Hydrolyzable tannins (ellagitannins), flavonoids, pentacyclic triterpenes and their glycosides in antimycobacterial extracts of the ethnopharmacologically selected Sudanese medicinal plant Combretum hartmannianum Schweinf.

In Sudanese traditional medicine, decoctions, macerations, and tonics of the stem and root of Combretum hartmannianum are used for the treatment of persistent cough, a symptom that could be related to tuberculosis (TB). To verify these traditional uses, extracts from the stem wood, stem bark, and roots of C. hartmannianum were screened for their growth inhibitory effects against Mycobacterium smegmatis ATCC 14468. Methanol Soxhlet and ethyl acetate extracts of the root gave the strongest effects (MIC 312.5 and 625 µg/ml, respectively). HPLC-UV/DAD and UHPLC/QTOF-MS analysis of the ethyl acetate extract of the root led to the detection of 54 compounds, of which most were polyphenols and many characterized for the first time in C. hartmannianum. Among the major compounds were terflavin B and its two isomers, castalagin, corilagin, tellimagrandin I and its derivative, (S)-flavogallonic acid dilactone, punicalagin, and methyl-ellagic acid xylopyranoside. In addition, di-, tri- and tetra-galloyl glucose, combregenin, terminolic acid, cordifoliside D, luteolin, and quercetin-3-O-galactoside-7-O-rhamnoside-(2→1)-O-ß-D-arabinopyranoside were characterized. Luteolin gave better growth inhibition against M. smegmatis (MIC 250 µg/ml) than corilagin, ellagic acid, and gallic acid (MIC 500-1000 µg/ml). Our study justifies the use of C. hartmannianum in Sudanese folk medicine against prolonged cough that could be related to TB infection. This study demonstrates that C. hartmannianum should be explored further for new anti-TB drug scaffolds and antibiotic adjuvants.

Mycobacterium tuberculosis Rv2145c Promotes Intracellular Survival by STAT3 and IL-10 Receptor Signaling.

Although Mycobacterium tuberculosis (Mtb) is an intracellular pathogen in phagocytic cells, the factors and mechanisms by which they invade and persist in host cells are still not well understood. Characterization of the bacterial proteins modulating macrophage function is essential for understanding tuberculosis pathogenesis and bacterial virulence. Here we investigated the pathogenic role of the Rv2145c protein in stimulating IL-10 production. We first found that recombinant Rv2145c stimulated bone marrow-derived macrophages (BMDMs) to secrete IL-10, IL-6 and TNF-α but not IL-12p70 and to increase the expression of surface molecules through the MAPK, NF-κB, and TLR4 pathways and enhanced STAT3 activation and the expression of IL-10 receptor in Mtb-infected BMDMs. Rv2145c significantly enhanced intracellular Mtb growth in BMDMs compared with that in untreated cells, which was abrogated by STAT3 inhibition and IL-10 receptor (IL-10R) blockade. Expression of Rv2145c in Mycobacterium smegmatis (M. smegmatis) led to STAT3-dependent IL-10 production and enhancement of intracellular growth in BMDMs. Furthermore, the clearance of Rv2145c-expressing M. smegmatis in the lungs and spleens of mice was delayed, and these effects were abrogated by administration of anti-IL-10R antibodies. Finally, all mice infected with Rv2145c-expressing M. smegmatis died, but those infected with the vector control strain did not. Our data suggest that Rv2145c plays a role in creating a favorable environment for bacterial survival by modulating host signals.

