Mycolic Acids Research Articles

Detection of mycobacterium tuberculosis by microscopic examination and gene expert in a suspected patient with pulmonary tuberculosis disease, in Dili City, East Timor

Tuberculosis is one of the most dangerous and deadly diseases, so control is needed to reduce the transmission rate of TB disease and mortality among the community, so the gene method is very appropriate to be able to help in diagnosing TB. Acid-resistant bacilli are bacteria that have a very thick lipid content so that the staining cannot be affected by other dye reactions, except by using phenol and through the heating process. Mycobacterium tuberculosis has a cell wall consisting of a waxy coating and mycolic acid; therefore, these bacteria can only color with BTA. Mycbakterium Tuberculosis can maintain the first color, namely Carbun Fuchin, and fight decolorization with alcoholic acid, so this bacterium is called an acid-resistant bacillus. The right examination method to diagnose TB disease is necessary to be able to follow the appropriate treatment so as to reduce transmission and death rates due to TB disease. The aim of this research is to analyze the difference in examination results using microscopes and Gene Xpert machines in Dili district in suspected patients with pulmonary tuberculosis.

Alleviation of monocyte exhaustion by BCG derivative mycolic acid

Monocyte exhaustion with sustained pathogenic inflammation and immune-suppression, a hallmark of sepsis resulting from systemic infections, presents a challenge with limited therapeutic solutions. This study identified Methoxy-Mycolic Acid (M-MA), a branched mycolic acid derived from Mycobacterium bovis Bacillus Calmette-Guérin (BCG), as a potent agent in alleviating monocyte exhaustion and restoring immune homeostasis. Co-treatment of monocytes with M-MA effectively blocked the expansion of Ly6Chi/CD38hi/PD-L1hi monocytes induced by LPS challenges and restored the expression of immune-enhancing CD86. M-MA treatment restored mitochondrial functions of exhausted monocytes and alleviated their suppressive activities on co-cultured Tcells. Independent of TREM2, M-MA blocks Src-STAT1-mediated inflammatory polarization and reduces the production of immune suppressors TAX1BP1 and PLAC8. Whole genome methylation analyses revealed M-MA's ability to erase the methylation memory of exhausted monocytes, particularly restoring Plac8 methylation. Together, our data suggest M-MA as an effective agent in restoring monocyte homeostasis with a therapeutic potential for treating sepsis.

CdbC: a disulfide bond isomerase involved in the refolding of mycoloyltransferases in Corynebacterium glutamicum cells exposed to oxidative conditions.

In Corynebacterium glutamicum cells, cdbC, which encodes a protein containing the CysXXCys motif, is regulated by the global redox-responsive regulator OsnR. In this study, we assessed the role of the periplasmic protein CdbC in disulfide bond formation and its involvement in mycomembrane biosynthesis. Purified CdbC efficiently refolded scrambled RNaseA, exhibiting prominent disulfide bond isomerase activity. The transcription of cdbC was decreased in cells grown in the presence of the reductant dithiothreitol (DTT). Moreover, unlike wild-type and cdbC-deleted cells, cdbC-overexpressing (P180-cdbC) cells grown in the presence of DTT exhibited retarded growth, abnormal cell morphology, increased cell surface hydrophobicity and altered mycolic acid composition. P180-cdbC cells cultured in a reducing environment accumulated trehalose monocorynomycolate, indicating mycomembrane deformation. Similarly, a two-hybrid analysis demonstrated the interaction of CdbC with the mycoloyltransferases MytA and MytB. Collectively, our findings suggest that CdbC, a periplasmic disulfide bond isomerase, refolds misfolded MytA and MytB and thereby assists in mycomembrane biosynthesis in cells exposed to oxidative conditions.

Rhodococcus indonesiensis sp. nov. a new member of the Rhodococcus ruber lineage isolated from sediment of a neutral hot spring and reclassification of Rhodococcus electrodiphilus (Ramaprasad et al. 2018) as a later heterotypic synonym of Rhodococcus ruber (Kruse 1896) Goodfellow and Alderson 1977 (Approved Lists 1980).

