Bacterial phenotypes mediated bymviAand their relationship to the mouse virulence ofSalmonella typhimurium

The focus of this study was the phenotypic characterization ofSalmonella typhimuriummutants lacking the function of the response regulatormviA. The inactivation ofmviA⁺(mviA: :kan) is shown to induce a significant change in the growth of most virulent strains, as reflected in the size of the colonies formed on agar plates. The colony phenotype observed in these strains has been designated as the small colony morphology (Scm⁺) phenotype. Mutants exhibiting the Scm⁺phenotype are shown to be significantly attenuated for virulence in susceptible (Ity^s) mice. The Scm⁺phenotype therefore provides anin vitrophenotypic marker formviA⁺activity. Further examination of Scm⁺mutants has revealed that they lack expression of a 55 kDa periplasmic protein which is detected in isogenicmviA⁺strains. This protein has been designatedmviA⁺relatedprotein A (MrpA) and was expressed in direct correlation with virulence in allS. typhimuriumstrains examined.     

Identification of major antigenic proteins ofPasteurella piscicida

Two different antigenic protein-coding clones (PPA1 and PPA2) were isolated using anti-Pasteurella piscicidarabbit serum from a genomic DNA library ofP. piscicidastrain KP9038. The PPA1 and PPA2 expressed 7 kDa and 45 kDa proteins inEscherichia coli, respectively, and the molecular sizes of these expressed proteins are the same as these of the major antigenic proteins ofP. piscicida. PPA1 encodes a protein of 83 amino acids residues, which is similar to the bacterial lipoprotein. Comparison of the predicted amino acid sequence of the PPA1-encoded 7 kDa protein ofP. piscicidawith previously reported bacterial lipoprotein sequence data revealed that it shares about 40% amino acid sequence identity. PPA2 has two large open reading frame (ORFs). The larger ORF (encoding 452 amino acid residues) encodes a homolog of DegQ protease, and the smaller ORF (371 amino acid residues) encodes a homolog of DegS protease. The antibodies reacted with the larger ORF-encoded 45 kDa DegQ homolog protein. The DegQ and DegS homolog proteins contain an export signal and a serine protease active site. The structural features of the PPA2-coding locus are similar to those of the loci inE. colifor thedegQanddegSserine protease genes. A sequence in the 3′ non-coding region ofVibrio hollisaethermostable hemolysin gene that is highly homologous with a similar located sequence in thePseudomonas putidap-cresol methylhydroxylase gene is also found in the 3′ non-coding region of thedegShomolog gene of the PPA2.     

The cell surface ofAeromonas salmonicidadeterminesin vitrosurvival in cultured brook trout (Salvelinus fontinalis) peritoneal macrophages

Aeromonas salmonicidastrains phenotypically differing in their A-layer, lipopolysaccharide, and macrophage cytotoxicity were testedin vitrofor survivability in brook trout (Salvelinus fontinalis) serum with or without antibodies, andin vivofollowing intraperitoneal injection. The ability of brook trout peritoneal macrophages to phagocytize and kill the different phenotypes was investigated in anin vitroassay. The virulent strain,A. salmonicida80204, which has the full complement of known virulence factors, as well as the recently described macrophage cytotoxin, was resistantin vitroto both the bactericidal activity of normal and immune serum, and to brook trout peritoneal macrophages.A. salmonicidaSS-70.1, which possesses the A-layer but lacks the cytotoxin, was resistant to the bactericidal activity of normal and immune serum but was avirulent and killed by macrophages. Phenotypes lacking the A-layer, regardless of whether or not they possessed the macrophage cytotoxin were avirulent, susceptible to normal and immune serum and the bactericidal activity of peritoneal macrophages.A. salmonicidavirulence expression requires both the A-layer and the macrophage cytotoxin.     

Characterization of HEp-2 cell projection formation induced by diffusely adherentEscherichia coli