Mycobacterial origin protein Rv0674 localizes into mitochondria, interacts with D-loop and regulates OXPHOS for intracellular persistence of Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) employs diverse strategies to survive inside the host macrophages. In this study, we have identified a conserved hypothetical protein of Mtb; Rv0674, which is present in the mitochondria of the host cell. The genetic knock-out of rv0674 (Mtb-KO) showed increased growth of Mtb. The intracellular infection with recombinant Mycobacterium smegmatis (MSMEG) expressing Rv0674 (MS_Rv0674), established that the protein is involved in promoting the apoptotic cell death of the macrophage. To investigate the mechanism incurred in mitochondria, we observed that the protein physically interacts with the control region (D-loop) of the mitochondrial DNA (LSP and HSP promoters of the loop) of the macrophages and facilitates the increased expression of mRNA in all the complexes of mitochondrial encoded OXPHOS subunits. The changes in OXPHOS levels corroborated with the ATP synthesis, mitochondrial membrane potential and superoxide production. The infection with MS_Rv0674 confirmed the role of this protein in effecting the intracellular infection. The fluorescent and confocal microscopy confirmed that the protein is localized in the mitochondria of infected macrophages and in the cells of BAL of TB patients. Together these findings indicate towards the novel function of the protein which is unlike to the earlier established mechanisms of mycobacterial physiology.

Role of LmeA, a Mycobacterial Periplasmic Protein, in Maintaining the Mannosyltransferase MptA and Its Product Lipomannan under Stress.

The mycobacterial cell envelope has a diderm structure, composed of an outer mycomembrane, an arabinogalactan-peptidoglycan cell wall, a periplasm, and an inner membrane. Lipomannan (LM) and lipoarabinomannan (LAM) are structural and immunomodulatory components of this cell envelope. LM/LAM biosynthesis involves a number of mannosyltransferases and acyltransferases, and MptA is an α1,6-mannosyltransferase involved in the final extension of the mannan chain. Recently, we reported the periplasmic protein LmeA being involved in the maturation of the mannan backbone in Mycobacterium smegmatis Here, we examined the role of LmeA under stress conditions. We found that lmeA transcription was upregulated under two stress conditions: stationary growth phase and nutrient starvation. Under both conditions, LAM was decreased, but LM was relatively stable, suggesting that maintaining the cellular level of LM under stress is important. Surprisingly, the protein levels of MptA were decreased in an lmeA deletion (ΔlmeA) mutant under both stress conditions. The transcript levels of mptA in the ΔlmeA mutant were similar to or even higher than those in the wild type, indicating that the decrease of MptA protein was a posttranscriptional event. The ΔlmeA mutant was unable to maintain the cellular level of LM under stress, consistent with the decrease in MptA. Even during active growth, overexpression of LmeA led the cells to produce more LM and become more resistant to several antibiotics. Altogether, our study reveals the roles of LmeA in the homeostasis of the MptA mannosyltransferase, particularly under stress conditions, ensuring the stable expression of LM and the maintenance of cell envelope integrity.IMPORTANCE Mycobacteria differentially regulate the cellular amounts of lipoglycans in response to environmental changes, but the molecular mechanisms of this regulation remain unknown. Here, we demonstrate that cellular lipoarabinomannan (LAM) levels rapidly decline under two stress conditions, stationary growth phase and nutrient starvation, while the levels of another related lipoglycan, lipomannan (LM), stay relatively constant. The persistence of LM under stress correlated with the maintenance of two key mannosyltransferases, MptA and MptC, in the LM biosynthetic pathway. We further showed that the stress exposures lead to the upregulation of lmeA gene expression and that the periplasmic protein LmeA plays a key role in maintaining the enzyme MptA and its product LM under stress conditions. These findings reveal new aspects of how lipoglycan biosynthesis is regulated under stress conditions in mycobacteria.

Mycobacterium tuberculosis Rv1096, facilitates mycobacterial survival by modulating the NF-κB/MAPK pathway as peptidoglycan N-deacetylase.

Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that can infect and replicate in macrophages. Peptidoglycan (PGN) is a major component of the mycobacterial cell wall and is recognized by host pattern recognition receptors (PRRs). Many bacteria modulate and evade the immune defenses of their hosts through PGN deacetylation. Rv1096 was previously characterized as a PGN N-deacetylase gene in Mtb. However, the underlying mechanism by which Rv1096 regulates host immune defenses during macrophage infection remains unclear. In the present study, we investigated the role of Rv1096 in evading host immunity using a recombinant M. smegmatis expressing exogenous Rv1096 and Rv1096-deleted Mtb strain H37Rv mutant. We found that Rv1096 promoted intracellular bacillary survival and inhibited the inflammatory response in M. smegmatis- or Mtb-infected macrophages. The inhibition of mycobacteria-induced inflammatory response in macrophages was at least partially due to NF-κB and MAPK activation downstream of TLR and NOD signaling pathways. Furthermore, we found that Rv1096 inhibitory effect on inflammatory response was associated with TLR2, TLR4 and NOD2. Finally, we demonstrated the PGN deacetylase activity of Rv1096 by Fourier transform IR and Rv1096 NODB deficient mutant. Our findings suggest that Rv1096 may deacetylate PGNs to evade PRRs recognition, thus protecting Mtb from host immune surveillance and clearance in macrophages.

Mycobacterium Von Willebrand factor protein MSMEG_3641 is involved in biofilm formation and intracellular survival.

Aim:Mycobacterium tuberculosis in vitro biofilm is associated with the virulence and persistence capability. Our aim is to delineate factors involved in biofilms development. Materials & methods: We performed transposon mutants screen and found that mutation of MSMEG_3641, a homolog of M. tuberculosis Rv1836c, can change M. smegmatis colony morphology and biofilm. Results: MSMEG_3641 contains a vWA domain that is highly conserved among Mycobacteria. The phenotypes of MSMEG_3641 mutants include disrupted biofilm, weakened migration ability and changed colony morphology. All phenotypes might be contributed to the enhanced cell wall permeability and declined cell aggregation ability. Conclusion: To our knowledge, this is the first report concerning the mycobacteria Von Willebrand factor domain function, especially in colony morphology and biofilm development.

Extra-ribosomal functions of Mtb RpsB in imparting stress resilience and drug tolerance to mycobacteria.

Emerging observations suggest that ribosomal proteins (RPs) play important extra-ribosomal roles in maintenance of cellular homeostasis. However, the mechanistic insights into these processes have not been extensively explored, especially in pathogenic bacteria. Here, we present our findings on potential extra-ribosomal functions of Mycobacterium tuberculosis (Mtb) RPs. We observed that Mtb RpsB and RpsQ are differentially localized to cell wall fraction in M. tuberculosis (H37Rv), while their M. smegmatis (Msm) homologs are primarily cytosolic. Cellular fractionation of ectopically expressed Mtb RPs in surrogate host (M. smegmatis) also shows their association with cell membrane/cell wall without any gross changes in cell morphology. M. smegmatis expressing Mtb RpsB exhibited altered redox homeostasis, decreased drug-induced ROS, reduced cell wall permeability and increased tolerance to various proteotoxic stress (oxidative stress, SDS and starvation). Mtb RpsB expression was also associated with increased resistance specifically towards Isoniazid, Ethionamide and Streptomycin. The enhanced drug tolerance was specific to Mtb RpsB and not observed upon ectopic expression of M. smegmatis homolog (Msm RpsB). Interestingly, C-terminus deletion in Mtb RpsB affected its localization and reversed the stress-resilient phenotypes. We also observed that M. tuberculosis (H37Rv) with upregulated RpsB levels had higher intracellular survival in macrophage. All these observations hint towards existence of moonlighting roles of Mtb RpsB in imparting stress resilience to mycobacteria. This work open avenues for further exploration of alternative pathways associated with fitness and drug tolerance in mycobacteria.

Cryo-EM Structures and Regulation of Arabinofuranosyltransferase AftD from Mycobacteria.

