Targeted protein degradation in mycobacteria uncovers antibacterial effects and potentiates antibiotic efficacy.

Proteolysis-targeting chimeras (PROTACs) represent a new therapeutic modality involving selectively directing disease-causing proteins for degradation through proteolytic systems. Our ability to exploit targeted protein degradation (TPD) for antibiotic development remains nascent due to our limited understanding of which bacterial proteins are amenable to a TPD strategy. Here, we use a genetic system to model chemically-induced proximity and degradation to screen essential proteins in Mycobacterium smegmatis (Msm), a model for the human pathogen M. tuberculosis (Mtb). By integrating experimental screening of 72 protein candidates and machine learning, we find that drug-induced proximity to the bacterial ClpC1P1P2 proteolytic complex leads to the degradation of many endogenous proteins, especially those with disordered termini. Additionally, TPD of essential Msm proteins inhibits bacterial growth and potentiates the effects of existing antimicrobial compounds. Together, our results provide biological principles to select and evaluate attractive targets for future Mtb PROTAC development, as both standalone antibiotics and potentiators of existing antibiotic efficacy.

Rv2231c, a unique histidinol phosphate aminotransferase from Mycobacterium tuberculosis, supports virulence by inhibiting host-directed defense.

Nitrogen metabolism of M. tuberculosis is critical for its survival in infected host cells. M. tuberculosis has evolved sophisticated strategies to switch between de novo synthesis and uptake of various amino acids from host cells for metabolic demands. Pyridoxal phosphate-dependent histidinol phosphate aminotransferase-HspAT enzyme is critically required for histidine biosynthesis. HspAT is involved in metabolic synthesis of histidine, phenylalanine, tyrosine, tryptophan, and novobiocin. We showed that M. tuberculosis Rv2231c is a conserved enzyme with HspAT activity. Rv2231c is a monomeric globular protein that contains α-helices and ß-sheets. It is a secretory and cell wall-localized protein that regulates critical pathogenic attributes. Rv2231c enhances the survival and virulence of recombinant M. smegmatis in infected RAW264.7 macrophage cells. Rv2231c is recognized by the TLR4 innate immune receptor and modulates the host immune response by suppressing the secretion of the antibacterial pro-inflammatory cytokines TNF, IL-12, and IL-6. It also inhibits the expression of co-stimulatory molecules CD80 and CD86 along with antigen presenting molecule MHC-I on macrophage and suppresses reactive nitrogen species formation, thereby promoting M2 macrophage polarization. Recombinant M. smegmatis expressing Rv2231c inhibited apoptosis in macrophages, promoting efficient bacterial survival and proliferation, thereby increasing virulence. Our results indicate that Rv2231c is a moonlighting protein that regulates multiple functions of M. tuberculosis pathophysiology to increase its virulence. These mechanistic insights can be used to better understand the pathogenesis of M. tuberculosis and to design strategies for tuberculosis mitigation.

Substrate and functional characterization of the lysine acetyltransferase MsKat and deacetylase MsCobB in Mycobacterium smegmatis.

Tuberculosis (TB) is a serious cause of infectious death worldwide. Recent studies have reported that about 30% of the Mtb proteome was modified post-translationally, indicating that their functions are essential for drug resistance, mycobacterial survival, and pathogenicity. Among them, lysine acetylation, reversibly regulated by acetyltransferase and deacetylase, has important roles involved in energy metabolism, cellular adaptation, and protein interactions. However, the substrate and biological functions of these two important regulatory enzymes remain unclear. Herein, we utilized the non-pathogenic M. smegmatis strain as a model and systematically investigated the dynamic proteome changes in response to the overexpressing of MsKat/MsCobB in mycobacteria. A total of 4179 proteins and 1236 acetylated sites were identified in our data. Further analysis of the dynamic changes involved in proteome and acetylome showed that MsKat/MsCobB played a regulatory role in various metabolic pathways and nucleic acid processes. After that, the quantitative mass spectrometric method was utilized and proved that the AMP-dependent synthetase, Citrate synthase, ATP-dependent specificity component of the Clp protease, and ATP-dependent DNA/RNA helicases were identified to be the substrates of MsKat. Overall, our study provided an important resource underlying the substrates and functions of the acetylation regulatory enzymes in mycobacteria. SIGNIFICANCE: In this study, we systematically analyzed the dynamic molecular changes in response to the MsKat/MsCobB overexpression in mycobacteria at proteome and lysine acetylation level by using a TMT-based quantitative proteomic approach. Pathways related with glycolysis, degradation of branched chain amino acids, phosphotransferase system were affected after disturbance of the two regulates enzymes involved in lysine acetylation. We also proved that AMP-dependent synthetase Clp protease, ATP-dependent DNA/RNA helicases and citrate synthase was the substrate of MsKat according to our proteomic data and biological validation. Together, our study underlined the substrates and functions of the acetylation regulatory enzymes in mycobacteria.

