The numerous sigma () factors present in Mycobacterium tuberculosis are indicative of the adaptability of this pathogen to different environmental conditions. In this report, we describe the M. tuberculosis B regulon and the phenotypes of an M. tuberculosis sigB mutant strain exposed to cell envelope stress, oxidative stress, and hypoxia. The sigB mutant was especially defective in survival under hypoxic conditions in vitro, but it was not attenuated for growth in THP-1 cells or during mouse and guinea pig infection.factors are components of RNA polymerases that bind to the enzyme's core subunits and give promoter specificity. The presence of 13 factors in Mycobacterium tuberculosis reflects the ability of this microorganism to adapt to various stress conditions that are likely encountered during host infection and that make M. tuberculosis a successful pathogen.B is closely related to the primary sigma factor A in terms of amino acid sequence (4). sigB, the structural gene for B , is induced by different stresses (12) and is positively regulated by three extracytoplasmic function sigma factors, E , H , and L (3,13,14). In vitro transcription studies showed that isolated mycobacterial RNA polymerases containing E , H , and L can transcribe sigB using the same transcription start site (3).F -containing RNA polymerase was also shown to transcribe sigB in these studies by using a different transcriptional start site. However, transcriptome studies with sigF mutants or strains overexpressing F show no changes in sigB expression (7, 11), suggesting that the F RNA polymerase transcription of sigB observed in biochemical experiments may not be physiological. The autoregulation of B has also been recently determined by primer extension and reverse transcription-PCR (RT-PCR) (11).In M. tuberculosis, the response to cell envelope is regulated by E and the response to oxidative stress and heat shock is regulated by H , and B is a component of both regulons (13,14). The fact that sigB expression is controlled by many regulatory pathways suggests that B plays a central role in the M. tuberculosis stress response. In this report, we describe the in vitro phenotype of an M. tuberculosis sigB mutant exposed to stress conditions related to the activation of E and H . To analyze the extent to which the response to stress of E and H is transmitted through B , we studied the B regulon activated by cell envelope and oxidative stress in vitro. We also evaluated the growth of the M. tuberculosis sigB mutant strain in THP-1 macrophage-like cells and in vivo in the mouse and guinea pig models of infection. MATERIALS AND METHODSBacterial strains, media, and growth conditions. Escherichia coli strains JM109 and GM161 were grown in Luria broth (LB) (Difco) at 37°C. Antibiotic concentrations used to isolate selectable markers in E. coli were as follows: kanamycin, 50 g/ml; streptomycin, 20 g/ml; and hygromycin, 50 g/ml. The M. tuberculosis strains created in this study were derivatives of M. tuberculosis H37Rv. All M. tuberculosis culture c...
The in vitro activities of human -defensin 3 (hBD-3) alone or combined with lysozyme, metronidazole, amoxicillin, and chlorhexidine were investigated with the oral bacteria Streptococcus mutans, Streptococcus sanguinis, Streptococcus sobrinus, Lactobacillus acidophilus, Actinobacillus actinomycetemcomitans, and Porphyromonas gingivalis. hBD-3 showed bactericidal activity against all of the bacterial species tested. The bactericidal effect was enhanced when the peptide was used in combination with the antimicrobial agents mentioned above.
Little is known about the effect of removable orthodontic appliances on oral colonisation by mutans streptococci (MS). In the present study, the frequency of isolation and serotype distribution of MS were evaluated in two groups of children, one undergoing therapy with removable appliances and the other not subjected to any kind of orthodontic treatment, respectively. Streptococci isolated from dental plaque samples from both groups of children were identified as mutans streptococci on the basis of their morphological and biochemical properties and were then serotyped in an enzyme immuno-assay using monoclonal antibodies. The number of subjects harbouring MS in their dental plaque was statistically higher in the group of orthodontic children without caries experience (CF) in comparison with CF children of the control group (10/12, 83.3% vs. 15/44, 34%). No clear difference was observed in the distribution of the different MS serotypes between the experimental and control group: S. mutans c,f serotype was the most frequently isolated in both groups of children followed by S. mutans serotype e and S. sobrinus serotype g. Such results suggest that the use of removable appliances may lead to the creation of new retentive areas and surfaces, which favour the local adherence and growth of MS. The data obtained stress the importance of a careful monitoring of patients treated orthodontically for risk of caries development.
Autophagy is a primordial eukaryotic pathway, which provides the immune system with multiple mechanisms for the elimination of invading pathogens including Mycobacterium tuberculosis (Mtb). As a consequence, Mtb has evolved different strategies to hijack the autophagy process. Given the crucial role of human primary dendritic cells (DC) in host immunity control, we characterized Mtb-DC interplay by studying the contribution of cellular microRNAs (miRNAs) in the post-transcriptional regulation of autophagy related genes. From the expression profile of de-regulated miRNAs obtained in Mtb-infected human DC, we identified 7 miRNAs whose expression was previously found to be altered in specimens of TB patients. Among them, gene ontology analysis showed that miR-155, miR-155* and miR-146a target mRNAs with a significant enrichment in biological processes linked to autophagy. Interestingly, miR-155 was significantly stimulated by live and virulent Mtb and enriched in polysome-associated RNA fraction, where actively translated mRNAs reside. The putative pair interaction among the E2 conjugating enzyme involved in LC3-lipidation and autophagosome formation-ATG3-and miR-155 arose by target prediction analysis, was confirmed by both luciferase reporter assay and Atg3 immunoblotting analysis of miR-155-transfected DC, which showed also a consistent Atg3 protein and LC3 lipidated form reduction. Late in infection, when miR-155 expression peaked, both the level of Atg3 and the number of LC3 puncta per cell (autophagosomes) decreased dramatically. In accordance, miR-155 silencing rescued autophagosome number in Mtb infected DC and enhanced autolysosome fusion, thereby supporting a previously unidentified role of the miR-155 as inhibitor of ATG3 expression. Taken together, our findings suggest how Mtb can manipulate cellular miRNA expression to regulate Atg3 for its own survival, and highlight the importance to develop novel therapeutic strategies against tuberculosis that would boost autophagy.
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