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.

Mycobacterium tuberculosis ESAT6 modulates host innate immunity by downregulating miR-222-3p target PTEN.

Tuberculosis (TB) remains a major cause of mortality and morbidity worldwide, and it is instant to discover novel anti-TB drugs due to the rapidly growing drug-resistance TB. Mycobacterium tuberculosis (Mtb) secreted effector ESAT6 plays a critical role in modulation miRNAs to regulate host defense mechanisms during Mtb infection, it can be a possible target for new tuberculosis drugs. The non-tuberculous mycobacteria Mycobacterium smegmatis (M. smegmatis) and Mtb have high gene homology but no pathogenicity. We used ESAT6 to interfere with macrophages or mice infected by M. smegmatis and determined that it enhanced the survival rate of bacteria and regulated miR-222-3p target PTEN. Expression of miR-222-3p reduced and PTEN enhanced with the progression of macrophages infected by M. smegmatis with ESAT6 co-incubation. MiR-222-3p overexpression diminished M. smegmatis survival and upregulated proinflammatory cytokines. VO-Ohpic trihydrate (PTEN inhibitor) reduced M. smegmatis survival and upregulated proinflammatory cytokines in vivo and in vitro, and VO-Ohpic trihydrate reversed the tissue damage of mouse organs caused by ESAT6. These results uncover an ESAT6 dependent role for miR-222-3p and its target PTEN in regulating host immune responses to bacterial infection and may provide a potential site for the development of anti-tuberculosis drugs that specifically antagonize the virulence of ESAT6.

A Bumpy Ride of Mycobacterial Phagosome Maturation: Roleplay of Coronin1 Through Cofilin1 and cAMP.

Phagosome-lysosome fusion in innate immune cells like macrophages and neutrophils marshal an essential role in eliminating intracellular microorganisms. In microbe-challenged macrophages, phagosome-lysosome fusion occurs 4 to 6 h after the phagocytic uptake of the microbe. However, live pathogenic mycobacteria hinder the transfer of phagosomes to lysosomes, up to 20 h post-phagocytic uptake. This period is required to evade pro-inflammatory response and upregulate the acid-stress tolerant proteins. The exact sequence of events through which mycobacteria retards phagolysosome formation remains an enigma. The macrophage coat protein Coronin1(Cor1) is recruited and retained by mycobacteria on the phagosome membrane to retard its maturation by hindering the access of phagosome maturation factors. Mycobacteria-infected macrophages exhibit an increased cAMP level, and based on receptor stimulus, Cor1 expressing cells show a higher level of cAMP than non-Cor1 expressing cells. Here we have shown that infection of bone marrow-derived macrophages with H37Rv causes a Cor1 dependent rise of intracellular cAMP levels at the vicinity of the phagosomes. This increased cAMP fuels cytoskeletal protein Cofilin1 to depolymerize F-actin around the mycobacteria-containing phagosome. Owing to reduced F-actin levels, the movement of the phagosome toward the lysosomes is hindered, thus contributing to the retarded phagosome maturation process. Additionally, Cor1 mediated upregulation of Cofilin1 also contributes to the prevention of phagosomal acidification, which further aids in the retardation of phagosome maturation. Overall, our study provides first-hand information on Cor1 mediated retardation of phagosome maturation, which can be utilized in developing novel peptidomimetics as part of host-directed therapeutics against tuberculosis.

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.

Cryo-EM structure of Mycobacterium smegmatis DyP-loaded encapsulin.

Encapsulins containing dye-decolorizing peroxidase (DyP)-type peroxidases are ubiquitous among prokaryotes, protecting cells against oxidative stress. However, little is known about how they interact and function. Here, we have isolated a native cargo-packaging encapsulin from Mycobacterium smegmatis and determined its complete high-resolution structure by cryogenic electron microscopy (cryo-EM). This encapsulin comprises an icosahedral shell and a dodecameric DyP cargo. The dodecameric DyP consists of two hexamers with a twofold axis of symmetry and stretches across the interior of the encapsulin. Our results reveal that the encapsulin shell plays a role in stabilizing the dodecameric DyP. Furthermore, we have proposed a potential mechanism for removing the hydrogen peroxide based on the structural features. Our study also suggests that the DyP is the primary cargo protein of mycobacterial encapsulins and is a potential target for antituberculosis drug discovery.

