The Cell Wall Lipid PDIM Contributes to Phagosomal Escape and Host Cell Exit of Mycobacterium tuberculosis.

The cell wall of Mycobacterium tuberculosis is composed of unique lipids that are important for pathogenesis. Indeed, the first-ever genetic screen in M. tuberculosis identified genes involved in the biosynthesis and transport of the cell wall lipid PDIM (phthiocerol dimycocerosates) as crucial for the survival of M. tuberculosis in mice. Here we show evidence for a novel molecular mechanism of the PDIM-mediated virulence in M. tuberculosis We characterized the DNA interaction and the regulon of Rv3167c, a transcriptional repressor that is involved in virulence regulation of M. tuberculosis, and discovered that it controls the PDIM operon. A loss-of-function genetic approach showed that PDIM levels directly correlate with the capacity of M. tuberculosis to escape the phagosome and induce host cell necrosis and macroautophagy. In conclusion, our study attributes a novel role of the cell wall lipid PDIM in intracellular host cell modulation, which is important for host cell exit and dissemination of M. tuberculosisIMPORTANCEMycobacterium tuberculosis is a major human pathogen that has coevolved with its host for thousands of years. The complex and unique cell wall of M. tuberculosis contains the lipid PDIM (phthiocerol dimycocerosates), which is crucial for virulence of the bacterium, but its function is not well understood. Here we show that PDIM expression by M. tuberculosis is negatively regulated by a novel transcriptional repressor, Rv3167c. In addition, we discovered that the escape of M. tuberculosis from its intracellular vacuole was greatly augmented by the presence of PDIM. The increased release of M. tuberculosis into the cytosol led to increased host cell necrosis. The discovery of a link between the cell wall lipid PDIM and a major pathogenesis pathway of M. tuberculosis provides important insights into the molecular mechanisms of host cell manipulation by M. tuberculosis.

Improved immunogenicity of a vaccination regimen combining a DNA vaccine encoding Brucella melitensis outer membrane protein 31 (Omp31) and recombinant Omp31 boosting.

In the present study, we report an attempt to improve the immunogenicity of the Omp31 antigen by a DNA prime-protein boost immunization regimen. We immunized BALB/c mice with an Omp31 DNA vaccine (pCIOmp31) followed by boosting with recombinant Omp31 (rOmp31) in incomplete Freund's adjuvant and characterized the resulting immune responses and the protective efficacy against Brucella ovis and B. melitensis infection. Immunoglobulin G1 (IgG1) and IgG2a titers were higher in sera from pCIOmp31/rOmp31-immunized mice than in sera from mice immunized with pCIOmp31 or rOmp31 alone. Splenocytes from pCIOmp31/rOmp31-immunized mice produced significantly higher levels of gamma interferon than did those from mice given rOmp31 alone. In contrast, interleukin 2 (IL-2) production levels were comparable between the two groups of immunized mice. Cells from all immunized mice produced undetectable levels of IL-4. Notably, rOmp31 stimulated IL-10 production in the pCIOmp31/rOmp31-immunized group but not in the pCIOmp31- or rOmp31-immunized group. Although the prime-boost regimen induced specific cytotoxic responses, these responses could not reach the levels achieved by the pCIOmp31 immunization. In conclusion, pCIOmp31 priming followed by rOmp31 boosting led to moderately improved protection against a challenge with B. ovis or B. melitensis.

A recombinant subunit vaccine based on the insertion of 27 amino acids from Omp31 to the N-terminus of BLS induced a similar degree of protection against B. ovis than Rev.1 vaccination.

