Whole Cell Screen for Inhibitors of pH Homeostasis in Mycobacterium tuberculosis

Bacterial pathogens like Mycobacterium tuberculosis (Mtb) encounter acidic microenvironments in the host and must maintain their acid-base homeostasis to survive. A genetic screen identified two Mtb strains that cannot control intrabacterial pH (pH_IB) in an acidic environment; infection with either strain led to severe attenuation in mice. To search for additional proteins that Mtb requires to survive at low pH, we introduced a whole-cell screen for compounds that disrupt pH_IB, along with counter-screens that identify ionophores and membrane perturbors. Application of these methods to a natural product library identified four compounds of interest, one of which may inhibit novel pathway(s). This approach yields compounds that may lead to the identification of pathways that allow Mtb to survive in acidic environments, a setting in which Mtb is resistant to most of the drugs currently used to treat tuberculosis.     

The Mycobacterium tuberculosis Rv2745c Plays an Important Role in Responding to Redox Stress

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the leading cause of death from an infectious disease worldwide. Over the course of its life cycle in vivo, Mtb is exposed to a plethora of environmental stress conditions. Temporal regulation of genes involved in sensing and responding to such conditions is therefore crucial for Mtb to establish an infection. The Rv2745c (clgR) gene encodes a Clp protease gene regulator that is induced in response to a variety of stress conditions and potentially plays a role in Mtb pathogenesis. Our isogenic mutant, Mtb:ΔRv2745c, is significantly more sensitive to in vitro redox stress generated by diamide, relative to wild-type Mtb as well as to a complemented strain. Together with the fact that the expression of Rv2745c is strongly induced in response to redox stress, these results strongly implicate a role for ClgR in the management of intraphagosomal redox stress. Additionally, we observed that redox stress led to the dysregulation of the expression of the σ^H/σ^E regulon in the isogenic mutant, Mtb:ΔRv2745c. Furthermore, induction of clgR in Mtb and Mtb:ΔRv2745c (comp) did not lead to Clp protease induction, indicating that clgR has additional functions that need to be elucidated. Our data, when taken together with that obtained by other groups, indicates that ClgR plays diverse roles in multiple regulatory networks in response to different stress conditions. In addition to redox stress, the expression of Rv2745c correlates with the expression of genes involved in sulfate assimilation as well as in response to hypoxia and reaeration. Clearly, the Mtb Rv2745c-encoded ClgR performs different functions during stress response and is important for the pathogenicity of Mtb in-vivo, regardless of its induction of the Clp proteolytic pathway.     

Molecular insights into the binding of coenzyme F420 to the conserved protein Rv1155 from Mycobacterium tuberculosis

Coenzyme F₄₂₀ is a deazaflavin hydride carrier with a lower reduction potential than most flavins. In Mycobacterium tuberculosis (Mtb), F₄₂₀ plays an important role in activating PA-824, an antituberculosis drug currently used in clinical trials. Although F₄₂₀ is important to Mtb redox metabolism, little is known about the enzymes that bind F₄₂₀ and the reactions that they catalyze. We have identified a novel F₄₂₀-binding protein, Rv1155, which is annotated in the Mtb genome sequence as a putative flavin mononucleotide (FMN)-binding protein. Using biophysical techniques, we have demonstrated that instead of binding FMN or other flavins, Rv1155 binds coenzyme F₄₂₀. The crystal structure of the complex of Rv1155 and F₄₂₀ reveals one F₄₂₀ molecule bound to each monomer of the Rv1155 dimer. Structural, biophysical, and bioinformatic analyses of the Rv1155–F₄₂₀ complex provide clues about its role in the bacterium.     

An Extracellular Disulfide Bond Forming Protein (DsbF) from Mycobacterium tuberculosis: Structural, Biochemical, and Gene Expression Analysis

