Tuberculosis is still a leading cause of death worldwide. The selection and spread of Mycobacterium tuberculosis multidrug-resistant (MDR-TB) and extensively drug-resistant strains (XDR-TB) is a severe public health problem. Recently, two different classes of chemical series, the benzothiazinones (BTZ) and the dinitrobenzamide (DNB) derivatives have been found to be highly active against M. tuberculosis, including XDR-TB strains. The target of BTZs is DprE1 protein which works in concert with DprE2 to form the heteromeric decaprenylphosphoryl-β-D-ribose 2′-epimerase, involved in Decaprenyl-Phospho-Arabinose (DPA) biosynthesis. Interestingly, it has been shown that the DNBs block the same pathway thus suggesting that both drugs could share the same target. Moreover, in Mycobacterium smegmatis the overexpression of the NfnB nitroreductase led to the inactivation of the BTZs by reduction of a critical nitro-group to an amino-group. In this work several spontaneous M. smegmatis mutants resistant to DNBs were isolated. Sixteen mutants, showing high levels of DNB resistance, exhibited a mutation in the Cys394 of DprE1. Using fluorescence titration and mass spectrometry it has been possible to monitor the binding between DprE1 and DNBs, achieving direct evidence that MSMEG_6382 is the cellular target of DNBs in mycobacteria. Additionally, M. smegmatis mutants having low levels of resistance to DNBs harbor various mutations in MSMEG_6503 gene encoding the transcriptional repressor of the nitroreductase NfnB. By LC/MS2 analysis it has been demonstrated that NfnB is responsible for DNB inactivation. Taken together, our data demonstrate that both DNB and BTZ drugs share common resistance mechanisms in M. smegmatis.
ABSTRACT Legionella pneumophila expresses two peroxide-scavenging alkyl hydroperoxide reductase systems (AhpC1 and AhpC2D) that are expressed differentially during the bacterial growth cycle. Functional loss of the postexponentially expressed AhpC1 system is compensated for by increased expression of the exponentially expressed AhpC2D system. In this study, we used an acrylamide capture of DNA-bound complexes (ACDC) technique and mass spectrometry to identify proteins that bind to the promoter region of the ahpC2D operon. The major protein captured was an ortholog of OxyR (OxyR Lp ). Genetic studies indicated that oxyR Lp was an essential gene expressed postexponentially and only partially complemented an Escherichia coli oxyR mutant (GS077). Gel shift assays confirmed specific binding of OxyR Lp to ahpC2D promoter sequences, but not to promoters of ahpC1 or oxyR Lp ; however, OxyR Lp weakly bound to E. coli OxyR-regulated promoters ( katG , oxyR , and ahpCF ). DNase I protection studies showed that the OxyR Lp binding motif spanned the promoter and transcriptional start sequences of ahpC2 and that the protected region was unchanged by treatments with reducing agents or hydrogen peroxide (H 2 O 2 ). Moreover, the OxyR Lp (pBADLp oxyR )-mediated repression of an ahpC2 - gfp reporter construct in E. coli GS077 (the oxyR mutant) was not reversed by H 2 O 2 challenge. Alignments with other OxyR proteins revealed several amino acid substitutions predicted to ablate thiol oxidation or conformational changes required for activation. We suggest these mutations have locked OxyR Lp in an active DNA-binding conformation, which has permitted a divergence of function from a regulator of oxidative stress to a cell cycle regulator, perhaps controlling gene expression during postexponential differentiation.
ABSTRACT Tuberculosis remains a major health problem due to the emergence of drug-resistant strains of Mycobacterium tuberculosis . Some models have provided valuable information about drug resistance and efficacy; however, the translation of these results into effective human treatments has mostly proven unsuccessful. In this study, we adapted high-content screening (HCS) technology to investigate the activities of antitubercular compounds in the context of an in vitro granuloma model. We observed significant shifts in the MIC 50 s between the activities of the compounds under extracellular and granuloma conditions.
A critical feature of Mycobacterium tuberculosis, the causative agent of human tuberculosis (TB), is its ability to survive and multiply within macrophages, making these host cells an ideal niche for persisting microbes. Killing the intracellular tubercle bacilli is a key requirement for efficient tuberculosis treatment, yet identifying potent inhibitors has been hampered by labor-intensive techniques and lack of validated targets. Here, we present the development of a phenotypic cell-based assay that uses automated confocal fluorescence microscopy for high throughput screening of chemicals that interfere with the replication of M. tuberculosis within macrophages. Screening a library of 57,000 small molecules led to the identification of 135 active compounds with potent intracellular anti-mycobacterial efficacy and no host cell toxicity. Among these, the dinitrobenzamide derivatives (DNB) showed high activity against M. tuberculosis, including extensively drug resistant (XDR) strains. More importantly, we demonstrate that incubation of M. tuberculosis with DNB inhibited the formation of both lipoarabinomannan and arabinogalactan, attributable to the inhibition of decaprenyl-phospho-arabinose synthesis catalyzed by the decaprenyl-phosphoribose 2' epimerase DprE1/DprE2. Inhibition of this new target will likely contribute to new therapeutic solutions against emerging XDR-TB. Beyond validating the high throughput/content screening approach, our results open new avenues for finding the next generation of antimicrobials.
