In vitro activity of bedaquiline against rapidly growing nontuberculous mycobacteria

Bedaquiline (BDQ) has been proven to be effective in the treatment of multidrug-resistant tuberculosis. We hypothesized that BDQ could be a potential agent to treat nontuberculous mycobacterial (NTM) infection. The objective of this study was to evaluate the in vitro activity of BDQ against rapidly growing mycobacteria by assessing the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) against 18 NTM strains. For MIC determination we performed the resazurin microtitre assay broth dilution, and for the MBC the c.f.u. was determined. BDQ exhibited a strong inhibitory effect against most NTM tested; however, for some NTM strains the MBC was significantly higher than the MIC. A new finding is that Mycobacterium flavescens has a mutation in the gene atpE associated with natural resistance to BDQ. These preliminary promising results demonstrate that BDQ could be potentially useful for the treatment of NTM

The transcriptome of Mycobacterium tuberculosis in a lipid-rich dormancy model through RNAseq analysis

Tuberculosis (TB) is currently the number one killer among infectious diseases worldwide. Lipids are abundant molecules during the infectious cycle of Mycobacterium tuberculosis (Mtb) and studies better mimicking its actual metabolic state during pathogenesis are needed. Though most studies have focused on the mycobacterial lipid metabolism under standard culture conditions, little is known about the transcriptome of Mtb in a lipid environment. Here we determined the transcriptome of Mtb H37Rv in a lipid-rich environment (cholesterol and fatty acid) under aerobic and hypoxic conditions, using RNAseq. Lipids significantly induced the expression of 368 genes. A main core lipid response was observed involving efflux systems, iron caption and sulfur reduction. In co-expression with ncRNAs and other genes discussed below, may act coordinately to prepare the machinery conferring drug tolerance and increasing a persistent population. Our findings could be useful to tag relevant pathways for the development of new drugs, vaccines and new strategies to control TB

Detection of a putative TetR-like gene related to Mycobacterium bovis BCG growth in cholesterol using a gfp-transposon mutagenesis system

In vitro transposition is a powerful genetic tool for identifying mycobacterial virulence genes and studying virulence factors in relation to the host. Transposon shuttle mutagenesis is a method for constructing stable insertions in the genome of different microorganisms including mycobacteria. Using an IS1096 derivative, we have constructed the Tngfp, a transposon containing a promoterless green fluorescent protein (gfp) gene. This transposon was able to transpose randomly in Mycobacterium bovis BCG. Bacteria with a single copy of the gfp gene per chromosome from an M. bovis BCG::Tngfp library were analyzed and cells exhibiting high levels of fluorescence were detected by flow cytometry. Application of this approach allowed for the selection of a mutant, BCG_2177c::Tngfp (BCG-Tn), on the basis of high level of long-standing fluorescence at stationary phase. This BCG-Tn mutant showed some particular phenotypic features compared to the wild type strain, mainly during stationary phase, when cholesterol was used as a sole carbon source, thus supporting the relationships of the targeted gene with the regulation of cholesterol metabolism in this bacteria. This approach showed that Tngfp is a potentially useful tool for studying the involvement of the targeted loci in metabolic pathways of mycobacteria

Mycobacterial transcriptional signals: requirements for recognition by RNA polymerase and optimal transcriptional activity

Majority of the promoter elements of mycobacteria do not function well in other eubacterial systems and analysis of their sequences has established the presence of only single conserved sequence located at the −10 position. Additional sequences for the appropriate functioning of these promoters have been proposed but not characterized, probably due to the absence of sufficient number of strong mycobacterial promoters. In the current study, we have isolated functional promoter-like sequences of mycobacteria from the pool of random DNA sequences. Based on the promoter activity in Mycobacterium smegmatis and score assigned by neural network promoter prediction program, we selected one of these promoter sequences, namely A(37) for characterization in order to understand the structure of housekeeping promoters of mycobacteria. A(37)–RNAP complexes were subjected to DNase I footprinting and subsequent mutagenesis. Our results demonstrate that in addition to −10 sequences, DNA sequence at −35 site can also influence the activity of mycobacterial promoters by modulating the promoter recognition by RNA polymerase and subsequent formation of open complex. We also provide evidence that despite exhibiting similarities in −10 and −35 sequences, promoter regions of mycobacteria and Escherichia coli differ from each other due to differences in their requirement of spacer sequences between the two positions

First insights into the genetic diversity of Mycobacterium tuberculosis isolates from HIV-infected Mexican patients and mutations causing multidrug resistance

