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In ruminants Mycobacterium avium subspecies paratuberculosis (MAP) is the causative organism of a chronic granulomatous inflammatory bowel disease called Johne's disease (JD). Some researchers have hypothesised that MAP is also associated with Crohn's disease (CD), an inflammatory bowel disease in humans that shares some histological features of JD. Despite numerous attempts to demonstrate causality by researchers, direct microbiological evidence of MAP involvement in CD remains elusive. Importantly, it has not been possible to reliably and reproducibly demonstrate mycobacteria in the tissue of CD patients. Past attempts to visualise mycobacteria in tissue may have been hampered by the use of stains optimised for Mycobacterium tuberculosis complex (MTB) and the lack of reliable bacteriological culture media for both non-tuberculous mycobacteria (NTM) and cell-wall-deficient mycobacteria (CWDM). Here we describe a Ziehl-Neelsen (ZN) staining method for the demonstration of CWDM in resected tissue from patients with Crohn's disease, revealing the association of CWDM in situ with host tissue reactions, and posit this as a cause of the tissue inflammation. Using the ZN stain described we demonstrated the presence of CWDM in 18 out of 18 excised tissue samples from patients diagnosed as having Crohn's disease, and in zero samples out of 15 non-inflammatory bowel disease controls.

Mycobacterium tuberculosis (Mtb) cells are known to synthesize very long chain (C60-90) structurally complex mycolic acids with various functional groups. In this study, we applied linear ion-trap (LIT) multiple-stage mass spectrometry (MSn), combined with high-resolution mass spectrometry to study the mechanisms underlying the fragmentation processes of mycolic acid standards desorbed as lithiated adduct ions by ESI. This is followed by structural characterization of a Mtb mycolic acid family (Bovine strain). Using the insight fragmentation processes gained from the study, we are able to achieve a near complete characterization of the whole mycolic acid family, revealing the identity of the α-alkyl chain, the location of the functional groups including methyl, methoxy, and keto groups along the meroaldehyde chain in each lipid species. This study showcased the power of LIT MSn toward structural determination of complex lipids in a mixture, which would be otherwise very difficult to define using other analytical techniques.

Little is known about the prevalence, functionality and ecological roles of temperate phages for members of the mycolic acid producing bacteria, the Mycolata. While many lytic phages infective for these organisms have been isolated, and assessed for their suitability for use as biological control agents of activated sludge foaming, no studies have investigated how temperate phages might be induced for this purpose. Bioinformatic analysis using the PHAge Search Tool (PHAST) on Mycolata whole genome sequence data in GenBank for members of the genera Gordonia, Mycobacterium, Nocardia, Rhodococcus, and Tsukamurella revealed 83% contained putative prophage DNA sequences. Subsequent prophage inductions using mitomycin C were conducted on 17 Mycolata strains. This led to the isolation and genome characterization of three novel Caudovirales temperate phages, namely GAL1, GMA1, and TPA4, induced from Gordonia alkanivorans, Gordonia malaquae, and Tsukamurella paurometabola, respectively. All possessed highly distinctive dsDNA genome sequences.

Tuberculosis is a prominent aliment throughout the world and a leading cause of mortality among infectious diseases. Drug development for multi-drug resistance and reducing the current therapy time is the top priority. Mycobacterial membrane protein large 3 (MmpL3) is a promising target with high potential, however, it has not been explored to its greatest potential. It is a membrane transporter that translocates trehalose-monomycolate which is a precursor for the synthesis of mycolic acid that is essential for the synthesis of the bacterial cell wall and is pathogenic in nature. In this review, we have discussed the current development of MmpL3 inhibitors, different scaffolds, their derivatives, and their synthetic schemes and provide insight into the challenges in developing these inhibitors.

Isoniazid (isonicotinic acid hydrazide, INH) is an effective frontline antituberculosis drug. INH targets several Mycobacterium tuberculosis processes, including mycolic acid biosynthesis, DNA synthesis, and redox potential. M. tuberculosis responds to INH stress by altering the expression level of crucial genes involved in various pathways. In this study, we summarize the induced gene expression pattern of active M. tuberculosis upon INH exposure. Most genes triggered by INH are involved in processes such as mycolic acid biosynthesis, a compensatory response, stress response, and drug efflux. These patterns are absent in dormant M. tuberculosis. The differential INH response pattern can inform future novel measures against M. tuberculosis.

Primary infection with Mycobacterium tuberculosis (Mtb) results in the formation of a densely packed granulomatous response that essentially limits the entry and efficacy of immune effector cells. Furthermore, the physical nature of the granuloma does not readily permit the entry of therapeutic agents to sites where organisms reside. The Mtb cell wall mycolic acid, trehalose 6,6′-dimycolate (TDM), is a physiologically relevant molecule for modelling macrophage-mediated events during the establishment of the tuberculosis-induced granuloma pathogenesis. At present, there are no treatments for tuberculosis that focus on modulating the host’s immune responses. Previous studies showed that lactoferrin (LF), a natural iron-binding protein proven to modulate inflammation, can ameliorate the cohesiveness of granuloma. This led to a series of studies that further examined the effects of recombinant human LF (rHLF) on the histological progression of TDM-induced pathology. Treatment with rHLF demonstrated significant reduction in size and number of inflammatory foci following injections of TDM, together with reduced levels pulmonary pro-inflammatory cytokines TNF-α and IL-1β. LF facilitated greater penetration of fluoroquinolone to the sites of pathology. Mice treated with TDM alone demonstrated exclusion of ofloxacin to regions of inflammatory response, whereas the animals treated with rHLF demonstrated increased penetration to inflammatory foci. Finally, recent findings support the hypothesis that this mycobacterial mycolic acid can specifically recruit M1-like polarized macrophages; rHLF treatment was shown to limit the level of this M1-like phenotypic recruitment, corresponding highly with decreased inflammatory response.

Background: mycobacterial cells contain complex mixtures of mycolic acid esters. These can be used as antigens recognised by antibodies in the serum of individuals with active tuberculosis, caused by Mycobacterium tuberculosis. In high burden populations, a significant number of false positives are observed; possibly these antigens are also recognised by antibodies generated by other mycobacterial infections, particularly ubiquitous ‘environmental mycobacteria’. This suggests similar responses may be observed in a low burden TB population, particularly in groups regularly exposed to mycobacteria. Methods: ELISA using single synthetic trehalose mycolates corresponding to major classes in many mycobacteria was used to detect antibodies in serum of individuals with no known mycobacterial infection, comprising farmers, abattoir workers, and rural and urban populations. Results: serum from four Welsh or Scottish cohorts showed lower (with some antigens significantly lower) median responses than those reported for TB negatives from high-burden TB populations, and significantly lower responses than those with active TB. A small fraction, particularly older farmers, showed strong responses. A second study examined BCG vaccinated and non-vaccinated farmers and non-farmers. Farmers gave significantly higher median responses than non-farmers with three of five antigens, while there was no significant difference between vaccinated or non-vaccinated for either farmer or non-farmer groups. Conclusions: this initial study shows that serodiagnosis with mycobacterial lipid antigens can detect antibodies in a population sub-group that is significantly exposed to mycobacteria, in an assay that is not interfered with by vaccination. Given the links between mycobacterial exposure and a range of immune system diseases, further understanding such responses may provide a new opportunity for monitoring public health and directing treatment.