Mycobacterium tuberculosis causes tuberculosis, a disease that kills over 1 million people each year. Its cell envelope is a common antibiotic target and has a unique structure due, in part, to two lipidated polysaccharides-arabinogalactan and lipoarabinomannan. Arabinofuranosyltransferase D (AftD) is an essential enzyme involved in assembling these glycolipids. We present the 2.9-Å resolution structure of M. abscessus AftD, determined by single-particle cryo-electron microscopy. AftD has a conserved GT-C glycosyltransferase fold and three carbohydrate-binding modules. Glycan array analysis shows that AftD binds complex arabinose glycans. Additionally, AftD is non-covalently complexed with an acyl carrier protein (ACP). 3.4- and 3.5-Å structures of a mutant with impaired ACP binding reveal a conformational change, suggesting that ACP may regulate AftD function. Mutagenesis experiments using a conditional knockout constructed in M. smegmatis confirm the essentiality of the putative active site and the ACP binding for AftD function.

Role of a 21-kDa iron-regulated protein IrpA in the uptake of ferri-exochelin by Mycobacterium smegmatis.

AIMS: To characterize the 21-kDa iron-regulated cell wall protein in Mycobacterium smegmatis co-expressed with the siderophores mycobactin, exochelin and carboxymycobactin upon iron limitation. METHODS AND RESULTS: Mycobacterium smegmatis, grown in the presence of 0·02 µg Fe ml-1 (low iron) produced high levels of all the three siderophores, which were repressed in bacteria supplemented with 8 µg Fe ml-1 (high iron). Exochelin, the major extracellular siderophore was the first to rise and was expressed at high levels during log phase of growth. Carboxymycobactin, a minor component in log phase iron-starved M. smegmatis continued to rise when cultured for longer periods, reaching levels greater than exochelin. Iron-starved bacteria expressed a 21-kDa iron-regulated protein (IrpA) that was identified as Clp protease subunit (MSMEG_3671) and characterized as a receptor for ferri-exochelin. CONCLUSIONS: Ferri-exochelin is the preferred siderophore in M. smegmatis and this ferri-exochelin: IrpA machinery is absent in Mycobacterium tuberculosis. SIGNIFICANCE AND IMPACT OF THE STUDY: Exochelin machinery is functional in M. smegmatis and the carboxymycobactin-mycobactin machinery is the sole iron uptake system in M. tuberculosis. The absence of the ferri-exochelin: IrpA system in the pathogen signifies the importance of the carboxymycobactin-mycobactin system machinery in M. tuberculosis.

Effects of Mycobacterium tuberculosis Rv1096 on mycobacterial cell division and modulation on macrophages.

Mycobacterium tuberculosis is capable of escaping the clearance of immune system mainly due to its complex constituents of cell wall. Certain studies show that glycoproteins are involved in immune evasion and act as virulence factors. Peptidoglycan deacetylase Rv1096 is a member of mannosylated proteins. Previously, we reported Rv1096 protein contributed to the resistance of Mycobacterium smegmatis (M. smegmatis) to lysozyme, but more characterization of this protein is required where further intracellular function is unknown. Here, Rv1096 was heterologously over-expressed in the fast-growing and nonpathogenic M. smegmatis (named as M. smegmatis/Rv1096). We observed the morphological alterations in M. smegmatis/Rv1096 including an elongated rod-like shape and increased amounts of Z-rings, which implied that Rv1096 facilitated the cell growth and division. Moreover, a series of assays concerning the interaction between M. smegmatis/Rv1096 and host were carried out. The results showed that M. smegmatis/Rv1096 evaded the killing of macrophages due to the inhibition of phagosome-lysosome fusion, nicotinamide adenine dinucleotide phosphate oxidase activity and reactive oxygen species production. The secretion of interleukin-12 and tumor necrosis factor-α was also impaired by Rv1096. In addition, five putative interaction partners of Rv1096 were identified, which possibly cooperated with Rv1096 in cell division and immune regulation. These results suggested that Rv1096 had effects on mycobacterial division and might act as a virulence factor to mediate the immune evasion in macrophage during mycobacterial infection.