<p>Abstract</p> <p>Background</p> <p>The prevalence of infections with <it>Mycobacterium tuberculosis </it>(MTb) and nontuberculous mycobacteria (NTM) species in HIV-infected patients in Mexico is unknown. The aims of this study were to determine the frequency of MTb and NTM species in HIV-infected patients from Mexico City, to evaluate the genotypic diversity of the <it>Mycobacterium tuberculosis </it>complex strains, to determine their drug resistance profiles by colorimetric microplate Alamar Blue assay (MABA), and finally, to detect mutations present in <it>kat</it>G, <it>rpo</it>B and <it>inh</it>A genes, resulting in isoniazid (INH) and rifampin (RIF) resistance.</p> <p>Results</p> <p>Of the 67 mycobacterial strains isolated, 48 were identified as MTb, 9 as <it>M. bovis</it>, 9 as <it>M. avium </it>and 1 as <it>M. intracellulare</it>. IS<it>6110</it>-RFLP of 48 MTb strains showed 27 profiles. Spoligotyping of the 48 MTb strains yielded 21 patterns, and 9 <it>M. bovis </it>strains produced 7 patterns. Eleven new spoligotypes patterns were found. A total of 40 patterns were produced from the 48 MTb strains when MIRU-VNTR was performed. Nineteen (39.6%) MTb strains were resistant to one or more drugs. One (2.1%) multidrug-resistant (MDR) strain was identified. A novel mutation was identified in a RIF-resistant strain, GAG → TCG (Glu → Ser) at codon 469 of <it>rpo</it>B gene.</p> <p>Conclusions</p> <p>This is the first molecular analysis of mycobacteria isolated from HIV-infected patients in Mexico, which describe the prevalence of different mycobacterial species in this population. A high genetic diversity of MTb strains was identified. New spoligotypes and MIRU-VNTR patterns as well as a novel mutation associated to RIF-resistance were found. This information will facilitate the tracking of different mycobacterial species in HIV-infected individuals, and monitoring the spread of these microorganisms, leading to more appropriate measures for tuberculosis control.</p

The lack of the TetR-like repressor gene BCG_2177c (Rv2160A) may help mycobacteria overcome intracellular redox stress and survive longer inside macrophages when surrounded by a lipid environment

Mycobacteria, like other microorganisms, survive under different environmental variations by expressing an efficient adaptive response, oriented by regulatory elements, such as transcriptional repressors of the TetR family. These repressors in mycobacteria also appear to be related to cholesterol metabolism. In this study, we have evaluated the effect of a fatty acid (oleic–palmitic–stearic)/cholesterol mixture on some phenotypic and genotypic characteristics of a tetR-mutant strain (BCG_2177c mutated gene) of M. bovis BCG, a homologous of Rv2160A of M. tuberculosis. In order to accomplish this, we have analyzed the global gene expression of this strain by RNA-seq and evaluated its neutral-lipid storage capacity and potential to infect macrophages. We have also determined the macrophage response by measuring some pro- and anti-inflammatory cytokine expressions. In comparison with wild-type microorganisms, we showed that the mutation in the BCG_2177c gene did not affect the growth of M. bovis BCG in the presence of lipids but it probably modified the structure/composition of its cell envelope. Compared to with dextrose, an overexpression of the transcriptome of the wild-type and mutant strains was observed when these mycobacteria were cultured in lipids, mainly at the exponential phase. Twelve putative intracellular redox balance maintenance genes and four others coding for putative transcriptional factors (including WhiB6 and three TetR-like) were the main elements repeatedly overexpressed when cultured in the presence of lipids. These genes belonged to the central part of what we called the “genetic lipid signature” for M. bovis BCG. We have also found that all these mycobacteria genotypic changes affected the outcome of BCG-infected macrophages, being the mutant strain most adapted to persist longer inside the host. This high persistence result was also confirmed when mutant-infected macrophages showed overexpression of the anti-inflammatory cytokine TGF-ß versus pro-inflammatory cytokines. In summary, the lack of this TetR-like repressor expression, within a lipid environment, may help mycobacteria overcome intracellular redox stress and survive longer inside their host

Measurement of the Rates of Synthesis of Three Components of Ribosomes of Mycobacterium fortuitum: A Theoretical Approach to qRT-PCR Experimentation

BACKGROUND: Except for the ribosomal protein L12 (rplL), ribosomal proteins are present as one copy per ribosome; L12 (rplL) is unusual because it is present as four copies per ribosome. Thus, the strategies used by Mycobacterium fortuitum to regulate ribosomal protein synthesis were investigated, including evaluations of the rates of chain elongations of 16S rRNA, rplL and ribosomal protein S12 (rpsL). METHODOLOGY: RNA was isolated from cell cultures and cDNA was prepared. The numbers of cDNA copies of 16S rRNA, precursor-16S rRNA and transcripts of rpsL and rplL were quantified by qRT-PCR and then related to the rates of 16S rRNA, rpsL and rplL chain elongations by means of a mathematical framework for coupled transcription/translation. PRINCIPAL FINDINGS: The rates of synthesis of 16S rRNA, rpsL and rplL respectively were found to be approximately 50 x 10(3) nucleotides h(-1), 1.6 x 10(3) amino acid residues h(-1) and 3.4 x 10(3) amino acid residues h(-1). The number of transcripts of rplL was approximately twice that of rpsL. These data account for the presence of one copy of rpsL and four copies of rplL per ribosome, and reveal that the rate of M. fortuitum ribosome synthesis was closer to that of M. tuberculosis than to E. coli. Except for rplJ, the elongation rate obtained for rpsL was inferred to be appropriate for all other proteins present as one copy per ribosome. SIGNIFICANCE: The results obtained provide the basis for a comprehensive view of the kinetics of ribosome synthesis, and of the ways that bacterial cells utilize genes encoding ribosomal proteins. The methodology also applies to proteins involved in transcription, energy generation and to bacterial proteins in general. The method proposed for measuring the fidelity of cDNA preparations is intrinsically much more sensitive than procedures that measure the integrity of 16S rRNA