Disulfide bond forming (Dsb) proteins ensure correct folding and disulfide bond formation of secreted proteins. Previously, we showed that Mycobacterium tuberculosis DsbE (Mtb DsbE, Rv2878c) aids in vitro oxidative folding of proteins. Here, we present structural, biochemical, and gene expression analyses of another putative Mtb secreted disulfide bond isomerase protein homologous to Mtb DsbE, Mtb DsbF (Rv1677). The X-ray crystal structure of Mtb DsbF reveals a conserved thioredoxin fold although the active-site cysteines may be modeled in both oxidized and reduced forms, in contrast to the solely reduced form in Mtb DsbE. Furthermore, the shorter loop region in Mtb DsbF results in a more solvent-exposed active site. Biochemical analyses show that, similar to Mtb DsbE, Mtb DsbF can oxidatively refold reduced, unfolded hirudin and has a comparable pK_a for the active-site solvent-exposed cysteine. However, contrary to Mtb DsbE, the Mtb DsbF redox potential is more oxidizing and its reduced state is more stable. From computational genomics analysis of the M. tuberculosis genome, we identified a potential Mtb DsbF interaction partner, Rv1676, a predicted peroxiredoxin. Complex formation is supported by protein coexpression studies and inferred by gene expression profiles, whereby Mtb DsbF and Rv1676 are upregulated under similar environments. Additionally, comparison of Mtb DsbF and Mtb DsbE gene expression data indicates anticorrelated gene expression patterns, suggesting that these two proteins and their functionally linked partners constitute analogous pathways that may function under different conditions.     

Responses of Listeria monocytogenes to Acid Stress and Glucose Availability Revealed by a Novel Combination of Fluorescence Microscopy and Microelectrode Ion-Selective Techniques

Fluorescence ratio imaging microscopy and microelectrode ion flux estimation techniques were combined to study mechanisms of pH homeostasis in Listeria monocytogenes subjected to acid stress at different levels of glucose availability. This novel combination provided a unique opportunity to measure changes in H⁺ at either side of the bacterial membrane in real time and therefore to evaluate the rate of H⁺ flux across the bacterial plasma membrane and its contribution to bacterial pH homeostasis. Responses were assessed at external pHs (pH_o) between 3.0 and 6.0 for three levels of glucose (0, 1, and 10 mM) in the medium. Both the intracellular pH (pH_i) and net H⁺ fluxes were affected by the glucose concentration in the medium, with the highest absolute values corresponding to the highest glucose concentration. In the presence of glucose, the pH_i remained above 7.0 within a pH_o range of 4 to 6 and decreased below pH_o 4. Above pH_o 4, H⁺ extrusion increased correspondingly, with the maximum value at pH_o 5.5, and below pH_o 4, a net H⁺ influx was observed. Without glucose in the medium, the pH_i decreased, and a net H⁺ influx was observed below pH_o 5.5. A high correlation (R = 0.75 to 0.92) between the pH_i and net H⁺ flux changes is reported, indicating that the two processes are complementary. The results obtained support other reports indicating that membrane transport processes are the main contributors to the process of pH_i homeostasis in L. monocytogenes subjected to acid stress.     

Mycobacterium tuberculosis requires SufT for Fe-S cluster maturation,metabolism, and survival in vivo

Iron-sulfur (Fe-S) cluster proteins carry out essential cellular functions in diverse organisms, including the human pathogen Mycobacterium tuberculosis (Mtb). The mechanisms underlying Fe-S cluster biogenesis are poorly defined in Mtb. Here, we show that Mtb SufT (Rv1466), a DUF59 domain-containing essential protein, is required for the Fe-S cluster maturation. Mtb SufT homodimerizes and interacts with Fe-S cluster biogenesis proteins; SufS and SufU. SufT also interacts with the 4Fe-4S cluster containing proteins; aconitase and SufR. Importantly, a hyperactive cysteine in the DUF59 domain mediates interaction of SufT with SufS, SufU, aconitase, and SufR. We efficiently repressed the expression of SufT to generate a SufT knock-down strain in Mtb (SufT-KD) using CRISPR interference. Depleting SufT reduces aconitase’s enzymatic activity under standard growth conditions and in response to oxidative stress and iron limitation. The SufT-KD strain exhibited defective growth and an altered pool of tricarboxylic acid cycle intermediates, amino acids, and sulfur metabolites. Using Seahorse Extracellular Flux analyzer, we demonstrated that SufT depletion diminishes glycolytic rate and oxidative phosphorylation in Mtb. The SufT-KD strain showed defective survival upon exposure to oxidative stress and nitric oxide. Lastly, SufT depletion reduced the survival of Mtb in macrophages and attenuated the ability of Mtb to persist in mice. Altogether, SufT assists in Fe-S cluster maturation and couples this process to bioenergetics of Mtb for survival under low and high demand for Fe-S clusters.     