Diffusely adherentEscherichia coli(DAEC) are diarrheagenicE. coliwhose pathogenetic mechanisms are largely unknown. DAEC have been shown to induce an unusual phenotype upon adherence to HEp-2 cells in culture characterized by the induction of long thin membrane processes extending from the cell surface. In addition, DAEC have been shown to be protected from the bactericidal effects of gentamicin when incubated with HEp-2 cells. In our studies, we found that three DAEC strains induced formation of eukaryotic cell processes and were protected from gentamicin killing after a 3 h incubation. Pre-incubation of HEp-2 cells with colchicine or cytochalasin D prior to infection with DAEC strain C1845 resulted in decreased projection formation, suggesting that the effect was dependent upon microfilament and microtubule rearrangement. When the standard gentamicin protection assay was extended for an additional 3 h incubation in the presence of gentamicin, a greater number of DAEC survived gentamicin treatment, more eukaryotic projections were seen in association with the bacteria and the bacteria were actually observed to be «embedded» within these projections. Projection formation was not observed when the bacteria were separated from the cells by a permeable membrane or when the inoculum was inactivated by ultraviolet irradiation. Transposon TnphoAmutants of C1845 were screened for decreased gentamicin protection. All three mutants which were deficient in gentamicin protection demonstrated less projection formation. Insertion mutations affecting gentamicin protection were localized to both the chromosome (two) and a plasmid (one). Eukaryotic projections are a novel interaction of DAEC with epithelial cells, may play a role of the survival of the bacteria against host defenses and may contribute to DAEC pathogenesis. The effect is dependent upon epithelial cell contact and requires multiple bacterial genes.     

Contribution of plasminogen activator urokinase to in vitro cytotoxicity of diphtheria toxin

Nicking of diphtheria toxin (DT), i.e. proteolytic cleavage at an arginine-rich region within the first disulphide loop, is a prerequisite to the intoxication process. We show that protease(s) required in this process was synthesized and secreted by the sensitive cells and that antibodies against plasminogen activator urokinase (uPA) decreased the in vitro cytotoxicity of DT on Vero cells. Our results demonstrate that uPA secreted by Vero cells cultured in vitro is one of the cellular proteases involved in the cleavage and activation of diphtheria toxin.     

Characterization of surface layer proteins from different Clostridium difficile clinical isolates

In a previous study we suggested that two surface proteins of a Clostridium difficile strain were involved in the formation of a regularly assembled surface layer (S-layer) external to the cell wall. In the present paper sixC. difficile strains isolated from cases and healthy carriers were studied. By using freeze-etching and negative staining techniques two superimposed structurally different lattices were detected on the cell surface of the different C. difficile strains. In each strain, the outer S-layer lattice was arranged in a square symmetry and the inner S-layer lattice in hexagonal symmetry. The S-layer proteins from the different strains were isolated and characterized. Each strain showed two distinct S-layer glycoproteins ranging in molecular mass 36–56 kDa. Antigenic cross-reactivity among the S-layer proteins of higher molecular masses extracted from each strain was demonstrated whereas no antigenic relationship was observed among the different S-layer proteins of lower molecular masses. N-terminal sequence analysis showed the presence of common structural motifs conserved among the high S-layer proteins as well as among the low S-layer proteins. These data indicate that the presence of S-layer on C. difficile strains is common and that its glycoprotein subunits show a certain degree of heterogeneity.     

Localization of thein vivoexpression of P and F1 fimbriae in chickens experimentally inoculated with pathogenicEscherichia coli

Escherichia colicausing septicemia in poultry often possess F1 (type 1) and/or P fimbriae which may be involved in bacterial colonization and infection. To investigate the expression of these fimbriaein vivo, two pathogenicE. colistrains with different fimbrial profiles, TK3 (fim⁺/pap⁺) and MT78 (fim⁺/pap⁻), were administered to 2-week-old chickens by either the intratracheal or caudal thoracic air sac inoculation route. Antibodies specific for native F1 fimbriae were detected by ELISA and immunodot in the serum of chickens inoculated with either strain MT78 or strain TK3, irrespective of the route of inoculation. Antibodies specific for P fimbriae of serotype F11 were detected by ELISA and immunoblotting in the serum of chickens inoculated by either route with strain TK3. F1, but not P fimbriae, were expressed by bacteria colonizing the trachea of chickens inoculated by the air sac route with strain MT78 or TK3, as demonstrated by examination of frozen tissue sections using immunofluorescence. F1 fimbriae were also expressed by bacteria colonizing the air sacs and lungs, but not by bacteria in the blood or other internal organs, of chickens inoculated with either strain. P fimbriae were expressed by bacteria colonizing the air sacs, lungs, kidney, blood, and pericardial fluid, but not by bacteria colonizing the trachea, of chickens inoculated with strain TK3. Fimbriae-like structures were observed by electron microscopy on bacteria adhering to the epithelial cells of the air sacs of chickens inoculated with strain TK3. These results demonstrate that both strains MT78 and TK3 undergoin vivophase variation with respect to their fimbrial profiles and site of bacterial colonization in different organs of infected chickens and suggest that F1 fimbriae are important for initial bacterial colonization of the upper respiratory tract whereas P fimbriae are important for later stages of the infection.     

