Identification of a copper-binding metallothionein in pathogenic mycobacteria

A screen of a genomic library from Mycobacterium tuberculosis (Mtb) identified a small, unannotated open reading frame (MT0196) that encodes a 4.9-kDa, cysteine-rich protein. Despite extensive nucleotide divergence, the amino acid sequence is highly conserved among mycobacteria that are pathogenic in vertebrate hosts. We synthesized the protein and found that it preferentially binds up to six Cu(I) ions in a solvent-shielded core. Copper, cadmium and compounds that generate nitric oxide or superoxide induced the gene's expression in Mtb up to 1,000-fold above normal expression. The native protein bound copper within Mtb and partially protected Mtb from copper toxicity. We propose that the product of the MT0196 gene be named mycobacterial metallothionein (MymT). To our knowledge, MymT is the first metallothionein of a Gram-positive bacterium with a demonstrated function.     

Genome-wide screen for Mycobacterium tuberculosis genes that regulate host immunity

In spite of its highly immunogenic properties, Mycobacterium tuberculosis (Mtb) establishes persistent infection in otherwise healthy individuals, making it one of the most widespread and deadly human pathogens. Mtb's prolonged survival may reflect production of microbial factors that prevent even more vigorous immunity (quantitative effect) or that divert the immune response to a non-sterilizing mode (qualitative effect). Disruption of Mtb genes has produced a list of several dozen candidate immunomodulatory factors. Here we used robotic fluorescence microscopy to screen 10,100 loss-of-function transposon mutants of Mtb for their impact on the expression of promoter-reporter constructs for 12 host immune response genes in a mouse macrophage cell line. The screen identified 364 candidate immunoregulatory genes. To illustrate the utility of the candidate list, we confirmed the impact of 35 Mtb mutant strains on expression of endogenous immune response genes in primary macrophages. Detailed analysis focused on a strain of Mtb in which a transposon disrupts Rv0431, a gene encoding a conserved protein of unknown function. This mutant elicited much more macrophage TNFα, IL-12p40 and IL-6 in vitro than wild type Mtb, and was attenuated in the mouse. The mutant list provides a platform for exploring the immunobiology of tuberculosis, for example, by combining immunoregulatory mutations in a candidate vaccine strain.     

Response to copper stress in Streptomyces lividans extends beyond genes under direct control of a copper-sensitive operon repressor protein (CsoR)

A copper-sensitive operon repressor protein (CsoR) has been identified in Streptomyces lividans (CsoR^Sl) and found to regulate copper homeostasis with attomolar affinity for Cu(I). Solution studies reveal apo- and Cu^I-CsoR^Sl to be a tetramer assembly, and a 1.7-Å resolution crystal structure of apo-CsoR^Sl reveals that a significant conformational change is necessary to enable Cu(I) binding. In silico prediction of the CsoR regulon was confirmed in vitro (EMSA) and in vivo (RNA-seq), which highlighted that next to the csoR gene itself, the regulon consists of two Cu(I) efflux systems involving a CopZ-like copper metallochaperone protein and a CopA P₁-type ATPase. Although deletion of csoR has only minor effects on S. lividans development when grown under high copper concentrations, mutations of the Cu(I) ligands decrease tolerance to copper as a result of the Cu(I)-CsoR mutants failing to disengage from the DNA targets, thus inhibiting the derepression of the regulon. RNA-seq experiments carried out on samples incubated with exogenous copper and a ΔcsoR strain showed that the set of genes responding to copper stress is much wider than anticipated and largely extends beyond genes targeted by CsoR. This suggests more control levels are operating and directing other regulons in copper homeostasis beside the CsoR regulon.     

Rapid Molecular Detection of Multidrug-Resistant Tuberculosis by PCR-Nucleic Acid Lateral Flow Immunoassay

Several existing molecular tests for multidrug-resistant tuberculosis (MDR-TB) are limited by complexity and cost, hindering their widespread application. The objective of this proof of concept study was to develop a simple Nucleic Acid Lateral Flow (NALF) immunoassay as a potential diagnostic alternative, to complement conventional PCR, for the rapid molecular detection of MDR-TB. The NALF device was designed using antibodies for the indirect detection of labeled PCR amplification products. Multiplex PCR was optimized to permit the simultaneous detection of the drug resistant determining mutations in the 81-bp hot spot region of the rpoB gene (rifampicin resistance), while semi-nested PCR was optimized for the S315T mutation detection in the katG gene (isoniazid resistance). The amplification process additionally targeted a conserved region of the genes as Mycobacterium tuberculosis (Mtb) DNA control. The optimized conditions were validated with the H37Rv wild-type (WT) Mtb isolate and Mtb isolates with known mutations (MT) within the rpoB and katG genes. Results indicate the correct identification of WT (drug susceptible) and MT (drug resistant) Mtb isolates, with the least limit of detection (LOD) being 10⁴ genomic copies per PCR reaction. NALF is a simple, rapid and low-cost device suitable for low resource settings where conventional PCR is already employed on a regular basis. Moreover, the use of antibody-based NALF to target primer-labels, without the requirement for DNA hybridization, renders the device generic, which could easily be adapted for the molecular diagnosis of other infectious and non-infectious diseases requiring nucleic acid detection.     

