Project description:Comparison of gene expression profile of the whiB4 mutant strain of Mycobacterium tuberculosis with the wild type Mycobacterium tuberculosis H37RV Mtb WhiB4 mutant mRNA was compared with the mRNA of wtMtb H37RV under aerobic conditons Aerbic conditions OD600 nm of 0.4, MtbWhiB4KO vs wtMtb, biological replicates: 3 wt Mtb H37RV and 3 MtbWhiB4 KO
Project description:BACKGROUND: A yeast strain lacking the two genes SSA1 and SSA2, which encode cytosolic molecular chaperones, acquires thermotolerance as well as the mild heat-shocked wild-type yeast strain. We investigated the genomic response at the level of mRNA expression to the deletion of SSA1/2 in comparison with the mild heat-shocked wild-type using cDNA microarray. RESULTS: Yeast cDNA microarray analysis revealed that genes involved in the stress response, including molecular chaperones, were up-regulated in a similar manner in both the ssa1/2 deletion mutant and the mild heat-shocked wild-type. Genes involved in protein synthesis were up-regulated in the ssa1/2 deletion mutant, but were markedly suppressed in the mild heat-shocked wild-type. The genes involved in ubiquitin-proteasome protein degradation were also up-regulated in the ssa1/2 deletion mutant, whereas the unfolded protein response (UPR) genes were highly expressed in the mild heat-shocked wild-type. RT-PCR confirmed that the genes regulating protein synthesis and cytosolic protein degradation were up-regulated in the ssa1/2 deletion mutant. At the translational level, more ubiquitinated proteins and proteasomes were detected in the ssa1/2 deletion mutant, than in the wild-type, confirming that ubiquitin-proteasome protein degradation was up-regulated by the deletion of SSA1/2. CONCLUSION: These results suggest that the mechanism for rescue of denatured proteins in the ssa1/2 deletion mutant is different from that in the mild heat-shocked wild-type: Activated protein synthesis in the ssa1/2 deletion mutant supplies a deficiency of proteins by their degradation, whereas mild heat-shock induces UPR.
Project description:Mycobacterium tuberculosis genome contains four related sets of an operon called mce (mce1-4). The disruption of one of these operons, mce1, causes M. tuberculosis to become hypervirulent, whereas the mce3 and mce4 operon mutants are attenuated in mice. This study examined the phenotype of the mce2 operon mutant. The deletion of mce2 operon in M. tuberculosis H37Rv had no effect on bacterial growth in 7H9 liquid broth or survival within macrophages. However, RAW macrophage-like cells infected with the mutant strain were reduced in their ability to produce TNF-alpha, IL-6 and MCP-1. In C57BL/6 mouse lungs, the mce2 operon mutant and wild type H37Rv replicated similarly up to 20 weeks of infection. However, by 56 weeks of infection, all mice infected with the wild type H37Rv had died, while 80% of those infected with the mutant remained alive (P<0.0001). The proportion of affected lung parenchyma in mice infected with the mutant was substantially less than that of mice infected with the wild type for the same time periods of infection. These observations suggest that the mce2 operon mutant is attenuated, and that this attenuation is related not to the bacterial burden but to the mutant's decreased ability to elicit a type of immune response and lung pathology detrimental to the survival of the animal.
Project description:Tuberculosis is a worldwide health problem, given that one-third of the world's population is currently infected with Mycobacterium tuberculosis. Understanding the regulation of virulence on the molecular level will provide a better understanding of how M. tuberculosis can establish chronic infection. Using in vivo microarray analysis (IVMA), we previously identified a group of genes that are activated in BALB/c mouse lungs compared to in vitro cultures, including the rv0990c gene. Our analysis indicated that this gene is a member of the heat shock regulon and was activated under other stress conditions, including survival in macrophages or during the late phase of chronic tuberculosis in the murine lungs. Deletion of rv0990c from the genome of M. tuberculosis strain H37Rv affected the transcriptional profiles of many genes (n = 382) and operons involved in mycobacterial survival, including the dormancy regulon, ATP synthesis, respiration, protein synthesis, and lipid metabolism. Comparison of the proteomes of the mutant to those of the wild-type strain further confirmed the differential expression of 15 proteins, especially those involved in the heat shock response (e.g., DnaK and GrpE). Finally, the rv0990c mutant strain showed survival equivalent to that of the isogenic wild-type strain during active tuberculosis in guinea pigs, despite showing significant attenuation in BALB/c mice during the chronic phase of the disease. Overall, we suggest that rv0990c encodes a heat shock protein that plays an important role in mycobacterial virulence. Hence, we renamed rv0990c heat shock protein 22.5 (hsp22.5), reflecting its molecular mass.
Project description:Capreomycin, an important drug for the treatment of multidrug-resistant tuberculosis, is a macrocyclic peptide antibiotic produced by Saccharothrix mutabolis subspecies capreolus. The basis of resistance to this drug was investigated by isolating and characterizing capreomycin-resistant strains of Mycobacterium smegmatis and Mycobacterium tuberculosis. Colonies resistant to capreomycin were recovered from a library of transposon-mutagenized M. smegmatis. The transposon insertion site of one mutant was mapped to an open reading frame in the unfinished M. smegmatis genome corresponding to the tlyA gene (Rv1694) in the M. tuberculosis H37Rv genome. In M. smegmatis spontaneous capreomycin-resistant mutants, the tlyA gene was disrupted by one of three different naturally occurring insertion elements. Genomic DNAs from pools of transposon mutants of M. tuberculosis H37Rv were screened by PCR by using primers to the tlyA gene and the transposon to detect mutants with an insertion in the tlyA gene. One capreomycin-resistant mutant was recovered that contained the transposon inserted at base 644 of the tlyA gene. Complementation with the wild-type tlyA gene restored susceptibility to capreomycin in the M. smegmatis and M. tuberculosis tlyA transposon mutants. Mutations were found in the tlyA genes of 28 spontaneous capreomycin-resistant mutants generated from three different M. tuberculosis strains and in the tlyA genes of capreomycin-resistant clinical isolates. In in vitro transcription-translation assays, ribosomes from tlyA mutant but not tlyA(+) strains resist capreomycin inhibition of transcription-translation. Therefore, TlyA appears to affect the ribosome, and mutation of tlyA confers capreomycin resistance.