The development of an effective subunit vaccine against brucellosis is a research area of intense interest. The enzyme lumazine synthase from Brucella spp. (BLS) is highly immunogenic, presumably due to its decameric arrangement and remarkable stability. In this work we decided to develop a chimera with the scaffold protein BLS decorated with 10 copies of a known protective epitope derived from an outer membrane protein of 31kDa (Omp31) from Brucella spp. Vaccination of BALB/c mice with the chimera as a recombinant protein (rBLSOmp31) provided the best protection level against Brucella ovis, which was higher than the given by the co-delivery of both recombinant proteins (rBLS + rOmp31) and similar than the control vaccine Brucella melitensis strain Rev.1. Moreover rBLSOmp31 induced protection against Brucella melitensis but to a lesser degree than Rev.1. The chimera induced a strong humoral response against the inserted peptide. It also induced peptide- and BLS-specific T helper 1 and cytotoxic T responses. In conclusion, our results indicate that BLSOmp31 could be a useful candidate for the development of subunit vaccines against brucellosis since it elicits humoral, T helper and cytotoxic immune responses and protection against smooth and rough species of Brucella.

Vaccination with the recombinant Brucella outer membrane protein 31 or a derived 27-amino-acid synthetic peptide elicits a CD4+ T helper 1 response that protects against Brucella melitensis infection.

The immunogenicity and protective efficacy of the recombinant 31-kDa outer membrane protein from Brucella melitensis (rOmp31), administered with incomplete Freund's adjuvant, were evaluated in mice. Immunization of BALB/c mice with rOmp31 conferred protection against B. ovis and B. melitensis infection. rOmp31 induced a vigorous immunoglobulin G (IgG) response, with higher IgG1 than IgG2 titers. In addition, spleen cells from rOmp31-immunized mice produced interleukin 2 (IL-2) and gamma interferon, but not IL-10 or IL-4, after in vitro stimulation with rOmp31, suggesting the induction of a T helper 1 (Th1) response. Splenocytes from rOmp31-vaccinated animals also induced a specific cytotoxic-T-lymphocyte activity, which led to the in vitro lysis of Brucella-infected macrophages. In vitro T-cell subset depletion indicated that rOmp31 immunization elicited specific CD4+ T cells that secrete IL-2 and gamma interferon, while CD8+ T cells induced cytotoxic-T-lymphocyte activity. In vivo depletion of T-cell subsets showed that the rOmp31-elicited protection against B. melitensis infection is mediated by CD4+ T cells while the contribution of CD8+ T cells may be limited. We then evaluated the immunogenicity and protective efficacy of a known exposed region from Omp31 on the Brucella membrane, a peptide that contains amino acids 48 to 74 of Omp31. Immunization with the synthetic peptide in adjuvant did not elicit a specific humoral response but elicited a Th1 response mediated by CD4+ T cells. The peptide in adjuvant induced levels of protection similar to those induced by rOmp31 against B. melitensis but less protection than was induced by rOmp31 against B. ovis. Our results indicate that rOmp31 could be a useful candidate for the development of subunit vaccines against B. melitensis and B. ovis.

A DNA vaccine coding for the Brucella outer membrane protein 31 confers protection against B. melitensis and B. ovis infection by eliciting a specific cytotoxic response.

The development of an effective subunit vaccine against brucellosis is a research area of intense interest. The outer membrane proteins (Omps) of Brucella spp. have been extensively characterized as potential immunogenic and protective antigens. This study was conducted to evaluate the immunogenicity and protective efficacy of the B. melitensis Omp31 gene cloned in the pCI plasmid (pCIOmp31). Immunization of BALB/c mice with pCIOmp31 conferred protection against B. ovis and B. melitensis infection. Mice vaccinated with pCIOmp31 developed a very weak humoral response, and in vitro stimulation of their splenocytes with recombinant Omp31 did not induced the secretion of gamma interferon. Splenocytes from Omp31-vaccinated animals induced a specific cytotoxic-T-lymphocyte activity, which leads to the in vitro lysis of Brucella-infected macrophages. pCIOmp31 immunization elicited mainly CD8(+) T cells, which mediate cytotoxicity via perforins, but also CD4(+) T cells, which mediate lysis via the Fas-FasL pathway. In vivo depletion of T-cell subsets showed that the pCIOmp31-induced protection against Brucella infection is mediated predominantly by CD8(+) T cells, although CD4(+)T cells also contribute. Our results demonstrate that the Omp31 DNA vaccine induces cytotoxic responses that have the potential to contribute to protection against Brucella infection. The protective response could be related to the induction of CD8(+) T cells that eliminate Brucella-infected cells via the perforin pathway.

