Mycolic Acids Research Articles

Method of extraction and proteome profiling of mycobacteria using liquid chromatography-high resolution mass spectrometry

Advances in massively parallel sequencing, of complete bacterial genomes, have led to many novel findings in the field of genomics. However, these data often lack correlation with expressed protein profiles. It has been demonstrated that even very closely related genomes, such as in mycobacteria, express drastically different phenotypes. These phenotypes often have major roles in pathogenicity. Therefore, it is just as important to have a method for examining the proteome of a bacterium as well as its genome. These studies are further complicated in mycobacteria due to the cell wall and mycolic acid. A comprehensive method for the identification and characterization of the whole mycobacterium protein profile is needed. In the present study, a simple, sensitive, and specific liquid chromatography tandem mass spectrometry method was developed for the extraction, purification and profiling the mycobacterial proteome in various species. During development, sonication and bead-beating cell lysis protocol was tested using 15% Acetonitrile and 6 M guanidine-HCl (GuHCl) as extraction solvent. Sonication lysis in 6 M GuHCl with glass beads was the preferred method for cell lysis. This method was developed using reverse phase liquid chromatography and a Q Exactive ™ Plus Orbitrap™ mass spectrometer for peptide and protein identification. Bottom-up liquid chromatography-mass spectrometry LC–MS analysis resulted in identification of greater than 2500 proteins.

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

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. The present study aimed to elucidate the antimycobacterial potential of Geraniol (Ger), monoterpene alcohol, against Mycobacterium smegmatis. 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. 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. Taken together, the current study demonstrates the antimycobacterial mechanisms of Ger, which may be exploited as an effective candidate of pharmacological interest.

Lipolytic enzymes inhibitors: A new way for antibacterial drugs discovery.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (M.tb) still remains the deadliest infectious disease worldwide with 1.5 million deaths in 2018, of which about 15% are attributed to resistant strains. Another significant example is Mycobacterium abscessus (M.abscessus), a nontuberculous mycobacteria (NTM) responsible for cutaneous and pulmonary infections, representing up to 95% of NTM infections in cystic fibrosis (CF) patients. M.abscessus is a new clinically relevant pathogen and is considered one of the most drug-resistant mycobacteria for which standardized chemotherapeutic regimens are still lacking. Together the emergence of M.tb and M.abscessus multi-drug resistant strains with ineffective and expensive therapeutics, have paved the way to the development of new classes of anti-mycobacterial agents offering additional therapeutic options. In this context, specific inhibitors of mycobacterial lipolytic enzymes represent novel and promising antibacterial molecules to address this challenging issue. The results highlighted here include a complete overview of the antibacterial activities, either in broth medium or inside infected macrophages, of two families of promising and potent anti-mycobacterial multi-target agents, i.e. oxadiazolone-core compounds (OX) and Cyclophostin & Cyclipostins analogs (CyC); the identification and biochemical validation of their effective targets (e.g., the antigen 85 complex and TesA playing key roles in mycolic acid metabolism) together with their respective crystal structures. To our knowledge, these are the first families of compounds able to target and impair replicating as well as intracellular bacteria. We are still impelled in deciphering their mode of action and finding new potential therapeutic targets against mycobacterial-related diseases.

Design, synthesis, antimycobacterial activity and molecular docking studies of novel 3- (N-substituted glycinamido) benzoic acid analogues as anti tubercular agents

We have recently identified mycolic acid methyl transferase (MmaA1) enzyme inhibitors as potential antitubercular agents using in silico modelling techniques. In continuation of that study, we synthesised a series of novel 3-(N-substituted glycinamido) benzoic acid derivatives with an aim to optimise the lead molecule. The newly synthesised compounds were evaluated for their in vitro antimycobacterial activity against M. tuberculosis H37Rv. Among these, 5 compounds A3, A4, A5, A6 and A10 exhibited most potent activity with an MIC value of 1.6 μg/ml. Further molecular docking studies were carried out to investigate the binding mode of the ligands with MmaA1 protein. The docking studies revealed that the ligands made strong interactions with the catalytic site residues TRP30, TYR 32, GLY 71, TRP 74, GLY 76, ALA 77 and GLU 136 of MmaA1 protein. Druglikeness and leadlikeness properties of the compounds were also studied using computational tools. The results of in silico and in vitro studies indicate that these novel compounds are propitious leads for tuberculosis therapy.