The Mycobacterium tuberculosis cell envelope is a critical interface between the host and pathogen and provides a protective barrier against the immune response and antibiotics. Cell envelope lipids are also mycobacterial virulence factors that influence the host immune response. The mycobacterial membrane protein large (MmpL) proteins transport cell envelope lipids and siderophores that are important for the basic physiology and pathogenesis of M. tuberculosis. We recently identified MmpL11 as a conserved transporter of mycolic acid-containing lipids including monomeromycolyl diacylglycerol (MMDAG), mycolate wax ester (MWE), and long-chain triacylglycerols (LC-TAGs). These lipids contribute to biofilm formation in M. tuberculosis and M. smegmatis, and non-replicating persistence in M. tuberculosis. In this report, we identified domains and residues that are essential for MmpL11TB lipid transporter activity. Specifically, we show that the D1 periplasmic loop and a conserved tyrosine are essential for the MmpL11 function. Intriguingly, we found that MmpL11 levels are regulated by the phosphorylation of threonine in the cytoplasmic C-terminal domain, providing the first direct evidence of the phospho-regulation of MmpL11 transporter activity in M. tuberculosis and M. smegmatis. Our results offer further insight into the function of MmpL transporters and regulation of mycobacterial cell envelope biogenesis.

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.

A novel actinobacterium, designated strain HNM0687T, was isolated from mangrove soil samples collected from Hainan Province, PR China and its polyphasic taxonomy was studied. Based on the results of 16S rRNA gene sequence analysis, strain HNM0687T was closely related to Gordonia bronchialis NBRC 16047T (98.7 %), Gordonia rhizosphera NBRC 16068T (98.2 %), Gordonia oryzae RS15-1ST (97.9 %), Gordonia polyisoprenivorans NBRC 16320T (97.7 %) and Gordonia sediminis AMA 120T (97.7 %). Genome-based comparisons revealed a clear distinction in average nucleotide identity values between strain HNM0687T and its closely related strains (74.4–78.3 %). Strain HNM0687T contained meso-diaminopimelic acid, arabinose and galactose in whole-cell hydrolysates. Mycolic acid was present. The menaquinones of strain HNM0687T were MK-9(H2) and MK-7(H2). The phospholipids of the isolate were composed of diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol. The major fatty acids were C16 : 0, C16 : 1 ω7c/C16 : 1 ω6c, C18 : 010-methyl (TBSA), C18 : 0 and C18 : 1 ω9c. Based on its genotypic, chemotaxonomic and phenotypic characteristics, it is concluded that strain HNM0687T represents a novel species of the genus Gordonia for which the name Gordonia mangrovi sp. nov. is proposed. The type strain is HNM0687T (=CCTCC AA 2019074 T=KCTC 49383 T).

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

Mycobacterium tuberculosis produces mycolic acids which are relevant for persistence, recalcitrance to antibiotics and defiance to host immunity. c-di-GMP is a second messenger involved in transition from planktonic cells to biofilms, whose levels are controlled by diguanylate cyclases (DGC) and phosphodiesterases (PDE). The transcriptional regulator dosR, is involved in response to low oxygen, a condition likely happening to a subset of cells within biofilms. Here, we found that in M. bovis BCG, expression of both BCG1416c and BCG1419c genes, which code for a DGC and a PDE, respectively, decreased in both stationary phase and during biofilm production. The kasA, kasB, and fas genes, which are involved in mycolic acid biosynthesis, were induced in biofilm cultures, as was dosR, therefore suggesting an inverse correlation in their expression compared with that of genes involved in c-di-GMP metabolism. The relative abundance within trehalose dimycolate (TDM) of α-mycolates decreased during biofilm maturation, with methoxy mycolates increasing over time, and keto species remaining practically stable. Moreover, addition of synthetic c-di-GMP to mid-log phase BCG cultures reduced methoxy mycolates, increased keto species and practically did not affect α-mycolates, showing a differential effect of c-di-GMP on keto- and methoxy-mycolic acid metabolism.

Tuberculosis is one of the leading causes of death across the world. The treatment regimens for tuberculosis are well established, but still the control of the disease faces many challenges such as lengthy treatment protocols, drug resistance and toxicity. In the present work, mycolic acid methyl transferase (MmaA1), a protein involved in the maturation of mycolic acids in the biochemical pathway of the Mycobacterium, was studied for novel drug discovery. The homology model of the MmaA1 protein was built and validated by using computational techniques. The MmaA1 protein has 286 amino acid residues consisting of 10 α-helices and 7 β-sheets. The active site of the MmaA1 protein was identified using CASTp, SiteMap and PatchDock. Virtual screening studies were performed with two small molecule ligand databases: Asinex synergy and Diverse_Elite_Gold_Platinum databases having a total of 43,446 molecules and generated 1,30,814 conformers against the predicted and validated active site of the MmaA1 protein. Binding analysis showed that the residues ASP 19, PHE 22, TRP 30, TYR 32, TRP 74 and ALA 77 of MmaA1 protein have consistent interactions with the ligands. The hit ligands were further filtered by in silico ADME properties to eliminate potentially toxic molecules. Of the top 10 molecules, 3-(2-morpholinoacetamido)-N-(1,4-dihydro-4-oxoquinazolin-6-yl) benzamide was synthesised and screened for in vitro anti-TB activity against Mtb H37Rv using MABA assay. The compound and its intermediates exhibited good in vitro anti-TB activity which can be taken up for future lead optimisation studies. Structure based virtual screening study was performed using a validated homology model against small molecules from two virtual compound libraries. Synthesised the lead compound 3-(2-morpholinoacetamido)-N-(1,4-dihydro-4-oxoquinazolin-6-yl)benzamide obtained from virtual screening. In vitro activity against Mtb H37Rv has given a promising result.

In Mycobacterium tuberculosis, mycolic acids and their glycerol, glucose, and trehalose esters (“cord factor”) form the main part of the mycomembrane. Despite their first isolation almost a century ago, full stereochemical evaluation is lacking, as is a scalable synthesis required for accurate immunological, including vaccination, studies. Herein, we report an efficient, convergent, gram‐scale synthesis of four stereo‐isomers of a mycolic acid and its glucose ester. Binding to the antigen presenting protein CD1b and T cell activation studies are used to confirm the antigenicity of the synthetic material. The absolute stereochemistry of the syn‐methoxy methyl moiety in natural material is evaluated by comparing its optical rotation with that of synthetic material., The total synthesis of four methoxy mycolic acid diastereomers is described. Key steps involve a Suzuki–Fu cross‐coupling, an anti‐selective Abiko–Masamune asymmetric aldol reaction, and an enantioselective Charette cyclopropanation. CD1b‐free mycolic acid tetramer staining experiments clearly favoured one diastereomer over the other three.

Co-culture is an efficient strategy for natural product discovery. We have used mycolic acid-containing bacteria (MACB) Tsukamurella pumonis TP-B0596 to induce secondary metabolism by actinomycetes and have found several natural products. We also observed that MACB attached to the mycelium of Streptomyces lividans forming coaggregates during combined-culture. This stimulated interest in the interactions among actinomycetes and MACB, and we found that soil isolated cultures contained a mixture of actinomycetes and MACB. Our previously observed interactions were the result of selective screening and combination of bacteria in the lab, which warranted investigation of the existence of these interactions in the natural soil environment. Therefore, in this paper, we report the interaction between a co-isolated natural pair of actinomycetes and MACB in terms of morphology and metabolic changes. A natural pair of actinomycetes and MACB co-aggregated in liquid culture and showed metabolic changes. Interestingly, co-aggregated actinomycetes and MACB were re-isolated from soil with no obvious morphological colony differences from the colony of a single strain. The results demonstrate that there is a stochastic chance of picking colonies containing co-aggregated actinomycetes and MACB, which suggests that the pair can exist in co-aggregate form in the soil environment and interact with each other.

