The Erp protein is anchored at the surface by a carboxy-terminal hydrophobic domain and is important for cell-wall structure in Mycobacterium smegmatis

Erp (Exported Repetitive Protein), also known as P36, Pirg and Rv3810, is a member of a mycobacteria-specific family of extracellular proteins. In pathogenic species, the erp gene has been described as a virulence factor. The Erp proteins comprise three domains. The N- and C-terminal domains are similar in all mycobacterial species, while the central domain consists of a repeated module that differs considerably between species. Here we show that the Erp protein is loosely attached to the surface and that the carboxy-terminal domain, which displays hydrophobic features, anchors Erp at the surface of the bacillus. The hydrophobic region is not necessary for the complementation of the altered colony morphology of a Mycobacterium smegmatis erp- mutant but proved to be necessary to achieve resistance to detergent at wild-type levels.     

Simultaneous coexpression of Borrelia burgdorferi Erp proteins occurs through a specific,erp locus-directed regulatory mechanism

An individual Borrelia burgdorferi bacterium can encode as many as 13 different Erp (OspE/F-related) proteins from mono-and bicistronic loci that are carried on up to 10 separate plasmids. We demonstrate through multilabel immunofluorescence analyses that individual bacteria simultaneously coexpress their entire Erp protein repertoire. While it has been proposed that B. burgdorferi controls expression of Erp and other plasmid-encoded proteins through changes in DNA topology, we observed regulated Erp expression in the absence of detectable differences in DNA supercoiling. Likewise, inhibition of DNA gyrase had no detectable effect on Erp expression. Furthermore, expression of loci physically adjacent to erp loci was observed to be independently regulated. It is concluded that Erp expression is regulated by a mechanism(s) directed at erp loci and not by a global, plasmid-wide mechanism.     

Disruption of response regulator gene, devR, leads to attenuation in virulence of Mycobacterium tuberculosis

The devR-devS two-component system of Mycobacterium tuberculosis was identified earlier and partially characterized in our laboratory. A devR::kan mutant of M. tuberculosis was constructed by allelic exchange. The devR mutant strain showed reduced cell-to-cell adherence in comparison to the parental strain in laboratory culture media. This phenotype was reversed on complementation with a wild-type copy of devR. The devR mutant and parental strains grew at equivalent rates within human monocytes either in the absence or in the presence of lymphocytic cells. The expression of DevR was not modulated upon entry of M. tuberculosis into human monocytes. However, guinea pigs infected with the mutant strain showed a significant decrease in gross lesions in lung, liver and spleen; only mild pathological changes in liver and lung; and a nearly 3 log lower bacterial burden in spleen compared to guinea pigs infected with the parental strain. Our results suggest that DevR is required for virulence in guinea pigs but is not essential for entry, survival and multiplication of M. tuberculosis within human monocytes in vitro. The attenuation in virulence of the devR mutant in guinea pigs together with DevR-DevS being a bona fide signal transduction system indicates that DevR plays a critical and regulatory role in the adaptation and survival of M. tuberculosis within tissues.     

Mycobacterium tuberculosis inhibits neutrophil apoptosis, leading to delayed activation of naive CD4 T cells

Mycobacterium tuberculosis promotes its replication by inhibiting the apoptosis of infected macrophages. A proapoptotic M.?tuberculosis mutant lacking nuoG, a subunit of the type I NADH dehydrogenase complex, exhibits attenuated growth in?vivo, indicating that this virulence mechanism is essential. We show that M.?tuberculosis also suppresses neutrophil apoptosis. Compared to wild-type, the nuoG mutant spread to a larger number of lung phagocytic cells. Consistent with the shorter lifespan of infected neutrophils, infection with the nuoG mutant resulted in fewer bacteria per infected neutrophil, accelerated bacterial acquisition by dendritic cells, earlier trafficking of these dendritic cells to lymph nodes, and faster CD4 T?cell priming. Neutrophil depletion abrogated accelerated CD4 T?cell priming by the nuoG mutant, suggesting that inhibiting neutrophil apoptosis delays adaptive immunity in tuberculosis. Thus, pathogen modulation of apoptosis is beneficial at multiple levels, and enhancing phagocyte apoptosis promotes CD4 as well as CD8 T?cell responses.     