Genomic Virulence Features of Two Novel Species Nocardia barduliensis sp. nov. and Nocardia gipuzkoensis sp. nov., Isolated from Patients with Chronic Pulmonary Diseases.

Strains 335427T and 234509T, isolated from two 76-year-old patients with chronic pulmonary diseases, were the subject of polyphasic taxonomic studies and comparative genomic analyses for virulence factors. The 16 rRNA gene sequence similarity between strains 335427T and 234509T and their closest phylogenetic neighbors Nocardia asiatica NBRC 100129T and Nocardia abscessus NBRC 100374T were 99.5% and 100%, respectively. Digital DNA–DNA hybridization values between the aforementioned studied strains were well below the 70% threshold for assigning prokaryotic strains to a novel species. Strains 335427T and 234509T have genome sizes of 8.49 Mpb and 8.07 Mpb, respectively, with G + C content of 68.5%. Isolate 335427T has C16:0, C18:1 ω9c, C18:0 and C18:0 10 methyl as major fatty acids (>15%) and mycolic acids formed of 52–54 carbon atoms. However, only C18:1 ω9c was detected for isolate 234509T, which had mycolic acids with 44–56 carbon. Based on phenotypic and genetic data, strains 335427T (DSM 109819T = CECT 9924T) and 234509T (DSM 111366T = CECT 30129T) merit recognition as novel species, which are named Nocardia barduliensis sp. nov. and Nocardia gipuzkoensis sp. nov., respectively. All the strains studied had homologous VF-associated genes to those described in M. tuberculosis, including experimentally verified virulence genes in humans related to tuberculosis. The narGHIJ (nitrate reduction pathway) and gvpAFGOJLMK (gas vesicles) genetic maps of strains 335427T, 234509T, NBRC 100129T and NBRC 100374T showed the same syntenic block and raise the question of whether their functions are interlinked during the infection of the human host. However, further research is required to decipher the role of the gas vesicle in the pathogenicity mechanism of Nocardia spp.

An integrated computational investigation to unveil the structural impacts of mutation on the InhA structural gene of Mycobacterium tuberculosis

Growing concern about the difficulty in diagnosis and treatments of drug-resistant tuberculosis falls under the major global health issues. There is an urgent need for finding novel strategies to develop drugs or bioactive molecules against the global threat of Mycobacterium tuberculosis (MTB). Isoniazid (INH) is a front line drug against tuberculosis; it primarily targets the enoyl-acyl carrier protein reductase (InhA), a potent drug target in the mycolic acid pathway of MTB. To gain deeper insight into the impact of INH resistant mutation and its influence on the structural dynamics of InhA, combined conformational dynamics and residue interaction network (RIN) studies were performed. The molecular dynamics investigation provided a hint about the structural changes altering protein activity. The principal component analysis (PCA) based free energy landscape plot highlighted the highest stable part of wild-type (WT) and mutant structures. Intriguingly, the mutation at the 78th position of InhA from its native residue valine to alanine increases the structural stability with higher NADH binding affinity. The MM-PBSA based binding energy calculations confirm that electrostatic interactions played a critical role in the binding of NADH at the binding site of InhA. The calculated binding energy score, as well as potential hydrogen bonds and salt bridge networks, proved the strong binding of mutant InhA as compared to WT. Further, the mutation potentially altered the protein network topology, thereby subsequently affected the landscape of NADH binding. The present study is an attempt to understand the structural and functional impact associated with a drug-resistant mutation (V78A) thus it will be helpful in designing potent inhibitors against drug-resistant tuberculosis.