As part of a screening programme for antibiotic-producing bacteria, a novel Actinomadura species was discovered from a soil sample collected in Santorini, Greece. Preliminary 16S rRNA gene sequence comparisons highlighted Actinomadura macra as the most similar characterised species. However, whole-genome sequencing revealed an average nucleotide identity (ANI) value of 89% with A. macra, the highest among related species. Further phenotypic and chemotaxonomic analyses confirmed that the isolate represents a previously uncharacterised species in the genus Actinomadura, for which the name Actinomadura graeca sp. nov. is proposed (type strain 32-07T). The G+C content of A. graeca 32–07 is 72.36%. The cell wall contains DL-diaminopimelic acid, intracellular sugars are glucose, ribose and galactose, the predominant menaquinone is MK-9(H6), the major cellular lipid is phosphatidylinositol and fatty acids consist mainly of hexadecanoic acid. No mycolic acid was detected. Furthermore, A. graeca 32–07 has been confirmed as a novel producer of the non-ribosomal peptide antibiotic zelkovamycin and we report herein a provisional description of the unique biosynthetic gene cluster.

Our previous research has demonstrated a promising capacity of streptomycetes isolated from red soils to produce novel secondary metabolites, most of which, however, remain to be explored. Co-culturing with mycolic acid-containing bacteria (MACB) has been used successfully in activating the secondary metabolism in Streptomyces. Here, we co-cultured 44 strains of red soil-derived streptomycetes with four MACB of different species in a pairwise manner and analyzed the secondary metabolites. The results revealed that each of the MACB strains induced changes in the metabolite profiles of 35–40 streptomycetes tested, of which 12–14 streptomycetes produced “new” metabolites that were not detected in the pure cultures. Moreover, some of the co-cultures showed additional or enhanced antimicrobial activity compared to the pure cultures, indicating that co-culture may activate the production of bioactive compounds. From the co-culture-induced metabolites, we identified 49 putative new compounds. Taking the co-culture of Streptomyces sp. FXJ1.264 and Mycobacterium sp. HX09-1 as a case, we further explored the underlying mechanism of co-culture activation and found that it most likely relied on direct physical contact between the two living bacteria. Overall, our results verify co-culture with MACB as an effective approach to discover novel natural products from red soil-derived streptomycetes.

The mycobacterial membrane protein large 3 (MmpL3) transporter is essential and required for shuttling the lipid trehalose monomycolate (TMM), a precursor of mycolic acid (MA)-containing trehalose dimycolate (TDM) and mycolyl arabinogalactan peptidoglycan (mAGP), in Mycobacterium species, including Mycobacterium tuberculosis and Mycobacterium smegmatis. However, the mechanism that MmpL3 uses to facilitate the transport of fatty acids and lipidic elements to the mycobacterial cell wall remains elusive. Here, we report 7 structures of the M. smegmatis MmpL3 transporter in its unbound state and in complex with trehalose 6-decanoate (T6D) or TMM using single-particle cryo-electron microscopy (cryo-EM) and X-ray crystallography. Combined with calculated results from molecular dynamics (MD) and target MD simulations, we reveal a lipid transport mechanism that involves a coupled movement of the periplasmic domain and transmembrane helices of the MmpL3 transporter that facilitates the shuttling of lipids to the mycobacterial cell wall., Mycobacterial membrane protein Large 3 (MmpL3) is a transporter required for shuttling trehalose monomycolate. Structures of M. smegmatis MmpL3 with and without substrate reveal the mechanism by which MmpL3 transports this essential precursor of lipids for the mycobacterial cell wall.

The final step in mycolic acid biosynthesis in Mycobacterium tuberculosis is catalysed by mycolyl reductase encoded by the Rv2509 gene. Sequence analysis and homology modelling indicate that Rv2509 belongs to the short‐chain fatty acid dehydrogenase/reductase (SDR) family, but with some distinct features that warrant its classification as belonging to a novel family of short‐chain dehydrogenases. In particular, the predicted structure revealed a unique α‐helical C‐terminal region which we demonstrated to be essential for Rv2509 function, though this region did not seem to play any role in protein stabilisation or oligomerisation. We also show that unlike the M. smegmatis homologue which was not essential for growth, Rv2509 was an essential gene in slow‐growing mycobacteria. A knockdown strain of the BCG2529 gene, the Rv2509 homologue in Mycobacterium bovis BCG, was unable to grow following the conditional depletion of BCG2529. This conditional depletion also led to a reduction of mature mycolic acid production and accumulation of intermediates derived from 3‐oxo‐mycolate precursors. Our studies demonstrate novel features of the mycolyl reductase Rv2509 and outline its role in mycobacterial growth, highlighting its potential as a new target for therapies., The Mycobacterium tuberculosis gene Rv2509 encodes a reductase enzyme involved in the last step of synthesis of mycolic acids, a key cell wall lipid. By generating a predicted model for the protein encoded by Rv2509, we show that the enzyme has distinct structural motifs including a distinct C‐terminal region, which we show by complementation studies to be vital for function. The gene is also essential for growth and viability underlining its potential as a new drug target.

Isoxyl (ISO) and thiacetazone (TAC) are two antitubercular prodrugs that abolish mycolic acid biosynthesis and kill Mycobacterium tuberculosis (Mtb) through the inhibition of the essential type II fatty acid synthase (FAS-II) dehydratase HadAB. While mutations preventing ISO and TAC either from being converted to their active form or from covalently modifying their target are the most frequent spontaneous mutations associated with high-level resistance to both drugs, the molecular mechanisms underlying the high-level ISO and TAC resistance of Mtb strains harboring missense mutations in the second, nonessential, FAS-II dehydratase HadBC have remained unexplained. Using a combination of genetic, biochemical, and biophysical approaches and molecular dynamics simulation, we here show that all four reported resistance mutations in the HadC subunit of HadBC alter the stability and/or specific activity of the enzyme, allowing it in two cases (HadBCV85I and HadBCK157R) to compensate for a deficiency in HadAB in whole Mtb bacilli. The analysis of the mycolic acid profiles of Mtb strains expressing the mutated forms of HadC further points to alterations in the activity of the mycolic acid biosynthetic complex and suggests an additional contributing resistance mechanism whereby HadC mutations may reduce the accessibility of HadAB to ISO and TAC. Collectively, our results highlight the importance of developing optimized inhibitors of the dehydration step of FAS-II capable of inhibiting both dehydratases simultaneously, a goal that may be achievable given the structural resemblance of the two enzymes and their reliance on the same catalytic subunit HadB.

A common technique used to differentiate bacterial species and to determine evolutionary relationships is sequencing their 16S ribosomal RNA genes. However, this method fails when organisms exhibit high similarity in these sequences. Two such strains that have identical 16S rRNA sequences are Mycobacterium indicus pranii (MIP) and Mycobacterium intracellulare. MIP is of significance as it is used as an adjuvant for protection against tuberculosis and leprosy; in addition, it shows potent anti-cancer activity. On the other hand, M. intracellulare is an opportunistic pathogen and causes severe respiratory infections in AIDS patients. It is important to differentiate these two bacterial species as they co-exist in immuno-compromised individuals. To unambiguously distinguish these two closely related bacterial strains, we employed Raman and resonance Raman spectroscopy in conjunction with multivariate statistical tools. Phenotypic profiling for these bacterial species was performed in a kinetic manner. Differences were observed in the mycolic acid profile and carotenoid pigments to show that MIP is biochemically distinct from M. intracellulare. Resonance Raman studies confirmed that carotenoids were produced by both MIP as well as M. intracellulare, though the latter produced higher amounts. Overall, this study demonstrates the potential of Raman spectroscopy in differentiating two closely related mycobacterial strains. Graphical abstract.