Sedimentation velocity analysis of heterogeneous protein-protein interactions: Lamm equation modeling and sedimentation coefficient distributions c(s).

We describe algorithms for solving the Lamm equations for the reaction-diffusion-sedimentation process in analytical ultracentrifugation, and examine the potential and limitations for fitting experimental data. The theoretical limiting case of a small, uniformly distributed ligand rapidly reacting with a larger protein in a "constant bath" of the ligand is recapitulated, which predicts the reaction boundary to sediment with a single sedimentation and diffusion coefficient. As a consequence, it is possible to express the sedimentation profiles of reacting systems as c(s) distribution of noninteracting Lamm equation solutions, deconvoluting the effects of diffusion. For rapid reactions, the results are quantitatively consistent with the "constant bath" approximation, showing c(s) peaks at concentration-dependent positions. For slower reactions, the deconvolution of diffusion is still partially successful, with c(s) resolving peaks that reflect the populations of sedimenting species. The transition between c(s) peaks describing reaction boundaries of moderately strong interactions (K(D) approximately 10(-6) M) or resolving sedimenting species was found to occur in a narrow range of dissociation rate constant between 10(-3) and 10(-4) s(-1). The integration of the c(s) peaks can lead to isotherms of species populations or s-value of the reaction boundary, respectively, which can be used for the determination of the equilibrium binding constant.

Studying multiprotein complexes by multisignal sedimentation velocity analytical ultracentrifugation.

Protein interactions can promote the reversible assembly of multiprotein complexes, which have been identified as critical elements in many regulatory processes in cells. The biophysical characterization of assembly products, their number and stoichiometry, and the dynamics of their interactions in solution can be very difficult. A classical first-principle approach for the study of purified proteins and their interactions is sedimentation velocity analytical ultracentrifugation. This approach allows one to distinguish different protein complexes based on their migration in the centrifugal field without isolating reversibly formed complexes from the individual components. An important existing limitation for systems with multiple components and assembly products is the identification of the species associated with the observed sedimentation rates. We developed a computational approach for integrating multiple optical signals into the sedimentation coefficient distribution analysis of components, which combines the size-dependent hydrodynamic separation with discrimination of the extinction properties of the sedimenting species. This approach allows one to deduce the stoichiometry and to assign the identity of the assembly products without prior assumptions of the number of species and the nature of their interaction. Although chromophoric labels may be used to enhance the spectral resolution, we demonstrate the ability to work label-free for three-component protein mixtures. We observed that the spectral discrimination can synergistically enhance the hydrodynamic resolution. This method can take advantage of differences in the absorbance spectra of interacting solution components, for example, for the study of protein-protein, protein-nucleic acid or protein-small molecule interactions, and can determine the size, hydrodynamic shape, and stoichiometry of multiple complexes in solution.

Brucella lumazine synthase elicits a mixed Th1-Th2 immune response and reduces infection in mice challenged with Brucella abortus 544 independently of the adjuvant formulation used.