Isolation and characterization of a 70 kDa protein fromMycobacterium avium

Mycobacterium aviumcomplex (MAC) is an intracellular pathogen which causes disseminated bacterial infection in immunocompromised individuals. This organism predominantly infects macrophages. Attachment of MAC to macrophages is the first step prior to invasion. We have previously shown that a 70 kDa protein ofM. avium(Ma) is one of nine monocyte-binding proteins. In the present study, we have purified this protein from sonic extracts of Ma and studied some of its properties. The N-terminal sequence of this protein was identified and found to exhibit a strong homology to the 70 kDa heat shock protein (hsp) ofM. leprae(Ml) andM. tuberculosis(Mtb). This protein was found to be present on the surface of the organism and was able to inhibit the attachment of intact Ma to human monocyte derived macrophages (MDM) up to 49% in anin vitroattachment assay using intact fluorescein isothiocyanate (FITC)-labelled Ma. Bovine serum albumin (BSA) and recombinant 70 kDa hsp from Mtb, which were used as controls, inhibited this attachment by 9.8 and 18%, respectively. These results suggest that the 70 kDa protein may have a role in the attachment of intact Ma to MDM. When tested in lymphocyte activation assays, this protein did not appear to significantly stimulate proliferation. However, it was found to stimulate the production of tumor necrosis factor (TNF)-αby MDM. This protein may be one of several Ma antigens that trigger host immune response by binding to MDM and stimulating the production of inflammatory cytokines such as TNF-αby these cells.     

Detection of the phosphorylcholine epitope in streptococci,Haemophilusand pathogenicNeisseriaeby immunoblotting

The phosphorylcholine (PC) determinant inStreptococcus pneumoniaeis known to be linked to the cell wall polysaccharides (C-Ps) and to the lipoteichoic acid (LTA) (Forssman antigen) of the plasma membrane. Western blotting with two PC specific murine monoclonal antibodies (MAbs) designated 145,F-2 (IgM) and 147,A-1 (IgA) showed a similar ladder-like pattern for all examined strains ofS. pneumoniaeandStreptococcus mitis. Purified antigens run in parallel indicated that this ladder pattern is due to the PC of LTA. Unlike other techniques, Western blotting thus enables the identification of only one of the streptococcal structures carrying the PC epitope. Gram-negative organisms were also examined, and six of 11Haemophilus influenzaestrains reacted with the MAbs. For this species, unlike the streptococci, only one fast moving band was detected. Analyses by thin-layer chromatography (TLC) detected the PC epitope in lipopolysaccharide (LPS) fraction fromH. influenzae. Some strains of theNeisseriaceaefamily were also positive by Western blotting, but TLC and immunostaining did not detect the PC determinant in LPS.     

Canary finches (Serinus canaria) as an avian infection model for Lyme borreliosis

To investigate if passerine birds can be used as an avian model for Lyme borreliosis, theBorrelia burgdorferi sensu strictostrain Sh2-82 was inoculated subcutaneously on Canary finches (Serinus canaria). Spirochaetes could be detected in the blood by direct microscopy and an immunofluorescence assay (IFA) up to 2 weeks after the inoculation. DNA amplification (PCR) revealedB. burgdorferiDNA in the serum for up to 3 weeks, in the faeces up to 4 weeks, and from several internal organs at autopsy 3 months after the inoculation. Of particular interest was the finding ofB. burgdorferiDNA in the liver of all birds at the end of the experiment, indicating a persistent infection. Four weeks after the primary infection, all infected birds showed an increase in antiBorreliaIgG antibodies and after 6 weeks all birds had seroconverted. Except for a brief episode of diarrhoea, none of the birds showed any signs of disease. This study shows that passerine birds experience short-term spirochaetemia after infection withB. burgdorferi, but show few clinical symptoms. This suggests that, although these birds can act as reservoirs ofB. burgdorferi s.s., they may be of less importance as long lasting amplifiers for this pathogen.     

Cloning and molecular analysis of genes encoding two immunodominant antigensofEhrlichia risticii