A polyphasic study was designed to determine the taxonomic status of isolate CSLK01-03T, which was recovered from an Indonesian neutral hot spring and provisionally assigned to the genus Rhodococcus. The isolate was found to have chemotaxonomic, cultural and morphological properties typical of rhodococci. It has a rod-coccus lifecycle and grows from 10 to 39 °C, from pH 6.5 to 8.0 and in the presence of 0-10 % (w/v) sodium chloride. Whole-organism hydrolysates contain meso-diaminopimelic acid, arabinose and galactose, the predominant menaquinone is MK-8 (H2), the polar lipid pattern consists of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol mannosides, phosphatidylmethylethanolamine and two unidentified components, it produces mycolic acids, and C16:0 is the major fatty acid. Whole-genome analyses show that the isolate and Rhodococcus electrodiphilus LMG 29881T (GenBank accession: JAULCK000000000) have genome sizes of 5.5 and 5.1 Mbp, respectively. These strains and Rhodococcus aetherivorans DSM 44752T and Rhodococcus ruber DSM 43338T form well-supported lineages in 16S rRNA and whole-genome trees that are close to sister lineages composed of the type strains of Rhodococcus rhodochrous and related Rhodococcus species. The isolate can be distinguished from its closest evolutionary neighbours using combinations of cultural and phenotypic features, and by low DNA-DNA hybridization values. Based on these data it is proposed that isolate CSLK01-03T (=CCMM B1310T=ICEBB-06T=NCIMB 15214T) be classified in the genus Rhodococcus as Rhodococcus indonesiensis sp. nov. The genomes of the isolate and its closest phylogenomic relatives are rich in biosynthetic gene clusters with the potential to synthesize new natural products, notably antibiotics. In addition, whole-genome-based taxonomy revealed that Rhodococcus electrodiphilus LMG 29881T and Rhodococcus ruber DSM 43338T belong to a single species. It is, therefore, proposed that R. electrodiphilus be recognized as a heterotypic synonym of R. ruber.

Actinoplanes oblitus sp. nov., producing the glycopeptide antibiotic A477.

In 1973, Eli Lilly and Company described the filamentous actinomycete producing the glycopeptide antibiotic A477 as an Actinoplanes species on the basis of its morphological and physiological features and deposited it as NRRL 3884T. In this paper, we report that the phylogenetic analysis based on the 16S rRNA gene sequence and the whole genome phylogenomic study indicate that NRRL 3884T forms a distinct monophyletic line within the genus Actinoplanes, being most closely related to Actinoplanes octamycinicus NBRC 14524T [99.6 % 16S rRNA gene similarity, 89.4 % average nucleotide identity (ANI), 46.0 % digital DNA-DNA hybridization (dDDH)] and Actinoplanes ianthinogenes NBRC 13996T (98.8 % 16S rRNA gene similarity, 89.0 % ANI, 47.0 % dDDH). NRRL 3884T forms an extensively branched, non-fragmented vegetative mycelium; either sterile aerial hyphae or regular subglobose sporangia are formed depending on cultivation conditions. The cell wall contains meso-2,6-diaminopimelic acid and 2,6-diamino-3-hydroxypimelic acid and the diagnostic sugars are glucose, mannose and ribose with a minor amount of rhamnose. The predominant menaquinone (MK) is MK-9(H4), with minor amounts of MK-9(H2), MK-9(H6) and MK-9(H8). Mycolic acids are absent. The diagnostic phospholipids are diphosphatidylglycerol and phosphatidylethanolamine. The major cellular fatty acids are anteiso-C17 : 0, iso-C16 : 0 and iso-C15 : 0, with moderate amounts of anteiso-C15 : 0 and iso-C17 : 0. The genomic G+C content is 71.5 mol%. Significant differences in the genomic, morphological, chemotaxonomic and biochemical data between NRRL 3884T and the two most closely related Actinoplanes type strains clearly demonstrate that NRRL 3884T represents a novel species of the genus Actinoplanes, for which the name Actinoplanes oblitus sp. nov. is proposed. The type strain is NRRL 3884T (=DSM 116196T).

Permeability of TB drugs through the mycolic acid monolayer: a tale of two force fields.

