1,721,041 research outputs found
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
A 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
Increase in non-tuberculous mycobacteria isolated from humans in Tuscany, Italy, from 2004 to 2016
No full textIntroduction: Non-tuberculous mycobacteria (NTM) include all Mycobacterium species other than Mycobacterium tuberculosis complex and Mycobacterium leprae. NTM are a group of over 150 environmental species; they are generally endowed with low pathogenicity to human, however some species are associated with a variety of human diseases: respiratory tract infection are the most frequent, followed by lymphadenitis in children, disseminated infections in severely immunocompromised patients and skin infections. In Italy, the prevalence of NTM in human infections is largely unknown. The aim of the present survey is to report the epidemiology and recent trend of NTM infections in a region of central Italy, Tuscany, over the last 13 years, and provide a review of the recent literature on NTM isolation rates in different geographic regions.
Materials and Methods: The complete collection of NTM strains isolated from 50.150 clinical specimens at the Laboratory of Clinical Mycobacteriology of Pisa University Hospital, Italy, from 1 January 2004 to 31 December 2016 was included.
Results: In our setting, in the period 2004-2016 a total of 215 patients had cultures positive for NTM. The number of NTM isolates increased considerably from 5 isolates in 2004 to 36 in 2016; a sharp increase occurred in the last 5 years. Overall, 18 NTM species were isolated; the most common were M. avium, M. intracellulare and M. gordonae detected in respectively in 37.7%, 15.8% and 13.5% of NTM patients. Notably, M. kansasii, a pulmonary pathogen not reported before 2012, was repeatedly isolated in the last 5 years, representing 7,1% of total NTM isolates. In general, NTM isolates were largely prevalent in people older than 60 (59.5%); patients aged 1-10 year-old almost exclusively yielded M. avium and M. intracellulare. Of the 215 NTM clinical isolates, 77.2% were from respiratory specimens, 10.7% from lymph nodes, 2.3% from blood (yielding exclusively M. avium), and the remaining 9.8% from other clinical specimens.
Conclusions: The present study provides a snapshot of the prevalent NTM species in our setting and shows an increase in NTM isolation rate, which is in keeping with the general increase in NTM infections reported worldwide in the past two decades, although the distribution of the NTM prevalent species differs by geographic region
Genes of Mycobacterium tuberculosis H37Rv downregulated in the attenuated strain H37Ra are restricted to M. tuberculosis complex species
No full textBy comparing gene expression of virulent Mycobacterium tuberculosis H37Rv and attenuated strain H37Ra, we previously detected six genes that appear to be markedly downregulated in the attenuated strain compared with the virulent one. Three of these genes, i.e. Rv1345, Rv2770c, and Rv0288, code for proteins that can be 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 (Rindi et al., 1999). In this paper we searched for the above mentioned genes in Pvu II-digested genomic DNA of a number of mycobacterial species by southern blot analysis employing PCR-generated probes in high-stringency conditions. Hybridization signals were only found in species belonging to the M. tuberculosis complex, i.e., M. tuberculosis, M. bovis, including the BCG strain, and M. microti, but not in other mycobacterial species, including M. avium, M. intracellulare, M. malmoense, M. xenopi, M. kansasii, M. simiae, M. marinum, M. scrofulaceum, M. gordonae, M. fortuitum, and M. smegmantis. These results indicate that genes Rv1345, Rv2770c, Rv0288, Rv2336, Rv1320c, and Rv2819c are associated with the most virulent mycobacteria and further support their potential role in M. tuberculosis virulence
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
Genetic diversity of human isolates of Mycobacterium bovis assessed by Spoligotyping and Variable-Number-Tandem-Repeat genotyping
No full textA collection of clinical isolates including 9 Mycobacterium bovis bacille Calmette-Guérin (BCG), 37 M. bovis and 1 isolate identified as M. bovis/caprae intermediate, recovered from humans in Tuscany, Italy, from 1990 to 2009, was genotyped by spoligotyping and Variable Number Tandem Repeat (VNTR) typing. Spoligotyping detected 15 unique profiles; the "BCG-like" SIT482/SB0120 spoligotype was largely prevalent accounting for 63.8% of isolates. VNTR typing, based on the 15 VNTR loci commonly tested for Mycobacterium tuberculosis, detected 29 unique profiles; only 8 VNTR loci (VNTR 43, MIRU 04, QUB-11b, ETR-A, VNTR 47, MIRU 31, QUB-26 and VNTR 53) provided a satisfactory allelic diversity in the VNTR analysis. Combined together, spoligotyping and VNTR typing yielded 33 unique patterns and 5 clusters including a total of 19 isolates. Clustered isolates, further typed for additional 9 VNTR loci, finally yielded 3 distinct clusters including 3 M. bovis BCG isolates each, and 1 cluster of 6 M. bovis isolates. Minimum spanning tree analysis showed that, in spite of the many distinct VNTR profiles, most M. bovis isolates displayed a high phylogenetic proximity, due to the variation of a single VNTR allele, thus indicating that the population of human M. bovis isolates in our setting is relatively homogeneous and conserved
Polimorfismo ed espressione del gene ppe44 in isolati clinici di Mycobacterium tuberculosis
No full textLe proteine PPE di Mycobacterium tuberculosis, definite sulla base del motivo aminoacidico Pro-Pro-Glu, costituiscono una famiglia di 69 proteine polimorfiche ricche in glicina ritenute una probabile fonte di variabilità antigenica del bacillo tubercolare. Nel nostro laboratorio, nell’ambito di ricerche sul ruolo immunologico della proteina PPE44 nell’infezione tubercolare, è stato studiato l’eventuale polimorfismo e l’espressione del gene ppe44 in isolati clinici rappresentativi delle principali linee filogenetiche di M. tuberculosis. L’analisi PCR-RFLP mediante tre diversi enzimi di restrizione ha mostrato profili di digestione identici in tutti gli isolati; inoltre, la sequenza nucleotidica del gene ppe44 degli isolati non ha evidenziato mutazioni, ad eccezione di una sostituzione nucleotidica in posizione 581 (TTC→TCC, Phe→Ser) ritrovata unicamente negli isolati di genotipo Beijing. L’espressione di ppe44 negli isolati clinici, determinata mediante real-time RT-PCR quantitativa, normalizzata rispetto al gene di riferimento costitutivamente espresso sigA e confrontata con quella del ceppo di riferimento H37Rv, è risultata notevolmente variabile negli isolati clinici, rivelandosi paragonabile a quella di H37Rv in circa il 25% degli isolati, significativamente aumentata nel 60% degli isolati e diminuita nel rimanente 15%; gli isolati di genotipo Beijing hanno mostrato elevati livelli di espressione di ppe44. Tali risultati dimostrano quindi una sostanziale conservazione del gene ppe44 ed, al tempo stesso, una marcata variabilità dell’espressione di ppe44 tra gli isolati clinici. Per valutare se tale variabilità di espressione sia potenzialmente in grado di influenzare la risposta immunitaria dell’ospite, è stata determinata la risposta anticorpale anti-PPE44 in pazienti con tubercolosi in atto ed in soggetti sani con infezione tubercolare latente. E’ stata così dimostrata un’ampia variabilità nella risposta immunitaria verso PPE44 in pazienti con infezione tubercolare sia per quanto riguarda i titoli anticorpali, sia per quanto riguarda la proporzione dei soggetti rispondenti. Tali risultati suggeriscono che l’espressione differenziale dei geni ppe possa costituire una fonte di variabilità antigenica che può quindi avere implicazioni nella immunopatogenesi della tubercolosi
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
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