Rv2223c, an acid inducible carboxyl-esterase of Mycobacterium tuberculosis enhanced the growth and survival of Mycobacterium smegmatis.

Aim: To elucidate the role of Rv2223c in Mycobacterium tuberculosis. Methods: Purified recombinant Rv2223c protein was characterized. Expression of rv2223c in the presence of different stress environment and subcellular localization were performed in M. tuberculosis H37Ra and Mycobacterium smegmatis (MS_2223c). Effect of its overexpression on growth rate, infection and intracellular survival in THP-1/PBMC cells were studied. Results: rRv2223c demonstrated esterase activity with preference for pNP-octanoate and hydrolyzed trioctanoate to di- and mono-octanoate. Expression of rv2223c was upregulated in acidic and nutritive stress conditions. rRv2223c was identified in extracellular and cell wall fractions. MS_2223c exhibited enhanced growth, survival during in vitro stress, infection and intracellular survival. Conclusions: Rv2223c is a secretary, carboxyl-esterase, with enhanced expression under acidic and nutritive stress condition and might help in intracellular survival of bacteria.

Nudix hydrolases with Coenzyme A (CoA) and acyl-CoA pyrophosphatase activities confer growth advantage to Mycobacterium smegmatis.

Nudix hydrolase family proteins hydrolyse toxic by-products of cellular metabolism such as mutagenic nucleoside triphosphates, sugar nucleotides and signalling molecules. We studied the substrate specificities of Nudix hydrolases encoded by rv3672c and rv3040c from Mycobacterium tuberculosis and their respective homologues, msmeg_6185 and msmeg_2327 from M. smegmatis. The rv3672c- and msmeg_6185-encoded proteins (Rv3672 and MSMEG_6185, respectively) showed CoA pyrophosphatase (CoAse) activity that converted acyl-CoA to adenosine-3',5'-diphosphate (3', 5'-ADP) and 4-acyl phosphopantetheine. The efficiencies of Rv3672 and MSMEG_6185 in hydrolysing CoA derivatives were found to be higher than those of the Rv3040 and MSMEG_2327 (encoded by rv3040c and msmeg_2327, respectively). Further, amongst the substrates tested, Rv3672 and MSMEG_6185 used CoA and oxidized CoA as the most preferred substrates. Use of the M. smegmatis model showed that the expression of msmeg_6185 occurs in the log and stationary phases but declines during the late stationary phase and becomes undetectable during hypoxia. The co-culture competition experiments performed between the wild-type and Δmsmeg_6185 strains of M. smegmatis in different carbon sources revealed that the presence of msmeg_6185 provided growth fitness advantage to M. smegmatis, irrespective of the carbon source, implicating its function in regulation for the optimal physiological levels of acyl-CoAs in the cell.

Mycofactocin Is Associated with Ethanol Metabolism in Mycobacteria.