Mycobacterium smegmatis MSMEG_0129 is a nutrition-associated regulator that interacts with CarD and ClpP2.

Mycobacterium smegmatis MSMEG_0129 and Rv0164, its homologue in Mycobacterium tuberculosis, are single START-domain proteins essential for bacterial growth and survival, but their biochemical activities and biological roles remain undetermined. Here, we probed the possible functions of MSMEG_0129 and its underlying mechanisms by determining its cellular location, searching for its interaction partners and monitoring its transcription profile. MSMEG_0129, and Rv0164 by extension, were found to be cytosolic proteins rather than secreted components as previously understood. Increases in MSMEG_0129 expression at physiological levels accelerated bacterial growth in a proportional manner, but additional growth acceleration was not observed when MSMEG_0129 was overexpressed up to 20 fold. MSMEG_0129 is a short-lived protein, unstable at both the mRNA and protein levels. Co-IP and GST pull-down assays showed that MSMEG_0129 interacts with the ClpP2 protease and a global transcription factor, CarD, their expression being correlated with that of MSMEG_0129. Nutrient deficiency led to the downregulation of MSMEG_0129 but upregulation of CarD. However, in the context of constitutive MSMEG_0129 overexpression under nutrient-rich or starvation conditions, the mRNA level of CarD was reduced 3 fold. Conversely, expression of ClpP2 decreased with MSMEG_0129 downregulation under starvation conditions, but increased 4-8 fold when MSMEG_0129 was overexpressed. Our data suggest that MSMEG_0129, and Rv0164 by analogy, are likely to be nutrition sensing factors that regulate mycobacterial growth and may be involved in signal transfer under nutrient deficiency, possibly via physical and regulatory interactions with CarD and ClpP2.

Synthesis and Antibacterial Activity of Difluoromethyl Cinnamoyl Amides.

Series of novel amides of isoferulic acid, where the phenolic hydroxyl was replaced by a difluoromethyl group, were synthesized and their in vitro antibacterial activities assayed against fourteen bacterial strains (six Gram-positive and eight Gram-negative). A one-pot methodology was developed to obtain the 3'-(difluoromethyl)-4'-methoxycinnamoyl amides using Deoxofluor® as a fluorinating agent. The N-isopropyl, N-isopentyl, and N-(2-phenylethyl) amides 11b, 11d and 11g were the most active and selective against Mycobacterium smegmatis (MIC = 8 µg/mL) with 11b and 11g displaying negligible or no cytotoxicity against HepG2 and A549 cells. Thirteen analogs of N-isopropylamide 11b were also synthesized and their antibacterial activity assayed. Results show that the difluoromethyl moiety enhanced antibacterial activity and selectivity towards M. smegmatis, changing the microorganism inhibition profile of the parent compound. The selectivity exhibited by some of the compounds towards M. smegmatis makes them potential leads in the search for new narrow spectrum antibiotics against M. tuberculosis.

Monoterpenoid Geraniol Improves Anti-mycobacterial Drug Efficiency by Interfering with Lipidome and Virulence of Mycobacteria.

