170,066 research outputs found
Identification of one insertion site of IS6110 in Mycobacterium tuberculosis H37Ra and analysis of the RvD2 deletion in M. tuberculosis clinical isolates
No full textMycobacterium tuberculosis H37Rv and the attenuated strain H37Ra were used as a model to investigate the virulence properties of M. tuberculosis at the genetic level. To test whether transposition of the insertion element IS6110 might be involved in the loss of virulence of strain H37Ra, the nucleotide sequence of a differential IS6110-positive restriction fragment detected in strain H37Ra, but not in strain H37Rv, was determined. The region flanking the 3' end of the IS6110 element showed partial sequence homology with internal sequences of M. tuberculosis H37Rv genes plcA, plcB and plcC, each one coding for phospholipase C, a well-known bacterial virulence factor. A 100% homology was found between the IS6110-flanking region and an internal sequence of M. bovis plcD, a further phospholipase C gene that is truncated and partly lost in strain H37Rv in the so-called RvD2 deletion. This result indicates that the differential restriction fragment of strain H37Ra originally stems from the plcD gene interrupted by the insertion of the IS6110 element. The occurrence of the RvD2 deletion was then investigated in 45 clinical isolates of M. tuberculosis by Southern blot. The deletion was demonstrated in 15 isolates; the entire RvD2 region (including the undisrupted plcD gene) was detected in 29 isolates, whereas only one isolate showed the RvD2 region in which the plcD gene was interrupted by an IS6110 insertion. It is concluded that disruption of the plcD gene and deletion of the RvD2 region by IS6110 insertion have no consequence for the virulence of M. tuberculosis, although the role of phospholipase C as a virulence factor of M. tuberculosis remains debatable
Molecular typing of Mycobacterium avium isolates by sequencing of the 16S-23S rDNA internal transcribed spacer and comparison with IS 1245-based fingerprinting
No full textThe nucleotide sequence of the variable 16S-23S rDNA internal transcribed spacer (ITS) was determined in 32 strains of Mycobacterium avium, including 29 clinical isolates, two environmental isolates and the reference strain M. avium ATCC 35712. The results were compared with those obtained by the IS1245-based restriction fragment length polymorphism (RFLP) assay. The strains belonged to three distinct ITS sequevars, Mav-A, Mav-B and Mav-C. Sixteen of 17 isolates of the Mav-B sequevar were from HIV-positive patients; the Mav-A sequevar included six and five isolates from HIV-positive and HIV-negative individuals, respectively, as well as the two environmental isolates and the M. avium reference strain ATCC 35712; only one isolate, from a HIV-infected patient, belonged to the Mav-C sequevar. IS1245-RFLP analysis of M. avium isolates of sequevars Mav-A and Mav-B showed that isolates occurring in clusters of identical or highly related RFLP patterns generally belong to the same sequevar, and that M. avium strains belonging to the same sequevar may present distinct and unrelated IS1245-RFLP patterns. The question of the molecular markers specific for M. avium clones pathogenic for man is discussed
Immune responses to protein PPE44 (Rv2770c) in mice infected with Mycobacterium bovis BCG
No full textBackground
The PPE protein family of Mycobacterium tuberculosis includes 69 glycine-rich proteins. Their role in tuberculosis is unknown, but it has been speculated that they may have an important immunological significance. In this investigation, the immunogenicity of the ppe44 (Rv2770c) gene product was evaluated in mice infected with Mycobacterium bovis BCG. Moreover, the protective efficacy of a DNA vaccine expressing ppe44 was evaluated in mice.
Methods
The gene ppe44 of M. tuberculosis H37Rv was cloned in E. coli and the purified recombinant protein (rPPE44) was used to evaluate antibody, cytokine and DTH responses in BALB/c mice infected with M. bovis BCG. The potential protective efficacy of PPE44 was evaluated in BALB/c and C57Bl/6 mice immunized intramuscularly with a DNA vaccine expressing ppe44 (ppe44-DNA) and challenged intravenously with M. bovis BCG.
Results
BALB/c mice infected subcutaneously developed high titers of anti-rPPE44 IgG1, but no IgG2a antibodies. Cultures of rPPE44-primed cells from draining lymph nodes and spleen produced low levels of IFN-; a moderate degree of DTH was observed following rPPE44 intracutaneous challenge. Similar results were obtained in mice infected intravenously.
BALB/c mice immunized with ppe44-DNA developed anti-rPPE44 IgG1 predominant over IgG2a antibodies; rPPE44-primed spleen cell cultures did not produce significant levels of IFN-. This regime of immunization conferred a moderate protection against an intravenous challenge with M. bovis BCG, as shown by the reduction of CFUs in the lungs compared to control mice immunized with the empty vector. C57Bl/6 mice immunized with ppe44-DNA developed comparable titers of anti-rPPE44 IgG1 and IgG2c antibodies; rPPE44-primed spleen cell cultures produced IFN-but no IL-4. After an intravenous challenge with M. bovis BCG, a one-log10 reduction of CFUs was detected in the lungs of ppe44-DNA immunized animals.