Unexpected Role for IL-17 in Protective Immunity against Hypervirulent Mycobacterium tuberculosis HN878 Infection

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), infects one third of the world''s population. Among these infections, clinical isolates belonging to the W-Beijing appear to be emerging, representing about 50% of Mtb isolates in East Asia, and about 13% of all Mtb isolates worldwide. In animal models, infection with W-Beijing strain, Mtb HN878, is considered “hypervirulent” as it results in increased mortality and causes exacerbated immunopathology in infected animals. We had previously shown the Interleukin (IL) -17 pathway is dispensable for primary immunity against infection with the lab adapted Mtb H37Rv strain. However, it is not known whether IL-17 has any role to play in protective immunity against infection with clinical Mtb isolates. We report here that lab adapted Mtb strains, such as H37Rv, or less virulent Mtb clinical isolates, such as Mtb CDC1551, do not require IL-17 for protective immunity against infection while infection with Mtb HN878 requires IL-17 for early protective immunity. Unexpectedly, Mtb HN878 induces robust production of IL-1β through a TLR-2-dependent mechanism, which supports potent IL-17 responses. We also show that the role for IL-17 in mediating protective immunity against Mtb HN878 is through IL-17 Receptor signaling in non-hematopoietic cells, mediating the induction of the chemokine, CXCL-13, which is required for localization of T cells within lung lymphoid follicles. Correct T cell localization within lymphoid follicles in the lung is required for maximal macrophage activation and Mtb control. Since IL-17 has a critical role in vaccine-induced immunity against TB, our results have far reaching implications for the design of vaccines and therapies to prevent and treat emerging Mtb strains. In addition, our data changes the existing paradigm that IL-17 is dispensable for primary immunity against Mtb infection, and instead suggests a differential role for IL-17 in early protective immunity against emerging Mtb strains.     

DNA Clasping by Mycobacterial HU: The C-Terminal Region of HupB Mediates Increased Specificity of DNA Binding

Background

HU a small, basic, histone like protein is a major component of the bacterial nucleoid. E. coli has two subunits of HU coded by hupA and hupB genes whereas Mycobacterium tuberculosis (Mtb) has only one subunit of HU coded by ORF Rv2986c (hupB gene). One noticeable feature regarding Mtb HupB, based on sequence alignment of HU orthologs from different bacteria, was that HupB_Mtb bears at its C-terminal end, a highly basic extension and this prompted an examination of its role in Mtb HupB function.

Methodology/Principal Findings

With this objective two clones of Mtb HupB were generated; one expressing full length HupB protein (HupB_Mtb) and another which expresses only the N terminal region (first 95 amino acid) of hupB (HupB_MtbN). Gel retardation assays revealed that HupB_MtbN is almost like E. coli HU (heat stable nucleoid protein) in terms of its DNA binding, with a binding constant (K_d) for linear dsDNA greater than 1000 nM, a value comparable to that obtained for the HUαα and HUαβ forms. However CTR (C-terminal Region) of HupB_Mtb imparts greater specificity in DNA binding. HupB_Mtb protein binds more strongly to supercoiled plasmid DNA than to linear DNA, also this binding is very stable as it provides DNase I protection even up to 5 minutes. Similar results were obtained when the abilities of both proteins to mediate protection against DNA strand cleavage by hydroxyl radicals generated by the Fenton''s reaction, were compared. It was also observed that both the proteins have DNA binding preference for A:T rich DNA which may occur at the regulatory regions of ORFs and the oriC region of Mtb.

Conclusions/Significance

These data thus point that HupB_Mtb may participate in chromosome organization in-vivo, it may also play a passive, possibly an architectural role.     

A Mycobacterium tuberculosis Sigma Factor Network Responds to Cell-Envelope Damage by the Promising Anti-Mycobacterial Thioridazine

Background

Novel therapeutics are urgently needed to control tuberculosis (TB). Thioridazine (THZ) is a candidate for the therapy of multidrug and extensively drug-resistant TB.

Methodology/Principal Findings

We studied the impact of THZ on Mycobacterium tuberculosis (Mtb) by analyzing gene expression profiles after treatment at the minimal inhibitory (1x MIC) or highly inhibitory (4x MIC) concentrations between 1–6 hours. THZ modulated the expression of genes encoding membrane proteins, efflux pumps, oxido-reductases and enzymes involved in fatty acid metabolism and aerobic respiration. The Rv3160c-Rv3161c operon, a multi-drug transporter and the Rv3614c/3615c/3616c regulon, were highly induced in response to THZ. A significantly high number of Mtb genes co-expressed with σ^B (the σ^B regulon) was turned on by THZ treatment. σ^B has recently been shown to protect Mtb from envelope-damage. We hypothesized that THZ damages the Mtb cell-envelope, turning on the expression of the σ^B regulon. Consistent with this hypothesis, we present electron-microscopy data which shows that THZ modulates cell-envelope integrity. Moreover, the Mtb mutants in σ^H and σ^E, two alternate stress response sigma factors that induce the expression of σ^B, exhibited higher sensitivity to THZ, indicating that the presence and expression of σ^B allows Mtb to resist the impact of THZ. Conditional induction of σ^B levels increased the survival of Mtb in the presence of THZ.

