoidalSalmonella. Analysis of the genetic organization of pKDSC50 suggests that the plasmid is composed of several virulence-associated genes, which include the spvRABCD genes, plasmid replication and maintenance genes, and one insertion sequence element. A second virulence-associated region including the pef(plasmid-encoded fimbria) operon and rck (resistance to complement killing) gene, which has been identified on the virulence plasmid of S. enterica serovar Typhimurium, was absent. Two different replicon regions, similar to the RepFIIA and RepFIB replicons, were found. Both showed high similarity to those of the pO157 plasmid of enterohemorrhagic Escherichia coliO157:H7 and the enteropathogenic E. coli (EPEC) adherence factor plasmid harbored by EPEC strain B171 (O111:NM), as well as the virulence plasmids of Salmonella serovars Typhimurium and Enteritidis. Comparative analysis of the nucleotide sequences of the 50-kb virulence plasmid of serovar Choleraesuis and the 94-kb virulence plasmid of serovar Typhimurium revealed that 47 out of 48 ORFs of the virulence plasmid of serovar Choleraesuis are highly homologous to the corresponding ORFs of the virulence plasmid of serovar Typhimurium, suggesting a common ancestry.

Evidence of correlation between 50-kilobase plasmid of Salmonella choleraesuis and its virulence.

A plasmid of 50 kilobases (kb) was found in all six strains of Salmonella choleraesuis studied, originally isolated from septicemic swine or man. A derivative cured of the 50 kb plasmid was isolated after treatment with novobiocin, and its virulence compared to that of the parent by intraperitoneal injection into mice. The lethality of the parent strain was found higher than that of the cured strain. With a sublethal number of cells of the parent strain injected, the spleen, liver and lymph nodes in various sites were markedly enlarged, and the bacteria were isolated from the heart blood. In contrast to this, neither enlargement of the organs nor recovery of organisms was observed with the cured strain. The Tn1-tagged 50 kb plasmid was next introduced by transformation into the cured strain. The virulence of the plasmid-reintroduced strain was higher than that of the cured strain, and in fact identical to that of the parent. We conclude that the 50 kb plasmid of S. choleraesuis is closely related with the mouse virulence of the organism.

Salmonella type III effector SpvC, a phosphothreonine lyase, contributes to reduction in inflammatory response during intestinal phase of infection

Salmonella phosphothreonine lyase SpvC inactivates the dual‐phosphorylated host mitogen‐activated protein kinases (MAPK) through β‐elimination. While SpvC can be secreted in vitro by both Salmonella pathogenicity island (SPI)‐1 and SPI‐2 type III secretion systems (T3SSs), translocation of this protein into the host cell cytosol has only been demonstrated by SPI‐2 T3SS. In this study, we show that SpvC can be delivered into the host cell cytoplasm by both SPI‐1 and SPI‐2 T3SSs. Dephosphorylation of the extracellular signal‐regulated protein kinases (ERK) was detected in an SPI‐1 T3SS‐dependent manner 2 h post infection. Using a mouse model for Salmonella enterocolitis, which was treated with streptomycin prior to infection, we observed that mice infected with Salmonella enterica serovar Typhimurium strains lacking the spvC gene showed pronounced colitis when compared with mice infected with the wild‐type strain 1 day after infection. The effect of SpvC on the development of colitis was characterized by reduced mRNA levels of the pro‐inflammatory cytokines and chemokines, and reduced inflammation with less infiltration of neutrophils. Furthermore, the reduction in inflammation by SpvC resulted in increased bacterial dissemination in spleen of mice infected with Salmonella. Collectively, our findings suggest that SpvC exerts as an anti‐inflammatory effector and the attenuation of intestinal inflammatory response by SpvC is involved in systemic infection of Salmonella.