A membrane protein preserves intrabacterial pH in intraphagosomal Mycobacterium tuberculosis

Acidification of the phagosome is considered to be a major mechanism used by macrophages against bacteria, including Mycobacterium tuberculosis (Mtb). Mtb blocks phagosome acidification, but interferon-gamma (IFN-gamma) restores acidification and confers antimycobacterial activity. Nonetheless, it remains unclear whether acid kills Mtb, whether the intrabacterial pH of any pathogen falls when it is in the phagosome and whether acid resistance is required for mycobacterial virulence. In vitro at pH 4.5, Mtb survived in a simple buffer and maintained intrabacterial pH. Therefore, Mtb resists phagolysosomal concentrations of acid. Mtb also maintained its intrabacterial pH and survived when phagocytosed by IFN-gamma-activated macrophages. We used transposon mutagenesis to identify genes responsible for Mtb's acid resistance. A strain disrupted in Rv3671c, a previously uncharacterized gene encoding a membrane-associated protein, was sensitive to acid and failed to maintain intrabacterial pH in acid in vitro and in activated macrophages. Growth of the mutant was also severely attenuated in mice. Thus, Mtb is able to resist acid, owing in large part to Rv3671c, and this resistance is essential for virulence. Disruption of Mtb's acid resistance and intrabacterial pH maintenance systems is an attractive target for chemotherapy.     

Characterization of a novel cell wall-anchored protein with carboxylesterase activity required for virulence in Mycobacterium tuberculosis

Pooled mutant competition assays have shown that the Mycobacterium tuberculosis MT2282 gene (Rv2224c, annotated as encoding a proteinase) is required for bacterial survival in mice. To understand the mechanism of this requirement, we conducted a genetic and biochemical study of the MT2282 gene and its product. MT2282 encodes a member of the microbial esterase/lipase family with active site consensus sequences of G-X-S-X-G, and we have concluded that the MT2282 protein is, in fact, a cell wall-associated carboxylesterase rather than a proteinase, as initially annotated. The MT2282 gene product preferentially hydrolyzes ester bonds of substrates with intermediate carbon chain length. Purified MT2282 is a monomer with enzymatic catalysis properties that fit in the Michaelis-Menten kinetic model. Esterase activity was inhibited by paraoxon and dichlorvos. Replacement of Ser215, Asp450, and His477 by Ala in the consensus motifs completely abolishes esterase activity, suggesting that Ser215-Asp450-His477 forms a catalytic triad with Ser215 as an active site residue. To evaluate the role of the MT2282 in pathogenesis, the gene was deleted from the M. tuberculosis genome. BALB/c mouse aerosol infections showed reduced colony-forming unit loads in lungs and spleens and less lung pathology for the DeltaMT2282 mutant. High dose intravenous infection of mice with the mutant resulted in a significantly delayed time to death compared with the wild type or complemented mutant. These results indicate that MT2282 encodes a cell wall-associated carboxylesterase, which is required for full virulence of M. tuberculosis. We propose that MT2282 (Rv2224c) and its adjacent paralogous gene MT2281 (Rv2223c) be named caeA and caeB respectively, for carboxylesterase A and B.     

Differential regulation of protein kinase C isoforms of macrophages by pathogenic and non-pathogenic mycobacteria

Given the fact that Mycobacterium tuberculosis (Mtb) may respond to the intracellular milieu of the macrophage with the induction of environmentally regulated genes required for survival and growth of the bacteria we assumed that the protein kinases may also be the factors in Mycobacterium-macrophage interaction. Since, protein kinases play a major role in various critical cellular processes including regulation of immune responses, we describe the fate of expression and phosphorylation of protein kinase C in macrophage cell lines exposed to Mtb H37Rv and raised the question whether the change in the events of expression and phosphorylation are the results of direct interaction of bacilli with macrophages and/or, are also indirectly mediated by specific cytokines that are induced in response to exposure. Our results show that only novel PKCs are phosphorylated during infection of macrophages by pathogenic and non-pathogenic mycobacteria and the alteration is a result of direct host-bacilli association which is independent of cytokines as mediators. Expression of PKC-alpha (conventional PKC isoform) was down regulated by Mtb H37Rv. In contrast the non-pathogenic fast grower Mycobacterium smegmatis (MS) increased the expression and phosphorylation of PKC-alpha. PKC-alpha was also increased in macrophages treated with serum of mice immunized with Mtb H37Rv. The study has shown that pathogenic and non-pathogenic mycobacteria categorically select the type of protein kinases C for activation/deactivation.