Ehrlichia risticii, the causative agent of Potomac horse fever, is an obligate intracellular rickettsial organism. To understand the role of 55 and 51 kilodalton immunodominant antigens ofE. risticiiin strain variation, their genes from the 25-D and 90-12 strains were cloned, sequenced, and expressed inE. coli.Sequence analysis revealed that the gene for the 55 kDa antigen was present in a heat shock operon along with the gene for a 10 kDa protein. Homology searches indicated that the 55 kDa antigen and the 10 kDa protein were homologues ofE. coliGroEL and GroES proteins, respectively. There was no nucleotide sequence difference between the genes of the 55 kDa antigen, nor between the entire operons, from both strains ofE. risticii.The sequence-based estimation of the sizes of the putative mature 51 kDa antigens of the 90-12 and 25-D strains were 52.7 kDa and 52.9 kDa, respectively. The 51 kDa antigens from the 90-12 and 25-D strains shared a 98% identity in their deduced amino acid sequences. The difference in some of the amino acids may be responsible for variation in their mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, where the 51 kDa antigen of the 25-D strain migrates towards a 2 kDa lower molecular weight region. In Western blots, a 155 kDa protein that appeared to be a trimer product of the 51 kDa antigen was identified. The 55 and 51 kDa antigens were overexpressed inE. coliusing a commercial expression system, pRSET A,B,C (Invitrogen Inc., San Diego, CA, U.S.A.). The purified recombinant proteins cross-reacted with antisera toE. canisandE. sennetsu.     

Characterization of Borrelia burgdorferi sensu lato isolates by pulsed-field gel electrophoresis after MluI restriction of genomic DNA

At least three Borrelia species (Borrelia afzelii, Borrelia garinii and Borrelia burgdorferi sensu stricto) cause disease in humans, but Borrelia spielmanii, Borrelia valaisiana, Borrelia lusitaniae and Borrelia bissettii have also been reported to be rare or potential causes of human disease in Europe. Pulsed-field gel electrophoresis after MluI restriction of the genomic DNA (MluI large restriction fragment patterns, LRFPs) represents one of several approaches that have been used to assess Borrelia genotypic characteristics. The aim of the present report was to analyze the value of MluI-LRFP for identification of B. burgdorferi sensu lato at a species level and for further species subtype delineation. Results of the present study are based on 1487 B. afzelii strains, 285 B. garinii strains, 29 B. burgdorferi sensu stricto strains, 23 B. valaisiana strains, 8 B. spielmanii strains and 3 B. lusitaniae strains. Using MluI-LRFP, we were able to delineate all Borrelia species included in the study. Each of the six examined Borrelia species displayed unique MluI-LRFPs that enabled straightforward separation of strains into particular species, and also of strains within species. The subtypes of B. afzelii (Mla2 and Mla3), B. spielmanii (Mls1 and Mls2) and B. lusitaniae (Mll1 and Mll2) uncovered in the present analysis have not been reported previously. MluI-LRFP represents a highly specific and reproducible method for Borrelia identification.     

Molecular characterization of Korean Pepper mottle virus isolates and its relationship to symptom variations

The symptom variations among Korean Pepper mottle virus (PepMoV) isolates infecting pepper, tomato and potato were described and the cause of variations in relation to molecular variability were investigated. In addition, the entire genome of the 13 PepMoV isolates, collected from five provinces (Kyonggi, Chungnam, Gyeongnam, Jeonbuk and Jeonnam) in Korea, were determined and compared including the previously reported Korean-Vb isolate and 2 other PepMoV isolates isolated from America (CA and FL). Our results showed that the nucleotide sequence of all Korean isolates tested were nearly identical (98–99%) and only 94% similar to American isolates. In general, the complete nucleotide sequences and deduced polyprotein sequences indicated low genetic variation among isolates showing 0.1–3% nucleotide changes per site. However, based on ratio between nucleotide diversity values in nonsynonymous and synonymous position (dN/dS ratio) surprisingly, P1 and 6K2 genes showed relatively high nucleotide substitution ratio (0.8 and 1.0 nucleotide, respectively). When the 6K2 amino acid were aligned, there were 15 amino acid substitutions found in PepMoV-infected potato and only 1 amino acid change from two isolates of PepMoV-infected bell pepper. Interestingly, three isolates including isolate numbers 731, 205135 and 205136 that possessed different aa changes at 6K2 region also showed distinct symptom differentiation in indicator hosts and cosegregated in the phylogenetic analysis. These results further proved previous studies that P1 and 6K2 genes with other proteins might have some involvement on host specificity and pathogenicity.     

Survival rate of mice after transient colonization withEscherichia coliclones carrying variant Shiga-like toxin type II operons

We have previously employed a streptomycin-treated mouse model to demonstrate thatEscherichia coliclones expressing particular variant Shiga-like toxin type II operons differ in oral virulence, as judged by median survival time. Differences in virulence were not seen between all toxin variants, including two which differed significantly in cytotoxicity for Vero cells. In the present study, we have modified the animal model by withdrawing antibiotic selection and reintroducing normal mouse intestinal flora at various times after oral challenge with three different variant SLT-II-producing clones. This resulted in a transient colonization more akin to that seen in natural human infections. This has enabled detection of significant differences in survival rate between mice challenged withE. coliclones producing different SLT-II variants, which were not observed when colonization was maintained at high levels.     