The immunogenicity and protective efficacy of recombinant lumazine synthase from Brucella spp. (rBLS) administered with different adjuvants was evaluated in mice. Mice were immunized with rBLS in the absence or the presence of aluminum hydroxide gel (BLS-Al), monophosphoryl lipid A (BLS-MPA), or incomplete Freund's adjuvant (BLS-IFA). rBLS per se induced a vigorous immunoglobulin G (IgG) response, with high titers of IgG1 as well as IgG2. All the adjuvants increased this response; the BLS-IFA formulation was the most effective at inducing BLS-specific IgG antibodies. In addition, after in vitro stimulation with rBLS, spleen cells from BLS-IFA-, BLS-Al-, or BLS-MPA-immunized mice proliferated and produced interleukin-2 (IL-2), gamma interferon (IFN-gamma), IL-10, and IL-4, suggesting the induction of a mixed Th1-Th2 response. Immunization with rBLS protected mice against challenge with B. abortus 544. The levels of protection in the spleen were similar for all adjuvants, but only BLS-Al and BLS-IFA were effective in the liver. Our results indicate that BLS might be a useful candidate for the development of subunit vaccines against brucellosis, since it elicits antigen-specific cellular responses, with production of IFN-gamma and protection, independently of the adjuvant formulation used.

Diminished production of T helper 1 cytokines correlates with T cell unresponsiveness to Brucella cytoplasmic proteins in chronic human brucellosis.

This study evaluated the cellular immune response against Brucella species cytoplasmic protein (CP) in peripheral blood mononuclear cells (PBMC) of 25 patients with brucellosis. In vitro proliferation and cytokine gene expression and production were investigated. PBMC from 14 patients proliferated in response to CP (responder patients [RPs]) and cells from 11 patients did not (nonresponder patients [NRPs]). CP-specific interleukin (IL)-2 and interferon-gamma were significantly induced in PBMC from RPs, compared with cells from NRPs. No significant differences were found in the production of IL-10 between the 2 groups. CP did not induce IL-4 production. A close relationship was observed between the clinical status of the patients and the T cell response against CP. Patient with acute infections responded to CP and induced production of T helper 1 (Th1) cytokines, whereas chronically infected patients did not. Diminished production of Th1 cytokines may contribute to T cell unresponsiveness in chronic human brucellosis.

A DNA vaccine encoding lumazine synthase from Brucella abortus induces protective immunity in BALB/c mice.

This study was conducted to evaluate the immunogenicity of the Brucella abortus lumazine synthase (BLS) gene cloned into the pcDNA3 plasmid, which is driven by the cytomegalovirus promoter. Injection of plasmid DNA carrying the BLS gene (pcDNA-BLS) into BALB/c mice elicited both humoral and cellular immune responses. Antibodies to the encoded BLS included immunoglobulin G1 (IgG1) IgG2a, IgG2b, IgG3, and IgM isotypes. Animals injected with pcDNA-BLS exhibited a dominance of IgG2a over IgG1. In addition, spleen cells from vaccinated animals produced interleukin-2 and gamma interferon but not IL-10 or IL-4 after in vitro stimulation with recombinant BLS (rBLS), suggesting the induction of a Th1 response. Protection was evaluated by comparing the levels of infection in the spleens of vaccinated mice challenged with B. abortus 544. Immunization with pcDNA-BLS- reduced the bacterial burden relative to those in the control groups. Mice immunized with rBLS produced a significant humoral response but did not show a specific cellular response or any protection from challenge. Altogether, these data suggest that pcDNA-BLS is a good immunogen for the production of humoral and cell-mediated responses in mice and is a candidate for use in future studies of vaccination against brucellosis.

Diagnostic usefulness of antibodies against ribosome recycling factor from Brucella melitensis in human or canine brucellosis.

