Virulence plasmid of Rhodococcus equi contains inducible gene family encoding secreted proteins.
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Rhodococcus equi causes severe pyogranulomatous pneumonia in foals. This facultative intracellular pathogen produces similar lesions in immunocompromised humans, particularly in AIDS patients. Virulent strains of R. equi bear a large plasmid that is required for intracellular survival within macrophages and for virulence in foals and mice. Only two plasmid-encoded proteins have been described previously; a 15- to 17-kDa surface protein designated virulence-associated protein A (VapA) and an antigenically related 20-kDa protein (herein designated VapB). These two proteins are not expressed by the same R. equi isolate. We describe here the substantial similarity between VapA and VapB. Moreover, we identify three additional genes carried on the virulence plasmid, vapC, -D, and -E, that are tandemly arranged downstream of vapA. These new genes are members of a gene family and encode proteins that are approximately 50% homologous to VapA, VapB, and each other. vapC, -D, and -E are found only in R. equi strains that express VapA and are highly conserved in VapA-positive isolates from both horses and humans. VapC, -D, and -E are secreted proteins coordinately regulated by temperature with VapA; the proteins are expressed when R. equi is cultured at 37 degrees C but not at 30 degrees C, a finding that is compatible with a role in virulence. As secreted proteins, VapC, -D, and -E may represent targets for the prevention of rhodococcal pneumonia. An immunologic study using VapA-specific antibodies and recombinant Vap proteins revealed no evidence of cross-reactivity despite extensive sequence similarity over the carboxy terminus of all four proteins. (+info)
Depletion of CCR5-expressing cells with bispecific antibodies and chemokine toxins: a new strategy in the treatment of chronic inflammatory diseases and HIV.
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The chemokine receptor CCR5 is expressed on the majority of T cells and monocytes in the inflammatory infiltrate of diseases such as rheumatoid arthritis, renal diseases, and multiple sclerosis. In contrast, little expression of CCR5 is found on peripheral blood leukocytes. A specific depletion of CCR5(+) cells could therefore be a useful strategy to reduce the cellular infiltrate in chronic inflammations. Moreover, CCR5 is the major coreceptor for M-tropic HIV-1 strains. Depletion of CCR5(+) leukocytes may help to eliminate cells latently infected with HIV-1. We designed two constructs that specifically destroy chemokine receptor-positive cells. The first construct, a bispecific Ab, binds simultaneously to CCR5 and CD3. Thereby it redirects CD3(+) T cells against CCR5(+) target cells. The Ab specifically depletes CCR5(+) T cells and monocytes, but is inactive against cells that do not express CCR5. Furthermore, ex vivo the bispecific Ab eliminated >95% of CCR5(+) monocytes and T cells from the synovial fluid of patients with arthritis. Also, we designed a fusion protein of the chemokine RANTES and a truncated version of Pseudomonas. exotoxin A. The fusion protein binds to CCR5 and down-modulates the receptor from the cell surface. The chemokine toxin completely destroyed CCR5(+) Chinese hamster ovary cells at a concentration of 10 nM, whereas no cytotoxic effect was detectable against CCR5(-) Chinese hamster ovary cells. Both constructs efficiently deplete CCR5-positive cells, appear as useful agents in the treatment of chronic inflammatory diseases, and may help to eradicate HIV-1 by increasing the turnover of latently infected cells. (+info)
Crystal structure of the hexameric traffic ATPase of the Helicobacter pylori type IV secretion system.
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The type IV secretion system of Helicobacter pylori consists of 10--15 proteins responsible for transport of the transforming protein CagA into target epithelial cells. Secretion of CagA crucially depends on the hexameric ATPase, HP0525, a member of the VirB11-PulE family. We present the crystal structure of a binary complex of HP0525 bound to ADP. Each monomer consists of two domains formed by the N- and C-terminal halves of the sequence. ADP is bound at the interface between the two domains. In the hexamer, the N- and C-terminal domains form two rings, which together form a chamber open on one side and closed on the other. A model is proposed in which HP0525 functions as an inner membrane pore, the closure and opening of which is regulated by ATP binding and ADP release. (+info)
Genetic transformation of HeLa cells by Agrobacterium.
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Agrobacterium tumefaciens is a soil phytopathogen that elicits neoplastic growths on the host plant species. In nature, however, Agrobacterium also may encounter organisms belonging to other kingdoms such as insects and animals that feed on the infected plants. Can Agrobacterium, then, also infect animal cells? Here, we report that Agrobacterium attaches to and genetically transforms several types of human cells. In stably transformed HeLa cells, the integration event occurred at the right border of the tumor-inducing plasmid's transferred-DNA (T-DNA), suggesting bona fide T-DNA transfer and lending support to the notion that Agrobacterium transforms human cells by a mechanism similar to that which it uses for transformation of plants cells. Collectively, our results suggest that Agrobacterium can transport its T-DNA to human cells and integrate it into their genome. (+info)
implication of the structure of the Helicobacter pylori cag pathogenicity island in induction of interleukin-8 secretion.