Introduction. ML1899 is conserved in all mycobacterium sp. and is a middle member of mle-ML1898 operon involved in mycolic acid modification. Aim. In the present study attempts were made to characterize ML1899 in detail. Methodology. Bioinformatics tools were used for prediction of active-site residues, antigenic epitopes and a three-dimensional model of protein. The gene was cloned, expressed and purified as His-tagged protein in Escherichia coli for biophysical/biochemical characterization. Recombinant protein was used to treat THP-1 cells to study change in production of nitric oxide (NO), reactive oxygen species (ROS), cytokines and chemokines using flowcytometry/ELISA. Results. In silico analysis predicted ML1899 as a member of α/β hydrolase family with GXSXG-motif and Ser126, His282, Asp254 as active-site residues that were confirmed by site-directed mutagensis. ML1899 exhibited esterase activity. It hydrolysed pNP-butyrate as optimum substrate at pH 8.0 and 50 °C with 5.56 µM−1 min−1 catalytic efficiency. The enzyme exhibited stability up to 60 °C temperature and between pH 6.0 to 9.0. K m, V max and specific activity of ML1899 were calculated to be 400 µM, 40 µmoles min−1 ml−1 and 27 U mg− 1, respectively. ML1899 also exhibited phospholipase activity. The protein affected the survival of macrophages when treated at higher concentration. ML1899 enhanced ROS/NO production and up-regulated pro-inflammatory cytokines and chemokine including TNF-α, IFN-γ, IL-6 and IL-8 in macrophages. ML1899 was also observed to elicit humoral response in 69 % of leprosy patients. Conclusion. These results suggested that ML1899, an esterase could up-regulate the immune responses in favour of macrophages at a low concentration but kills the THP-1 macrophages cells at a higher concentration.

The main effectors in tumor control are the class I MHC molecule-restricted CD8+ cytotoxic T lymphocytes (CTLs). Tumor-specific CTL induction can be regulated by dendritic cells (DCs) expressing both tumor-derived epitopes and co-stimulatory molecules. Immunosuppressive tolerogenic DCs, having down-regulated co-stimulatory molecules, are seen within the tumor mass and can suppress tumor-specific CTL induction. The tolerogenic DCs expressing down-regulated XCR1+CD141+ appear to be induced by tumor-derived soluble factors or dexamethasone, while the immunogenic DCs usually express XCR1+CD141+ molecules with a cross-presentation function in humans. Thus, if tolerogenic DCs can be reactivated into immunogenic DCs with sufficient co-stimulatory molecules, tumor-specific CD8+ CTLs can be primed and activated in vivo. In the present study, we converted human tolerogenic CD141+ DCs with enhanced co-stimulatory molecule expression of CD40, CD80, and CD86 through stimulation with non-toxic mycobacterial lipids such as mycolic acid (MA) and lipoarabinomannan (LAM), which synergistically enhanced both co-stimulatory molecule expression and interleukin (IL)-12 secretion by XCR1+CD141+ DCs. Moreover, MA and LAM-stimulated DCs captured tumor antigens and presented tumor epitope(s) in association with class I MHCs and sufficient upregulated co-stimulatory molecules to prime naïve CD3+ T cells to become CD8+ tumor-specific CTLs. Repeat CD141+ DC stimulation with MA and LAM augmented the secretion of IL-12. These findings provide us a new method for altering the tumor environment by converting tolerogenic DCs to immunogenic DCs with MA and LAM from Mycobacterium tuberculosis.

A novel group of agents known as the indole-2-carboxamides (often referred to as indoleamides) have been shown to demonstrate high antimycobacterial activity. Studies have demonstrated that the best indoleamides possess desirable ADME/Tox properties, with less adverse effects and increased efficacy against both MDR-TB (multi-drug resistant TB) and XDR-TB (extensively drug-resistant TB). The primary mechanism of killing Mycobacterium tuberculosis (Mtb) by indoleamides is by disrupting the function of the essential mycolic acid transporter MmpL3 protein (Mycobacterial membrane protein Large 3). Therefore, targeting this essential mycobacterial transporter by small molecules opens new possibility for the development of novel and effective anti-TB agents. In the present study, we characterized the effects of indoleamides in altering the viability of Mtb in an in vitro granuloma model using immune cells derived from healthy subjects and those with type 2 diabetes mellitus (T2DM). Our results indicate that treatment with the best indoleamide 3 resulted in a significant reduction in the viability of Mtb in both THP-1 macrophages as well as in granulomas derived from healthy individuals and subjects with T2DM. Graphical Abstract.

The taxonomic position of an actinomycete designated AMA 120T, isolated from mangrove sediment, was clarified by phenotypic, chemotaxonomic and phylogenetic studies. The 16S rRNA gene sequence revealed that strain AMA 120T was most closely related to Gordonia rhizosphera NBRC 16068T (98.9 %), Gordonia polyisoprenivorans NBRC 16320T (98.1 %) and Gordonia bronchialis NBRC 16047T (98.1 %). A fragment of the gyrB gene of strain AMA 120T formed a distinct phyletic line with G. rhizosphera NBRC 16068T (95.4 %). Strain AMA 120T contained meso-diaminopimelic acid, arabinose and galactose as cell-wall components, and MK-9(H2) was the predominant menaquinone. The polar lipid profile for this strain consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside and two unidentified phospholipids. Mycolic acid was present. The major fatty acids were C16 : 0, C18 : 1ω9c and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c). The DNA–DNA relatedness values between AMA 120T and close species were below 70 %. There was an obvious distinction in the average nucleotide identity distribution between strain AMA 120T and its closely related strains at around 75–92%. The DNA G+C content of strain AMA 120T was 66.6 mol%. These results, coupled with the phenotypic and chemotaxonomic data, indicated that strain AMA 120T represents a novel species of the genus Gordonia , for which the name Gordonia sediminis sp. nov. is proposed. The type strain is AMA 120T (=TBRC 7109T=NBRC 113236T).

Caseous lymphadenitis (CLA) is a chronic bacterial disease that causes economic losses in small ruminant throughout the world. Corynebacterium pseudotuberculosis is the causative agent of CLA characterized by large abscesses internally/externally, abortions, weight loss, and decreased wool production in sheep and goat farming. There is paucity of information on Corynebacterium pseudotuberculosis and its immunogen mycolic acid responses in male goat during the course of CLA. As a result, this study was designed to investigate the clinical responses and pathological changes of the reproductive tract and its associated lymph nodes in male goats following inoculation with Corynebacterium pseudotuberculosis and its immunogen mycolic acids. A total of 12 healthy crossbred Boer male goats aged 16–20 months were divided into three groups, 1, 2, and 3, of four in each group. Group 1 (control group) was inoculated with 2 ml of sterile phosphate-buffered solution (PBS) via intradermal route, group 2 was inoculated with 2 ml 1 × 109 colony forming unit (cfu) of C. pseudotuberculosis intradermally and group 3 was inoculated with 2 ml of mycolic acid (1 g/ml) intradermally. The bucks were observed and assessed for changes in clinical responses daily for 60 days post challenged. At the end of the experiment, animals were slaughtered and organs of the reproductive tract and lymph nodes were harvested for histopathological investigations. In this study, all experimental bucks survived throughout the study period with some evidence of clinical manifestation such as pyrexia, weight loss, and enlargement or rupture of the lymph nodes. Microscopically, C. pseudotuberculosis and mycolic acid challenged groups showed significant cellular changes in the reproductive system and lymph nodes. Group 2 showed abscessation of the lymph nodes while group 3 did not indicate any abscess formation in the lymph nodes. For mycolic acid extract, the result revealed only mild to moderate clinic-pathological responses in bucks with no abscess formation in the lymph nodes. The result obtained added knowledge in the field of CLA research in small ruminants. In conclusion, this research revealed that C. pseudotuberculosis has a negative effect on reproductive performances of bucks and affects the clinical responses and abscess formation.