Streptomyces smaragdinus sp. nov., isolated from the gut of the fungus growing-termite Macrotermes natalensis.

The taxonomic position of a novel aerobic, Gram-positive actinobacteria, designated strain RB5T, was determined using a polyphasic approach. The strain, isolated from the gut of the fungus-farming termite Macrotermes natalensis, showed morphological, physiological and chemotaxonomic properties typical of the genus Streptomyces . Based on 16S rRNA gene sequence analysis, the closest phylogenetic neighbour of RB5T was Streptomyces polyrhachis DSM 42102T (98.87 %). DNA–DNA hybridization experiments between strain RB5T and S. polyrhachis DSM 42102T resulted in a value of 27.4 % (26.8 %). The cell wall of strain RB5T contained ll-diaminopimelic acid as the diagnostic amino acid. Mycolic acids and diagnostic sugars in whole-cell hydrolysates were not detected. The strain produced the following major phospholipids: diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol-mannoside and phosphatidylserine. The menaquinone profile showed hexa- and octahydrogenated menaquinones containing nine isoprene units [MK-9(H6) and MK-9(H8)]. The strain exhibited a fatty acid profile containing the following major fatty acids: 12-methyltridecanoic acid (iso-C14 : 0) 12-methyltetradecanoic acid (anteiso-C15 : 0), 13-methyltetradecanoic acid (iso-C15 : 0) and 14-methylpentadecanoic acid (iso-C16 : 0). Here, we propose a novel species of the genus Streptomyces – Streptomyces smaragdinus with the type strain RB5T (=VKM Ac-2839T=NRRL B65539T).

Oldest evidence of tuberculosis in Argentina: A multidisciplinary investigation in an adult male skeleton from Saujil, Tinogasta, Catamarca (905–1030 CE)

The Mycobacterium tuberculosis complex (MTC) has affected South American populations since ca. 200 years BCE. In Argentina, possible cases date from ca. 1000–1400 Common Era (CE). This paper describes the oldest (905–1030 CE) confirmed case of tuberculosis (TB) in a young adult male from Lomitas de Saujil (Tinogasta, Catamarca, Argentina). Osteolytic lesions on the bodies of the lower spine were macroscopically and radiographically identified. Bilateral new bone formation was seen on the visceral vertebral third of several ribs and in long bones, compatible with hypertrophic osteoarthropathy. Representative rib and hand bones gave profiles for MTC-specific C27–C32 mycocerosic acid lipid biomarkers; these were strongest in one heavily-lesioned lower rib, which also had MTC-diagnostic C76–C89 mycolic acids and positive amplification of MTC-typical IS6110 aDNA fragments. During the first millennium CE, the intense social interaction, the spatial circumscription of villages among the pre-Hispanic societies in the mesothermal valleys of Catamarca and the fluid contacts with the Eastern lowlands, valleys and puna, were factors likely to favor disease transmission. It is proposed that TB arrived from northern Chile and dispersed towards the northeast into the Yocavil valley, where several cases of TB infection were macroscopically identified for a later chronology.

Mycobacterium alvei (ω-1)-methoxy mycolic acids: Absolute stereochemistry and synthesis

Cells of Mycobacterium alvei are known to contain a unique set of mycolic acids with a (ω-1)-methoxy group; although the various enzymes in the biosynthesis of other types of mycolic acid have been widely studied, the biosynthetic route to this substituent is unclear. We now define the stereochemistry of the (ω-1)-methoxy fragment as R, and describe the synthesis of a major R-(ω-1)-methoxy-mycolic acid and its sugar esters, and of two natural M. alvei diene mycolic acids.

Pseudonocardia acidicola sp. nov., a novel actinomycete isolated from peat swamp forest soil.