Tuberculosis (TB) treatment becomes challenging due to the unique cell wall structure of Mycobacterium tuberculosis (M. tb). Among various components of the M.tb cell wall, mycolic acid (MA) is of particular interest because it is speculated to exhibit extremely low permeability for most of the drug molecules, thus helping M.tb to survive against medical treatment. However, no quantitative assessment of the thermodynamic barrier encountered by various well-known TB drugs in the mycolic acid monolayer has been performed so far using computational tools. On this premise, our present work aims to probe the permeability of some first and second line TB drugs, namely ethambutol, ethionamide, and isoniazid, through the modelled mycolic acid monolayer, using molecular dynamics (MD) simulation with two sets of force field (FF) parameters, namely GROMOS 54A7-ATB (GROMOS) and CHARMM36 (CHARMM) FFs. Our findings indicate that both FFs provide consistent results in terms of the mode of drug-monolayer interactions but significantly differ in the drug permeability through the monolayer. The mycolic acid monolayer generally exhibited a higher free energy barrier of crossing with CHARMM FF, while with GROMOS FF, better stability of drug molecules on the monolayer surface was observed, which can be attributed to the greater electrostatic potential at the monolayer-water interface, found for the later. Although both the FF parameters predicted the highest resistance against ethambutol (permeability values of 8.40 × 10-34 cm s-1 and 9.61 × 10-31 cm s-1 for the CHARMM FF and the GROMOS FF, respectively), results obtained using GROMOS were found to be consistent with the water solubility of drugs, suggesting it to be a slightly better FF for modelling drug-mycolic acid interactions. Therefore, this study enhances our understanding of TB drug permeability and highlights the potential of the GROMOS FF in simulating drug-mycolic acid interactions.

Effect of Mycolic Acids on Host Immunity and Lipid Metabolism.

Mycolic acids constitute pivotal constituents within the cell wall structure of Mycobacterium tuberculosis. Due to their structural diversity, the composition of mycolic acids exhibits substantial variations among different strains, endowing them with the distinctive label of being the 'signature' feature of mycobacterial species. Within Mycobacterium tuberculosis, the primary classes of mycolic acids include α-, keto-, and methoxy-mycolic acids. While these mycolic acids are predominantly esterified to the cell wall components (such as arabinogalactan, alginate, or glucose) of Mycobacterium tuberculosis, a fraction of free mycolic acids are secreted during in vitro growth of the bacterium. Remarkably, different types of mycolic acids possess varying capabilities to induce foamy macro-phages and trigger immune responses. Additionally, mycolic acids play a regulatory role in the lipid metabolism of host cells, thereby exerting influence over the progression of tuberculosis. Consequently, the multifaceted properties of mycolic acids shape the immune evasion strategy employed by Mycobacterium tuberculosis. A comprehensive understanding of mycolic acids is of paramount significance in the pursuit of developing tuberculosis therapeutics and unraveling the intricacies of its pathogenic mechanisms.

Virtual screening and identification of promising therapeutic compounds against drug-resistant Mycobacterium tuberculosis β-ketoacyl-acyl carrier protein synthase I (KasA)

The emergence of new Mycobacterium tuberculosis (Mtb) strains resistant to the key drugs currently used in the clinic for tuberculosis treatment can substantially reduce the probability of therapy success, causing the relevance and importance of studies on the development of novel potent antibacterial agents targeting different vulnerable spots of Mtb. In this study, 28,860 compounds from the library of bioactive molecules were screened to identify novel potential inhibitors of β-ketoacyl-acyl carrier protein synthase I (KasA), one of the key enzymes involved in the biosynthesis of mycolic acids of the Mtb cell wall. In doing so, we used a structure-based virtual screening approach to drug repurposing that included high-throughput docking of the C171Q KasA enzyme with compounds from the library of bioactive molecules including the FDA-approved drugs and investigational drug candidates, assessment of the binding affinity for the docked ligand/C171Q KasA complexes, and molecular dynamics simulations followed by binding free energy calculations. As a result, post-modeling analysis revealed 6 top-ranking compounds exhibiting a strong attachment to the malonyl binding site of the enzyme, as evidenced by the values of binding free energy which are significantly lower than those predicted for the KasA inhibitor TLM5 used in the calculations as a positive control. In light of the data obtained, the identified compounds are suggested to form a good basis for the development of new antitubercular molecules of clinical significance with activity against the KasA enzyme of Mtb. Communicated by Ramaswamy H. Sarma

Disruption of a kasB homolog gene (kasB) causes attenuation of cell invasion and virulence of Nocardia seriolae.