Conclusions/Significance

THZ targets different pathways and can thus be used as a multi-target inhibitor itself as well as provide strategies for multi-target drug development for combination chemotherapy. Our results show that the Mtb sigma factor network comprising of σ^H, σ^E and σ^B plays a crucial role in protecting the pathogen against cell-envelope damage.     

Mycobacterium tuberculosis Rv2179c Protein Establishes a New Exoribonuclease Family with Broad Phylogenetic Distribution

Ribonucleases (RNases) maintain the cellular RNA pool by RNA processing and degradation. In many bacteria, including the human pathogen Mycobacterium tuberculosis (Mtb), the enzymes mediating several central RNA processing functions are still unknown. Here, we identify the hypothetical Mtb protein Rv2179c as a highly divergent exoribonuclease. Although the primary sequence of Rv2179c has no detectable similarity to any known RNase, the Rv2179c crystal structure reveals an RNase fold. Active site residues are equivalent to those in the DEDD family of RNases, and Rv2179c has close structural homology to Escherichia coli RNase T. Consistent with the DEDD fold, Rv2179c has exoribonuclease activity, cleaving the 3′ single-strand overhangs of duplex RNA. Functional orthologs of Rv2179c are prevalent in actinobacteria and found in bacteria as phylogenetically distant as proteobacteria. Thus, Rv2179c is the founding member of a new, large RNase family with hundreds of members across the bacterial kingdom.     

Potential of Mycobacterium tuberculosis resuscitation-promoting factors as antigens in novel tuberculosis sub-unit vaccines

Novel vaccines are needed to control tuberculosis (TB), the bacterial infectious disease that together with malaria and HIV is worldwide responsible for high levels of morbidity and mortality. TB can result from the reactivation of an initially controlled latent infection by Mycobacterium tuberculosis (Mtb). Mtb proteins for which a possible role in this reactivation process has been hypothesized are the five homologs of the resuscitation-promoting factor of Micrococcus luteus, namely Mtb Rv0867c (rpfA), Rv1009 (rpfB), Rv1884c (rpfC), Rv2389c (rpfD) and Rv2450c (rpfE). Analysis of the immune recognition of these 5 proteins following Mtb infection or Mycobacterium bovis BCG vaccination of mice showed that Rv1009 (rpfB) and Rv2389c (rpfD) are the most antigenic in the tested models. We therefore selected rpfB and rpfD for testing their vaccine potential as plasmid DNA vaccines. Elevated cellular immune responses and modest but significant protection against intra-tracheal Mtb challenge were induced by immunization with the rpfB encoding DNA vaccine. The results indicate that rpfB is the most promising candidate of the five rpf-like proteins of Mtb in terms of its immunogenicity and protective efficacy and warrants further analysis for inclusion as an antigen in novel TB vaccines.     

Mycobacterium tuberculosis IMPDH in Complexes with Substrates,Products and Antitubercular Compounds

Tuberculosis (TB) remains a worldwide problem and the need for new drugs is increasingly more urgent with the emergence of multidrug- and extensively-drug resistant TB. Inosine 5’-monophosphate dehydrogenase 2 (IMPDH2) from Mycobacterium tuberculosis (Mtb) is an attractive drug target. The enzyme catalyzes the conversion of inosine 5’-monophosphate into xanthosine 5’-monophosphate with the concomitant reduction of NAD⁺ to NADH. This reaction controls flux into the guanine nucleotide pool. We report seventeen selective IMPDH inhibitors with antitubercular activity. The crystal structures of a deletion mutant of MtbIMPDH2 in the apo form and in complex with the product XMP and substrate NAD⁺ are determined. We also report the structures of complexes with IMP and three structurally distinct inhibitors, including two with antitubercular activity. These structures will greatly facilitate the development of MtbIMPDH2-targeted antibiotics.     