Structural and functional characterization of FabG4 from Mycolicibacterium smegmatis.

The rise in antimicrobial resistance is a global health crisis and necessitates the development of novel strategies to treat infections. For example, in 2022 tuberculosis (TB) was the second leading infectious killer after COVID-19, with multi-drug-resistant strains of TB having an ∼40% fatality rate. Targeting essential biosynthetic pathways in pathogens has proven to be successful for the development of novel antimicrobial treatments. Fatty-acid synthesis (FAS) in bacteria proceeds via the type II pathway, which is substantially different from the type I pathway utilized in animals. This makes bacterial fatty-acid biosynthesis (Fab) enzymes appealing as drug targets. FabG is an essential FASII enzyme, and some bacteria, such as Mycobacterium tuberculosis, the causative agent of TB, harbor multiple homologs. FabG4 is a conserved, high-molecular-weight FabG (HMwFabG) that was first identified in M. tuberculosis and is distinct from the canonical low-molecular-weight FabG. Here, structural and functional analyses of Mycolicibacterium smegmatis FabG4, the third HMwFabG studied to date, are reported. Crystal structures of NAD+ and apo MsFabG4, along with kinetic analyses, show that MsFabG4 preferentially binds and uses NADH when reducing CoA substrates. As M. smegmatis is often used as a model organism for M. tuberculosis, these studies may aid the development of drugs to treat TB and add to the growing body of research that distinguish HMwFabGs from the archetypal low-molecular-weight FabG.

Genetically encoded transcriptional plasticity underlies stress adaptation in Mycobacterium tuberculosis.

Transcriptional regulation is a critical adaptive mechanism that allows bacteria to respond to changing environments, yet the concept of transcriptional plasticity (TP) - the variability of gene expression in response to environmental changes - remains largely unexplored. In this study, we investigate the genome-wide TP profiles of Mycobacterium tuberculosis (Mtb) genes by analyzing 894 RNA sequencing samples derived from 73 different environmental conditions. Our data reveal that Mtb genes exhibit significant TP variation that correlates with gene function and gene essentiality. We also find that critical genetic features, such as gene length, GC content, and operon size independently impose constraints on TP, beyond trans-regulation. By extending our analysis to include two other Mycobacterium species -- M. smegmatis and M. abscessus -- we demonstrate a striking conservation of the TP landscape. This study provides a comprehensive understanding of the TP exhibited by mycobacteria genes, shedding light on this significant, yet understudied, genetic feature encoded in bacterial genomes.

Recombinant mycobacterial DNA-binding protein 1 with post-translational modifications boosts IFN-gamma production from BCG-vaccinated individuals' blood cells in combination with CpG-DNA.

Tuberculosis remains a large health threat, despite the availability of the tuberculosis vaccine, BCG. As BCG efficacy gradually decreases from adolescence, BCG-Prime and antigen-booster may be an efficient strategy to confer vaccine efficacy. Mycobacterial DNA-binding protein 1 (MDP1, namely Rv2986c, hupB or HU) is a major Mycobacterium tuberculosis protein that induces vaccine-efficacy by co-administration with CpG DNA. To produce MDP1 for booster-vaccine use, we have created recombinant MDP1 produced in both Escherichia coli (eMDP1) and Mycolicibacterium smegmatis (mMDP1), an avirulent rapid-growing mycobacteria. We tested their immunogenicity by checking interferon (IFN)-gamma production by stimulated peripheral blood cells derived from BCG-vaccinated individuals. Similar to native M. tuberculosis MDP1, we observed that most lysin resides in the C-terminal half of mMDP1 are highly methylated. In contrast, eMDP1 had less post-translational modifications and IFN-gamma stimulation. mMDP1 stimulated the highest amount of IFN-gamma production among the examined native M. tuberculosis proteins including immunodominant MPT32 and Antigen 85 complex. MDP1-mediated IFN-gamma production was more strongly enhanced when combined with a new type of CpG DNA G9.1 than any other tested CpG DNAs. Taken together, these results suggest that the combination of mMDP1 and G9.1 possess high potential use for human booster vaccine against tuberculosis.