Nocardia seriolae is the primary aetiological agent of nocardiosis in fish, which causes mass mortality in freshwater and marine fish. β-ketoacyl-ACP synthase (KAS) is one of the essential enzymes in the synthesis of mycolic acids (MASs) in Mycobacterium spp. and has been chosen as the target for therapeutic intervention in mycobacterial diseases. In the present study, a kasB homologue gene (kasB) was identified in the genome of N. seriolae, and the gene-deficient mutant (ΔkasB) was generated based on a clinical isolate, XSYC-Ns. Compared to the wild-type (WT) strain, the ΔkasB showed a measurably growth defect invitro but retained the acid-fastness in acid-fast staining. Observation of the cell ultrastructure showed some alterations in the cell wall of the ΔkasB strain. Compared to its original strain, the cell wall lipid layer seemed sparser, and a wider electron-transparent zone was observed in the cell wall of ΔkasB strain. Moreover, the ΔkasB strain showed impaired ability of cell invasion as well as intracellular survival in the cell line originating from the head-kidney of the large yellow croaker (LYC-hK), compared to its original strain. In addition, the deficiency of ΔkasB significantly attenuated the virulence of N. seriolae in largemouth bass. The present study suggested that the ΔkasB gene might be involved in the synthesis of extracellular cell-wall lipids in N. seriolae and play a crucial role in its pathogenicity.

Cytotoxicity and gene expression studies in understanding the mechanism on antimycobacterial properties of methanolic leaf extract of Acalypha indica Linn.

Despite the availability of effective antituberculosis medication, tuberculosis (TB) remains a serious global health concern, raising the demand for low-after effect drug alternative. This study aims to explore the anti-mycobacterial potency of Acalypha indica Linn. on Mycobacterium tuberculosis using Mycobacterium smegmatis as a model and thereby uncover its mechanism. Characterization of bioactive phytocompounds and their cumulative effects were studied through both biochemical and molecular approaches. The antimycobacterial efficacy was screened by cell viability assay and IC50. FE-SEM and qRT-PCR were used to study the effect of A. indica leaf methanolic extract (AILME) on cell integrity and mycolic acid synthesis pathway. This study reports AILME to be the most effective solvent-extract combination which showed >40 % cell inhibition from 25 µg mL−1 onwards at 24 hr. AILME showed >80 % antioxidant potential, lowest IC50 of 48.24 µg mL−1 and a MIC value of 200 µg mL−1 at 24 hr. The FE-SEM analysis of the AILME treated bacterial cells showed significant morphological damage which supports the results of antimycobacterial activity. The gene expression study revealed that AILME inhibited the expression of inhA and mtrA genes involved in mycolic acid biosynthesis pathway and cell wall integrity, respectively. The bioactive phytocompounds characterised through GCMS and HR-LCMS revealed about nineteen potent anti-TB compounds like malic acid, catechin, pyrrolidine, hexadecanoic acid, quercetin etc. that might supervise the role behind the antimycobacterial effect of the herb. This study reveals that AILME possesses strong antimycobacterial action by impeding the formation of mycolic acid biosynthesis pathway and disorganization of cell wall. Presence of anti-TB phytocompounds in abundance suggest its potential chemotherapeutic nature against M. tuberculosis.

Computational insights into potential marine natural products as selective inhibitors of Mycobacterium tuberculosis InhA: A structure-based virtual screening study

