The virulence plasmids of the equine virulent strains Rhodococcus equi ATCC 33701 and 103 were sequenced, and their genetic structure was analyzed. p33701 was 80,610 bp in length, and p103 was 1 bp shorter; their sequences were virtually identical. The plasmids contained 64 open reading frames (ORFs), 22 of which were homologous with genes of known function and 3 of which were homologous with putative genes of unknown function in other species. Putative functions were assigned to five ORFs based on protein family characteristics. The most striking feature of the virulence plasmids was the presence of a 27,536-bp pathogenicity island containing seven virulence-associated protein (vap) genes, including vapA. These vap genes have extensive homology to vapA, which encodes a thermoregulated and surface-expressed protein. The pathogenicity island contained a LysR family transcriptional regulator and a two-component response regulator upstream of six of the vap genes. The vap genes were present as a cluster of three (vapA, vapC, and vapD), as a pair (vapE and vapF), or individually (vapG; vapH). A region of extensive direct repeats of unknown function, possibly associated with thermoregulation, was present immediately upstream of the clustered and the paired genes but not the individual vap genes. There was extensive homology among the C-terminal halves of all vap genes but not generally among the N-terminal halves. The remainder of the plasmid consisted of a large region which appears to be associated with conjugation functions and a large region which appears to be associated with replication and partitioning functions.Rhodococcus equi is an important pulmonary pathogen of foals and is increasingly isolated from pneumonic infections and other infections in human immunodeficiency virus (HIV)-infected patients (19,33). Isolates from foals possess a large virulence plasmid, varying in size from 80 to 90 kb (45,47,49). Isolates lacking the plasmid are avirulent to foals (16,51). Little is known about the function of the plasmid apart from its encoding a virulence-associated surface protein (VapA) (45,49), the presence of a family of four vap genes (5), and the origin of replication (53). Infection with R. equi bacteria carrying the virulence plasmid may lead to immunomodulation in foals by causing failure to mount an effective Th1-based cellular immune response, but the basis of this effect is undefined (17). The expression of VapA is thermoregulated (Ն34°C) and pH regulated (41, 42), so that in this respect the plasmid has similarities to the virulence plasmids of pathogenic Yersinia species, such as Yersinia pestis, and of Shigella species (11,22,30). The plasmid is of significant interest, since it is associated with survival of the bacterium inside macrophages (16,21,33). Understanding its structure and function may therefore yield insights not only into the basis of virulence of this organism but also into the mechanisms of macrophage survival of other facultative intracellular pathogens, including Mycobacterium tub...
Serotype 4b strains of the food-borne pathogen Listeria monocytogenes are responsible for a large portion of sporadic listeric infections and all major food-borne listeriosis outbreaks in humans. Hybridomas were produced from three fusions with lymphocytes of ND4 mice immunized either with the insoluble antigens of L. monocytogenes serotype 4b or with formalin-killed bacterial cells and screened for monoclonal antibodies (mAbs) reactive to L. monocytogenes serotype 4b. A set of 35 mAbs was identified by ELISA as having reactivity with both the insoluble antigen fraction and the whole-cell antigens. Thirteen of these mAbs belonged to immunoglobulin subclass G1 (IgG1), fifteen were IgG2a and seven mAbs were IgM. Only 20 out of the 35 mAbs were capable of detecting protein bands of various sizes ranging from 20 to 88 kDa in Western blots. Two of these mAbs, M2365 and M2367, were capable of binding to cell-surface antigens of live L. monocytogenes serotype 4b, as demonstrated by immunofluorescence microscopy and immunogold transmission electron microscopy. Immunofluorescence microscopy showed that M2365 and M2367 failed to bind to the cell surfaces of Escherichia coli O157 : H7, Salmonella enterica (serotype Typhimurium DT104) or Campylobacter jejuni. Evaluation of the cross-reactions of all 35 mAbs with whole-cell antigens of E. coli O157 : H7, S. Typhimurium, C. jejuni and Listeria innocua by ELISA indicated that the majority of the mAbs, including M2365 and M2367, did not cross-react with E. coli O157 : H7, S. Typhimurium or C. jejuni and showed no or a very weak reaction with L. innocua. Furthermore, M2365 and M2367 showed no reaction with whole-cell antigens derived from L. monocytogenes serotypes 1/2a, 1/2b and 3a, and from Listeria grayi, Listeria ivanovii and Listeria seeligeri, in an ELISA. Collectively, these data suggest that M2365 and M2367 have potential use in the development of immunological methods of laboratory diagnosis for L. monocytogenes serotype 4b in clinical or food samples.
The role of the humoral immune response in protective immunity against listerial infection has been overlooked and is essentially unknown. This study aimed to discover the protein targets of Listeria monocytogenes that elicit an antibody response following infection in a rabbit model. A genomic expression library for L. monocytogenes was constructed and differentially screened to identify genes encoding proteins that reacted with antiserum from rabbits infected with live L. monocytogenes serotype 4b (RαL), but not with that from animals immunized with heat-killed bacteria (RαK). Thirty-one clones expressing proteins that reacted exclusively with RαL were identified and sequenced. Sequence analysis, together with Western blot analysis of the proteins expressed from positive clones, led to the identification of eight L. monocytogenes proteins as targets of humoral immune responses during listerial infection: three internalin members (InlA, InlD and InlC2) and five novel proteins of unknown function (designated IspA, IspB, IspC, IspD and IspE, respectively). Exhibition of humoral immune responses to these proteins in actively infected rabbits but not in animals receiving heat-killed L. monocytogenes suggested that they were induced or significantly upregulated in vivo during infection and thus are important in Listeria pathogenesis. With the exception of antibodies to InlA, this is the first demonstration of antibodies to the other seven proteins in infected hosts. These immunogenic proteins may be useful candidates for elucidation of the role of antibodies in protective immunity in the context of listerial infection, as well as potential targets for serodiagnostic reagents and vaccine and drug development.
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