Hypoxia Is Not a Main Stress When Mycobacterium tuberculosis Is in a Dormancy-Like Long-Chain Fatty Acid Environment

The capacity of Mycobacterium tuberculosis (Mtb) to sense, respond and adapt to a variable and hostile environment within the host makes it one of the most successful human pathogens. During different stages of infection, Mtb is surrounded by a plethora of lipid molecules and current evidence points out the relevance of fatty acids during the infectious process. In this study, we have compared the transcriptional response of Mtb to hypoxia in cultures supplemented with a mix of even long-chain fatty acids or dextrose as main carbon sources. Using RNA sequencing, we have identified differential expressed genes in early and late hypoxia, defined according to the in vitro Wayne and Hayes model, and compared the results with the exponential phase of growth in both carbon sources. We show that the number of genes over-expressed in the lipid medium was quite low in both, early and late hypoxia, relative to conditions including dextrose, with the exception of transcripts of stable and non-coding RNAs, which were more expressed in the fatty acid medium. We found that sigB and sigE were over-expressed in the early phase of hypoxia, confirming their pivotal role in early adaptation to low oxygen concentration independently of the carbon source. A drastic contrast was found with the transcriptional regulatory factors at early hypoxia. Only 2 transcriptional factors were over-expressed in early hypoxia in the lipid medium compared to 37 that were over-expressed in the dextrose medium. Instead of Rv0081, known to be the central regulator of hypoxia in dextrose, Rv2745c (ClgR), seems to play a main role in hypoxia in the fatty acid medium. The low level of genes associated to the stress-response during their adaptation to hypoxia in fatty acids, suggests that this lipid environment makes hypoxia a less stressful condition for the tubercle bacilli. Taken all together, these results indicate that the presence of lipid molecules shapes the metabolic response of Mtb to an adaptive state for different stresses within the host, including hypoxia. This fact could explain the success of Mtb to establish long-term survival during latent infection

Influence of organic molecules on the aggregation of TiO2 nanoparticles in acidic conditions

Engineered nanoparticles released into the environment may interact with natural organic matter (NOM). Surface complexation affects the surface potential, which in turn may lead to aggregation of the particles. Aggregation of synthetic TiO2 (anatase) nanoparticles in aqueous suspension was investigated at pH 2.8 as a function of time in the presence of various organic molecules and Suwannee River fulvic acid (SRFA), using dynamic light scattering (DLS) and high-resolution transmission electron microscopy (TEM). Results showed that the average hydrodynamic diameter and ?-potential were dependent on both concentration and molecular structure of the organic molecule. Results were also compared with those of quantitative batch adsorption experiments. Further, a time study of the aggregation of TiO2 nanoparticles in the presence of 2,3-dihydroxybenzoic acid (2,3-DHBA) and SRFA, respectively, was performed in order to observe changes in ?-potential and particle size over a time period of 9 months. In the 2,3-DHBA-TiO2 system, ?-potentials decreased with time resulting in charge neutralization and/or inversion depending on ligand concentration. Aggregate sizes increased initially to the micrometer size range, followed by disaggregation after several months. No or very little interaction between SRFA and TiO2 occurred at the lowest concentrations tested. However, at the higher concentrations of SRFA, there was an increase in both aggregate size and the amount of SRFA adsorbed to the TiO2 surface. This was in correlation with the ?-potential that decreased with increased SRFA concentration, leading to destabilization of the system. These results stress the importance of performing studies over both short and long time periods to better understand and predict the long-term effects of nanoparticles in the environment

Rapid changes in gene expression: DNA determinants of promoter regulation by the concentration of the transcription initiating NTP in Bacillus subtilis

In bacteria, rapid changes in gene expression can be achieved by affecting the activity of RNA polymerase with small molecule effectors during transcription initiation. An important small molecule effector is the initiating nucleoside triphosphate (iNTP). At some promoters, an increasing iNTP concentration stimulates promoter activity, while a decreasing concentration has the opposite effect. Ribosomal RNA (rRNA) promoters from Gram-positive Bacillus subtilis are regulated by the concentration of their iNTP. Yet, the sequences of these promoters do not emulate the sequence characteristics of [iNTP]-regulated rRNA promoters of Gram-negative Escherichia coli. Here, we identified the 3′-promoter region, corresponding to the transcription bubble, as key for B. subtilis rRNA promoter regulation via the concentration of the iNTP. Within this region, the conserved −5T (3 bp downstream from the −10 hexamer) is required for this regulation. Moreover, we identified a second class of [iNTP]-regulated promoters in B. subtilis where the sequence determinants are not limited to the transcription bubble region. Overall, it seems that various sequence combinations can result in promoter regulation by [iNTP] in B. subtilis. Finally, this study demonstrates how the same type of regulation can be achieved with strikingly different promoter sequences in phylogenetically distant species