Several factors are associated with the emergence of drug resistance mechanisms, such as impermeable cell walls, gene mutations, and drug efflux systems. Consequently, bacteria acquire resistance, leading to a decrease in drug efficacy. A new and innovative strategy is required to combat drug resistance in tuberculosis (TB) effectively. Therefore, targeting the mycolic acid biosynthesis pathway, which is involved in synthesising mycolic acids (MAs), essential structural components responsible for mycobacterial pathogenicity, has garnered interest in TB research and the concept of drug resistance. In this context, InhA, which plays a crucial role in the fatty acid synthase-II (FAS-II) system of the MA biosynthetic pathway, was selected as a druggable target for screening investigation. To identify potential lead molecules against InhA, diverse marine natural products (MNPs) were collected from the comprehensive marine natural products database (CMNPD). Virtual screening studies aided in selecting potential lead molecules that best fit within the substrate-binding pocket (SBP) of InhA, forming crucial hydrogen bond interaction with the catalytic residue Tyr158. Three MNPs, CMNPD30814, CMNPD1702, and CMNPD27355, were chosen as prospective alternative molecules due to their favorable pharmacokinetic properties and lack of toxicity according to ProTox-II predictions. Additionally, improved reactivity of the MNPs was observed in the results of density functional theory (DFT) studies. Furthermore, comparative molecular dynamics simulation (MDS), principal component (PC)-based free energy landscape (FEL) analysis, and molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) were employed to show enhanced structural stability, increased H-bond potential, and high binding affinity toward the target InhA. Moreover, the hot spot residues that contributed to the high binding energy profile and anchored the stability of the complexes were revealed with their individual interaction energy. The computational insights from this study provide potential avenues to combat TB through the multifaceted mode of action of these marine lead molecules, which can be further explored in future experimental investigations.

Biophysical Interaction Landscape of Mycobacterial Mycolic Acids and Phenolic Glycolipids with Host Macrophage Membranes.

Lipidic adjuvant formulations consisting of immunomodulatory mycobacterial cell wall lipids interact with host cells following administration. The impact of this cross-talk on the host membrane's structure and function is rarely given enough consideration but is imperative to rule out nonspecific perturbation underlying the adjuvant. In this work, we investigated changes in the plasma membranes of live mammalian cells after exposure to mycobacterial mycolic acid (MA) and phenolic glycolipids, two strong candidates for lipidic adjuvant therapy. We found that phenolic glycolipid 1 softened the plasma membrane, lowering membrane tension and stiffness, but MA did not significantly change the membrane characteristics. Further, phenolic glycolipid 1 had a fluidizing impact on the host plasma membrane, increasing the fluidity and the abundance of fluid-ordered-disordered coexisting lipid domains. Notably, lipid diffusion was not impacted. Overall, MA and, to a lesser extent, phenolic glycolipid 1, due to minor disruption of host cell membranes, may serve as appropriate lipids in adjuvant formulations.

Reassessing the putative molecular Target(s) of potent antitubercular 2-(Alkylsulfonyl)acetamides

Simple alkyl-sulfonylacetamides have potent antitubercular activity and significantly decrease mycolic acid levels in mycobacteria. Although these compounds were originally designed to inhibit the ketoacyl synthase domain of fatty acid synthase, structure-activity relationships and biochemical evidence do not fully support fatty acid synthase as the target. In 2004, an enzyme family involved in the activation and transfer of fatty acids as acyl-adenylates was identified in mycobacteria, separate from the universal acetyl-CoA carrier mechanism. These fatty acyl-AMP ligases (FAAL), encoded by the FadD family play important roles in the biosynthesis of mycolic acids along with fatty acid metabolism and are hypothesised here to be the molecular target of the sulfonylacetamides. Due to structural similarities with the ligase's natural substrate, it is believed these compounds are exerting action via competitive inhibition of these highly potent molecular targets. The primary aim of this investigation was to synthesize an extended library of sulfonylacetamide derivatives, building upon existing structural activity relations to validate the molecular mechanism with the aid of molecular modelling, while also attempting to explore novel structural isosteres for further drug design and development. Sulfonylacetamide derivatives were modified based on the putative molecular target resulting in derivatives with improved activities towards Mycobacteriumtuberculosis (H37Rv). The most active novel derivatives reported were 19, 22b, 22c and 46 displaying MIC90 levels of 1.4, 16.0, 13.0 and 5.9 μg/mL, respectively.

Gordonia metallireducens sp. nov., a tellurite- and selenite-resistant bacterium isolated from the sediment of an acid mine drainage stream.