A novel actinobacterium, designated strain K10HN5T, was isolated from a peat soil sample collected from Kantulee peat swamp forest, Surat Thani Province, Thailand and its taxonomic position was determined using a polyphasic approach. Strain K10HN5T contained meso-diaminopimelic acid, arabinose, galactose, glucose and ribose in its whole-cell hydrolysates. The predominant menaquinone was MK-8(H4). The major fatty acids were iso-C16 : 0, iso-C15 : 0 and iso-C16 : 1H. Mycolic acids were not present. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylmethylethanolamine, hydroxyphosphatidylethanolamine, hydroxyphosphatidylmethylethanolamine and phosphatidylinositol. The 16S rRNA gene sequence analysis indicated that it was closely related to Pseudonocardia bannensis DSM 45300T (97.9 %) and Pseudonocardia xinjiangensis JCM 11839T (97.9 %). Strain K10HN5T exhibited low average nucleotide identity and digital DNA-DNA hybridization values with P. bannensis DSM 45300T (82.6, 28.7 %) and P. xinjiangensis JCM11839T (76.3, 22.2 %). The DNA G+C content of strain K10HN5T was 72.4 mol%. Based on polyphasic data, strain K10HN5T represents a novel species of the genus Pseudonocardia, for which the name Pseudonocardia acidicola sp. nov. is proposed. The type strain is K10HN5T (=TBRC 10048T=NBRC 113897T).

Antrihabitans stalactiti gen. nov., sp. nov., a new member of the family Nocardiaceae isolated from a cave.

Two Gram-stain-positive, strictly aerobic, non-spore-forming actinobacterial strains, designated YC2-7T and YC5-17, were isolated from the Yongcheondonggul (larva cave) in Jeju, Republic of Korea and their taxonomic ranks were examined by a polyphasic approach. The 16S rRNA gene tree showed that the novel isolates occupied an independent position separated from recognized genera of the family Nocardiaceae. In the 92 core gene-based phylogenomic analysis, strain YC2-7T was loosely associated with the type strain of Aldersonia kummingensis with 66.2 % average amino acid identity. The 16S rRNA gene sequence simairity between the isolate and members of the family Nocardiaceae was below 96.7 %. The cell-wall peptidoglycan was meso-diaminopimelic acid as a diagnostic diamino acid. Whole-cell sugars consisted of arabinose, galactose and glucose. The predominant menaquinone was MK-8(H4, ω-cycl). The major polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannoside. The cellular fatty acids consisted mainly of saturated and unsaturated components with small amounts of tuberculostearic acid. Mycolic acids of 52-58 carbon atoms were present. The DNA G+C content of the genome was 63.8 mol%. On the basis of combination of morphological and chemotaxonomic differences, in addition to phylogenetic distinctness, the novel isolates are considered to constitute members of a novel species of a new genus in the family Nocardiaceae, for which the name Antrihabitans stalactiti gen. nov., sp. nov. is proposed. The type strain is YC2-7T (=KACC 19965T=DSM 108733T).

Nocardia aurantiaca sp. nov., isolated from soil in Thailand.

A novel actinomycete strain, CT2-14T, belonging to the genus Nocardia, was isolated from a soil sample collected from Phichit Province, Thailand. The taxonomic position of the strain was characterized using a polyphasic approach. The strain grew at 15-40 °C (optimum, 28-37 °C), pH 6-11 (optimum, pH 6-8) and on an International Streptomyces Project 2 with 4 % (w/v) NaCl agar plate. Meso-diaminopimelic acid was detected in the cell-wall peptidoglycan. Ribose, arabinose and galactose were detected in its whole-cell hydrolysates. Mycolic acids were present. The strain contained C16 : 0, summed feature 3, C17 : 0 10-methyl and C18 : 1 ω9c as the major fatty acids and MK-8(H4ω-cycl) as the major menaquinone. The major polar lipids were phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylinositol mannosides. Strain CT2-14T showed the highest 16S rRNA gene similarity to Nocardia veterana JCM 11307T (98.4 %), Nocardia africana JCM 11438T (98.2 %) and Nocardia kruczakiae JCM 13032T (98.0 %). The draft genome of strain CT2-14T was 7.37 Mb with 6685 coding sequences with an average G+C content of 67.9 mol %. Based on the phylogenomic tree analysis, the strain was closely related to Nocardia niigatensis NBRC 100131T. On the basis of polyphasic and genome analyses, strain CT2-14T represented a novel species of the genus Nocardia for which the name Nocardia aurantiaca sp. nov. is proposed. The type strain is CT2-14T (=JCM 33775T=TISTR 2838T).