Nude rat (F344/N-rnu) tuberculosis

As many mononuclear cells from Mycobacterium tuberculosis-infected lung tissues are not available for fluorescence-activated cell sorter (FACS) analysis and the tuberculin test is not feasible in a mouse tuberculosis model, we attempted to develop a rat tuberculosis model. We have previously reported that rat tuberculosis is associated with granulomas that lack central necrosis. In order to develop a better animal model of tuberculosis in immunocompromised humans (tuberculosis associated with HIV infection or tuberculosis of the elderly), we infected F344/N-rnu nude rats with M. tuberculosis via the airborne route. The animals developed pulmonary granulomas with central necrosis encapsulated by dense collagen fibres, closely resembling those of human tuberculosis. The nude rats died of disseminated tuberculosis by the 85th day after aerosol infection, while F344 wild-type rats did not. Interestingly, T-cells that were reactive with anti-CD4 antibody and anti-CD8 antibody, indicating the presence of remnant thymus, were observed in the infected lung tissues of the nude rats. Therefore, T-cell precursors may be present in nude rats. The nude rat tuberculosis model mimics tuberculosis in immunocompromised humans and may provide a suitable model for immunological studies in vivo.     

Studies on neutral exopolysaccharides produced by the ectomycorrhiza Thelephora terrestris

We developed an in vitro tissue-culture model to analyze the process involved in mycobacterial spread through lung epithelial cell monolayers. A549 cells were infected with low numbers of viable Mycobacterium tuberculosis bacilli expressing the gfp gene. Subsequent addition of a soft agarose overlay prevented the dispersal of the bacilli from the initial points of attachment. By fluorescence microscopy the bacteria were observed to infect and grow within the primary target cells; this was followed by lysis of the infected cells and subsequent infection of adjacent cells. This process repeated itself until an area of clearing (plaque formation) was observed. The addition of amikacin after initial infection did not prevent intracellular growth; however, subsequent plaque formation was not observed. Plaque formation was also observed after infection with Mycobacterium bovis BCG bacilli, but the plaques were smaller than those formed after infection with M. tuberculosis. These observations reinforce the possibility that cell-to-cell spreading of M. tuberculosis bacilli, particularly early in the course of infection within lung macrophages, pneumocytes, and other cells, may be an important component in the infectious process.     

Guinea pig model of Mycobacterium tuberculosis latent/dormant infection

Although guinea pigs are considered one of the best animal models of tuberculosis, little data exist describing latent or dormant tuberculosis infection in these animals. Here we address this issue using a streptomycin auxotrophic mutant of Mycobacterium tuberculosis. This mutant grows unimpaired in the presence of streptomycin but in its absence shifts to latency/dormancy (lack growth and over-expression of alpha-crystallin). To establish infection animals are inoculated with the mutant followed by daily administration of streptomycin (three weeks), which allows initial microbial multiplication in the animal's tissues. Withdrawal of streptomycin establishes latency/dormancy and few viable organisms are recovered from the animals' lungs and spleen six months later. During the infectious process guinea pigs steadily gained weight and presented no clinical signs (scuff fur and lethargy) of disease. Histopathology of organs mimicked tuberculous lesions in humans and PBMC from infected animals strongly responded to stimulation with PPD. Finally, tuberculin skin test (a hallmark of latent infection diagnosis) performed in infected animals was strongly positive (>or=15 mm induration). These results point to an interesting and reliable model of latent/dormant tuberculosis infection in guinea pigs.     

A family of genes located on four separate 32-kilobase circular plasmids in Borrelia burgdorferi B31.

We have identified four loci in Borrelia burgdorferi B31 that contain open reading frames capable of encoding six proteins that are related to the antigenic proteins OspE and OspF. We have designated these proteins Erp, for OspEF-related protein, and named their respective genes erp. The erpA and erpB genes are linked, as are erpC and erpD, and the pairs probably constitute two operons. The erpG and erpH genes appear to be monocistronic. The ErpA and ErpC proteins are expressed by B. burgdorferi B31 in culture and are recognized by a polyclonal antiserum raised against the OspE protein of B. burgdorferi N40. The four erp loci are each located on different 32-kb circular plasmids that contain additional DNA sequences that are homologous to each other and to an 8.3-kb circular plasmid of B. burgdorferi sensu lato Ip2l. All four 32-kb plasmids can be maintained within a single bacterium, which may provide a model for the study of plasmid replication and segregation in B. burgdorferi.     