Corynebacterium pseudotuberculosis is the causative agent of caseous lymphadenitis, a debilitating chronic disease of sheep and goats. Little is known about the buck's reproductive pathophysiology with respect to inoculation with Corynebacterium pseudotuberculois and its immunogen mycolic acid extract. Therefore, this present study was designed to determine the concentration of testosterone hormone, pro-inflammatory cytokines, and semen quality of the experimental animals. A total of 12 bucks, divided into groups 1, 2, and 3 (Negative control group, Positive control group and Mycolic acid group respectively), were enrolled in this study. Following inoculation, all goats were observed for clinical responses and monitored for 60 days post-challenge and were then sacrificed. Blood samples were collected via the jugular once before inoculation and on a weekly basis post-challenge. Semen samples were collected 2 weeks post-challenge and prior to the sacrifice of the experimental animals. During the post inoculation period of 60 days, the concentration of testosterone hormone for group 2 was increased significantly (p 0.05) in weeks 5, 6, and 9 but decreased in weeks 2 and 7 post inoculation. In group 3, the mean concentration of testosterone was increased significantly (p 0.05) in weeks 5, 6, 7, and 9 post inoculation but decreased in week 2. The concentration of interleukin 6 (IL 6) in treated group 2 did not show any significant difference (p 0.05) but increased significantly (p 0.05) in week 2 post inoculation in group 3. For concentration of interleukin 1β (IL1β) in both treated groups 2 and 3 showed significant difference (p 0.05) in weeks 2 and 3 post inoculation. The tumor necrosis factor-alpha (TNF-α) concentration in both treated groups 2 and 3 did not show any significant difference (p 0.05) as compared to group 1. The concentration of interferon-γ (IFNγ) significantly increased (p 0.05) for group 2 for weeks 2, 3, 4, and 5 where else for group 3 was not in significant difference (p 0.05) compared to group 1. Both group 2 and group 3 showed a reduction in semen qualities as compared to group 1, but the severity was more intense in group 2 if compared to group 3. In conclusion, therefore, the present study concluded that the mycolic acid group revealed significant responses of testosterone hormone concentration, semen quality, and its related pro-inflammatory cytokines in bucks following infection but the severity lesser compared to Corynebacterium pseudotuberculosis group.

Bacterial secondary metabolites (SM) are rich sources of drug leads, and in particular, numerous metabolites have been isolated from actinomycetes. It was revealed by recent genome sequence projects that actinomycetes harbor much more secondary metabolite-biosynthetic gene clusters (SM-BGCs) than previously expected. Nevertheless, large parts of SM-BGCs in actinomycetes are dormant and cryptic under the standard culture conditions. Therefore, a widely applicable methodology for cryptic SM-BGC activation is required to obtain novel SM. Recently, it was discovered that co-culturing with mycolic-acid-containing bacteria (MACB) widely activated cryptic SM-BGCs in actinomycetes. This “combined-culture” methodology (co-culture methodology using MACB as the partner of actinomycetes) is easily applicable for a broad range of actinomycetes, and indeed, 33 novel SM have been successfully obtained from 12 actinomycetes so far. In this review, the development, application, and mechanistic analysis of the combined-culture method were summarized.

A slow-growing, scotochromogenic mycobacterial strain (24T) was isolated from the sputum of a Chinese male human. Phylogenetic analysis using the 16S rRNA gene assigned strain 24T to the Mycobacterium gordonae complex, which includes Mycobacterium gordonae and Mycobacterium paragordonae . The phenotypic characteristics, unique mycolic acid profile and the results of phylogenetic analysis based on hsp65 and rpoB sequences strongly supported the taxonomic status of strain 24T as a representative of a species distinct from the other members of the M. gordonae complex. The genomic G+C content of strain 24T was 65.40mol%. Genomic comparisons showed that strain 24T and M. gordonae ATCC 14470T had an average nucleotide identity (ANI) value of 81.00 % and a DNA–DNA hybridization (DDH) value of 22.80 %, while the ANI and DDH values between strain 24Tand M. paragordonae 49 061T were 80.98 and 22.80 %, respectively. In terms of phylogenetic, phenotypic and chemotaxonomic features, strain 24T is distinguishable from its closest phylogenetic relatives and represents a novel species of the genus Mycobacterium , therefore the name Mycobacterium vicinigordonae sp. nov. is proposed. The type strain is 24T (=CMCC 93559T=DSM 105979T).

Mycobacteria species can differ from one another in the rate of growth, presence of pigmentation, the colony morphology displayed on solid media, as well as other phenotypic characteristics. However, they all have in common the most relevant character of mycobacteria: its unique and highly hydrophobic cell wall. Mycobacteria species contain a membrane-covalent linked complex that includes arabinogalactan, peptidoglycan, and long-chains of mycolic acids with types that differ between mycobacteria species. Additionally, mycobacteria can also produce lipids that are located, non-covalently linked, on their cell surfaces, such as phthiocerol dimycocerosates (PDIM), phenolic glycolipids (PGL), glycopeptidolipids (GPL), acyltrehaloses (AT), or phosphatidil-inositol mannosides (PIM), among others. Some of them are considered virulence factors in pathogenic mycobacteria, or critical antigenic lipids in host-mycobacteria interaction. For these reasons, there is a significant interest in the study of mycobacterial lipids due to their application in several fields, from understanding their role in the pathogenicity of mycobacteria infections, to a possible implication as immunomodulatory agents for the treatment of infectious diseases and other pathologies such as cancer. Here, a simple approach to extract and analyze the total lipid content and the mycolic acid composition of mycobacteria cells grown in a solid medium using mixtures of organic solvents is presented. Once the lipid extracts are obtained, thin-layer chromatography (TLC) is performed to monitor the extracted compounds. The example experiment is performed with four different mycobacteria: the environmental fast-growing Mycolicibacterium brumae and Mycolicibacterium fortuitum, the attenuated slow-growing Mycobacterium bovis bacillus Calmette-Guerin (BCG), and the opportunistic pathogen fast-growing Mycobacterium abscessus, demonstrating that methods shown in the present protocol can be used to a wide range of mycobacteria.

SynopsisBackgroundThe major global health threat tuberculosis is caused by Mycobacterium tuberculosis (Mtb). Mtb has a complex cell envelope – a partially covalently linked composite of polysaccharides, peptidoglycan and lipids, including a mycolic acid layer – which conveys pathogenicity but also protects against antibiotics. Given previous successes in treating gram-positive and -negative infections with cell wall degrading enzymes, we investigated such approach for Mtb.Objectives(i) Development of an Mtb microtiter growth inhibition assay that allows undisturbed cell envelope formation, to overcome the invalidation of results by typical clumped Mtb-growth in surfactant-free assays. (ii) Exploring anti-Mtb potency of cell wall layer-degrading enzymes. (iii) Investigation of the concerted action of several such enzymes.MethodsWe inserted a bacterial luciferase-operon in an auxotrophic Mtb strain to develop a microtiter assay that allows proper evaluation of cell wall degrading anti-Mtb enzymes. We assessed growth-inhibition by enzymes (recombinant mycobacteriophage mycolic acid esterase (LysB), fungal α-amylase and human and chicken egg white lysozymes) and combinations thereof, in presence or absence of biopharmaceutically acceptable surfactant.ResultsOur biosafety level-2 assay identified both LysB and lysozymes as potent Mtb-inhibitors, but only in presence of surfactant. Moreover, most potent disruption of the mycolic acid hydrophobic barrier was obtained by the highly synergistic combination of LysB, α-amylase and polysorbate 80.ConclusionsSynergistically acting cell wall degrading enzymes are potently inhibiting Mtb – which sets the scene for the design of specifically tailored antimycobacterial (fusion) enzymes. Airway delivery of protein therapeutics has already been established and should be studied in animal models for active TB.

