关键词： Mycobacterium   PDIM   GNAT   acetylation   MbtK  

摘要： Phagosomal lysis is essential for mycobacterial infection of macrophages. Acetylation is a protein modification mediated enzymatically by N-acetyltransferases (NATs) that impacts bacterial pathogenesis and physiology. To identify NATs required for lytic activity, we leveraged Mycobacterium marinum, a nontubercular pathogen and a model for Mycobacterium tuberculosis. M. marinum hemolysis is a proxy for phagolytic activity. We generated M. marinum strains with deletions in conserved NAT genes and screened for hemolytic *** conserved lysine acetyltransferases (KATs) contributed to hemolysis, which is mediated by the ESX-1 secretion system and by the virulence lipid phthiocerol dimycocerosate (PDIM). Using thin-layer chromatography, we found that MbtK, a conserved acyl-transferase required for mycobactin siderophore synthesis and virulence, was required for PDIM and phenolic glycolipid (PGL) production. Exogenous addition of sodium propionate or Mycobactin J restored PDIM/PGL production in the Delta mbtKstrain. The Delta mbtKM. marinum strain was attenuated in macrophage and Galleria mellonella infection models. Constitutive expression of either eis or papA5, which encode a KAT required for aminoglycoside resistance and a PDIM/PGL biosynthetic enzyme, rescued PDIM/PGL production, and virulence of the triangle mbtKstrain. Eis N-terminally acetylated PapA5 in vitro, providing a plausible mechanism for restored lipid production. Overall, our study establishes connections between the MbtK and Eis NATs, and between iron uptake and PDIM and PGL synthesis in M. marinum. Our findings underscore the multifunctional nature of mycobacterial NATs and their connection to key virulence pathways.

关键词： Autophagy   cytosol access   EsxA   ESX-1   inflammasome   lysosome   PDIM  

摘要： Mycobacterium tuberculosis (M. tb) employs diverse virulence factors to evade immune defenses and persist intracellularly. The ESAT-6 secretion system-1 (ESX-1) type VII secretion system (T7SS) releases EsxA, EspA, and EspB, inducing phagosomal rupture and cytosolic access while triggering host defenses, including galectin recruitment and stress granule formation. To counteract host responses, M. tb utilizes phthiocerol dimycocerosates (PDIMs) to inhibit autophagy and LC3-associated phagocytosis (LAP) by suppressing NADPH oxidase (NOX2) recruitment and reactive oxygen species (ROS) production. Additionally, CspA blocks LC3 lipidation, impairing LAP activation and phagosome maturation. EsxG and EsxH interfere with ESCRT-mediated phagosomal repair, further enhancing intracellular survival. Cytosolic M. tb is ubiquitinated by host E3 ligases, marking it for selective autophagy (xenophagy), yet M. tb evades degradation by manipulating autophagic flux. Simultaneously, M. tb-derived DNA activates the cyclic GMP-AMP synthase-stimulator of interferon response cGAMP interactor 1 (CGAS-STING1) axis, leading to type I interferon (IFN) signaling and inflammasome activation, which drive IL-1B and IL-18 secretion, necrosis, and pyroptosis, facilitating bacterial dissemination. Additionally, exosomes released during infection disseminate bacterial components, modulating immune responses systemically. This review uniquely integrates current findings on the coordinated actions of ESX-1 T7SS and PDIMs in mediating phagosomal rupture and immune evasion, offering a unified framework for understanding M. tb's intracellular survival strategies. By bridging lipid- and protein-mediated virulence mechanisms and their impact on host autophagy, inflammasome activation, and phagosomal repair pathways, this work provides novel insights into therapeutic targets aimed at restoring host immune function.

关键词： B cells   LC3-associated phagocytosis   macrophages   phthiocerol dimycocerosate   tertiary lymphoid follicle  

摘要： Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB), remains a significant global health challenge. Mtb is transmitted by respiratory aerosols and infects a variety of myeloid populations. Our recent study shows that the Mtb virulence lipid phthiocerol dimycocerosate (PDIM) promotes the intracellular survival of Mtb in macrophages by inhibiting NADPH oxidase, thereby impairing LC3-associated phagocytosis, and in vivo PDIM also antagonizes canonical macroautophagy/autophagy. In addition, mice defective in autophagy in myeloid cells fail to develop B-cell follicles in the lungs during chronic infection. Here, we present a summary of our recent publication, highlighting the most significant findings and discussing how they provide new insight into the role of autophagy and the diversity of lung myeloid cells in the pathogenesis of Mtb.