Nanopore Identification of N-Acetylation by Hydroxylamine Deacetylation (NINAHD).

N-Acetyl modification, a chemical modification commonly found on biomacromolecules, plays a crucial role in the regulation of cell activities and is related to a variety of diseases. However, due to the instability of N-acetyl modification, accurate and rapid identification of N-acetyl modification with a low measurement cost is still technically challenging. Here, based on hydroxylamine deacetylation and nanopore single molecule chemistry, a universal sensing strategy for N-acetyl modification has been developed. Acetohydroxamic acid (AHA), which is produced by the hydroxylamine deacetylation reaction and serves as a reporter for N-acetylation identification, is specifically sensed by a phenylboronic acid (PBA)-modified Mycobacterium smegmatis porin A (MspA). With this strategy, N-acetyl modifications on RNA, DNA, proteins, and glycans were identified, demonstrating its generality. Specifically, histones can be treated with hydroxylamine deacetylation, from which the generated AHA can represent the amount of N-acetyl modification detected by a nanopore sensor. The unique event features of AHA also demonstrate the robustness of sensing against other interfering analytes in the environment.

RIP-seq reveals RNAs that interact with RNA polymerase and primary sigma factors in bacteria.

Bacteria have evolved structured RNAs that can associate with RNA polymerase (RNAP). Two of them have been known so far-6S RNA and Ms1 RNA but it is unclear if any other types of RNAs binding to RNAP exist in bacteria. To identify all RNAs interacting with RNAP and the primary σ factors, we have established and performed native RIP-seq in Bacillus subtilis, Corynebacterium glutamicum, Streptomyces coelicolor, Mycobacterium smegmatis and the pathogenic Mycobacterium tuberculosis. Besides known 6S RNAs in B. subtilis and Ms1 in M. smegmatis, we detected MTS2823, a homologue of Ms1, on RNAP in M. tuberculosis. In C. glutamicum, we discovered novel types of structured RNAs that associate with RNAP. Furthermore, we identified other species-specific RNAs including full-length mRNAs, revealing a previously unknown landscape of RNAs interacting with the bacterial transcription machinery.

The C-terminus is essential for the stability of the mycobacterial channel protein MspA.

Outer membrane proteins perform essential functions in uptake and secretion processes in bacteria. MspA is an octameric channel protein in the outer membrane of Mycobacterium smegmatis and is structurally distinct from any other known outer membrane protein. MspA is the founding member of a family with more than 3000 homologs and is one of the most widely used proteins in nanotechnological applications due to its advantageous pore structure and extraordinary stability. While a conserved C-terminal signal sequence is essential for folding and protein assembly in the outer membrane of Gram-negative bacteria, the molecular determinants of these processes are unknown for MspA. In this study, we show that mutation and deletion of methionine 183 in the highly conserved C-terminus of MspA and mutation of the conserved tryptophan 40 lead to a complete loss of protein in heat extracts of M. smegmatis. Swapping these residues partially restores the heat stability of MspA indicating that methionine 183 and tryptophan 40 form a conserved sulfur-π electron interaction, which stabilizes the MspA monomer. Flow cytometry showed that all MspA mutants are surface-accessible demonstrating that oligomerization and membrane integration in M. smegmatis are not affected. Thus, the conserved C-terminus of MspA is essential for its thermal stability, but it is not required for protein assembly in its native membrane, indicating that this process is mediated by a mechanism distinct from that in Gram-negative bacteria. These findings will benefit the rational design of MspA-like pores to tailor their properties in current and future applications.

An intramolecular cross-talk in D29 mycobacteriophage endolysin governs the lytic cycle and phage-host population dynamics.

D29 mycobacteriophage encodes LysA endolysin, which mediates mycobacterial host cell lysis by targeting its peptidoglycan layer, thus projecting itself as a potential therapeutic. However, the regulatory mechanism of LysA during the phage lytic cycle remains ill defined. Here, we show that during D29 lytic cycle, structural and functional regulation of LysA not only orchestrates host cell lysis but also is critical for maintaining phage-host population dynamics by governing various phases of lytic cycle. We report that LysA exists in two conformations, of which only one is active, and the protein undergoes a host peptidoglycan-dependent conformational switch to become active for carrying out endogenous host cell lysis. D29 maintains a pool of inactive LysA, allowing complete assembly of phage progeny, thus helping avoid premature host lysis. In addition, we show that the switch reverses after lysis, thus preventing exogenous targeting of bystanders, which otherwise negatively affects phage propagation in the environment.