First Report of Tsukamurella conjunctivitidis CAPD-related Peritonitis

First Report of Tsukamurella conjunctivitidis CAPD-related Peritonitis

MmpL3 Inhibition: A New Approach to Treat Nontuberculous Mycobacterial Infections.

Outside of Mycobacterium tuberculosis and Mycobacterium leprae, nontuberculous mycobacteria (NTM) are environmental mycobacteria (>190 species) and are classified as slow- or rapid-growing mycobacteria. Infections caused by NTM show an increased incidence in immunocompromised patients and patients with underlying structural lung disease. The true global prevalence of NTM infections remains unknown because many countries do not require mandatory reporting of the infection. This is coupled with a challenging diagnosis and identification of the species. Current therapies for treatment of NTM infections require multidrug regimens for a minimum of 18 months and are associated with serious adverse reactions, infection relapse, and high reinfection rates, necessitating discovery of novel antimycobacterial agents. Robust drug discovery processes have discovered inhibitors targeting mycobacterial membrane protein large 3 (MmpL3), a protein responsible for translocating mycolic acids from the inner membrane to periplasm in the biosynthesis of the mycobacterial cell membrane. This review focuses on promising new chemical scaffolds that inhibit MmpL3 function and represent interesting and promising putative drug candidates for the treatment of NTM infections. Additionally, agents (FS-1, SMARt-420, C10) that promote reversion of drug resistance are also reviewed.

Actinomadura rubteroloni sp. nov. and Actinomadura macrotermitis sp. nov., isolated from the gut of the fungus growing-termite Macrotermes natalensis.

The taxonomic positions of two novel aerobic, Gram-positive actinobacteria, designated strains RB29T and RB68T, were determined using a polyphasic approach. Based on 16S rRNA gene sequence analysis, the closest phylogenetic neighbours of RB29T were identified as Actinomadura rayongensis DSM 102126T (99.2 % similarity) and Actinomadura atramentaria DSM 43919T (98.7 %), and for strain RB68T was Actinomadura hibisca DSM 44148T (98.3 %). Digital DNA–DNA hybridization (dDDH) between RB29T and its closest phylogenetic neighbours, A. rayongensis DSM 102126T and A. atramentaria DSM 43919T, resulted in similarity values of 53.2 % (50.6–55.9 %) and 26.4 % (24.1–28.9 %), respectively. Additionally, the average nucleotide identity (ANI) was 93.2 % (94.0 %) for A. rayongensis DSM 102126T and 82.3 % (78.9 %) for A. atramentaria DSM 43919T. dDDH analysis between strain RB68T and A. hibisca DSM 44148T gave a similarity value of 24.5 % (22.2–27.0 %). Both strains, RB29T and RB68T, revealed morphological characteristics and chemotaxonomic features typical for the genus Actinomadura , such as the presence of meso-diaminopimelic acid in the cell wall, galactose and glucose as major sugar components within whole-cell hydrolysates and the absence of mycolic acids. The major phospholipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannoside. Predominant menaquinones were MK-9(H6) and MK-9(H8) for RB29T and MK-9(H4) and MK-9(H6) for RB68T. The main fatty acids were identified as 10-methyloctadecanoic acid (10-methyl C18:0), 14-methylpentadecanoic acid (iso-C16:0), hexadecanoic acid (C16:0) and cis-9-octadecanoic acid (C18 : 1 ω9c). Here, we propose two novel species of the genus Actinomadura : Actinomadura rubteroloni sp. nov. with the type strain RB29T (=CCUG 72668T=NRRL B-65537T) and Actinomadura macrotermitis sp. nov. with the type strain RB68T (=CCUG 72669T=NRRL B-65538T).

