Genetic characterization of a new type IV-A pilus gene cluster found in both classical and El Tor biotypes of Vibrio cholerae.
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The Vibrio cholerae genome contains a 5.4-kb pil gene cluster that resembles the Aeromonas hydrophila tap gene cluster and other type IV-A pilus assembly operons. The region consists of five complete open reading frames designated pilABCD and yacE, based on the nomenclature of related genes from Pseudomonas aeruginosa and Escherichia coli K-12. This cluster is present in both classical and El Tor biotypes, and the pilA and pilD genes are 100% conserved. The pilA gene encodes a putative type IV pilus subunit. However, deletion of pilA had no effect on either colonization of infant mice or adherence to HEp-2 cells, demonstrating that pilA does not encode the primary subunit of a pilus essential for these processes. The pilD gene product is similar to other type IV prepilin peptidases, proteins that process type IV signal sequences. Mutational analysis of the pilD gene showed that pilD is essential for secretion of cholera toxin and hemagglutinin-protease, mannose-sensitive hemagglutination (MSHA), production of toxin-coregulated pili, and colonization of infant mice. Defects in these functions are likely due to the lack of processing of N termini of four Eps secretion proteins, four proteins of the MSHA cluster, and TcpB, all of which contain type IV-A leader sequences. Some pilD mutants also showed reduced adherence to HEp-2 cells, but this defect could not be complemented in trans, indicating that the defect may not be directly due to a loss of pilD. Taken together, these data demonstrate the effectiveness of the V. cholerae genome project for rapid identification and characterization of potential virulence factors. (+info)
Effect of temperature on growth, hemagglutination, and protease activity of Porphyromonas gingivalis.
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Bacteria persisting in periodontal pockets are exposed to elevated temperatures during periods of inflammation. Temperature is an environmental factor that can modulate gene expression. Consequently, in the present study we examined the effect of temperature on the expression of virulence determinants by the periodontopathogen, Porphyromonas gingivalis. P. gingivalis W50 was grown in a complex medium under hemin excess at pH 7.0 and at a constant temperature of either 37, 39, or 41 degrees C; cultures were monitored for protease and hemagglutinin activity. P. gingivalis grew well at all three temperatures. An increase in growth temperature from 37 to 39 degrees C resulted in a 65% reduction in both total arginine- and lysine-specific activities (P < 0.01). A further rise in growth temperature to 41 degrees C led to even greater reductions in arginine-specific (82%; P < 0.001) and lysine-specific (73%; P < 0. 01) activities. These reductions were also associated with an altered distribution of individual arginine-specific enzyme isoforms. At 41 degrees C, there was a disproportionate reduction in the level of the heterodimeric RI protease, which also contains adhesin domains. The reduction also correlated with a markedly diminished hemagglutination activity of cells, especially in those grown at 41 degrees C, and a reduced immunoreactivity with a monoclonal antibody which recognizes gene products involved in hemagglutination. Thus, as the environmental temperature increased, P. gingivalis adopted a less aggressive phenotype, while retaining cell population levels. The coordinate down-regulation of virulence gene expression in response to an environmental cue linked to the intensity of the host inflammatory response is consistent with the clinically observed cyclical nature of disease progression in periodontal diseases. (+info)
Structural and functional studies of the measles virus hemagglutinin: identification of a novel site required for CD46 interaction.
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The entry of measles virus (MV) into human cells is mediated by the initial attachment of the viral hemagglutinin (HA) to the complement regulatory protein CD46. Two subdomains, one each within CD46 short consensus repeats (SCRs) 1 and 2, are responsible for this interaction. However, little is known about the regions within MV HA needed for a high-affinity CD46 interaction. To better define the HA-CD46 interaction, we took three approaches: chimeric domain swapping, peptide scanning, and alanine scanning mutagenesis. Chimeras of MV HA and the closely related rinderpest virus (RPV) HA were generated and tested for cell surface expression and the ability to hemadsorb CD46+ red blood cells (RBC). Exchanges with the N terminus of RPV were tolerated as MV HA could be replaced with RPV HA up to amino-acid position 154. However, both larger swaps with RPV and a small RPV HA replacement at the C terminus aborted cell-surface expression. Peptide scanning with 51 overlapping peptides derived from three MV HA regions showed one peptide, corresponding to MV HA amino acids 468-487, blocked hemagglutination of African green monkey (AGM) RBCs and inhibited MV infection of Chinese hamster ovary cells (CHO) expressing human CD46. Alanine scanning mutants mapped sites on the MV HA that were not required for trafficking to the cell surface or function in hemagglutination as well as a novel site required for CD46 interaction, amino acids 473-477. (+info)
Characterization of nonenterotoxigenic Escherichia coli strains producing F17 fimbriae isolated from diarrheic lambs and goat kids.
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Forty-five ovine and caprine nonenterotoxigenic Escherichia coli strains producing F17-related fimbriae were characterized with respect to the fimbrial structural subunit and adhesin subtypes produced. In addition, several characteristics related to the virulence of strains producing F17 fimbriae were studied. Most of the strains (73%) possessed the f17cA structural subunit gene, whereas the f17aA and f17dA genes were detected only on three (6%) and two (4%) strains, respectively. The f17bA gene was not detected. All but one of these strains possessed the f17G genes of the adhesin subfamily II. The only strain having the f17G gene of subfamily I possessed the structural subunit gene f17dA. Sequencing of the f17A and f17G genes of four selected strains confirmed the association of f17cA and f17dA structural subunit genes with the f17G genes of the adhesin subfamily II. These results indicated that adhesins of the subfamily II are prominent among ovine and caprine isolates and that they are indistinctly associated with the F17 structural subunit subtypes on these field strains. CS31A- and CNF2-related genes were not detected. Most of the strains adhered in vitro to ovine intestinal brush borders (36 of 45) and agglutinated the erythrocytes of different species in the presence of D-mannose (39 of 45). F17-positive strains produced colicin V (57%) and were resistant to the bactericidal effect of serum (91%) in significantly higher percentages than F17-negative strains (34% produced colicin V, and 66% were serum resistant). Thus, most of the studied ovine and caprine strains showed phenotypic characteristics of septicemic strains. (+info)
Characterization of the relationship between polysaccharide intercellular adhesin and hemagglutination in Staphylococcus epidermidis.