Borrelia burgdorferi EbfC, a novel, chromosomally encoded protein, binds specific DNA sequences adjacent to erp loci on the spirochete's resident cp32 prophages

All examined isolates of the Lyme disease spirochete, Borrelia burgdorferi, naturally maintain numerous variants of a prophage family as circular cp32 episomes. Each cp32 carries a locus encoding one or two different Erp outer membrane, surface-exposed lipoproteins. Many of the Erp proteins bind a host complement regulator, factor H, which is hypothesized to protect the spirochete from complement-mediated killing. We now describe the isolation and characterization of a novel, chromosomally encoded protein, EbfC, that binds specific DNA sequences located immediately 5' of all erp loci. This is one of the first site-specific DNA-binding proteins to be identified in any spirochete. The location of the ebfC gene on the B. burgdorferi chromosome suggests that the cp32 prophages have evolved to use this bacterial host protein for their own benefit and that EbfC probably plays additional roles in the bacterium. A wide range of other bacteria encode homologs of EbfC, none of which have been well characterized, so demonstration that B. burgdorferi EbfC is a site-specific DNA-binding protein has broad implications across the eubacterial kingdom.     

Secretory proteins of Mycobacterium habana induce a protective immune response against experimental tuberculosis

The capacity of proteins of Mycobacterium habana TMC 5135 secreted into culture medium during the mid-exponential growth phase (secretory proteins, SPs) to induce protective immunity against Mycobacterium tuberculosis H37Rv was studied in the mouse model. Mice immunized with SPs followed by a challenge with M. tuberculosis H37Rv showed lesser M. tuberculosis bacilli in their lung and spleen and survived longer than unimmunized controls. The findings suggest that SP antigens of M. habana are protective against tuberculosis infection.     

Conserved Immune Recognition Hierarchy of Mycobacterial PE/PPE Proteins during Infection in Natural Hosts

The Mycobacterium tuberculosis genome contains two large gene families encoding proteins of unknown function, characterized by conserved N-terminal proline and glutamate (PE and PPE) motifs. The presence of a large number of PE/PPE proteins with repetitive domains and evidence of strain variation has given rise to the suggestion that these proteins may play a role in immune evasion via antigenic variation, while emerging data suggests that some family members may play important roles in mycobacterial pathogenesis. In this study, we examined cellular immune responses to a panel of 36 PE/PPE proteins during human and bovine infection. We observed a distinct hierarchy of immune recognition, reflected both in the repertoire of PE/PPE peptide recognition in individual cows and humans and in the magnitude of IFN-γ responses elicited by stimulation of sensitized host cells. The pattern of immunodominance was strikingly similar between cattle that had been experimentally infected with Mycobacterium bovis and humans naturally infected with clinical isolates of M. tuberculosis. The same pattern was maintained as disease progressed throughout a four-month course of infection in cattle, and between humans with latent as well as active tuberculosis. Detailed analysis of PE/PPE responses at the peptide level suggests that antigenic cross-reactivity amongst related family members is a major determinant in the observed differences in immune hierarchy. Taken together, these results demonstrate that a subset of PE/PPE proteins are major targets of the cellular immune response to tuberculosis, and are recognized at multiple stages of infection and in different disease states. Thus this work identifies a number of novel antigens that could find application in vaccine development, and provides new insights into PE/PPE biology.     

Comparative genomics of metabolic pathways in Mycobacterium species: gene duplication, gene decay and lateral gene transfer

The genus Mycobacterium comprises significant pathogenic species that infect both humans and animals. One species within this genus, Mycobacterium tuberculosis, is the primary killer of humans resulting from bacterial infections. Five mycobacterial genomes belonging to four different species (M. tuberculosis, Mycobacterium bovis, Mycobacterium leprae and Mycobacterium avium ssp. paratuberculosis) have been sequenced to date and another 14 mycobacterial genomes are at various stages of completion. A comparative analysis of the gene products of key metabolic pathways revealed that the major differences among these species are in the gene products constituting the cell wall and the gene families encoding the acidic glycine-rich (PE/PPE/PGRS) proteins. Mycobacterium leprae has evolved by retaining a minimal gene set for most of the gene families, whereas M. avium ssp. paratuberculosis has acquired some of the virulence factors by lateral gene transfer.