Bacillus Calmette-Guérin (BCG) vaccination has proven to be efficient in immunologically naïve infants; however, it has not been investigated that maternal natural exposure to Mycobacterium and/or BCG vaccine could influence the characteristics of immune responses to BCG in newborns. In this study, we analyzed whether the maternal immune status to M tuberculosis (M tb) can affect neonatal immunity to BCG using a mouse model. Neonates were obtained from mice that were previously exposed to live BCG, to live M avium, or to heat-killed M tb H37Rv, and from naïve control mothers. One week after birth, the neonates were divided into two subgroups: one group immunized with live BCG via the subcutaneous route and the other group of neonates sham-treated. Interferon-gamma (IFNγ) secretion in response to in vitro stimulation with heat-killed BCG or purified protein derivative (PPD) was examined. Protection against M tb infection was evaluated by challenging mice nasally with live M tb H37Rv followed by counting colonies from spleen and lung homogenates. BCG-immunized neonates showed increased IFNγ secretion in response to heat-killed BCG or PPD. All mice in BCG-immunized neonates subgroups showed reduced bacterial burden (colony forming unit) in the lungs when compared with control naive neonate mice. However, no statistically significant difference was observed when comparing BCG-immunized mice born from mothers previously exposed to M avium or immunized with either heat-killed H37Rv or live BCG and mice born from naïve mothers. The maternal immune status to M tb does not appear to impact on the immunogenicity of BCG vaccine in their progeny in our experimental conditions.
ABSTRACT Adaptive regulation of gene expression in response to environmental changes is a general property of bacterial pathogens. By screening an ordered transposon mutagenesis library of Mycobacterium tuberculosis , we have identified three mutants containing a transposon in the coding sequence or in the 5′ regions of genes coding for two-component signal transduction systems ( trcS , regX3 , prrA ). The intracellular multiplication capacity of the three mutants was investigated in mouse bone marrow-derived macrophages. Only the prrA mutant showed a defect in intracellular growth during the early phase of infection, and this defect was fully reverted when the mutant was complemented with prrA-prrB wild-type copies. The mutant phenotype was transient, as after 1 week this strain recovered full growth capacity to reach levels similar to that of the wild type at day 9. Moreover, a transient induction of prrA promoter activity was observed during the initial phase of macrophage infection, as shown by a prrA promoter -gfp fusion in M. bovis BCG infecting the mouse macrophages. The concordant transience of the prrA mutant phenotype and prrA promoter activity indicates that the PrrA-PrrB two-component system is involved in the environmental adaptation of M. tuberculosis , specifically in an early phase of the intracellular growth, and that, similar to other facultative intracellular parasites, M. tuberculosis can use genes temporarily required at different stages in the course of macrophage infection.
Macrophages are reservoirs for replicating mycobacterium during tuberculosis (TB) infections. In this study, small molecules to be developed as anti-tubercular treatments were investigated for their ability to kill intracellular bacteria in in vitro macrophage models. High-content imaging technologies offer a high-throughput method to quantify a drug's ability to inhibit Mycobacterium tuberculosis intracellular invasion and multiplication in host cells. Dedicated image analysis enables the automated quantification of infected macrophages, and compounds that inhibit mycobacteria proliferation can be tested using this method. Furthermore, the implementation of the assay in 384-well microtiter plates combined with automated image acquisition and analysis allows large-scale screening of compound libraries in M. tuberculosis-infected macrophages. Here we describe a high-throughput and high-content workflow and detail its utility for the development of new TB drugs.
Hsp60 mediates invasion of HeLa cells, blocks organelle trafficking, and is the most abundant protein secreted into the phagosomes of host cells throughout the course of intracellular multiplication. In Escherichia coli the heat shock response is controlled by sigma factor 32 (RpoH) encoded by rpoH. Moreover, we do not know to what extent RpoH is either expressed or required for gene expression. In a study the authors focused on controlling levels of Hsp60 production by modulating expression of rpoH in order to evaluate the role of heat-shock protein (Hsp60) and the heat shock response in pathogenesis. The authors developed three strategies to alter rpoH expression: (i) construct an rpoH knockout mutant by allelic replacement; (ii) replace the endogenous rpoH promoter with an isopropyl- β-D-thio-galactopyranoside (IPTG)-inducible promoter; and (iii) to knock down levels of RpoH and HtpAB with antisense RNA. Either the rpoH gene is located in a dead zone for recombination or more likely the introduction of strong promoters into this locus causes constitutive expression of downstream genes associated with cell division (ftsYEX). Preliminary data presented in a report in this chapter suggest that antisense can be used to knock down protein expression levels in L. pneumophila.
Two-component systems are major regulatory systems for bacterial adaptation to environmental changes. During the infectious cycle of Mycobacterium tuberculosis, adaptation to an intracellular environment is critical for multiplication and survival of the micro-organism within the host. The M. tuberculosis prrA gene, encoding the regulator of the two-component system PrrA–PrrB, has been shown to be induced upon macrophage phagocytosis and to be transiently required for the early stages of macrophage infection. In order to study the mechanisms of regulation of the PrrA–PrrB two-component system, PrrA and the cytoplasmic part of the PrrB histidine kinase were produced and purified as hexahistidine-tagged recombinant proteins. Electrophoretic mobility shift assays indicated that PrrA specifically binds to the promoter of its own operon, with increased affinity upon phosphorylation. Moreover, induction of fluorescence was observed after phagocytosis of a wild-type M. tuberculosis strain containing the gfp reporter gene under the control of the prrA–prrB promoter, while this induction was not seen in a prrA/B mutant strain containing the same construct. These results indicate that the early intracellular induction of prrA depends on the autoregulation of this two-component system.
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