A possible mechanism for host-specific pathogenesis ofSalmonellaserovars

We have identified the complement receptors on human and murine macrophages involved in the recognition ofSalmonellaserovars, and investigated their relevance to the intracellular survival.S. typhiwas capable of surviving within human monocyte-derived macrophages, whereasS. typhimuriumwas not. Conversely,S. typhimurium, but notS. typhi, resisted intracellular killing by murine macrophages, demonstrating that the intracellular survival ofSalmonellaserovars is host-dependent. In the presence of serum opsonin, human monocyte-derived macrophages recognizedS. typhiandS. typhimuriumvia complement receptor type 1 (CR1) and type 3 (CR3), respectively. In contrast, murine macrophages recognizedS. typhiandS. typhimuriumvia CR3 and CR1, respectively. These findings demonstrate that the intracellular fate ofSalmonellaserovars following phagocytosis may depend on the type of complement receptors involved in their recognition, in that CR1-mediated recognition is closely correlated to subsequent intracellular survival. The Tn5 insertion mutant ofS. typhimuriumwhich lacks the ability to interact with CR1 was sensitive to intracellular killing by murine macrophagesin vitro, and was much less virulent to micein vivo, confirming the relevance of CR1-mediated bacterial recognition to the pathogenicity ofS. typhimuriumfor mice. These results suggest that selective recognition ofSalmonellaserovars through CR1 may lead to their subsequent intracellular survival, and is responsible for the host-specific pathogenesis ofSalmonellaserovars.     

Cloning and molecular analysis of theIsi1(rfaF) gene ofNeisseria meningitidiswhich encodes a heptosyl-2-transferase involved in LPS biosynthesis: evaluation of surface exposed carbohydrates in LPS mediated toxicity for human endothelial cells

Neisseria meningitidis, but notHaemophilus influenzae, damage cultured human endothelial cells. We have undertaken a study to generate genetically and structurally defined lipopolysaccharide (LPS) mutant strains of meningococci for functional studies to assess the role of surface exposed oligosaccharides in imparting specificity of toxic damage to human endothelial cells. TheIsi1gene, which had been shown to be involved in LPS biosynthesis ofNeisseria gonorrhoeae, was amplified by PCR and cloned. Nucleotide sequence analysis confirmed the identity of the clone and revealed homology withIsi1ofN. gonorrhoeaeand therfaFgene ofSalmonella typhimuriumwhich encodes a heptosyl-2-transferase involved in LPS biosynthesis. The identity of the clonedIsi1gene, as a functionalrfaFhomologue, was confirmed by the complementation of aS. typhimurium rfaFmutant using a P22 phage sensitivity test. AnIsi1mutant meningococcal strain was constructed, and structural analysis of the mutant LPS molecule revealed a single heptose in the core structure, consistent with a heptosyl-2-transferase deficient mutant. In order to investigate the relative cytotoxicities of meningococci expressing native and altered LPS, wild type,Isi1, andgalEstrains were compared in cytotoxicity assays using human umbilical vein endothelial cells (Huvecs) in culture. Analysis using Huvecs derived from several individuals (cords) showed that the three phenotypes were almost equally cytotoxic. Removal of the terminal portion (galEmutant) or the majority (Isimutant) of the oligosaccharide did not effect LPS-mediated cytopathic damage to Huvecs in a culture suggesting that the oligosaccharide portion did not play a major role in cytotoxicity.     

Characterization of recombinant Bordetella pertussis adenylate cyclase toxins carrying passenger proteins

Bordetella pertussis secretes a calmodulin-activated adenylate cyclase toxin, CyaA, that is able to deliver its N-terminal catalytic domain (400 amino acid residues) into the cytosol of eukaryotic target cells, directly through the cytoplasmic membrane. We have previously shown that CyaA can be used as a vehicle to deliver CD8⁺ T-cell epitopes, inserted within the catalytic domain of the toxin, into antigen-presenting cells and can trigger specific class I-restricted cytotoxic T-cell (CTL) responses in vivo. To explore the tolerance of CyaA to insertion of polypeptides of larger size, we constructed and characterized different recombinant CyaA toxins with protein inserts of 87 to 206 amino acids in length. Several of these recombinant CyaA toxins were found to be invasive. Furthermore, we showed that the unfolding of the passenger protein is a prerequisite for the translocation of the recombinant toxins into eukaryotic cells. Our results highlight the remarkable tolerance of the CyaA toxin and suggest that CyaA might be used to deliver proteins into eukaryotic cells.