PROTECTION AGAINST SHIGA TOXIN 1 CHALLENGE BY IMMUNIZATION OF MICE WITH PURIFIED MUTANT SHIGA TOXIN 1

ABSTRACT Shiga toxin 1 (Stx1) of enterohemorrhagic Escherichia coli O157:H7 was cloned, and four mutant Stx1s were constructed by site-directed mutagenesis with PCR. The wild-type and mutant Stx1s with amino acid replacements at positions 167 and 170 of the A subunit were purified by one-step affinity chromatography with commercially available Globotriose Fractogel, and the mutant Stxs were used for the immunization of mice. The mutant toxins were nontoxic to Vero cells in vitro and to mice in vivo and induced the immunoglobulin G antibody against the wild-type Stx1, which neutralized the cytotoxicity of Stx1. The induced antibody titers depended on the mutation at position 170 of the A subunit. The mice immunized with the mutant Stx1s were protected against a challenge of approximately 100 times the 50% lethal dose of the wild-type Stx1, suggesting that the mutant toxins are good candidates for toxoid vaccines for infection by Stx1-producing E. coli.

Intracellular expression of the Salmonella plasmid virulence protein, SpvB, causes apoptotic cell death in eukaryotic cells

The spv genes carried on the Salmonella virulence plasmid are commonly associated with severe systemic infection in experimental animals. The SpvB virulence-associated protein has been shown to ADP-ribosylate actin, and this enzymatic activity is essential for virulence in mice. Here, we present evidence that intracellular expression of SpvB protein induces not only disruption of actin filaments but also apoptotic cell death in eukaryotic cells.

Chromosomal and Extrachromosomal Synthesis of Exfoliative Toxin from Staphylococcus hyicus

Evidence for the existence of two molecular species of exfoliative toxin (ET) synthesized by Staphylococcus hyicus (SHET) under chromosomal and plasmid control is presented. Serological evidence that these molecular species of toxins are distinct from each other is given. The molecular weights of SHET from plasmidless strain P-1 (SHETA) and from plasmid-carrying strains P-10 and P-23 (SHETB) were almost equal. Both of the serotypes of SHET exhibited exfoliation in 1-day-old chickens. The plasmid-cured (P(-)) substrains (P-23C1 and P-23C2) of S. hyicus P-23 did not cause exfoliation in 1-day-old chickens, whereas P(-) substrains (P-10C1 and P-10C2) of strain P-10 caused exfoliation, but they decreased their exfoliative activity. These findings suggest that SHETB was synthesized along with SHETA by strain P-10, whereas the P-23 strain synthesized SHETB alone. The plasmid-carrying strain (P-23) as well as the plasmidless strain (P-1) exhibited the typical clinical signs of exudative epidermitis in pigs. However, plasmid-cured (P(-)) substrains of P-23 (P23C1 and P23C2) did not exhibit the typical clinical signs of exudative epidermitis. These findings suggest that SHETA is synthesized under chromosomal control and SHETB is synthesized under plasmid control and that SHET-producing strains can be divided into three groups: SHETA-producing strains, SHETB-producing strains, and strains producing both toxins.

Phytohabitans suffuscus gen. nov., sp. nov., an actinomycete of the family Micromonosporaceae isolated from plant roots.

An actinomycete strain, K07-0523(T), was isolated from the roots of an orchid collected in Okinawa prefecture, Japan. 16S rRNA gene sequence analysis indicated that the new strain belonged to the family Micromonosporaceae and the similarity values between strain K07-0523(T) and the type species of 24 genera in the family Micromonosporaceae were 93.3-97.7 %. Strain K07-0523(T) contained D-glutamic acid, glycine, D-alanine, meso-diaminopimelic acid, hydroxydiaminopimelic acid and L-lysine in the cell wall. The major menaquinones were MK-9(H(6)), MK-10(H(4)) and MK-10(H(6)). Galactose, glucose, mannose, ribose and xylose were present in the whole-cell sugars. The acyl type of the peptidoglycan was glycolyl. Major fatty acids were anteiso-C(17 : 0), iso-C(17 : 0), iso-C(16 : 0) and iso-C(15 : 0). Phosphatidylethanolamine was detected as the major phospholipid and corresponded to phospholipid type II. The G+C content of the genomic DNA was 73 mol%. On the basis of phylogenetic and chemotaxonomic data, the new strain represents a member of a new genus and novel species, namely Phytohabitans suffuscus gen. nov., sp. nov., in the family Micromonosporaceae. The type strain of the type species is K07-0523(T) (=DSM 45306(T)=NBRC 105367(T)).