Nocardia macrotermitis sp. nov. and Nocardia aurantia sp. nov., isolated from the gut of the fungus-growing termite Macrotermes natalensis.

The taxonomic positions of two novel aerobic, Gram-stain-positive Actinobacteria, designated RB20T and RB56T, were determined using a polyphasic approach. Both were isolated from the fungus-farming termite Macrotermes natalensis. Results of 16S rRNA gene sequence analysis revealed that both strains are members of the genus Nocardia with the closest phylogenetic neighbours Nocardia miyunensis JCM12860T (98.9 %) and Nocardia nova DSM44481T (98.5 %) for RB20T and Nocardia takedensis DSM 44801T (98.3 %), Nocardia pseudobrasiliensis DSM 44290T (98.3 %) and Nocardia rayongensis JCM 19832T (98.2 %) for RB56T. Digital DNA–DNA hybridization (DDH) between RB20T and N. miyunensis JCM12860T and N. nova DSM 44481T resulted in similarity values of 33.9 and 22.0 %, respectively. DDH between RB56T and N. takedensis DSM44801T and N. pseudobrasiliensis DSM44290T showed similarity values of 20.7 and 22.3 %, respectively. In addition, wet-lab DDH between RB56T and N. rayongensis JCM19832T resulted in 10.2 % (14.5 %) similarity. Both strains showed morphological and chemotaxonomic features typical for the genus Nocardia , such as the presence of meso-diaminopimelic acid (A2pm) within the cell wall, arabinose and galactose as major sugar components within whole cell-wall hydrolysates, the presence of mycolic acids and major phospholipids (diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol), and the predominant menaquinone MK-8 (H4, ω-cyclo). The main fatty acids for both strains were hexadecanoic acid (C16 : 0), 10-methyloctadecanoic acid (10-methyl C18 : 0) and cis-9-octadecenoic acid (C18 : 1 ω9c). We propose two novel species within the genus Nocardia : Nocardia macrotermitis sp. nov. with the type strain RB20T (=VKM Ac-2841T=NRRL B65541T) and Nocardia aurantia sp. nov. with the type strain RB56T (=VKM Ac-2842T=NRRL B65542T).

The aceE involves in mycolic acid synthesis and biofilm formation in Mycobacterium smegmatis

BackgroundThe integrity of cell wall structure is highly significant for the in vivo survival of mycobacteria. We hypothesized that changes in morphology may indicate changes in cell wall metabolism and identified an aceE gene mutant (aceE-mut) which presented a deficient colony morphology on 7H10 agar by screening transposon mutagenesis in Mycolicibacterium smegmatis, basonym Mycobacterium smegmatis (M. smegmatis). This study aimed to identify the functional role of aceE gene in cell wall biosynthesis in M. smegmatis.ResultsWe observed that the colony morphology of aceE-mut was quite different, smaller and smoother on the solid culture medium than the wild-type (WT) strain during the transposon library screening of M. smegmatis. Notably, in contrast with the WT, which aggregates and forms biofilm, the aceE-mut lost its ability of growing aggregately and biofilm formation, which are two very important features of mycobacteria. The morphological changes in the aceE-mut strain were further confirmed by electron microscopy which indicated smoother and thinner cell envelope images in contrast with the rough morphology of WT strains. Additionally, the aceE-mut was more fragile to acidic stress and exhibited a pronounced defects in entering the macrophages as compared to the WT. The analysis of mycolic acid (MA) using LC-MS indicated deficiency of alpha-MA and epoxy-MA in aceE-mut strain whereas complementation of the aceE-mut with a wild-type aceE gene restored the composition of MA.ConclusionsOver all, this study indicates that aceE gene plays a significant role in the mycolic acid synthesis and affects the colony morphology, biofilm formation of M. smegmatis and bacteria invasion of macrophage.