Project description:The Mycobacterium tuberculosis dosR gene (Rv3133c) is part of an operon, Rv3134c-Rv3132c, and encodes a response regulator that has been shown to be upregulated by hypoxia and other in vitro stress conditions and may be important for bacterial survival within granulomatous lesions found in tuberculosis. DosR is activated in response to hypoxia and nitric oxide by DosS (Rv3132c) or DosT (Rv2027c). We compared the virulence levels of an M. tuberculosis dosR-dosS deletion mutant (DeltadosR-dosS [DeltadosR-S]), a dosR-complemented strain, and wild-type H37Rv in rabbits, guinea pigs, and mice infected by the aerosol route and in a mouse hollow-fiber model that may mimic in vivo granulomatous conditions. In the mouse and the guinea pig models, the DeltadosR-S mutant exhibited a growth defect. In the rabbit, the DeltadosR-S mutant did not replicate more than the wild type. In the hollow-fiber model, the mutant phenotype was not different from that of the wild-type strain. Our analyses reveal that the dosR and dosS genes are required for full virulence and that there may be differences in the patterns of attenuation of this mutant between the animal models studied.
Project description:The proteins belonging to the WhiB superfamily are small global transcriptional regulators typical of actinomycetes. In this paper, we characterize the role of WhiB5, a Mycobacterium tuberculosis protein belonging to this superfamily. A null mutant was constructed in M. tuberculosis H37Rv and was shown to be attenuated during both progressive and chronic mouse infections. Mice infected with the mutant had smaller bacillary burdens in the lungs but a larger inflammatory response, suggesting a role of WhiB5 in immunomodulation. Most interestingly, the whiB5 mutant was not able to resume growth after reactivation from chronic infection, suggesting that WhiB5 controls the expression of genes involved in this process. The mutant was also more sensitive than the wild-type parental strain to S-nitrosoglutathione (GSNO) and was less metabolically active following prolonged starvation, underscoring the importance of GSNO and starvation in development and maintenance of chronic infection. DNA microarray analysis identified 58 genes whose expression is influenced by WhiB5, including sigM, encoding an alternative sigma factor, and genes encoding the constituents of two type VII secretion systems, namely, ESX-2 and ESX-4.
Project description:Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis, is among the top 10 leading causes of death worldwide. The treatment course for TB is challenging; it requires antibiotic administration for at least 6 months, and bacterial drug resistance makes treatment even more difficult. Understanding the mechanisms of resistance is important for improving treatment. To investigate new mechanisms of isoniazid (INH) resistance, we obtained three INH-resistant (INH-R) M. tuberculosis clinical isolates collected by the Taiwan Centers for Disease Control (TCDC) and sequenced genes known to harbor INH resistance-conferring mutations. Then, the relationship between the mutations and INH resistance of these three INH-R isolates was investigated. Sequencing of the INH-R isolates identified three novel katG mutations resulting in R146P, W341R, and L398P KatG proteins, respectively. To investigate the correlation between the observed INH-R phenotypes of the clinical isolates and these katG mutations, wild-type katG from H37Rv was expressed on a plasmid (pMN437-katG) in the isolates, and their susceptibilities to INH were determined. The plasmid expressing H37Rv katG restored INH susceptibility in the two INH-R isolates encoding the W341R KatG and L398P KatG proteins. In contrast, no phenotypic change was observed in the KatG R146P isolate harboring pMN437-katG. H37Rv isogenic mutant with W341R KatG or L398P KatG was further generated. Both showed resistant to INH. In conclusion, W341R KatG and L398P KatG conferred resistance to INH in M. tuberculosis, whereas R146P KatG did not affect the INH susceptibility of M. tuberculosis.
Project description:Posttranscriptional modifications of bacterial rRNA serve a variety of purposes, from stabilizing ribosome structure to preserving its functional integrity. Here, we investigated the functional role of one rRNA modification in particular-the methylation of guanosine at position 518 (G518) of the 16S rRNA in Mycobacterium tuberculosis. Based on previously reported evidence that G518 is located 5 Å; from proline 44 of ribosomal protein S12, which interacts directly with the mRNA wobble position of the codon:anticodon helix at the A site during translation, we speculated that methylation of G518 affects protein translation. We transformed reporter constructs designed to probe the effect of functional lesions at one of the three codon positions on translational fidelity into the wild-type strain, H37Rv, and into a ΔgidB mutant, which lacks the methyltransferase (GidB) that methylates G518. We show that mistranslation occurs less in the ΔgidB mutant only in the construct bearing a lesion in the wobble position compared to H37Rv. Thus, the methylation of G518 allows mistranslation to occur at some level in order for translation to proceed smoothly and efficiently. We also explored the role of methylation at G518 in altering the susceptibility of M. tuberculosis to streptomycin (SM). Using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), we confirmed that G518 is not methylated in the ΔgidB mutant. Furthermore, isothermal titration calorimetry experiments performed on 70S ribosomes purified from wild-type and ΔgidB mutant strains showed that methylation significantly enhances SM binding. These results provide a mechanistic explanation for the low-level, SM-resistant phenotype observed in M. tuberculosis strains that contain a gidB mutation.