Mycofactocin (MFT) belongs to the class of ribosomally synthesized and posttranslationally modified peptides conserved in many ActinobacteriaMycobacterium tuberculosis assimilates cholesterol during chronic infection, and its in vitro growth in the presence of cholesterol requires most of the MFT biosynthesis genes (mftA, mftB, mftC, mftD, mftE, and mftF), although the reasons for this requirement remain unclear. To identify the function of MFT, we characterized MFT biosynthesis mutants constructed in Mycobacterium smegmatis, M. marinum, and M. tuberculosis We found that the growth deficit of mft deletion mutants in medium containing cholesterol-a phenotypic basis for gene essentiality prediction-depends on ethanol, a solvent used to solubilize cholesterol. Furthermore, functionality of MFT was strictly required for growth of free-living mycobacteria in ethanol and other primary alcohols. Among other genes encoding predicted MFT-associated dehydrogenases, MSMEG_6242 was indispensable for M. smegmatis ethanol assimilation, suggesting that it is a candidate catalytic interactor with MFT. Despite being a poor growth substrate, ethanol treatment resulted in a reductive cellular state with NADH accumulation in M. tuberculosis During ethanol treatment, mftC mutant expressed the transcriptional signatures that are characteristic of respirational dysfunction and a redox-imbalanced cellular state. Counterintuitively, there were no differences in cellular bioenergetics and redox parameters in mftC mutant cells treated with ethanol. Therefore, further understanding of the function of MFT in ethanol metabolism is required to identify the cause of growth retardation of MFT mutants in cholesterol. Nevertheless, our results establish the physiological role of MFT and also provide new insights into the specific functions of MFT homologs in other actinobacterial systems.IMPORTANCE Tuberculosis is caused by Mycobacterium tuberculosis, and the increasing emergence of multidrug-resistant strains renders current treatment options ineffective. Although new antimycobacterial drugs are urgently required, their successful development often relies on complete understanding of the metabolic pathways-e.g., cholesterol assimilation-that are critical for persistence and for pathogenesis of M. tuberculosis In this regard, mycofactocin (MFT) function appears to be important because its biosynthesis genes are predicted to be essential for M. tuberculosisin vitro growth in cholesterol. In determining the metabolic basis of this genetic requirement, our results unexpectedly revealed the essential function of MFT in ethanol metabolism. The metabolic dysfunction thereof was found to affect the mycobacterial growth in cholesterol which is solubilized by ethanol. This knowledge is fundamental in recognizing the bona fide function of MFT, which likely resembles the pyrroloquinoline quinone-dependent ethanol oxidation in acetic acid bacteria exploited for industrial production of vinegar.

Mannopyranoside Glycolipids Inhibit Mycobacterial and Biofilm Growth and Potentiate Isoniazid Inhibition Activities in M.†smegmatis.

Lipomannan and lipoarabinomannan are integral components of the mycobacterial cell wall. Earlier studies demonstrated that synthetic arabinan and arabinomannan glycolipids acted as inhibitors of mycobacterial growth, in addition to exhibiting inhibitory activities of mycobacterial biofilm. Herein, it is demonstrated that synthetic mannan glycolipids are better inhibitors of mycobacterial growth, whereas lipoarabinomannan has a higher inhibition efficiency to biofilm. Syntheses of mannan glycolipids with a graded number of mannan moieties and an arabinomannan glycolipid are conducted by chemical methods and subsequent mycobacterial growth and biofilm inhibition studies are conducted on Mycobacterium smegmatis. Growth inhibition of (73±3) % is observed with a mannose trisaccharide containing a glycolipid, whereas this glycolipid did not promote biofilm inhibition activity better than that of arabinomannan glycolipid. The antibiotic supplementation activities of glycolipids on growth and biofilm inhibitions are evaluated. Increases in growth and biofilm inhibitions are observed if the antibiotic is supplemented with glycolipids, which leads to a significant reduction of inhibition concentrations of the antibiotic.

Elucidating the role of (p)ppGpp in mycobacterial persistence against antibiotics.

Bacterial persistence, the ability of bacteria to survive high concentrations of antibiotics for extended periods of time, is an important contributing factor to therapy failure and development of chronic and recurrent infections. Several recent studies have suggested that this persistence is mediated primarily by (p)ppGpp, through its interactions with toxin-antitoxin modules and polyphosphates. In this study, we address whether these key players play a role in mycobacterial persistence against antibiotics. We targeted these specific pathways in Mycobacterium smegmatis by constructing deletion strains of (p)ppGpp synthetase/hydrolase (relA), polyphosphate kinases (ppk1 and ppk2), exopolyphosphatases (ppx1 and ppx2), and the lon protease. None of these mutant strains exhibited altered levels of persisters against isoniazid and ciprofloxacin, when compared with wild-type strain. Even under conditions in which the stringent response usually gets activated, these strains displayed wild-type persister levels. Interestingly, we also found that unlike Escherichia coli, maintaining M. smegmatis in exponential phase by repeated passaging does not eliminate persisters suggesting that at least against the antibiotics tested, stationary-phase dependent persisters (type I) are not the major contributors. Thus, our data demonstrate that multiple mechanisms of antibiotic persistence exist and that these vary widely among different bacterial species. © 2018 IUBMB Life, 70(9):836-844, 2018.