Deciphering the molecular mechanism and regulation of formaldehyde detoxification in Mycobacterium smegmatis.

The build-up of formaldehyde, a highly reactive molecule is cytotoxic and must be eliminated for the organism's survival. Formaldehyde detoxification system is found in nearly all organisms including both pathogenic and non-pathogenic mycobacteria. MscR, a formaldehyde dehydrogenase from Mycobacterium smegmatis (Msm), is an indispensable part of this system and forms a bicistronic operon with its downstream uncharacterized gene, fmh. We here show that Fmh, a putative metallo-beta-lactamase, is essential in tolerating higher amounts of formaldehyde when co-overexpressed with mscR in vivo. Our NMR studies indicate that MscR, along with Fmh, enhances formate production through a mycothiol (MSH)-dependent pathway, emphasizing the importance of Fmh in detoxifying formaldehyde. Although another aldehyde dehydrogenase, MSMEG_1543, induces upon formaldehyde addition, it is not involved in its detoxification. We also show that the expression of the mscR operon is constitutive and remains unchanged upon formaldehyde addition, as displayed by the promoter activity of PmscR and by the transcript and protein levels of MscR. Furthermore, we establish the role of a thiol-responsive sigma factor SigH in formaldehyde detoxification. We show that SigH, and not SigE, is crucial for formaldehyde detoxification, even though it does not directly regulate mscR operon expression. In addition, sensitivity to formaldehyde in sigH-knockout could be alleviated by overexpression of mscR. Taken together, our data demonstrate the importance of MSH-dependent pathways in detoxifying formaldehyde in a mycobacterial system. An absence of such MSH-dependent proteins in eukaryotes and its complete conservation in M. tuberculosis, the causative agent of tuberculosis, further unravel new drug targets for this pathogen.IMPORTANCEExtensive research has been done on formaldehyde detoxification in different bacteria. However, our current understanding of the mechanisms underlying this process in mycobacteria remains exceedingly little. We previously showed that MscR, a formaldehyde dehydrogenase from Mycobacterium smegmatis, plays a pivotal role in this detoxification pathway. Here, we present a potential S-formyl-mycothiol hydrolase named Fmh, thought to be a metallo-beta-lactamase, which functions along with mycothiol (MSH) and MscR to enhance formate production within this detoxification pathway. Co-expression of Fmh with MscR significantly enhances the efficiency of formaldehyde detoxification in M. smegmatis. Our experiments establish that Fmh catalyzes the final step of this detoxification pathway. Although an alternative sigma factor SigH was found to be involved in formaldehyde detoxification, it did not directly regulate the expression of mscR. Since formaldehyde detoxification is essential for bacterial survival, we envisage this process to be a potential drug target for M. tuberculosis eradication.

Mycobacterial FtsEX-RipC interaction is required for normal growth and cell morphology in rifampicin and low ionic strength conditions.

Tuberculosis, a lung disease caused by Mycobacterium tuberculosis (Mtb), remains a major global health problem ranking as the second leading cause of death from a single infectious agent. One of the major factors contributing toward Mtb's success as a pathogen is its unique cell wall and its ability to counteract various arms of the host's immune response. A recent genome-scale study profiled a list of candidate genes that are predicted to be essential for Mtb survival of host-mediated responses. One candidate was FtsEX, a protein complex composed of an ATP-binding domain, FtsE, and a transmembrane domain, FtsX. FtsEX functions through interaction with a periplasmic hydrolase, RipC. Homologs of FtsEX exist in other bacteria and have been linked with playing a key role in regulating peptidoglycan hydrolysis during cell elongation and division. Here, we report on Mycobacterium smegmatis, FtsE, FtsX, and RipC and their protective roles in stressful conditions. We demonstrate that the individual genes of FtsEX complex and RipC are not essential for survival in normal growth conditions but conditionally essential in low-salt media and antibiotic-treated media. Growth defects in these conditions were characterized by short and bulgy cells as well as elongated filamentous cells. Our results suggest that FtsE, FtsX, and RipC are required for both normal cell elongation and division and ultimately for survival in stressful conditions. IMPORTANCE: Mycobacterial cell growth and division are coordinated with regulated peptidoglycan hydrolysis. Understanding cell wall gene complexes that govern normal cell division and elongation will aid in the development of tools to disarm the ability of mycobacteria to survive immune-like and antibiotic stresses. We combined genetic analyses and scanning electron microscopy to analyze morphological changes of mycobacterial FtsEX and RipC mutants in stressful conditions. We demonstrate that FtsE, FtsX, FtsEX, and RipC are conditionally required for the survival of Mycobacterium smegmatis during rifampicin treatment and in low-salt conditions. Growth defects in these conditions were characterized by short and bulgy cells as well as elongated filamentous cells. We also show that the FtsEX-RipC interaction is essential for the survival of M. smegmatis in rifampicin. Our results suggest that FtsE, FtsX, and RipC are required for normal cell wall regulation and ultimately for survival in stressful conditions.