摘要： The Mycobacterium tuberculosis virulence lipid phthiocerol dimycocerosate (PDIM) is exported by a complex mechanism that involves multiple proteins including the Resistance-Nodulation-Division (RND) transporter MmpL7 and the lipoprotein LppX. Here, we probe the role of the putative heterooligomeric ATP-Binding Cassette (ABC) transporter complex composed of DrrA, DrrB and DrrC in PDIM transport by constructing a set of individual null mutants of drrA, drrB and drrC in the vaccine strain Mycobacterium bovis BCG. Loss of all three, or individual drr genes, all resulted in a complete loss of PDIM export to the outer envelope of the mycobacterial cell. Furthermore, guided by a bioinformatic analysis we interrogated specific signature residues within the DrrABC to demonstrate that it is indeed an ABC transporter, and our modelling, together with the mutagenesis identify it as a member of the Type V family of ABC exporters. We identify several unique structural elements of the transporter, including a non-canonical C-terminally inserted domain (CTD) structure within DrrA, which may account for its functional properties.

摘要： Mycobacterium tuberculosis (Mtb) infects several lung macrophage populations, which have distinct abilities to restrict Mtb. What enables Mtb survival in certain macrophage populations is not well understood. Here we used transposon sequencing analysis of Mtb in wild-type and autophagy-deficient mouse macrophages lacking ATG5 or ATG7, and found that Mtb genes involved in phthiocerol dimycocerosate (PDIM) virulence lipid synthesis confer resistance to autophagy. Using ppsD mutant Mtb, we found that PDIM inhibits LC3-associated phagocytosis (LAP) by inhibiting phagosome recruitment of NADPH oxidase. In mice, PDIM protected Mtb from LAP and classical autophagy. During acute infection, PDIM was dispensable for Mtb survival in alveolar macrophages but required for survival in non-alveolar macrophages in an autophagy-dependent manner. During chronic infection, autophagy-deficient mice succumbed to infection with PDIM-deficient Mtb, with impairments in B-cell accumulation in lymphoid follicles. These findings demonstrate that PDIM contributes to Mtb virulence and immune evasion, revealing a contributory role for autophagy in B-cell responses.

摘要： Phthiocerol dimycocerosate (PDIM) is an essential virulence lipid of Mycobacterium tuberculosis. In vitro culturing rapidly selects for spontaneous PDIM-negative mutants that have attenuated virulence and increased cell wall permeability, thus impacting the relevance of experimental findings. PDIM loss can also reduce the efficacy of the BCG Pasteur vaccine. Here we show that vancomycin susceptibility can rapidly screen for M. tuberculosis PDIM production. We find that metabolic deficiency of methylmalonyl-CoA impedes the growth of PDIM-producing bacilli, selecting for PDIM-negative variants. Supplementation with odd-chain fatty acids, cholesterol or vitamin B12 restores PDIM-positive bacterial growth. Specifically, we show that propionate supplementation enhances PDIM-producing bacterial growth and selects against PDIM-negative mutants, analogous to in vivo conditions. Our study provides a simple approach to screen for and maintain PDIM production, and reveals how discrepancies between the host and in vitro nutrient environments can attenuate bacterial pathogenicity.

关键词： Mycobacterium   ESX-1   PDIM   secretion   proteomics  

摘要： The mycobacterial cell envelope is a major virulence determinant in pathogenic mycobacteria. Specific outer lipids play roles in pathogenesis, modulating the immune system and promoting the secretion of virulence factors. ESX-1 (ESAT-6 system-1) is a conserved protein secretion system required for mycobacterial pathogenesis. Previous studies revealed that mycobacterial strains lacking the outer lipid PDIM have impaired ESX-1 function during laboratory growth and infection. The mechanisms underlying changes in ESX-1 function are unknown. We used a proteo-genetic approach to measure phthiocerol dimycocerosate (PDIM)- and phenolic glycolipid (PGL)-dependent protein secretion in M. marinum, a non-tubercular mycobacterial pathogen that causes tuberculosis-like disease in ectothermic animals. Importantly, M. marinum is a well-established model for mycobacterial pathogenesis. Our findings showed that M. marinum strains without PDIM and PGL showed specific, significant reductions in protein secretion compared to the WT and complemented strains. We recently established a hierarchy for the secretion of ESX-1 substrates in four (I-IV) groups. Loss of PDIM differentially impacted secretion of Group III and IV ESX-1 substrates, which are likely the effectors of pathogenesis. Our data suggest that the altered secretion of specific ESX-1 substrates is responsible for the observed ESX-1-related effects in PDIM-deficient strains. IMPORTANCE Mycobacterium tuberculosis, the cause of human tuberculosis, killed an estimated 1.3 million people in 2022. Non-tubercular mycobacterial species cause acute and chronic human infections. Understanding how these bacteria cause disease is critical. Lipids in the cell envelope are essential for mycobacteria to interact with the host and promote disease. Strains lacking outer lipids are attenuated for infection, but the reasons are unclear. Our research aims to identify a mechanism for attenuation of mycobacterial strains without the PDIM and PGL ou

