Clara Espitia

A Heterologous DNA Priming-Mycobacterium bovis BCG Boosting Immunization Strategy Using Mycobacterial Hsp70, Hsp65, and Apa Antigens Improves Protection against Tuberculosis in Mice

ABSTRACT Tuberculosis is responsible for >2 million deaths a year, and the number of new cases is rising worldwide. DNA vaccination combined with Mycobacterium bovis bacillus Calmette Guérin (BCG) represents a potential strategy for prevention of this disease. Here, we used a heterologous prime-boost immunization approach using a combination of DNA plasmids and BCG in order to improve the efficacy of vaccination against Mycobacterium tuberculosis infection in mice. As model antigens, we selected the M. tuberculosis Apa (for alanine-proline-rich antigen) and the immunodominant Hsp65 and Hsp70 mycobacterial antigens combined with BCG. We demonstrated that animals injected with a combination of DNA vectors expressing these antigens, when boosted with BCG, showed increased specific antimycobacterial immune responses compared to animals vaccinated with BCG alone. More importantly, the protection achieved with this regimen was also significantly better than with BCG alone.

Mycobacterium tuberculosis glycoproteomics based on ConA-lectin affinity capture of mannosylated proteins.

A Mycobacterium tuberculosis culture filtrate enriched with mannose-containing proteins was resolved by 2-DE gel. After ConA ligand blotting, 41 proteins were identified by mass spectrometry as putative glycoproteins with 34 of them new probably mannosylated proteins. These results contribute to the construction of the ConA affinity glycoprotein database of M. tuberculosis, and provide useful information for understanding the biological role of glycoproteins in mycobacteria.

Expression, Secretion, and Glycosylation of the 45- and 47-kDa Glycoprotein of Mycobacterium tuberculosis in Streptomyces lividans

ABSTRACT The gene encoding the 45/47 kDa glycoprotein (Rv1860) of Mycobacterium tuberculosis was expressed in Streptomyces lividans under its own promoter and under the thiostrepton-inducible Streptomyces promoter PtipA. The recombinant protein was released into the culture medium and, like the native protein, migrated as a double band at 45 and 47 kDa in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels. However, in contrast to the native protein, only the 47-kDa recombinant protein could be labeled with concanavalin A (ConA). Carbohydrate digestion with jack bean α-d-mannosidase resulted in a reduction in the molecular mass of the recombinant protein upper band and completely eliminated ConA binding. Two-dimensional gel electrophoresis revealed only one isoelectric point for the recombinant protein. Comparative fingerprinting analysis of the individually purified upper and lower recombinant protein bands, treated under the same conditions with specific proteases, resulted in similar peptide patterns, and the peptides had the same N-terminal sequence, suggesting that migration of the recombinant protein as two bands in SDS-PAGE gels could be due to differences in glycosylation. Mass spectrometry analysis of the recombinant protein indicated that as in native protein, both the N-terminal and C-terminal domains of the recombinant protein are glycosylated. Furthermore, it was determined that antibodies of human tuberculosis patients reacted mainly against the carbohydrate residues of the glycoprotein. Altogether, these observations show that expression of genes for mycobacterial antigens in S. lividans is very useful for elucidation of the functional role and molecular mechanisms of glycosylation in bacteria.

Specific bacterial genotypes of Mycobacterium tuberculosis cause extensive dissemination and brain infection in an experimental model

Meningeal tuberculosis is a severe type of extrapulmonary disease, which is thought to begin with respiratory infection, followed by hematogenous dissemination and brain infection. Host genetic susceptibility factors and specific mycobacterial substrains could be involved in its development. From an epidemiological study in Colombia, we selected three Mycobacterium tuberculosis clinical strains isolated from the cerebrospinal fluid (CSF) of patients with meningeal tuberculosis, and used them to infect BALB/c mice through the intratracheal route. These strains showed a distinctive spoligotype pattern. The course of infection in terms of strain virulence (mice survival, bacillary loads in lungs), bacilli dissemination and extrapulmonary infection (bacilli loads in blood, brain, liver, kidney and spleen), and immune responses (cytokine expression determined by real time PCR in brain and lung) was studied and compared with that induced by the laboratory strain H37Rv and other five clinical strains isolated from patients with pulmonary TB. All the clinical isolates from meningeal TB patients disseminated extensively through the hematogenous route infecting the brain, producing inflammation in the cerebral parenchyma and meninges, whereas H37Rv and clinical isolates from pulmonary TB patients showed very limited efficiency to infect the brain. Thus, it seems that mycobacterial strains with a distinctive genotype are able to disseminate extensively after the respiratory infection and infect the brain.

Peptide mimotopes of Mycobacterium tuberculosis carbohydrate immunodeterminants.

Cell-surface saccharides of Mycobacterium tuberculosis appear to be crucial factors in tuberculosis pathogenicity and could be useful antigens in tuberculosis immunodiagnosis. In the present study, we report the successful antigenic and immunogenic mimicry of mannose-containing cell-wall compounds of M. tuberculosis by dodecamer peptides identified by phage-display technology. Using a rabbit antiserum raised against M. tuberculosis cell-surface saccharides as a target for biopanning, peptides with three different consensus sequences were identified. Phage-displayed and chemically synthesized peptides bound to the anticarbohydrate antiserum. Rabbit antibodies elicited against the peptide QEPLMGTVPIRAGGGS recognize the mannosylated M. tuberculosis cell-wall antigens arabinomannan and lipoarabinomannan, and the glycosylated recombinant protein alanine/proline-rich antigen. Furthermore, antibodies were also able to react with mannan from Saccharomyces cerevisiae, but not with phosphatidylinositol dimannosides or arabinogalactan from mycobacteria. These results suggest that the immunogenic peptide mimics oligomannosidic epitopes. Interestingly, this report provides evidence that, in contrast with previously known carbohydrate mimotopes, no aromatic residues are necessary in a peptide sequence for mimicking unusual glycoconjugates synthesized by mycobacteria. The possible usefulness of the identified peptide mimotopes as surrogate reagents for immunodiagnosis and for the study of functional roles of the native non-peptide epitopes is discussed.