Conclusions
PPE44 represents a novel mycobacterial antigen expressed during infection by M. bovis BCG in mice. Both subcutaneous and intravenous infections seem to polarize the immune response to PPE44 towards a Th2 phenotype. Immunization of mice with ppe44-DNA confers a partial protection against an intravenous challenge with M. bovis BCG. The protective efficacy of PPE44 needs to be further evaluated by other experimental models aimed at enhancing the Th1-type immune responsiveness
Search for genes potentially involved in Mycobacterium tuberculosis virulence by mRNA differential display
No full textAn mRNA differential display (DD) assay was developed
to compare gene expression between Mycobacterium
tuberculosis H37Rv and its avirulent mutant
H37Ra. The DD protocol made use of an oligo(dT) to
prime reverse-transcriptase (RT)-dependent transcription
of poly-A tailed mRNAs and a PCR amplification of
the RT products by using ten 12-base arbitrary primers
in all their pair combinations. This analysis yielded 745
and 708 bands, including 52 and 15 differentially generated
bands, in the strains H37Rv and H37Ra, respectively.
Six cDNAs that appeared to be expressed in
H37Rv, but not in H37Ra, were reamplified and cloned
and at least 10 inserts were sequenced for each cloned
cDNA. After resolving discrepant results, 6 inserts were
found highly homologous to M. tuberculosis H37Rv
genes. Three of these, i.e., genes Rv2770c, Rv1345, and
Rv0288, coding respectively for a member of the PPE
protein family, a probable polyketide synthase, and a
member of the protein family containing ESAT-6, have
been predictively associated to immunological or pathogenetic
aspects of M. tuberculosis infection; the other
genes, i.e., Rv2336, Rv1320c, and Rv2819c, code for proteins
with unknown functions. These results show that
mRNA DD methodology can represent a potential tool
for investigation of M. tuberculosis gene expression
Large Sequence Polymorphisms of the Euro-American lineage of Mycobacterium tuberculosis: a phylogenetic reconstruction and evidence for convergent evolution in the DR locus
No full textThe Euro-American lineage of the Mycobacterium tuberculosis complex consists of 10 sublineages, each defined by a deletion of a large genomic region (RD, region of difference); by spoligotyping, that probes the polymorphism of the Direct Repeat (DR) locus, the Euro-American strains are classified into 5 lineages (T, Haarlem, LAM, S and X) and 34 sublineages, but the relationships between the RD-defined sublineages and the spoligotype groupings are largely unclear. By testing a global sample of 158 Euro-American strains, mutually exclusive deletions of RD115, RD122, RD174, RD182, RD183, RD193, RD219, RD726 or RD761 were found in 122 strains; deletion of RD724, typical of strains from Central Africa, was not found. The RD-defined sublineages, tested for katG463/gyrA95 polymorphism, belonged to Principal Genotypic Group (PGG) 2, with the exception of RD219 sublineage belonging to PGG3; the 36 strains with no deletion were of either PGG2 or 3. Based on these polymorphisms, a phylogenetic reconstruction of the Euro-American lineage, that integrates the previously reported phylogeny, is proposed. Although certain deletions were found to be associated to certain spoligotype lineages (i.e., deletion RD115 to T and LAM, RD174 to LAM, RD182 to Haarlem, RD219 to T), our analysis indicates a general lack of concordance between RD-defined sublineages and spoligotype groupings. Moreover, of the 42 spoligotypes detected among the study strains, sixteen were shared by strains belonging to different RD sublineages. IS6110-RFLP analysis of strains sharing spoligotypes confirmed a poor genetic relatedness between strains of different RD sublineages. These findings provide evidence for the occurrence of a high degree of homoplasy in the DR locus leading to convergent evolution to identical spoligotypes. The incongruence between Large Sequence Polymorphism and spoligotype polymorphism argues against the use of spoligotyping for establishing phylogenetic relationships within the Euro-American lineage
Most human isolates of Mycobacterium avium Mav-A and Mav-B are strong producers of hemolysin, a putative virulence factor
No full textHemolysin was quantified in 58 isolates of Mycobacterium avium from human, animal, and environmental sources. Human Mav-A and Mav-B isolates were the strongest producers; in contrast, animal and environmental Mav-A isolates and human, animal, and environmental Mav-C organisms were low-level producers. Hemolysin production was not restricted to isolates causing invasive infections
Ricerca di geni di virulenza di Mycobacterium tuberculosis mediante differential display
No full textA duplex real-time PCR assay for detection of mutT4 and mutT2 mutations in Mycobacterium tuberculosis of Beijing genotype
No full textTo determine the polymorphism of mutT genes of Mycobacterium tuberculosis of Beijing genotype, we
developed a duplex real-time PCR assay based on hybridization probes for the Roche LightCycler instrument.
The assay rapidly detects mutations at codons 48 and 58 of genes mutT4 and at mutT2, respectively
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