Bioactive Pyridone Alkaloids from a Deep-Sea-Derived Fungus Arthrinium sp. UJNMF0008.

Eight new 4-hydroxy-2-pyridone alkaloids arthpyrones D⁻K (1⁻8), along with two known analogues apiosporamide (9) and arthpyrone B (10), were isolated from a deep-sea-derived fungus Arthrinium sp. UJNMF0008. The structures of the isolated compounds were elucidated on the basis of spectroscopic methods with that of 1 being established by chemical transformation and X-ray diffraction analysis. Compounds 1 and 2 bore an ester functionality linking the pyridone and decalin moieties first reported in this class of metabolites, while 3 and 4 incorporated a rare natural hexa- or tetrahydrobenzofuro[3,2-c]pyridin-3(2H)-one motif. Compounds 3⁻6 and 9 exhibited moderate to significant antibacterial activity against Mycobacterium smegmatis and Staphylococcus aureus with IC50 values ranging from 1.66⁻42.8 µM, while 9 displayed cytotoxicity against two human osteosarcoma cell lines (U2OS and MG63) with IC50 values of 19.3 and 11.7 µM, respectively.

Alternative ribosomal proteins are required for growth and morphogenesis of Mycobacterium smegmatis under zinc limiting conditions.

Zinc is an essential micronutrient required for proper structure and function of many proteins. Bacteria regularly encounter zinc depletion and have evolved diverse mechanisms to continue growth when zinc is limited, including the expression of zinc-independent paralogs of zinc-binding proteins. Mycobacteria have a conserved operon encoding four zinc-independent alternative ribosomal proteins (AltRPs) that are expressed when zinc is depleted. It is unknown if mycobacterial AltRPs replace their primary paralogs in the ribosome and maintain protein synthesis under zinc-limited conditions, and if such replacements contribute to their physiology. This study shows that AltRPs from Mycobacterium smegmatis are essential for growth when zinc ion is scarce. Specifically, the deletion mutant of this operon (ΔaltRP) is unable to grow in media containing a high-affinity zinc chelator, while growth of the wild type strain is unaffected under the same conditions. However, when zinc is gradually depleted during growth in zinc-limited medium, the ΔaltRP mutant maintains the same growth rate as seen for the wild type strain. In contrast to M. smegmatis grown with sufficient zinc supplementation that forms shorter cells when transitioning from logarithmic to stationary phase, M. smegmatis deficient for zinc elongates after the expression of AltRPs in late logarithmic phase. These zinc-depleted bacteria also exhibit a remarkable morphology characterized by a condensed chromosome, increased number of polyphosphate granules, and distinct appearance of lipid bodies and the cell wall compared to the zinc-replete cells. However, the ΔaltRP cells fail to elongate and transition into the zinc-limited morphotype, resembling the wild type zinc-replete bacteria instead. Therefore, the altRP operon in M. smegmatis has a vital role in continuation of growth when zinc is scarce and in triggering specific morphogenesis during the adaptation to zinc limitation, suggesting that AltRPs can functionally replace their zinc-dependent paralogs, but also contribute to mycobacterial physiology in a unique way.

Expression of Rv2031c-Rv2626c fusion protein in Mycobacterium smegmatis enhances bacillary survival and modulates innate immunity in macrophages.