A non-tuberculous mycobacterium, Mycobacterium abscessus is an emerging opportunistic pathogen associated with difficult to treat pulmonary infections, particularly in patients suffering from cystic fibrosis. It is capable of forming biofilms in vitro that result in an increase of already high levels of antibiotic resistance in this bacterium. Evidence that M. abscessus forms biofilm-like microcolonies in patient lungs and on medical devices further implicated the need to investigate this biofilm in detail. Therefore, in this study we characterized the M. abscessus pellicular biofilm, formed on a liquid-air interface, by studying its molecular composition, and its transcriptional profile in comparison to planktonic cells. Using scanning electron micrographs and fluorescence microscopy, we showed that M. abscessus biofilms produce an extracellular matrix composed of lipids, proteins, carbohydrates and extracellular DNA. Transcriptomic analysis of biofilms revealed an upregulation of pathways involved in the glyoxylate shunt, redox metabolism and mycolic acid biosynthesis. Genes involved in elongation and desaturation of mycolic acids were highly upregulated in biofilms and, mirroring those findings, biochemical analysis of mycolates revealed molecular changes and an increase in mycolic acid chain length. Together these results give us an insight into the complex structure of M. abscessus biofilms, the understanding of which may be adapted for clinical use in treatment of biofilm infections, including strategies for dispersing the extracellular matrix, allowing antibiotics to gain access to bacteria within the biofilm.

A Gram-positive, acid-fast and rapidly growing rod, designated S2-37 T , that could form yellowish colonies was isolated from soil samples collected from cotton cropping field located in the Xinjiang region of China. The draft genome of strain S2-37 T was 5.1 Mb in length, with a DNA G+C content of 68.4 mol%. 16S rRNA-directed phylogenetic analysis referred that strain S2-37 T was closely related to bacterial species belonging to the genus Mycolicibacterium and Mycobacterium . Multilocus sequence analysis of three genes (16S rRNA, hsp65 and rpoB ) revealed that strain S2-37 T shared high sequence similarities with Mycolicibacterium litorale CGMCC 4.5724 T (96.5%) and Mycobacterium neglectum CECT 8778 T (95.7%). Digital DNA-DNA hybridization (dDDH) and the average nucleotide identity (ANI) presented that strainS2-37 T displayed the highest values of 39.1% (35.7-42.6%) and 81.28% with M. litorale CGMCC 4.5724 T , respectively. And characterazition of conserved molecular signatures further confirmed that strain S2-37 T could be well classified into the genus Mycolicibacterium . The main fatty acids were identified as C 16:0 , C 18:0 , C 20:3 ω3 and C 22:6 ω3 . In addition, polar lipids profile was mainly composed of diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol. Results indicated that strain S2-37 T represented genetically and phenotypically distinct from its closest phylogenetic neighbour, M. litorale CGMCC 4.5724 T . Here, we propose a novel species of the genus Mycolicibacterium : Mycolicibacterium gossypii sp. nov. with the type strain S2-37 T (=JCM 34327T =CGMCC 1.18817T ).

A non-tuberculous mycobacterium (NTM), Mycobacterium abscessus is an emerging opportunistic pathogen associated with difficult to treat pulmonary infections, particularly in patients suffering from cystic fibrosis. It is capable of forming biofilms in vitro that result in an increase of already high levels of antibiotic resistance in this bacterium. Evidence that M. abscessus forms biofilm-like microcolonies in patient lungs and on medical devices further implicated the need to investigate this biofilm in detail. Therefore, in this study we characterized the M. abscessus pellicular biofilm, formed on a liquid-air interface, by studying its molecular composition, and its transcriptional profile in comparison to planktonic cells. Using scanning electron micrographs and fluorescence microscopy, we showed that M. abscessus biofilms produce an extracellular matrix composed of lipids, proteins, carbohydrates and eDNA. Transcriptomic analysis of biofilms revealed an upregulation of pathways involved in the glyoxylate shunt, redox metabolism and mycolic acid biosynthesis. Genes involved in elongation and desaturation of mycolic acids were highly upregulated in biofilms and, mirroring those findings, biochemical analysis of mycolates revealed molecular changes and an increase in mycolic acid chain length. Together these results give us an insight into the complex structure of M. abscessus biofilms, the understanding of which may be adapted for clinical use in treatment of biofilm infections, including strategies for dispersing the ECM, allowing antibiotics to gain access to bacteria within the biofilm.

The genome of the human intracellular pathogen Mycobacterium tuberculosis encodes an unusually large number of epoxide hydrolases, which are thought to be involved in lipid metabolism and detoxification reactions needed to endure the hostile environment of host macrophages. These enzymes therefore represent suitable targets for compounds such as urea derivatives, which are known inhibitors of soluble epoxide hydrolases. In this work, we studied in vitro the effect of the thiourea drug isoxyl on six epoxide hydrolases of M. tuberculosis using a fatty acid substrate. We show that one of the proteins inhibited by isoxyl is EphD, an enzyme involved in the metabolism of mycolic acids, key components of the mycobacterial cell wall. By analyzing mycolic acid profiles, we demonstrate the inhibition of EphD epoxide hydrolase activity by isoxyl and two other urea-based inhibitors, thiacetazone and AU1235, inside the mycobacterial cell.

Mycobacterium tuberculosis and its relatives, like many bacteria, have dynamic cell walls that respond to environmental stresses. Modulation of cell wall metabolism in stress is thought to be responsible for decreased permeability and increased tolerance to antibiotics. The signaling systems that control cell wall metabolism under stress, however, are poorly understood. Here, we examine the cell wall regulatory function of a key cell wall regulator, the Serine Threonine Phosphatase PstP, in the model organism Mycobacterium smegmatis. We show that the peptidoglycan regulator CwlM is a substrate of PstP. We find that a phospho-mimetic mutation, pstP T171E, slows growth, mis-regulates both mycolic acid and peptidoglycan metabolism in different conditions, and interferes with antibiotic tolerance. These data suggest that phosphorylation on PstP affects its activity against various substrates and is important in the transition between growth and stasis.ImportanceRegulation of cell wall assembly is essential for bacterial survival and contributes to pathogenesis and antibiotic tolerance in mycobacteria, including pathogens such as Mycobacterium tuberculosis. However, little is known about how the cell wall is regulated in stress. We describe a pathway of cell wall modulation in Mycobacterium smegmatis through the only essential Ser/Thr phosphatase, PstP. We showed that phosphorylation on PstP is important in regulating peptidoglycan metabolism in the transition to stasis and mycolic acid metabolism in growth. This regulation also affects antibiotic tolerance in growth and stasis. This work helps us to better understand the phosphorylation-mediated cell wall regulation circuitry in Mycobacteria.

The emergence of multi-drug resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis creates the urgency for new anti-tuberculosis drugs to improve the efficiency of current tuberculosis treatment. In the search for a new potential tuberculosis drug, we synthesized an isoindole based chemical library and screened a potential candidate with significant anti-tuberculosis activity. The compound named 2-hydroxy-4-(4-nitro-1,3-dioxoisoindolin-2-yl) benzoic acid (IDD-B40) showed strong activity against all the tested drug-susceptible and drug-resistant strains of M. tuberculosis, with the 50% minimum inhibitory concentrations (MIC50) of 0.39 μg/ml both in culture broth and inside Raw 264.7 cells. Also, IDD-B40, in combination with rifampicin, exhibited a direct synergistic effect against both XDR and H37Rv M. tuberculosis. Besides, IDD-B40 showed a better post-antibiotic effect (PAE) than did some first-line drugs and showed no significant cytotoxicity to any cell line tested, with a selectivity index of ≥ 128. Although IDD-B40 showed a result similar to isoniazid in the preliminary mycolic acid inhibition assay, it did not exhibit any effect against other mycolic acid-producing nontuberculous mycobacterial strains (NTM), and different non-mycobacterial pathogenic strains, so further studies are required to confirm the mode of action of IDD-B40. Considering its results against M. tuberculosis, IDD-B40 is a potential anti-tuberculosis drug candidate. However, further studies are required to evaluate its potential in vivo effect and therapeutic potential.