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To determine whether a relationship exists between biofilm formation and hemagglutination in Staphylococcus epidermidis, 20 skin isolates and 19 prosthetic valve endocarditis isolates were characterized for biofilm formation, hemagglutination, and the presence of a 357-bp polymerase chain reaction product within icaA. A strong association existed between biofilm formation, which has been linked to strains that produce polysaccharide intercellular adhesin (PIA), and hemagglutination. Strains that produced biofilm were significantly (P<.001) more likely to mediate hemagglutination (16 biofilm-positive/hemagglutination-positive strains and 19 biofilm-negative/hemagglutination-negative strains) within the 39 clinical strains tested. In addition, Staphylococcus carnosus TM300, a biofilm-negative, hemagglutination-negative strain, carrying the ica operon-containing plasmid pCN27, produced significant biofilm on glass and mediated hemagglutination (>/=1/128). It was concluded that production of PIA and hemagglutination are strongly associated and that PIA, at least in part, mediates hemagglutination in S. epidermidis. (+info)
Growth, cellular differentiation and virulence factor expression by Proteus mirabilis in vitro and in vivo.
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A uropathogenic strain of Proteus mirabilis was grown in vitro in human and mouse urine and brain-heart infusion broth (BHIB) and in vivo in subcutaneous open chambers (SOC) in mice, intraperitoneal diffusion chambers (IPC) in rats and by ascending urinary tract infection in mice in order to compare growth pattern, cellular differentiation and expression of virulence factors. Although the growth rate was slower in vivo than in vitro, the extent of growth was similar after 24 h. PR mirabilis differentiated into filamentous swarmer cells in all in-vitro culture conditions, but no filamentous cells were observed in either of the in-vivo chamber models. Transurethrally infected mice showed a rapid release or loss of filamentous cells and these could not be seen in kidney or bladder homogenates 7 days after infection. Bacteria showed increasing haemagglutination titres for fresh and tanned red blood cells after subculturing in BHIB, but bacteria grown in vivo did not show haemagglutination. An increasing resistance to normal serum was found when bacteria were grown in vivo. Significant haemolytic activity was detected with bacteria grown in BHIB and IPC, but almost no activity was found when bacteria had grown in urine. These findings improve the understanding of the role of P. mirabilis uropathogenic virulence factors in vivo. (+info)
Normal human serum contains natural antibodies reactive with autologous ABO blood group antigens.
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It is widely accepted that the serum of healthy individuals contains natural antibodies only against those blood group A or B antigens that are not expressed on the individual's red blood cells. The mechanisms involved in tolerance to autologous blood group antigens remain unclear. In the present study, we show that IgM and IgG antibodies reactive with autologous blood group antigens are present in the immunoglobulin fraction of normal human serum. Natural IgG anti-A antibodies purified by affinity chromatography from IgG of individuals of blood group A exhibited an affinity for A trisaccharide antigen in the micromolar range and agglutinated A red cells at sixfold higher concentrations than those required for agglutination with affinity-purified anti-A IgG of individuals of blood group B. Whereas autoantibodies reactive with self A and B antigens are readily detected in purified IgG and IgM fractions, their expression is restricted in whole serum as a result of complementary interactions between variable regions of antibodies. These observations suggest that tolerance to autologous ABO blood group antigens is dependent on peripheral control of antibody autoreactivity. (+info)
Genetic analyses of proteolysis, hemoglobin binding, and hemagglutination of Porphyromonas gingivalis. Construction of mutants with a combination of rgpA, rgpB, kgp, and hagA.
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Porphyromonas gingivalis produces arginine-specific cysteine proteinase (Arg-gingipain, RGP) and lysine-specific cysteine proteinase (Lys-gingipain, KGP) in the extracellular and cell-associated forms. Two separate genes (rgpA and rgpB) and a single gene (kgp) have been found to encode RGP and KGP, respectively. We constructed rgpA rgpB kgp triple mutants by homologous recombination with cloned rgp and kgp DNA interrupted by drug resistance gene markers. The triple mutants showed no RGP or KGP activity in either cell extracts or culture supernatants. The culture supernatants of the triple mutants grown in a rich medium had no proteolytic activity toward bovine serum albumin or gelatin derived from human type I collagen. Moreover, the mutants did not grow in a defined medium containing bovine serum albumin as the sole carbon/energy source. These results indicate that the proteolytic activity of P. gingivalis toward bovine serum albumin and gelatin derived from human type I collagen appears to be attributable to RGP and KGP. The hemagglutinin gene hagA of P. gingivalis possesses the adhesin domain regions responsible for hemagglutination and hemoglobin binding that are also located in the C-terminal regions of rgpA and kgp. A rgpA kgp hagA triple mutant constructed in this study exhibited no hemagglutination using sheep erythrocytes or hemoglobin binding activity, as determined by a solid-phase binding assay with horseradish peroxidase-conjugated human hemoglobin, indicating that the adhesin domains seem to be particularly important for P. gingivalis cells to agglutinate erythrocytes and bind hemoglobin, leading to heme acquisition. (+info)