Differential expression of PE and PE_PGRS genes in Mycobacterium tuberculosis strains

In this study, the expression of the Mycobacterium tuberculosis PE, PE_polimorphic GC-rich sequences (PGRS) gene family encoding approximately 99 glycine-rich proteins was assayed by reverse-transcriptase polymerase chain reaction (RT-PCR) in M. tuberculosis H37Rv, Mycobacterium canettii and two clinical isolates of M. tuberculosis. Restriction analyses and sequencing of the RT-PCR products showed that all the strains expressed the PE Rv1172c gene while the PE_PGRS Rv3652 gene was only expressed by one of the M. tuberculosis clinical isolates, and the PE_PGRS Rv0578c was not expressed by M. canettii. It was also determined that the PE_PGRS Rv0278c and Rv0279c were not expressed by any of the studied strains. The data presented in this report show that the PE, PE_PGRS genes are differentially expressed in M. tuberculosis strains during in vitro growth. These findings suggest that PE, PE_PGRS genes may play a role in protein variation between M. tuberculosis strains.

New insights into protein O-mannosylation in actinomycetes

Glycosylation is a common post-translational modification of surface exposed proteins and lipids present in all kingdoms of life. Information derived from bacterial genome sequencing, together with proteomic and genomic analysis has allowed the identification of the enzymatic glycosylation machinery. Among prokaryotes, O-mannosylation of proteins has been found in the actinomycetes and resembles protein O-mannosylation in fungi and higher eukaryotes. In this review we summarize the main features of the biosynthetic pathway of O-mannosylation in prokaryotes with special emphasis on the actinomycetes, as well as the biological role of the glycosylated target proteins.

Binding and Activation of Human Plasminogen by Mycobacterium tuberculosis

ABSTRACT The first evidence of the interaction of Mycobacterium tuberculosis with the plasminogen system is herein reported. By FACScan analysis and affinity blotting, lysine-dependent binding of plasminogen to M. tuberculosis was demonstrated. The binding molecules were 30-, 60-, and 66-kDa proteins present in cell wall and soluble protein extracts. The activation of plasminogen, which occurred only in presence of fibrin and was not inhibited by the host serpin, α2-antiplasmin, was also demonstrated.

Bioinformatic identification of Mycobacterium tuberculosis proteins likely to target host cell mitochondria: virulence factors?

BackgroundM. tuberculosis infection either induces or inhibits host cell death, depending on the bacterial strain and the cell microenvironment. There is evidence suggesting a role for mitochondria in these processes.On the other hand, it has been shown that several bacterial proteins are able to target mitochondria, playing a critical role in bacterial pathogenesis and modulation of cell death. However, mycobacteria–derived proteins able to target host cell mitochondria are less studied.ResultsA bioinformaic analysis based on available genomic sequences of the common laboratory virulent reference strain Mycobacterium tuberculosis H37Rv, the avirulent strain H37Ra, the clinical isolate CDC1551, and M. bovis BCG Pasteur strain 1173P2, as well as of suitable bioinformatic tools (MitoProt II, PSORT II, and SignalP) for the in silico search for proteins likely to be secreted by mycobacteria that could target host cell mitochondria, showed that at least 19 M. tuberculosis proteins could possibly target host cell mitochondria. We experimentally tested this bioinformatic prediction on four M. tuberculosis recombinant proteins chosen from this list of 19 proteins (p27, PE_PGRS1, PE_PGRS33, and MT_1866). Confocal microscopy analyses showed that p27, and PE_PGRS33 proteins colocalize with mitochondria.ConclusionsBased on the bioinformatic analysis of whole M. tuberculosis genome sequences, we propose that at least 19 out of 4,246 M. tuberculosis predicted proteins would be able to target host cell mitochondria and, in turn, control mitochondrial physiology. Interestingly, such a list of 19 proteins includes five members of a mycobacteria specific family of proteins (PE/PE_PGRS) thought to be virulence factors, and p27, a well known virulence factor. P27, and PE_PGRS33 proteins experimentally showed to target mitochondria in J774 cells. Our results suggest a link between mitochondrial targeting of M. tuberculosis proteins and virulence.

Recombinant BCG Vaccine Candidates

Given the variable protective efficacy provided by Mycobacterium bovis BCG (Bacillus Calmette-Guérin), there is a concerted effort worldwide to develop better vaccines that could be used to reduce the burden of tuberculosis. Recombinant BCG (rBCG) are vaccine candidates that offer some potential in this area. In this paper, we will discuss the molecular methods used to generate rBCG, and the results obtained with some of these new vaccines as compared with the conventional BCG vaccine in diverse animal models. Tuberculosis vaccine candidates based on rBCG are promising candidates, and some of them are now being tested in clinical trials.