This book chapter provides readers the step-by-step instruction for cell growth, lipid isolation, and lipid analysis to obtain the lipidome of Corynebacterium glutamicum (C. glutamicum) in the genus Corynebacterium, a biotechnologically important bacterium. We separate the lipid families by preparative HPLC with an analytical C-8 column, followed by linear ion-trap multiple stage mass spectrometry (LIT MSn) with high-resolution mass measurement to define the structures of cytidine diphosphate diacylglycerol (CDP-DAG), glucuronosyl diacylglycerol (GlcA-DAG), α-D-mannopyranosyl-(1 → 4)-α-D-glucuronyl diacylglycerol (Man-GlcA-DAG), 1-mycolyl-2-acyl-phosphatidylglycerol (MA-PG), and acyl trehalose monomycolate (acyl-TMM) whose structures have been previously mis-assigned or not defined by mass spectrometric means. We also define the structures of mycolic acid, phosphatidylglycerol, phosphatidylinositol, cardiolipin, trehalose dimycolate lipids in the cell wall. The similarity of the lipidome to that in the Mycobacterium genera is consistent with the notion that Corynebacterium and Mycobacterium are gram-positive bacteria belonging to the suborder Corynebacterineae.

Background Bacterial degradation/transformation of steroids is widely investigated to create biotechnologically relevant strains for industrial application. The strain of Nocardioides simplex VKM Ac-2033D is well known mainly for its superior 3-ketosteroid Δ1-dehydrogenase activity towards various 3-oxosteroids and other important reactions of sterol degradation. However, its biocatalytic capacities and the molecular fundamentals of its activity towards natural sterols and synthetic steroids were not fully understood. In this study, a comparative investigation of the genome-wide transcriptome profiling of the N. simplex VKM Ac-2033D grown on phytosterol, or in the presence of cortisone 21-acetate was performed with RNA-seq. Results Although the gene patterns induced by phytosterol generally resemble the gene sets involved in phytosterol degradation pathways in mycolic acid rich actinobacteria such as Mycolicibacterium, Mycobacterium and Rhodococcus species, the differences in gene organization and previously unreported genes with high expression level were revealed. Transcription of the genes related to KstR- and KstR2-regulons was mainly enhanced in response to phytosterol, and the role in steroid catabolism is predicted for some dozens of the genes in N. simplex. New transcription factors binding motifs and new candidate transcription regulators of steroid catabolism were predicted in N. simplex. Unlike phytosterol, cortisone 21-acetate does not provide induction of the genes with predicted KstR and KstR2 sites. Superior 3-ketosteroid-Δ1-dehydrogenase activity of N. simplex VKM Ac-2033D is due to the kstDs redundancy in the genome, with the highest expression level of the gene KR76_27125 orthologous to kstD2, in response to cortisone 21-acetate. The substrate spectrum of N. simplex 3-ketosteroid-Δ1-dehydrogenase was expanded in this study with progesterone and its 17α-hydroxylated and 11α,17α-dihydroxylated derivatives, that effectively were 1(2)-dehydrogenated in vivo by the whole cells of the N. simplex VKM Ac-2033D. Conclusion The results contribute to the knowledge of biocatalytic features and diversity of steroid modification capabilities of actinobacteria, defining targets for further bioengineering manipulations with the purpose of expansion of their biotechnological applications.

The major global health threat tuberculosis is caused by Mycobacterium tuberculosis. M. tuberculosis has a complex cell envelope-a partially covalently linked composite of polysaccharides, peptidoglycan, and lipids, including a mycolic acid layer -which conveys pathogenicity but also protects against antibiotics. Given previous successes in treating Gram-positive and -negative infections with cell wall-degrading enzymes, we investigated such an approach for M. tuberculosis. In this study, we aimed to (i) develop an M. tuberculosis microtiter growth inhibition assay that allows undisturbed cell envelope formation to overcome the invalidation of results by typical clumped M. tuberculosis growth in surfactant-free assays, (ii) explore anti-M. tuberculosis potency of cell wall layer-degrading enzymes, and (iii) investigate the concerted action of several such enzymes. We inserted a bacterial luciferase operon in an auxotrophic M. tuberculosis strain to develop a microtiter assay that allows proper evaluation of cell wall-degrading anti-M. tuberculosis enzymes. We assessed growth inhibition by enzymes (recombinant mycobacteriophage mycolic acid esterase [LysB], fungal alpha-amylase, and human and chicken egg white lysozymes) and combinations thereof in the presence or absence of biopharmaceutically acceptable surfactant. Our biosafety level 2 assay identified both LysB and lysozymes as potent M. tuberculosis inhibitors but only in the presence of surfactant. Moreover, the most potent disruption of the mycolic acid hydrophobic barrier was obtained by the highly synergistic combination of LysB, alpha-amylase, and polysorbate 80. Synergistically acting cell wall-degrading enzymes are potently inhibiting M. tuberculosis, which sets the scene for the design of specifically tailored antimycobacterial (fusion) enzymes. Airway delivery of protein therapeutics has already been established and should be studied in animal models for active TB.

The occurrence of nontuberculous mycobacteria in different hosts and their implication as obligate or opportunistic pathogens remain mainly unclear. Mycobacteriosis in pigs is usually associated with members of the Mycobacterium avium complex and, in particular, with ‘ Mycobacterium avium subsp. hominissuis ’. Here we describe a novel slow-growing mycobacterial species isolated from lymph nodes obtained from two sows housed in different Swiss farms. The animals presented chronic inappetence and mild diarrhoea. Gross pathology revealed focal caseous lymphadenopathy of the mesenteric lymph nodes. Complete genome sequencing of the two isolates from the two sows was performed. The genomes comprised 5.76 Mb and an average nucleotide identity score of 99.97 %. Whole genome sequence, mycolic acid and matrix-assisted laser desorption ionization-time of flight mass spectrometry analyses revealed that the two isolates were not related to any previously described Mycobacterium species. The closest related species was Mycobacterium parmense , a slow-growing scotochromogenic mycobacterium first isolated from a cervical lymph node of a 3-year-old child. The name proposed for the new species is Mycobacterium helveticum sp. nov. and 16-83T (=DSM 109965T= LMG 2019-02457T) is the type strain.

Tuberculosis (TB) remains a serious threat to whole human health. In particular, the drug resistance of Mycobacterium tuberculosis (Mtb) has become a huge challenge in clinical medicine, and it is extremely urgent to develop effective inhibitors with novel structures and mechanisms. Belonging to the Resistance, Nodulation and Division (RND) superfamily, Mycobacterial membrane proteins Large 3 (MmpL3) is mainly responsible for transporting mycolic acid outside cell membrane to form cell wall, and plays critical roles in iron acquisition which is vital to the survival of Mtb. As a potential Mtb target in recent years, its inhibitor research has attracted wide attention. A series of inhibitors (such as SQ109, AU1235, BM212, etc.) through experimental screening have been reported in succession, especially SQ109 has entered the clinical stage. In this paper, the structural biology information of target MmpL3 was summarized, and the structure-activity relationship (SAR) of inhibitors reported in recent years and their inhibitory mechanism both were reviewed, aiming to provide help for the rational design of MmpL3 inhibitors in the future.