The diagnostic usefulness of an enzyme-linked immunosorbent assay (ELISA) using a purified recombinant ribosome recycling factor from Brucella melitensis (CP24 antigen) was tested in human and canine infections caused by smooth and rough Brucella species, respectively. Anti-CP24 antibodies were detected in 9 (43%) of 21 consecutive cases of canine brucellosis and in 8 (53%) of 15 dogs followed for 60 days after the diagnosis of acute brucellosis. Among eight patients with acute brucellosis, anti-CP24 antibodies were detected in four in the 10 weeks following diagnosis, but the remaining four were negative during the whole follow-up (22 weeks). The frequency of anti-CP24 antibodies was also low among 24 patients with subacute brucellosis and 23 patients with chronic illness (29 and 26%, respectively). While all patients positive for anti-CP24 antibodies were also positive for antibodies to total cytoplasmic proteins of Brucella (CP), five were negative for antibodies to another cytoplasmic protein, the Brucella lumazine synthase (BLS). When a larger sample of 35 human sera negative for anti-BLS antibodies was assayed, 85.7% were positive for anti-CP24 antibodies, suggesting that the combined measurement of both reactivities could yield a higher sensitivity than any test alone. To test this hypothesis, an ELISA combining both antigens was designed. The percentage of positive results among chronic cases was higher for this assay than for the individual measurement of anti-CP24 or anti-BLS antibodies (83 versus 26 and 65%, respectively) and was closer to the value obtained for anti-CP antibodies (91%). The frequency of anti-CP24 antibodies is low in both canine and human brucellosis. In the latter case, however, an ELISA combining CP24 and BLS is more sensitive than assays measuring anti-CP24 or anti-BLS antibodies separately and almost as sensitive as the ELISA using CP.

Immunogenicity of the Brucella melitensis recombinant ribosome recycling factor-homologous protein and its cDNA.

A study was conducted to evaluate the immunogenicity of the Brucella melitensis ribosome recycling factor (RRF)-homologous protein (CP24). The CP24 gene was cloned, expressed in Escherichia coli and purified. The resulting purified recombinant protein (rCP24) produced delayed-type hypersensitivity (DTH) reactions in B. melitensis-infected mice but not in naive controls. Thus, we decided to characterise the immune responses generated with DNA vaccination (pcDNACP24) or immunisation with the rCP24 in adjuvant. Animals injected with pcDNACP24 exhibited a dominance of IgG2a to IgG1 while mice injected with rCP24 developed a higher response of IgG1 than IgG2a. Both immunisation protocols were capable of eliciting CP24-specific gamma interferon (IFN-gamma) producing cells. Spleen cells from pcDNACP24-immunised mice did not produce interleukin (IL)-4, IL-10 or up-regulation of IL-2 mRNA. Cells from rCP24-immunised mice produced IL-10, up-regulated IL-2 mRNA but did not produce IL-4. Neither immunisation with purified CP24 nor injection of pcDNACP24 protected mice against challenge with live smooth B. melitensis. However, the potential of CP24 for a Brucella diagnostic test based on an in vitro antigen (Ag)-specific IFN-gamma production or DTH test would be worth testing.

The 18-kDa cytoplasmic protein of Brucella species --an antigen useful for diagnosis--is a lumazine synthase.

Previous studies have shown that the detection of antibodies to an 18-kDa cytoplasmic protein of Brucella spp. is useful for the diagnosis of human and animal brucellosis. This protein has now been expressed in recombinant form in Escherichia coli. The recombinant protein is soluble only under reducing conditions, but alkylation with iodoacetamide renders it soluble in non-reducing media. As shown by gel exclusion chromatography, this soluble form arranges in pentamers of 90 kDa. The reactivity of human and animal sera against the recombinant protein was similar to that found with the native protein present in brucella cytoplasmic fraction, suggesting that the recombinant protein is correctly folded. The protein has low but significant homology (30%) with lumazine synthases involved in bacterial riboflavin biosynthesis, which also arrange as pentamers. Biological tests on the crude extract of the recombinant bacteria and on the purified recombinant protein showed that the biological activity of the Brucella spp. 18-kDa protein is that of lumazine synthase. Preliminary crystallographic analysis showed that the Brucella spp. lumazine synthase arranges in icosahedric capsids similar to those formed by the lumazine synthases of other bacteria. The high immunogenicity of this protein, potentially useful for the design of acellular vaccines, could be explained by this polymeric arrangement.