The accumulation of methylated porphyrins in dormant cells of Mycolicibacterium smegmatis is accompanied by a decrease in membrane fluidity and an impede of the functioning of the respiratory chain.

Transition of Mycolicibacterium smegmatis (Msm) and Mycobacterium tuberculosis to dormancy in vitro is accompanied by an accumulation of free methylated forms of porphyrins (tetramethyl coproporphyrin - TMC) localized in the cell wall of dormant bacteria. A study of the fluorescence anisotropy of BODIPY based fluorescent probes on individual cell level using confocal microscope revealed significant changes in this parameter for BODIPY FL C16 from 0.05 to 0.22 for vegetative and dormant Msm cells correspondingly. Similarly, the increase of TMC concentration in vegetative Msm cells grown in the presence of 5-aminolevulinic acid (a known inducer of porphyrin synthesis) resulted in an increase of BODIPY FL C16 anisotropy. These changes in TMC concentration and membrane fluidity were accompanied by an inhibition of the activity of the respiratory chain measured by oxygen consumption and a reduction of the DCPIP redox acceptor. During the first 8 h of the reactivation of the dormant Msm cells, the porphyrin content and probe fluorescent anisotropy returned to the level for vegetative bacteria. We suggested that upon transition to dormancy, an accumulation of TMC in membranes leads to a decrease in membrane fluidity, resulting in an inhibition of the respiratory chain activity. However, direct interactions of TMC with membrane bound enzymes cannot also be excluded. This, in turn, may result in the down regulation of many metabolic energy-dependent reactions as a part of mechanisms accompanying the transition to a hypometabolic state of mycobacteria.

Mycobacterial PE12 protein promotes bacterial survival through inhibiting cell apoptosis.

Mycobacterial PE_PGRS family proteins play key roles in pathogen-host interaction. However, the function of most PE_PGRS proteins remains unknown. In this study, we characterized the role of PE12 of Mycobacterium bovis (M. bovis) on bacterial growth, bacterial survival, and host cell apoptosis. Transcriptome sequencing of infected THP-1 cells was also performed. Compared to Ms_Vec, we found that M. bovis PE12 did not alter the colony morphology of M. smegmatis. The survival of Ms_PE12 was obviously higher than that of Ms_Vec. Furthermore, PE12 significantly suppressed the apoptosis of THP-1 induced by M. smegmatis infection. Transcriptome analysis results showed that there were 70 downregulated genes in the Ms_PE12 infection group in comparison with the Ms_Vec infection group, and these differentially expressed genes were enriched in 240 downregulated GO terms and 6 KEGG pathways. The downregulated expression genes are involved in cell adhesion, phagocytosis, apoptosis, inflammatory response, glycolysis and transmembrane transporter activity. Taken together, our study reveals that PE12 can suppress apoptosis and inhibit proinflammatory cytokine response. We propose that PE12 is related to macrophage phagocytosis and apoptosis, providing useful information to the pathogenic mechanisms of M. bovis.

Understanding the structural basis of the binding specificity of c-di-AMP to M. smegmatis RecA using computational biology approach.