Dormancy-associated antigens encoded by the dormancy survival regulon (DosR) genes are required for survival of Mycobacterium tuberculosis (Mtb) in macrophages. However, mechanisms underlying survival of Mtb in macrophages remains to be elucidated. A recombinant Mycobacterium smegmatis strain (rMs) expressing a fusion protein of two dormancy­associated antigens Rv2031c and Rv2626c from Mtb was constructed in the present study. In an in vitro culture, growth rate of rMs was lower compared with Ms. A total of 24 h following infection of murine macrophages with rMs or Ms, percentage of viable cells decreased and the number of bacteria in viable cells increased compared with Ms, demonstrating that virulence and intracellular survival of rMs were enhanced. Compared with macrophages infected with Ms, necrosis of macrophages infected with rMs was increased, while apoptosis was inhibited. Macrophages infected with rMs secreted more interferon­Î³ and interleukin­6, but fewer nitric oxide and tumor necrosis factor­α, compared with macrophages infected with Ms. The present study demonstrated that the fusion protein composed of dormancy­associated antigens Rv2031c and Rv2626c in Ms serves a physiological function of a dormancy­associated antigen and modulates innate immunity of host macrophages, therefore favoring intracellular bacillary survival.

RbpA and σB association regulates polyphosphate levels to modulate mycobacterial isoniazid-tolerance.

To facilitate survival under drug stresses, a small population of Mycobacterium tuberculosis can tolerate bactericidal concentrations of drugs without genetic mutations. These drug-tolerant mycobacteria can be induced by environmental stresses and contribute to recalcitrant infections. However, mechanisms underlying the development of drug-tolerant mycobacteria remain obscure. Herein, we characterized a regulatory pathway which is important for the tolerance to isoniazid (INH) in Mycobacterium smegmatis. We found that the RNA polymerase binding protein RbpA associates with the stress response sigma factor σB , to activate the transcription of ppk1, the gene encoding polyphosphate kinase. Subsequently, intracellular levels of inorganic polyphosphate increase to promote INH-tolerant mycobacteria. Interestingly, σB and ppk1 expression varied proportionately in mycobacterial populations and positively correlated with tolerance to INH in individual mycobacteria. Moreover, sigB and ppk1 transcription are both induced upon nutrient depletion, a condition that stimulates the formation of INH-tolerant mycobacteria. Over-expression of ppk1 in rbpA knockdown or sigB deleted strains successfully restored the number of INH-tolerant mycobacteria under both normal growth and nutrient starved conditions. These data suggest that RbpA and σB regulate ppk1 expression to control drug tolerance both during the logarithmic growth phase and under the nutrition starved conditions.

Discovery of novel bacterial topoisomerase I inhibitors by use of in silico docking and in vitro assays.

Topoisomerases are important targets for antibacterial and anticancer therapies. Bacterial topoisomerase I remains to be exploited for antibiotics that can be used in the clinic. Inhibitors of bacterial topoisomerase I may provide leads for novel antibacterial drugs against pathogens resistant to current antibiotics. TB is the leading infectious cause of death worldwide, and new TB drugs against an alternative target are urgently needed to overcome multi-drug resistance. Mycobacterium tuberculosis topoisomerase I (MtbTopI) has been validated genetically and chemically as a TB drug target. Here we conducted in silico screening targeting an active site pocket of MtbTopI. The top hits were assayed for inhibition of MtbTopI activity. The shared structural motif found in the active hits was utilized in a second round of in silico screening and in vitro assays, yielding selective inhibitors of MtbTopI with IC50s as low as 2 µM. Growth inhibition of Mycobacterium smegmatis by these compounds in combination with an efflux pump inhibitor was diminished by the overexpression of recombinant MtbTopI. This work demonstrates that in silico screening can be utilized to discover new bacterial topoisomerase I inhibitors, and identifies a novel structural motif which could be explored further for finding selective bacterial topoisomerase I inhibitors.