BACKGROUND: Tuberculosis (TB) remains a global infectious disorder for which efficient therapeutics are elusive. Nature is a source of novel pharmacologically active compounds with many potential drugs being derived directly or indirectly from plants, microorganisms and marine organisms. OBJECTIVE: The present study aimed to elucidate the antimycobacterial potential of Geraniol (Ger), monoterpene alcohol, against Mycobacterium smegmatis. METHODS: Disrupted membrane integrity was studied by membrane permeability assay and PI uptake. Cell surface phenotypes were studied by colony morphology, sliding motility and cell sedimentation rate. Lipidome profile was demonstrated by thin-layer chromatography and liquid chromatography-electrospray ionization mass spectrometry. Amendment in iron homeostasis was assessed by using iron chelator ferrozine and ferroxidase assay while genotoxicity was estimated with EtBr and DAPI staining. Biofilm formation was measured by staining, dry mass and metabolic activity using crystal violet. Cell adherence was examined microscopically and spectrophotometrically. RESULTS: We found the antimycobacterial activity of Ger to be 500 µg/ml against M. smegmatis. Underlying mechanisms revealed impaired cell surface phenotypes. Lipidomics analysis exposed profound decrement of mycolic acids, phosphatidylinositol mannosides and triacylglycerides which are crucial for MTB pathogenicity. We further explored that Ger impairs iron homeostasis and leads to genotoxic stress. Moreover, Ger inhibited the potential virulence attributes such as biofilm formation and cell adherence to both polystyrene surface and epithelial cells. Finally, we have validated all the disrupted phenotypes by RT-PCR which showed good correlation with the biochemical assays. CONCLUSION: Taken together, the current study demonstrates the antimycobacterial mechanisms of Ger, which may be exploited as an effective candidate of pharmacological interest.

Effect of peptidoglycan amidase MSMEG_6281 on fatty acid metabolism in Mycobacterium smegmatis.

Mycobacterium smegmatis MSMEG_6281, a peptidoglycan (PG) amidase, is essential in maintaining cell wall integrity. To address the potential roles during the MSMEG_6281-mediated biological process, we compared proteomes from wild-type M.smegmatis and MSMEG_6281 gene knockout strain (M.sm-ΔM_6281) using LC-MS/MS analysis. Peptide analysis revealed that 851 proteins were differentially produced with at least 1.2-fold changes, including some proteins involved in fatty acid metabolism such as acyl-CoA synthase, acyl-CoA dehydrogenase, MCE-family proteins, ATP-binding cassette (ABC) transporters, and MmpL4. Some proteins related to fatty acid degradation were enriched through protein-protein interaction analysis. Therefore, proteomic data showed that a lack of MSMEG_6281 affected fatty acid metabolism. Mycobacteria can produce diverse lipid molecules ranging from single fatty acids to highly complex mycolic acids, and mycobacterial surface-exposed lipids may impact biofilm formation. In this study, we also assessed the effects of MSMEG_6281 on biofilm phenotype using semi-quantitative and morphology analysis methods. These results found that M.sm-ΔM_6281 exhibited a delayed biofilm phenotype compared to that of the wild-type M.smegmatis, and the changes were recovered when PG amidase was rescued in a ΔM_6281::Rv3717 strain. Our results demonstrated that MSMEG_6281 impacts fatty acid metabolism and further interferes with biofilm formation. These results provide a clue to study the effects of PG amidase on mycobacterial pathogenicity.

Phase separation and clustering of an ABC transporter in Mycobacterium tuberculosis.

Phase separation drives numerous cellular processes, ranging from the formation of membrane-less organelles to the cooperative assembly of signaling proteins. Features such as multivalency and intrinsic disorder that enable condensate formation are found not only in cytosolic and nuclear proteins, but also in membrane-associated proteins. The ABC transporter Rv1747, which is important for Mycobacterium tuberculosis (Mtb) growth in infected hosts, has a cytoplasmic regulatory module consisting of 2 phosphothreonine-binding Forkhead-associated domains joined by an intrinsically disordered linker with multiple phospho-acceptor threonines. Here we demonstrate that the regulatory modules of Rv1747 and its homolog in Mycobacterium smegmatis form liquid-like condensates as a function of concentration and phosphorylation. The serine/threonine kinases and sole phosphatase of Mtb tune phosphorylation-enhanced phase separation and differentially colocalize with the resulting condensates. The Rv1747 regulatory module also phase-separates on supported lipid bilayers and forms dynamic foci when expressed heterologously in live yeast and M. smegmatis cells. Consistent with these observations, single-molecule localization microscopy reveals that the endogenous Mtb transporter forms higher-order clusters within the Mycobacterium membrane. Collectively, these data suggest a key role for phase separation in the function of these mycobacterial ABC transporters and their regulation via intracellular signaling.