Biofilms of the non-tuberculous Mycobacterium chelonae form an extracellular matrix and display distinct expression patterns

Mycobacterium chelonae is an environmental, non-tuberculous mycobacterial species, capable of causing infections in humans. Biofilm formation is a key strategy used by M. chelonae in colonising niches in the environment and in the host. We studied a water-air interface (pellicle) biofilm of M. chelonae using a wide array of approaches to outline the molecular structure and composition of the biofilm. Scanning electron micrographs showed that M. chelonae biofilms produced an extracellular matrix. Using a combination of biochemical analysis, Raman spectroscopy, and fluorescence microscopy, we showed the matrix to consist of proteins, carbohydrates, lipids and eDNA. Glucose was the predominant sugar present in the biofilm matrix, and its relative abundance decreased in late (established) biofilms. RNA-seq analysis of the biofilms showed upregulation of genes involved in redox metabolism. Additionally, genes involved in mycolic acid, other lipid and glyoxylate metabolism were also upregulated in the early biofilms.

The protein kinase PknB negatively regulates biosynthesis and trafficking of mycolic acids in mycobacteria

Mycobacterium tuberculosis is the causative agent of tuberculosis and remains one of the most widespread and deadliest bacterial pathogens in the world. A distinguishing feature of mycobacteria that sets them apart from other bacteria is the unique architecture of their cell wall, characterized by various species-specific lipids, most notably mycolic acids (MAs). Therefore, targeted inhibition of enzymes involved in MA biosynthesis, transport, and assembly has been extensively explored in drug discovery. Additionally, more recent evidence suggests that many enzymes in the MA biosynthesis pathway are regulated by kinase-mediated phosphorylation, thus opening additional drug-development opportunities. However, how phosphorylation regulates MA production remains unclear. Here, we used genetic strategies combined with lipidomics and phosphoproteomics approaches to investigate the role of protein phosphorylation in Mycobacterium The results of this analysis revealed that the Ser/Thr protein kinase PknB regulates the export of MAs and promotes the remodeling of the mycobacterial cell envelope. In particular, we identified the essential MmpL3 as a substrate negatively regulated by PknB. Taken together, our findings add to the understanding of how PknB activity affects the mycobacterial MA biosynthesis pathway and reveal the essential role of protein phosphorylation/dephosphorylation in governing lipid metabolism, paving the way for novel antimycobacterial strategies.

Designing novel inhibitors against cyclopropane mycolic acid synthase 3 (PcaA): targeting dormant state of Mycobacterium tuberculosis

Mycobacterium tuberculosis can sustain inside the host in dormant (non-replicating) state for years. It suppresses the host immune system by residing in the host alveolar macrophage, resulting in the development of latent tuberculosis. Despite many antibiotics available for the treatment of tuberculosis, the major hurdle in complete elimination is the ability of the bacilli to undergo dormancy and develop resistance against the existing drugs. Cyclopropanation of mycolic acids present in the cell wall of mycobacteria is required for its persistence and virulence. Cyclopropane synthases such as PcaA, CmaA1 and CmaA2, introduce site-specific modifications in mycolic acids. PcaA expression levels are high during dormancy and the gene mutants fails to persist, showing reduced survival in host macrophage. Hence, PcaA appears as a potential target to develop inhibitors against the dormant bacilli. In this study, we have identified compounds with maximum binding affinity against PcaA by in-silico virtual screening of anti-tuberculosis compounds and their structural analogues. In-silico docking followed molecular dynamic simulations and free energy calculations of the compounds with highest docking score in their respective libraries. This study reports novel inhibitors that can act as better anti-tuberculosis compounds targeting PcaA. Communicated by Ramaswamy H. Sarma