Tuberculosis (TB) continuously pose a major public health concern around the globe, with a mounting death toll of approximately 1.4 million in 2019. The reduced bioavailability, increased toxicity and resistance of several first-line and second-line anti-TB drugs such as isoniazid, ethionamide have necessitated the search for new medications. In this research, we have identified several novel chemical compounds with anti-TB properties using various computational tools like molecular docking analysis, drug-likeness evaluation, ADMET profiling, P450 site of metabolism prediction and molecular dynamics simulation study. This study involves fifty drug-like compounds with antibacterial activity that inhibit InhA and EthR involved in the synthesis of one of the major lipid components, mycolic acid, which is crucial for the viability of Mycobacterium tuberculosis. Among these fifty compounds, 3-[3-(4-Fluorophenyl)-1,2,4-oxadiazol-5-yl]-N-(2-methylphenyl) piperidine-1-carboxamide (C22) and 5-(4-Ethyl-phenyl)-2-(1H-tetrazol-5-ylmethyl)-2H-tetrazole (C29) were found to pass the two-step molecular docking, P450 site of metabolism prediction and pharmacokinetics filtering analysis successfully. Their binding stability for target proteins have been evaluated through RMSD, RMSF, Radius of gyration analysis from 10 ns Molecular Dynamics Simulation (MDS) run. Our identified drugs could be a capable therapeutic for Tuberculosis drug discovery, having said that more in vitro and in vivo testing is required to justify their potential as novel drug and mode of action.

Tuberculosis (Tb), caused by Mycobacterium tuberculosis (Mtb), is responsible for more than a million deaths annually. In the latent phase of infection, Mtb uses lipids as the source of carbon and energy for its survival. The lipid molecules are transported across the cell wall via multiple transport systems. One such set of widely present and less-studied transporters is the Mammalian-cell-entry (Mce) complexes. Here, we report the properties of the substrate-binding proteins (SBPs; MceA-F) of the Mce1 and Mce4 complexes from Mtb which are responsible for the import of mycolic acid/fatty acids, and cholesterol respectively. MceA-F are composed of four domains namely, transmembrane, MCE, helical and tail domains. Our studies show that MceA-F are predominantly monomeric when purified individually and do not form homohexamers unlike the reported homologs (MlaD, PqiB and LetB) from other prokaryotes. The crystal structure of MCE domain of Mtb Mce4A (MtMce4A39-140) determined at 2.9 Å shows the formation of an unexpected domain-swapped dimer in the crystals. Further, the purification and small-angle X-ray scattering (SAXS) analysis on MtMce1A, MtMce4A and their domains suggest that the helical domain requires hydrophobic interactions with the detergent molecules for its stability. Combining all the experimental data, we propose a heterohexameric arrangement of MtMceA-F SBPs, where the soluble MCE domain of the SBPs would remain in the periplasm with the helical domain extending to the lipid layer forming a hollow channel for the transport of lipids across the membranes. The tail domain would reach the cell surface assisting in lipid recognition and binding.

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.

The Mycobacterium tuberculosis H37Rv genome has been sequenced and annotated over 20 years ago, yet roughly half of the protein-coding genes still lack a predicted function. We characterized two genes of unknown function, rv3679 and rv3680, for which inconsistent findings regarding their importance for virulence in mice have been reported. We confirmed that a rv3679-80 deletion mutant (Δrv3679-80) was virulent in mice and discovered that Δrv3679-80 suffered from a glycerol-dependent recovery defect on agar plates following mouse infection. Glycerol also exacerbated killing of Δrv3679-80 by nitric oxide. Rv3679-Rv3680 have previously been shown to form a complex with ATPase activity and we demonstrate that the ability of M. tuberculosis to cope with elevated levels of glycerol and nitric oxide requires intact ATP-binding motifs in both Rv3679 and Rv3680. Inactivation of glycerol kinase or Rv2370c, a protein of unknown function, suppressed glycerol mediated toxicity in Δrv3679-80 Glycerol catabolism led to increased intracellular methylglyoxal pools and Δrv3679-80 was hypersusceptible to extracellular methylglyoxal suggesting that glycerol toxicity in Δrv3679-80 is caused by methylglyoxal. Rv3679 and Rv3680 interacted with Rv1509, and Rv3679 had numerous additional interactors including proteins of the type II fatty acid synthase (FASII) pathway and mycolic acid modifying enzymes linking Rv3679 to fatty acid or lipid synthesis. This work provides experimentally determined roles for Rv3679 and Rv3680 and stimulates future research on these and other proteins of unknown function.Importance A better understanding of the pathogenesis of tuberculosis requires a better understanding of gene function in M. tuberculosis This work provides the first functional insight into the Rv3679/Rv3680 ATPase complex. We demonstrate that M. tuberculosis requires this complex and specifically its ATPase activity to resist glycerol and nitric oxide toxicity. We provide evidence that the glycerol-derived metabolite methylglyoxal causes toxicity in the absence of Rv3679/Rv3680. We further show that glycerol-dependent toxicity is reversed when glycerol kinase (GlpK) is inactivated. Our work uncovered other genes of unknown function that interact with Rv3679 and/or Rv3680 genetically or physically, underscoring the importance of understanding uncharacterized genes.

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.

Background: Extrapulmonary tuberculosis (EPTB), accounting for 10%–20% of all cases of tuberculosis (TB), is known to be determined by host immunity. However, the contribution of bacterial factors to the development of EPTB has not been studied extensively. Mycolic acids are predominant lipids constituting the cell wall of Mycobacterium tuberculosis, and keto-mycolic acid is involved in the synthesis of foamy macrophages that facilitate persistence of mycobacteria. Hence, the present study was performed to gain an insight into variable expression of mycolic acids in clinical isolates of M. tuberculosis under stress. Methods: Pansusceptible clinical isolates of M. tuberculosis from patients with lymph node TB (LNTB) (n = 10) and pulmonary TB (PTB) (n = 10) were subjected to sodium dodecyl sulfate (SDS) stress, and the expression of mycolic acid and its biosynthetic genes was compared. Any bias arising due to the genotype of the clinical isolates was ruled out by performing single-nucleotide polymorphism cluster grouping (SCG), wherein no significant difference was observed between the SCG of LNTB or PTB isolates. Results: The expression of α-mycolic acid during the exposure to SDS was high in 7/10 (70%) LNTB and 6/10 (60%) PTB isolates. Methoxy mycolic acid showed an increased expression in 7/10 (70%) LNTB isolates and 4/10 (40%) PTB isolates. Increased expression of keto-mycolic acid on exposure with SDS was observed in 8/10 (80%) M. tuberculosis LNTB and 3/10 (30%) PTB isolates. Similarly, the mycolic acid synthesis gene, fas, was upregulated more in LNTB isolates than PTB isolates in vitro and ex vivo. SCG 3a was the most common SCG observed in 40% (8/20) of the isolates, followed by SCG 3b in 30% (6/20) of the isolates. There was no significant difference between the SCG of LNTB or PTB isolates. Conclusion: The higher expression of keto-mycolic acid in LNTB as against PTB isolates may indicate better survival in LNTB isolates in the presence of stress.

Background The 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. Results We 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. Conclusions Over 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.

Uncommon lipids in biotechnologically important Corynebacterium glutamicum and pathogen Corynebacterium striatum in genus Corynebacterium are isolated and identified by linear ion-trap multiple stage mass spectrometry (LIT MSn) with high resolution mass measurement. We redefined several lipid structures that were previously mis-assigned or not defined, including cytidine diphosphate diacylglycerol (CDP-DAG), glucuronosyl diacylglycerol (GlcA-DAG), (α- d -mannopyranosyl)-(1 → 4)-(α-D-glucuronyl diacyglycerol (Man-GlcA-DAG), 1-mycolyl-2-acyl-phosphatidylglycerol (MA-PG), acyl trehalose monomycolate (acyl-TMM). We also report the structures of mycolic acid, phosphatidylglycerol, phosphatidylinositol, cardiolipin, trehalose dimycolate lipids in which many isomeric structures are present. The LIT MSn approaches afford identification of the functional group, the fatty acid substituents and their regiospecificity in the molecules, revealing the biodiversities of the lipid species in two Corynebacterium strains that have played very different and important roles in human nutrition and health.