Nitrogen deprivation induces triacylglycerol accumulation, drug tolerance and hypervirulence in mycobacteria.

Mycobacteria share with other actinomycetes the ability to produce large quantities of triacylglycerol (TAG), which accumulate as intracytoplasmic lipid inclusions (ILI) also known as lipid droplets (LD). Mycobacterium tuberculosis (M. tb), the etiologic agent of tuberculosis, acquires fatty acids from the human host which are utilized to synthesize TAG, subsequently stored in the form of ILI to meet the carbon and nutrient requirements of the bacterium during long periods of persistence. However, environmental factors governing mycobacterial ILI formation and degradation remain poorly understood. Herein, we demonstrated that in the absence of host cells, carbon excess and nitrogen starvation promote TAG accumulation in the form of ILI in M. smegmatis and M. abscessus, used as surrogate species of M. tb. Based on these findings, we developed a simple and reversible in vitro model to regulate ILI biosynthesis and hydrolysis in mycobacteria. We also showed that TAG formation is tgs1 dependent and that lipolytic enzymes mediate TAG breakdown. Moreover, we confirmed that the nitrogen-deprived and ILI-rich phenotype was associated with an increased tolerance towards several drugs used for treating mycobacterial infections. Importantly, we showed that the presence of ILI substantially enhanced the bacterial burden and granuloma abundance in zebrafish embryos infected with lipid-rich M. abscessus as compared to embryos infected with lipid-poor M. abscessus, suggesting that ILI are actively contributing to mycobacterial virulence and pathogenesis.

Microenvironment of Mycobacterium smegmatis Culture to Induce Cholesterol Consumption Does Cell Wall Remodeling and Enables the Formation of Granuloma-Like Structures.

Pathogenic species of mycobacteria are known to use the host cholesterol during lung infection as an alternative source of carbon and energy. Mycobacteria culture in minimal medium (MM) has been used as an in vitro experimental model to study the consumption of exogenous cholesterol. Once in MM, different species of mycobacteria start to consume the cholesterol and initiate transcriptional and metabolic adaptations, upregulating the enzymes of the methylcitrate cycle (MCC) and accumulating a variety of primary metabolites that are known to be important substrates for cell wall biosynthesis. We hypothesized that stressful pressure of cultures in MM is able to induce critical adaptation for the bacteria which win the infection. To identify important modifications in the biosynthesis of the cell wall, we cultured the fast-growing and nonpathogenic Mycobacterium smegmatis in MM supplemented with or without glycerol and/or cholesterol. Different from the culture in complete medium Middlebrook 7H9 broth, the bacteria when cultured in MM decreased growth and changed in the accumulation of cell wall molecules. However, the supplementation of MM with glycerol and/or cholesterol recovered the accumulation of phosphatidylinositol mannosides (PIMs) and other phospholipids but maintained growth deceleration. The biosynthesis of lipomannan (LM) and of lipoarabinomannan (LAM) was significantly modulated after culture in MM, independently of glycerol and/or cholesterol supplementation, where LM size was decreased (LM13-25KDa) and LAM increased (LAM37-100KDa), when compared these molecules after bacteria culture in complete medium (LM17-25KDa and LAM37-50KDa). These changes modified the cell surface hydrophobicity and susceptibility against H2O2. The infection of J774 macrophages with M. smegmatis, after culture in MM, induced the formation of granuloma-like structures, while supplementation with cholesterol induced the highest rate of formation of these structures. Taken together, our results identify critical changes in mycobacterial cell wall molecules after culture in MM that induces cholesterol accumulation, helping the mycobacteria to increase their capacity to form granuloma-like structures.

PE_PGRS62 promotes the survival of Mycobacterium smegmatis within macrophages via disrupting ER stress-mediated apoptosis.