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Helicobacter pylori virulence is associated with the presence of the cag pathogenicity island (PAI). The cag PAI is involved in the ability to induce interleukin-8 (IL-8) secretion by human cells, which is implicated in the inflammatory response of the gastric mucosa to H. pylori infection. The aim of this study was to determine whether the genetic structure of the cag PAI is conserved and whether it is linked to IL-8 induction ability. Detection of specific markers (cagA, picB, cag13-cag14, virD4, and IS605) by PCR and dot blot hybridization and long-distance PCR determination of the presence of cagI, cagII, and the middle region of the cag PAI were performed on 153 strains isolated from adults suffering from ulcers (n = 79) or gastritis (n = 74). IL-8 induction ability was evaluated by coculture of the strains with HEp-2 cells. Eighty-three strains (54.3%) had an entire cag PAI, 12 strains (7.8%) had the cag PAI split in two, 49 strains (32%) had no cag PAI, and 9 strains exhibited other structural combinations. The presence of an entire cag PAI was statistically correlated with the presence of IS605 (P = 0.006) and the ability to induce IL-8 secretion but not with clinical presentation of the infection. The structure of the cag PAI appears to be rather conserved and is related to the proinflammatory power of a strain. The existence of strains inducing IL-8 secretion regardless of the cag PAI structure suggests that this region is not the only requirement for IL-8 secretion. (+info)
Helicobacter pylori pore-forming cytolysin orthologue TlyA possesses in vitro hemolytic activity and has a role in colonization of the gastric mucosa.
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Hemolysins have been found to possess a variety of functions in bacteria, including a role in virulence. Helicobacter pylori demonstrates hemolytic activity when cultured on unlysed blood agar plates which is increased under iron-limiting conditions. However, the role of an H. pylori hemolysin in virulence is unclear. Scrutiny of the H. pylori 26695 genome sequence suggests the presence of at least two distinct hemolysins, HP1086 and HP1490, in this strain. Previous studies have shown that the in vitro hemolytic activity of H. pylori is reduced when it is coincubated with dextran 5000, suggesting the presence of a pore-forming cytolysin. HP1086 has homology to pore-forming cytolysins (TlyA) from other bacterial species, and the introduction of the cloned H. pylori tlyA gene into a nonhemolytic Escherichia coli strain conferred hemolytic activity. An H. pylori tlyA defined mutant showed reduced in vitro hemolytic activity, which appears to be due to pore formation, as the hemolytic activity of the wild-type strain is reduced to the same level as the tlyA mutant by the addition of dextran 5000. The mutant also showed reduced adhesion to human gastric adenocarcinoma cells and failed to colonize the gastric mucosa of mice. These data clearly suggest a role in virulence for H. pylori TlyA, contrary to the suggestion that hemolytic activity is an in vitro phenomenon for this pathogen. (+info)
Composition and gene expression of the cag pathogenicity island in Helicobacter pylori strains isolated from gastric carcinoma and gastritis patients in Costa Rica.
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The composition and in vitro expression of the cag pathogenicity island genes in a group of Helicobacter pylori strains obtained from patients suffering from chronic gastritis-associated dyspepsia (n = 26) or gastric carcinoma (n = 17) were analyzed. No significant difference in the distribution of the 10 studied regions was found between the cases and the controls. Nine strains did not harbor any of the selected regions: eight (30.8%) isolated from patients with gastritis only and one (5.9%) from a patient with gastric carcinoma. No association was found between the number of repeated sequences at the 3' end of the cagA gene or the presence of tyrosine phosphorylation motifs and the clinical origin of the strains. The virB10 homolog gene was the sole gene studied to be significantly expressed more often in cancer strains than in gastritis strains (P = 0.03). (+info)
Interaction of the virulence protein VirF of Agrobacterium tumefaciens with plant homologs of the yeast Skp1 protein.
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The infection of plants by Agrobacterium tumefaciens leads to the formation of crown gall tumors due to the transfer of a nucleoprotein complex into plant cells that is mediated by the virulence (vir) region-encoded transport system (reviewed in [1-5]). In addition, A. tumefaciens secretes the Vir proteins, VirE2 and VirF, directly into plant cells via the same VirB/VirD4 transport system [6], and both assist there in the transformation of normal cells into tumor cells. The function of the 22 kDa VirF protein is not clear. Deletion of the virF gene in A. tumefaciens leads to diminished virulence [7, 8] and can be complemented by the expression of the virF gene in the host plant. This finding indicates that VirF functions within the plant cell [8]. Here, we report that the VirF protein is the first prokaryotic protein with an F box by which it can interact with plant homologs of the yeast Skp1 protein. The presence of the F box turned out to be essential for the biological function of VirF. F box proteins and Skp1p are both subunits of a class of E3 ubiquitin ligases referred to as SCF complexes. Thus, VirF may be involved in the targeted proteolysis of specific host proteins in early stages of the transformation process. (+info)