HCO3?-independent pH Regulation in Astrocytes in Situ Is Dominated by V-ATPase

The mechanisms of HCO₃⁻-independent intracellular pH (pH_i) regulation were examined in fibrous astrocytes within isolated neonatal rat optic nerve (RON) and in cultured cortical astrocytes. In agreement with previous studies, resting pH_i in cultured astrocytes was 6.82 ± 0.06 and inhibition of the V-ATPase H⁺ pump by Cl⁻ removal or via the selective inhibitor bafilomycin had only a small effect upon resting pH_i and recovery following an acid load. In contrast, resting pH_i in RON astrocytes was 7.10 ± 0.04, significantly less acidic than that in cultured cells (p < 0.001), and responded to inhibition of V-ATPase with profound acidification to the 6.3–6.5 range. Fluorescent immuno-staining and immuno-gold labeling confirmed the presence V-ATPase in the cell membrane of RON astrocyte processes and somata. Using ammonia pulse recovery, pH_i recovery in RON astrocyte was achieved largely via V-ATPase with sodium-proton exchange (NHE) playing a minor role. The findings indicate that astrocytes in a whole-mount preparation such as the optic nerve rely to a greater degree upon V-ATPase for HCO₃⁻-independent pH_i regulation than do cultured astrocytes, with important functional consequences for the regulation of pH in the CNS.     

The effect of fast created inbreeding on litter size and body weights in mice

This study was designed to reveal any differences in effects of fast created versus total inbreeding on reproduction and body weights in mice. A line selected for large litter size for 124 generations (H) and a control line (K) maintained without selection for the same number of generations were crossed (HK) and used as a basis for the experiment. Within the HK cross, full sib, cousin or random mating were practised for two generations in order to create new inbreeding (IB_F) at a fast rate. In the first generation of systematic mating, old inbreeding was regenerated in addition to creation of new inbreeding from the mating design giving total inbreeding (IB_T). The number of pups born alive (NBA) and body weights of the animals were then analysed by a model including both IB_T and IB_F. The IB_T of the dam was in the present study found to reduce the mean NBA with -0.48 (± 0.22) (p < 0.05) pups per 10% increase in the inbreeding coefficient, while the additional effect of IB_F was -0.42 (± 0.27). For the trait NBA per female mated, the effect of IB_T was estimated to be -0.45 (± 0.29) per 10% increase in the inbreeding coefficient and the effect of IB_F was -0.90 (± 0.37) (p < 0.05) pups. In the present study, only small or non-significant effects of IB_F of the dam could be found on sex-ratio and body weights at three and six weeks of age in a population already adjusted for IB_T.     

Horizontal Gene Transfer of PIB-Type ATPases among Bacteria Isolated from Radionuclide- and Metal-Contaminated Subsurface Soils

Aerobic heterotrophs were isolated from subsurface soil samples obtained from the U.S. Department of Energy's (DOE) Field Research Center (FRC) located at Oak Ridge, Tenn. The FRC represents a unique, extreme environment consisting of highly acidic soils with cooccurring heavy metals, radionuclides, and high nitrate concentrations. Four hundred isolates obtained from contaminated soil were assayed for heavy metal resistance, and a smaller subset was assayed for tolerance to uranium. The vast majority of the isolates were gram-positive bacteria and belonged to the high-G+C- and low-G+C-content genera Arthrobacter and Bacillus, respectively. Genomic DNA from a randomly chosen subset of 50 Pb-resistant (Pb^r) isolates was amplified with PCR primers specific for P_IB-type ATPases (i.e., pbrA/cadA/zntA). A total of 10 pbrA/cadA/zntA loci exhibited evidence of acquisition by horizontal gene transfer. A remarkable dissemination of the horizontally acquired P_IB-type ATPases was supported by unusual DNA base compositions and phylogenetic incongruence. Numerous Pb^r P_IB-type ATPase-positive FRC isolates belonging to the genus Arthrobacter tolerated toxic concentrations of soluble U(VI) (UO₂²⁺) at pH 4. These unrelated, yet synergistic, physiological traits observed in Arthrobacter isolates residing in the contaminated FRC subsurface may contribute to the survival of the organisms in such an extreme environment. This study is, to the best of our knowledge, the first study to report broad horizontal transfer of P_IB-type ATPases in contaminated subsurface soils and is among the first studies to report uranium tolerance of aerobic heterotrophs obtained from the acidic subsurface at the DOE FRC.