Mycobacterium tuberculosis, the leading causative agent of tuberculosis, remains one of the most deadly infectious pathogens. PE_PGRS proteins become a new focus as their species specificity in mycobacteria, especially in pathogenic mycobacteria. Despite intensive research, PE_PGRS proteins are still a mysterious aspect of mycobacterial pathogenesis with unknown mechanism. Herein, we focused on a PE_PGRS member from M. tuberculosis, PE_PGRS62, characterized by a surface-exposed protein function in disrupting phagolysosome maturation. Expression of PE_PGRS62 in Mycobacterium smegmatis, a nonpathogenic species naturally deficient in PE_PGRS genes, resulted in enhanced resistance to various in vitro stresses and cellular survival in macrophage. As a consequence, the cytokine profiles of macrophage were disturbed and cell apoptosis were inhibited via decreasing endoplasmic reticulum stress response.

Differential thermal stability, conformational stability and unfolding behavior of Eis proteins from Mycobacterium smegmatis and Mycobacterium tuberculosis.

Eis (Enhanced Intracellular Survival) is an important aminoglycoside N-acetyltransferase enzyme contributing to kanamycin resistance in Mtb clinical isolates. Eis proteins from M. tuberculosis (RvEis) and M. smegmatis (MsEis) have 58% identical and 69% similar amino acid sequences and acetylate aminoglycosides at multiple amines. Both the Eis proteins are hexameric and composed of two symmetric trimers. RvEis has remarkable structural stability and heat-stable aminoglycoside acetyltransferase activity. Although the structure and biochemical properties of MsEis have been studied earlier, the detailed characterization of its acetyltransferase activity and structural stability is lacking. In this study, we have performed comparative analysis of structural stability and aminoglycoside acetyltransferase activity of RvEis and MsEis proteins. Unlike RvEis, MsEis undergoes a three-state unfolding induced by heat or chemical denaturants and involves self-association of partially unfolded oligomers to form high molecular weight soluble aggregates. MsEis is highly susceptible to chemical denaturants and unfolds completely at lower concentrations of GdmCl and urea when compared to RvEis. In contrast to RvEis, the oligomeric forms of MsEis are SDS sensitive. However, SDS treatment resulted in increased helix formation in MsEis than RvEis. MsEis shows lesser thermostable activity with a decreased efficiency of kanamycin acetylation in comparison to RvEis. Furthermore, overexpression of MsEis does not provide thermal resistance to M. smegmatis unlike RvEis. Collectively, this study reveals that homologous proteins from pathogenic and nonpathogenic mycobacteria follow different modes of unfolding and demonstrate differential structural stability and activity despite highly similar sequences and oligomeric organization.

An NAD+ Phosphorylase Toxin Triggers Mycobacterium tuberculosis Cell Death.

Toxin-antitoxin (TA) systems regulate fundamental cellular processes in bacteria and represent potential therapeutic targets. We report a new RES-Xre TA system in multiple human pathogens, including Mycobacterium tuberculosis. The toxin, MbcT, is bactericidal unless neutralized by its antitoxin MbcA. To investigate the mechanism, we solved the 1.8 Å-resolution crystal structure of the MbcTA complex. We found that MbcT resembles secreted NAD+-dependent bacterial exotoxins, such as diphtheria toxin. Indeed, MbcT catalyzes NAD+ degradation in vitro and in vivo. Unexpectedly, the reaction is stimulated by inorganic phosphate, and our data reveal that MbcT is a NAD+ phosphorylase. In the absence of MbcA, MbcT triggers rapid M. tuberculosis cell death, which reduces mycobacterial survival in macrophages and prolongs the survival of infected mice. Our study expands the molecular activities employed by bacterial TA modules and uncovers a new class of enzymes that could be exploited to treat tuberculosis and other infectious diseases.

Lack of MSMEG_6281, a peptidoglycan amidase, affects cell wall integrity and virulence of Mycobacterium smegmatis.