Mycobacterium tuberculosis RecA (MtRecA), a protein involved in DNA repair, homologous recombination and SOS pathway, contributes to the development of multidrug resistance. ATP binding-site in RecA has been a drug target to disable RecA dependent DNA repair. For the first time, experiments have shown the existence and binding of c-di-AMP to a novel allosteric site in the C-terminal-Domain (CTD) of Mycobacterium smegmatis RecA (MsRecA), a close homolog of MtRecA. In addition, it was observed that the c-di-AMP was not binding to Escherichia coli RecA (EcRecA). This article analyses the possible interactions of the three RecA homologs with the various c-di-AMP conformations to gain insights into the structural basis of the natural preference of c-di-AMP to MsRecA and not to EcRecA, using the structural biology tools. The comparative analysis, based on amino acid composition, homology, motifs, residue types, docking, molecular dynamics simulations and binding free energy calculations, indeed, conclusively indicates strong binding of c-di-AMP to MsRecA. Having very similar results as MsRecA, it is highly plausible for c-di-AMP to strongly bind MtRecA as well. These insights from the in-silico studies adds a new therapeutic approach against TB through design and development of novel allosteric inhibitors for the first time against MtRecA.Communicated by Ramaswamy H. Sarma.

Deciphering the mannose transfer mechanism of mycobacterial PimE by molecular dynamics simulations.

Phosphatidyl-myo-inositol mannosides (PIMs), Lipomannan (LM), and Lipoarabinomannan (LAM) are essential components of the cell envelopes of mycobacteria. At the beginning of the biosynthesis of these compounds, phosphatidylinositol (PI) is mannosylated and acylated by various enzymes to produce Ac1/2PIM4, which is used to synthesize either Ac1/2PIM6 or LM/LAM. The protein PimE, a membrane-bound glycosyltransferase (GT-C), catalyzes the addition of a mannose group to Ac1PIM4 to produce Ac1PIM5, using polyprenolphosphate mannose (PPM) as the mannose donor. PimE-deleted Mycobacterium smegmatis (Msmeg) showed structural deformity and increased antibiotic and copper sensitivity. Despite knowing that the mutation D58A caused inactivity in Msmeg, how PimE catalyzes the transfer of mannose from PPM to Ac1/2PIM4 remains unknown. In this study, analyzing the AlphaFold structure of PimE revealed the presence of a tunnel through the D58 residue with two differently charged gates. Molecular docking suggested PPM binds to the hydrophobic tunnel gate, whereas Ac1PIM4 binds to the positively charged tunnel gate. Molecular dynamics (MD) simulations further demonstrated the critical roles of the residues N55, F87, L89, Y163, Q165, K197, L198, R251, F277, W324, H326, and I375 in binding PPM and Ac1PIM4. The mutation D58A caused a faster release of PPM from the catalytic tunnel, explaining the loss of PimE activity. Along with a hypothetical mechanism of mannose transfer by PimE, we also observe the presence of tunnels through a negatively charged aspartate or glutamate with two differently-charged gates among most GT-C enzymes. Common hydrophobic gates of GT-C enzymes probably harbor sugar donors, whereas, differently-charged tunnel gates accommodate various sugar-acceptors.

RNA G-quadruplexes inhibit translation of the PE/PPE transcripts in Mycobacterium tuberculosis.

The role of RNA G-quadruplexes (rG4s) in bacteria remains poorly understood. High G-quadruplex densities have been linked to organismal stress. Here we investigate rG4s in mycobacteria, which survive highly stressful conditions within the host. We show that rG4-enrichment is a unique feature exclusive to slow-growing pathogenic mycobacteria, and Mycobacterium tuberculosis (Mtb) transcripts contain an abundance of folded rG4s. Notably, the PE/PPE family of genes, unique to slow-growing pathogenic mycobacteria, contain over 50% of rG4s within Mtb transcripts. We found that RNA oligonucleotides of putative rG4s in PE/PPE genes form G-quadruplex structures in vitro, which are stabilized by the G-quadruplex ligand BRACO19. Furthermore, BRACO19 inhibits the transcription of PE/PPE genes and selectively suppresses the growth of Mtb but not Mycobacterium smegmatis or other rapidly growing bacteria. Importantly, the stabilization of rG4s inhibits the translation of Mtb PE/PPE genes (PPE56, PPE67, PPE68, PE_PGRS39, and PE_PGRS41) ectopically expressed in M. smegmatis or Escherichia coli. In addition, the rG4-mediated reduction in PE/PPE protein levels attenuates proinflammatory response upon infection of THP-1 cells. Our findings shed new light on the regulation of PE/PPE genes and highlight a pivotal role for rG4s in Mtb transcripts as regulators of post-transcriptional translational control. The rG4s in mycobacterial transcripts may represent potential drug targets for newer therapies.

