Publications (19) View all
-
Article: Identification of proteins from tuberculin purified protein derivative (PPD) by LC-MS/MS.
[show abstract] [hide abstract]
ABSTRACT: The tuberculin purified protein derivative (PPD) is a widely used diagnostic antigen for tuberculosis, however it is poorly defined. Most mycobacterial proteins are extensively denatured by the procedure employed in its preparation, which explains previous difficulties in identifying constituents from PPD to characterize their behaviour in B- and T-cell reactions. We here described a proteomics-based characterization of PPD from several different sources by LC-MS/MS, which combines the solute separation power of HPLC, with the detection power of a mass spectrometer. The technique is able to identify proteins from complex mixtures of peptide fragments. A total of 171 different proteins were identified among the four PPD samples (two bovine PPD and two avium PPD) from Brazil and UK. The majority of the proteins were cytoplasmic (77.9%) and involved in intermediary metabolism and respiration (24.25%) but there was a preponderance of proteins involved in lipid metabolism. We identified a group of 21 proteins that are present in both bovine PPD but were not detected in avium PPD preparation. In addition, four proteins found in bovine PPD are absent in Mycobacterium bovis BCG vaccine strain. This study provides a better understanding of the tuberculin PPD components leading to the identification of additional antigens useful as reagents for specific diagnosis of tuberculosis.Tuberculosis (Edinburgh, Scotland) 09/2009; 89(6):423-30. · 2.54 Impact Factor -
Article: Towards the immunoproteome of Neisseria meningitidis.
[show abstract] [hide abstract]
ABSTRACT: Despite the introduction of conjugated polysaccharide vaccines for many of the Neisseria meningitidis serogroups, neisserial infections continue to cause septicaemia and meningitis across the world. This is in part due to the difficulties in developing a, cross-protective vaccine that is effective against all serogroups, including serogroup B meningococci. Although convalescent N. meningitidis patients develop a natural long-lasting cross-protective immunity, the antigens that mediate this response remain unknown. To help define the target of this protective immunity we identified the proteins recognized by IgG in sera from meningococcal patients by a combination of 2D protein gels, western blots and mass spectrometry. Although a number of outer membrane antigens were identified the majority of the antigens were cytoplasmic, with roles in cellular processes and metabolism. When recombinant proteins were expressed and used to raise sera in mice, none of the antigens elicited a positive SBA result, however flow cytometry did demonstrate that some, including the ribosomal protein, RplY were localised to the neisserial cell surface.PLoS ONE 02/2009; 4(6):e5940. · 4.09 Impact Factor -
Article: Mycobacterium bovis BCG as a delivery system for the RAP-1 antigen from Babesia bovis.
[show abstract] [hide abstract]
ABSTRACT: Babesia bovis is the causative agent of babesiosis, a tick-borne disease that is a major cause of loss to livestock production in Latin America. Vaccination against Babesia species represents a major challenge against cattle morbidity and mortality in enzootic areas. The aim of this study was to evaluate the capacity of Bacille Calmette-Guerin (BCG) to deliver the rhoptry associated protein (RAP-1) antigen of B. bovis and to stimulate specific cellular and humoral immune responses in mice. Two of five mycobacterial expression vectors efficiently expressed the antigen. These constructs were subsequently studied in vivo following three immunization protocols. The construct with the greatest in vivo stability proved to be the one that induced the strongest immune responses. Our data support the hypothesis that specific T lymphocyte priming by rBCG can be employed as a component of a combined vaccine strategy to induce long-lasting humoral and cellular immune responsiveness towards B. bovis and encourage further work on the application of rBCG to the development of Babesia vaccines.Vaccine 02/2007; 25(6):1104-13. · 3.77 Impact Factor -
Article: Mutation in the P36 gene of Mycobacterium bovis provokes attenuation of the bacillus in a mouse model.
[show abstract] [hide abstract]
ABSTRACT: P36 is a member of a family of secreted proteins distributed throughout the genus Mycobacterium. The central domain of these proteins contains several amino acid PGLTS repeats, which differ considerably between species. P36, also called exported repetitive protein (Erp) in M. tuberculosis, has been shown to be associated with virulence since the disruption of its gene impaired multiplication of both virulent M. tuberculosis and M. bovis BCG in cultured macrophages and immunocompetent mice. In order to demonstrate that P36 is a putative virulence factor of wild-type Mycobacterium bovis we generated a P36 mutant by gene disruption and we evaluated its replication in spleen and lungs of infected mice. In this study, the mutant strain displays low levels of multiplication in mice, indicating that the P36 gene is important for in vivo growth of M. bovis.Tuberculosis 08/2005; 85(4):221-6. · 3.47 Impact Factor -
Article: Characterization of two novel Rhizobium leguminosarum bacteriophages from a field release site of genetically-modified rhizobia
[show abstract] [hide abstract]
ABSTRACT: Two Rhizobium leguminosarum biovar viceae bacteriophages with contrasting properties were isolated from a field site in which the survival of genetically modified R. leguminosarum inoculants had been monitored for several years. Inoculant strain RSM2004 was used as the indicator for phage isolation and propagation. One phage, RL1RES, was temperate and could not replicate in any of the 42 indigenous R. leguminosarum field isolates tested although nested PCR indicated that phage sequences were present in six of the isolates. The second phage, RL2RES, was virulent, capable of generalised transduction, contained DNA with modified cytosine residues, and was capable of infecting all field isolates tested although the GM inoculant strain CT0370 was resistant. Sequence with homology to RL2RES was detected by nested PCR in six of the 42 field-isolates. These were not the same isolates that showed homology to RL1RES. The implication of these findings for the survival of rhizobial inoculants, and the ecology of phages and their host bacteria, are discussed.Antonie van Leeuwenhoek 05/2001; 79(2):189-197. · 2.09 Impact Factor