Mycolyl-arabinogalactan-peptidoglycan (mAGP) is the major content of the mycobacterium cell wall structure and essential for mycobacterial survival. Peptidoglycan (PG) plays an important role in maintenance of cell division, cell wall integrity and pathogenesis. Mycobacterium smegmatis MSMEG_6281, a peptidoglycan amidase, is vital for mycobacterial cell division. However, the effects of MSMEG_6281on cell wall integrity and mycobacterial virulence remain unknown. In the current study, we demonstrate that MSMEG_6281gene knockout in M.smegmatis alters the microbiological characteristics. Our results revealed that MSMEG_6281gene knockout bacteria (M. sm-ΔM_6281) lost their acid-fastness, increased their sensitivity to lipophilic compounds and presented an abnormal morphology. Our results revealed that MSMEG_6281was related to maintaining the cell wall integrity. Furthermore, we investigated the effects of MSMEG_6281 inactivation on mycobacterial virulence using mice models infected by different M.smegmatis strains. MSMEG_6281 inactivation in the M sm-ΔM_6281 infected group caused less mycobacterial colonization, reduced pathological signs, decreased the anti-microbial enzymes production including iNOS and ß-defensins in mouse lungs. Moreover, IL-1ß and TLR2 expression were significantly down-regulated, while the production of IFN-γ and TNF-α was up-regulated. These findings indicated the diversity of host immune responses induced by different strains of M.smegmatis, suggesting that MSMEG_6281 inactivation impact mycobacterial virulence. In conclusion, the MSMEG_6281 protein plays important roles in maintaining cell wall integrity and mycobacterial virulence.

LipG a bifunctional phospholipase/thioesterase involved in mycobacterial envelope remodeling.

Tuberculosis caused by Mycobacterium tuberculosis is currently one of the leading causes of death from an infectious agent. The main difficulties encountered in eradicating this bacteria are mainly related to (i) a very complex lipid composition of the bacillus cell wall, (ii) its ability to hide from the immune system inside the granulomas, and (iii) the increasing number of resistant strains. In this context, we were interested in the Rv0646c (lipGMTB ) gene located upstream to the mmaA cluster which is described as being crucial for the production of cell wall components and required for the bacilli adaptation and survival in mouse macrophages. Using biochemical experiments combined with the construction of deletion and overexpression mutant strains in Mycobacterium smegmatis, we found that LipGMTB is a cytoplasmic membrane-associated enzyme that displays both phospholipase and thioesterase activities. Overproduction of LipGMTB decreases the glycopeptidolipids (GPL) level concomitantly to an increase in phosphatidylinositol (PI) which is the precursor of the PI mannoside (PIM), an essential lipid component of the bacterial cell wall. Conversely, deletion of the lipGMS gene in M. smegmatis leads to an overproduction of GPL, and subsequently decreases the strain susceptibility to various antibiotics. All these findings demonstrate that LipG is involved in cell envelope biosynthesis/remodeling, and consequently this enzyme may thus play an important role in mycobacterial physiology.

PE_PGRS3 of Mycobacterium tuberculosis is specifically expressed at low phosphate concentration, and its arginine-rich C-terminal domain mediates adhesion and persistence in host tissues when expressed in Mycobacterium smegmatis.

PE_PGRSs of Mycobacterium tuberculosis (Mtb) represent a family of complex and peculiar proteins whose role and function remain elusive. In this study, we investigated PE_PGRS3 and PE_PGRS4, two highly homologous PE_PGRSs encoded by two contiguous genes in the Mtb genome. Using a gene-reporter system in Mycobacterium smegmatis (Ms) and transcriptional analysis in Mtb, we show that PE_PGRS3, but not PE_PGRS4, is specifically expressed under low phosphate concentrations. Interestingly, PE_PGRS3, but not PE_PGRS4, has a unique, arginine-rich C-terminal domain of unknown function. Heterologous expression of PE_PGRS3 in Ms was used to demonstrate cellular localisation of the protein on the mycobacterial surface, where it significantly affects net surface charge. Moreover, expression of full-length PE_PGRS3 enhanced adhesion of Ms to murine macrophages and human epithelial cells and improved bacterial persistence in spleen tissue following infection in mice. Expression of the PE_PGRS3 functional deletion mutant lacking the C-terminal domain in Ms did not enhance adhesion to host cells, showing a phenotype similar to the Ms parental strain. Interestingly, enhanced persistence of Ms expressing PE_PGRS3 did not correlate with increased concentrations of inflammatory cytokines. These results point to a critical role for the ≈ 80 amino acids long, arginine-rich C-terminal domain of PE_PGRS3 in tuberculosis pathogenesis.