关键词： PDIM   Mycobacterium tuberculosis   Tn-seq   antibiotic resistance   drug tolerance   membrane permeability   nutrient limitation  

摘要： Tolerance of Mycobacterium tuberculosis to antibiotics contributes to the long duration of tuberculosis (TB) treatment and the emergence of drug-resistant strains. M. tuberculosis drug tolerance is induced by nutrient restriction, but the genetic determinants that promote antibiotic tolerance triggered by nutrient limitation have not been comprehensively identified. Here, we show that M. tuberculosis requires production of the outer membrane lipid phthiocerol dimycocerosate (PDIM) to tolerate antibiotics under nutrient-limited conditions. We developed an arrayed transposon (Tn) mutant library in M. tuberculosis Erdman and used orthogonal pooling and transposon sequencing (Tn-seq) to map the locations of individual mutants in the library. We screened a subset of the library (similar to 1,000 mutants) by Tn-seq and identified 32 and 102 Tn mutants with altered tolerance to antibiotics under stationary-phase and phosphate-starved conditions, respectively. Two mutants recovered from the arrayed library, ppgK::Tn and clpS::Tn, showed increased susceptibility to two different drug combinations under both nutrient-limited conditions, but their phenotypes were not complemented by the Tn-disrupted gene. Whole-genome sequencing revealed single nucleotide polymorphisms in both the ppgK::Tn and clpS::Tn mutants that prevented PDIM production. Complementation of the clpS::Tn ppsD Q291* mutant with ppsD restored PDIM production and antibiotic tolerance, demonstrating that loss of PDIM sensitized M. tuberculosis to antibiotics. Our data suggest that drugs targeting production of PDIM, a critical M. tuberculosis virulence determinant, have the potential to enhance the efficacy of existing antibiotics, thereby shortening TB treatment and limiting development of drug *** Mycobacterium tuberculosis causes 10 million cases of active TB disease and over 1 million deaths worldwide each year. TB treatment is complex, requiring at least 6 months of therapy with a combinat

关键词： Tuberculosis   Rifampicin   rpoB   Fitness   Virulence   PDIM  

摘要： Multidrug-resistant tuberculosis (isoniazid/rifampin[RIF]-resistant TB) ravages developing countries. Fitness is critical in clinical outcomes. Previous studies on RIF-resistant TB (RR-TB) showed competitive fitness gains and losses, with rpoB-S450L as the most isolated/fit mutation. This study measured virulence/resistance genes, phthiocerol dimycocerosate (PDIM) levels and their relationship with rpoB S450L ATCC25618 RR-TB strain fitness. After obtaining 10 different RR-TB GenoType MTBDRp1us 2.0-genotyped isolates (with nontyped, 5441, H445 and 5450 positions), only one S450L isolate (R9, rpoB-S450L ATCC 25618, RR 1 mu g/mL) was observed, with H445Y being the most common. A competitive fitness in vitro assay with wild-type (wt) ATCC 25618: R9 1:1 in 50 mL Middlebrook 7H9/OADC was performed, and generation time (G) in vitro and relative fitness were obtained. mRNA and PDIM were extracted on log and stationary phases. Fitness decreased in rpoB S450L and H445Y strains, with heterogeneous fitness cues in three biological replicas of rpoB-S450L: one high and two low fitness replicas. S450L strain had significant pknG increase. Compared with S450L, wt-rpoB showed increased polyketide synthase ppsA expression and high PDIM peak measured by HPLC-MS in log phase compared to S450L. This contrasts with previously increased PDIM in other RR-TB isolates.

关键词： cell wall   hydrophobicity   lipid   neutral red   PDIM   PGL  

摘要： Mycobacterium bovis bacillus Calmette-Guerin (BCG) efficacy as an immunotherapy tool can be influenced by the genetic background or immune status of the treated population and by the BCG substrain used. BCG comprises several substrains with genetic differences that elicit diverse phenotypic characteristics. Moreover, modifications of phenotypic characteristics can be influenced by culture conditions. However, several culture media formulations are used worldwide to produce BCG. To elucidate the influence of growth conditions on BCG characteristics, five different substrains were grown on two culture media, and the lipidic profile and physico-chemical properties were evaluated. Our results show that each BCG substrain displays a variety of lipidic profiles on the outermost surface depending on the growth conditions. These modifications lead to a breadth of hydrophobicity patterns and a different ability to reduce neutral red dye within the same BCG substrain, suggesting the influence of BCG growth conditions on the interaction between BCG cells and host cells.