Insight into the on/off switch that regulates expression of the MSMEG-3762/63 efflux pump in Mycobacterium smegmatis.

Drug resistance is one of the most difficult challenges facing tuberculosis (TB) control. Drug efflux is among the mechanisms leading to drug resistance. In our previous studies, we partially characterized the ABC-type MSMEG-3762/63 efflux pump in Mycobacterium smegmatis, which shares high percentage of identity with the Mycobacterium tuberculosis Rv1687/86c pump. MSMEG-3762/63 was shown to have extrusion activity for rifampicin and ciprofloxacin, used in first and second-line anti-TB treatments. Moreover, we described the functional role of the TetR-like MSMEG-3765 protein as a repressor of the MSMEG_3762/63/65 operon and orthologous Rv1687/86/85c in M. tuberculosis. Here we show that the operon is upregulated in the macrophage environment, supporting a previous observation of induction triggered by acid-nitrosative stress. Expression of the efflux pump was also induced by sub-inhibitory concentrations of rifampicin or ciprofloxacin. Both these drugs also prevented the binding of the MSMEG-3765 TetR repressor protein to its operator in the MSMEG_3762/63/65 operon. The hypothesis that these two drugs might be responsible for the induction of the efflux pump operon was assessed by bioinformatics analyses. Docking studies using a structural model of the regulator MSMEG-3765 showed that both antibiotics abolished the ability of this transcriptional repressor to recognize the efflux pump operon by interacting with the homodimer at different binding sites within the same binding pocket. Reduced binding of the repressor leads to induction of the efflux pump in M. smegmatis, and reduced efficacy of these two anti-mycobacterial drugs.

Overexpression of a membrane transport system MSMEG_1381 and MSMEG_1382 confers multidrug resistance in Mycobacterium smegmatis.

Mycobacterium tuberculosis is a leading cause of human mortality worldwide, and the emergence of drug-resistant strains demands the discovery of new classes of antimycobacterial that can be employed in the therapeutic pipeline. Previously, a secondary metabolite, chrysomycin A, isolated from Streptomyces sp. OA161 displayed potent bactericidal activity against drug-resistant clinical isolates of M. tuberculosis and different species of mycobacteria. The antibiotic inhibits mycobacterial topoisomerase I and DNA gyrase, leading to bacterial death, but the mechanisms that could cause resistance to this antibiotic are currently unknown. To further understand the resistance mechanism, using M. smegmatis as a model, spontaneous resistance mutants were isolated and subjected to whole-genome sequencing. Mutation in a TetR family transcriptional regulator MSMEG_1380 was identified in the resistant isolates wherein the gene was adjacent to an operon encoding membrane proteins MSMEG_1381 and MSMEG_1382. Sequence analysis and modeling studies indicated that MSMEG_1381 and MSMEG_1382 are components of the Mmp family of efflux pumps and over-expression of either the operon or individual genes conferred resistance to chrysomycin A, isoniazid, and ethambutol. Our study highlights the role of membrane transporter proteins in conferring multiple drug resistance and the utility of recombinant strains overexpressing membrane transporters in the drug screening pipeline.

The PPE2 protein of Mycobacterium tuberculosis is secreted during infection and facilitates mycobacterial survival inside the host.

Mycobacterium tuberculosis secrets various effector proteins to evade host immune responses for facilitating its intracellular survival. The bacterial genome encodes several unique PE/PPE family proteins, which have been implicated to play important role in mycobacterial pathogenesis. A member of this family, PPE2 have been shown to contain a monopartite nuclear localization signal (NLS) and a DNA binding domain. In this study, we demonstrate that PPE2 protein is present in the sera of mice infected with either M. smegmatis expressing PPE2 or a clinical strain of M. tuberculosis (CDC1551). It was found that exogenously added PPE2 can permeate through the macrophage cell membrane and eventually translocate into the nucleus which requires the presence of NLS which showed considerable homology to HIV-tat like cell permeable peptides. Exogenously added PPE2 could inhibit NO production and decreased mycobacterial survival in macrophages. PPE2-null mutant of M. tuberculosis failed to inhibit NO production and had poor survival in macrophages which could be rescued by complementation with full-length PPE2. PPE2-null mutants also had poor survival in the lungs of infected mice indicating that PPE2 even when present in the bloodstream can confer a survival advantage to mycobacteria.