Rv0613c/MSMEG_1285 Interacts with HBHA and Mediates Its Proper Cell-Surface Exposure in Mycobacteria.

Heparin-binding haemagglutinin (HBHA) is a surface-exposed virulence factor of Mycobacterium tuberculosis and is involved in the binding of mycobacteria to non-phagocytic cells, allowing for extra-pulmonary dissemination of the bacilli. Despite its surface exposure, HBHA is not produced as a pre-protein containing a typical cleavable N-terminal signal peptide and is thus likely secreted by a Sec-independent, as of yet unknown mechanism. Here, we used the bacterial adenylate cyclase two-hybrid system to identify the proteins encoded by rv0613c and mmpL14 as being able to interact with HBHA. Our study was focused on Rv0613c, as it showed more consistent interactions with HBHA than MmpL14. Deletion of its orthologous gene MSMEG_1285 in recombinant Mycobacterium smegmatis producing HBHA from M. tuberculosis resulted in the loss of proper surface exposure of HBHA, as evidenced by atomic force microscopy. Furthermore, the lack of MSMEG_1285 also abolished the clumping phenotype and rough colony morphology of the recombinant M. smegmatis and reduced its adherence to A549 epithelial cells. These phenotypes have previously been associated with surface-exposed HBHA. Thus, MSMEG_1285 is directly involved in the proper cell-surface exposure of HBHA. These observations identify MSMEG_1285/Rv0613c as the first accessory protein involved in the cell surface exposure of HBHA.

PhoPR Positively Regulates whiB3 Expression in Response to Low pH in Pathogenic Mycobacteria.

During infection, Mycobacterium tuberculosis colonizes macrophages or necrotic granulomas, in which low pH is one of the major challenges. The PhoPR two-component regulatory system and the cytosolic redox sensor WhiB3 both play important roles in the response to low pH by M. tuberculosis However, whether close association exists between PhoPR and WhiB3 remains unclear. In this study, the positive regulation of whiB3 by PhoPR in mycobacteria was characterized. We observed that the expression patterns of the whiB3 gene under acidic conditions are different among mycobacterial species, suggesting that the regulation of whiB3 differs among mycobacteria. A sequence analysis of the whiB3 promoters (whiB3p) from M. tuberculosis and two closely related species, namely, M. marinum and M. smegmatis, showed that the whiB3p regions from M. tuberculosis and M. marinum contain a new type of PhoP box that is absent in the M. smegmatiswhiB3p Direct binding of PhoP to whiB3p from M. tuberculosis and M. marinum but not that from M. smegmatis was validated by in vitro protein-DNA binding assays. The direct activation of whiB3 by PhoPR under acidic conditions was further verified by reverse transcription-quantitative PCR (qRT-PCR) analysis in M. marinum Moreover, mutating the residues important for the phosphorylation pathway of PhoPR in M. marinum abolished the activation of whiB3 expression by PhoPR under acidic conditions, suggesting that low pH triggers the phosphorylation of PhoPR, which in turn activates the transcription of whiB3 Since the PhoP box was only identified in whiB3p of pathogenic mycobacteria, we suggest that the PhoPR-whiB3 regulatory pathway may have evolved to facilitate mycobacterial infection.IMPORTANCE The low pH in macrophages is an important barrier for infection by microbes. The PhoPR two-component regulatory system is required for the response to low pH and plays a role in redox homeostasis in Mycobacterium tuberculosis WhiB3, a cytosolic redox-sensing transcriptional regulator, is also involved in these processes. However, there is no direct evidence to demonstrate the regulation of WhiB3 by PhoPR. In this study, we found that PhoPR directly activates whiB3 expression in response to low pH. An atypical PhoP box in the whiB3 promoters has been identified and is only found in pathogenic mycobacteria, which suggests that the PhoPR-whiB3 regulatory pathway may facilitate mycobacterial infection. This study provides novel information for further characterization of the PhoPR regulon.