A polyphasic taxonomic study was carried out on strain TSed Te1T, isolated from sediment of a stream contaminated with acid drainage from a coal mine. The bacterium forms pink-pigmented colonies and has a rod-coccus growth cycle, which also includes some coryneform arrangements. This bacterium is capable of growing in the presence of up to 750 μg ml-1 tellurite and 5000 μg ml-1 selenite, reducing each to elemental form. Nearly complete 16S rRNA gene sequence analysis associated the strain with Gordonia, with 99.5 and 99.3 % similarity to Gordonia namibiensis and Gordonia rubripertincta, respectively. Computation of the average nucleotide identity and digital DNA-DNA hybridization comparisons with the closest phylogenetic neighbour of TSed Te1T revealed genetic differences at the species level, which were further substantiated by differences in several physiological characteristics. The dominant fatty acids were C16 : 0, C18 : 1, C16 : 1 and tuberculostearic acid. The DNA G+C content was 67.6 mol%. Major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannoside, while MK-9(H2) was the only menaquinone found. Mycolic acids of C56-C60 were present. Whole-cell hydrolysates contained meso-diaminopimelic acid along with arabinose and galactose as the major cell-wall sugars. On the basis of the results obtained in this study, the bacterium was assigned to the genus Gordonia and represents a new species with the name Gordonia metallireducens sp. nov. The type strain is TSed Te1T (=NRRL B-65678T=DSM 114093T).

Nonomuraea corallina sp. nov., isolated from coastal sediment in Samila Beach, Thailand: insights into secondary metabolite synthesis as anticancer potential.

A novel marine actinomycete, designated strain MCN248T, was isolated from the coastal sediment in Songkhla Province, Thailand. Based on the 16S rRNA gene sequences, the new isolate was closely related to Nonomuraea harbinensis DSM45887T (99.2%) and Nonomuraea ferruginea DSM43553T (98.6%). Phylogenetic analyzes based on the 16S rRNA gene sequences showed that strain MCN248T was clustered with Nonomuraea harbinensis DSM45887T and Nonomuraea ferruginea DSM43553T. However, the digital DNA-DNA hybridization analyzes presented a low relatedness of 40.2% between strain MCN248T and the above closely related strains. This strain contained meso-diaminopimelic acid. The acyl type of the peptidoglycan was acetyl, and mycolic acids were absent. The major menaquinones were MK-9(H2) and MK-9(H4). The whole cell sugars consisted of madurose, ribose, mannose, and glucose. Diphosphatidylglycerol, hydroxyl-phosphatidylethanolamine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylglycerol were detected as the major phospholipids. The predominant cellular fatty acids were iso-C16:0 (40.4%), 10-methyl-C17:0 (22.1%), and C17:18c (10.9%). The DNA G + C content of the genomic DNA was 71.7%. With in silico analyzes, the antiSMASH platform uncovered a diverse 29 secondary metabolite biosynthesis arsenal, including non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) of strain MCN248T, with a high prevalence of gene cluster encoding pathways for the production of anticancer and cytotoxic compounds. Consistently, the crude extract could inhibit colorectal HCT-116 cancer cells at a final concentration of 50 μg/mL. Based on the polyphasic approach, strain MCN248 was designated as a novel species of the genus Nonomuraea, for which the name Nonomuraea corallina sp. nov. is proposed. The type strain of the type species is MCN248T (=NBRC115966T = TBRC17110T).

Mycobacterium bovis and its impact on human and animal tuberculosis.

Graphical abstract Mycobacterium bovis is a slow-growing (16–20 h generation time), Gram-positive and acid-fast bacterium member of the Mycobacterium tuberculosis complex pathogen group (MTBC). They are characterized by a complex, protective cell wall containing mycolic acids. The MTBCs are the causative agents of tuberculosis (TB). Following initial infection, subsequent pathological changes, and the progress of infection depend on the interplay between host defence mechanisms and mycobacterial virulence factors and the balance between the immunologic protective responses and the damaging inflammatory processes. Progression of the disease is characterized by the formation of typical caseous tuberculous granuloma (inflammatory mononuclear cell aggregates) because of the host's immune response to infection. The transmission and epidemiology of Mycobacterium bovis are complex and vary depending on the situation and ecosystem. In the UK, the spread of BTB in the UK cattle herd can occur by transmitting the disease from cattle to cattle and between badgers but also between badgers and cattle. The disease is thought to be primarily a respiratory disease with spread between individuals through mechanisms such as coughing or transfer of bacteria in respiratory secretions. It is also thought that environmental contamination may also lead to some transmission. The protective cell wall of the organism is believed to allow the organism to survive outside an animal host, which can then transfer to new hosts following subsequent environmental exposure. In some situations, ingestion of pathogens in food can lead to infection. The relative contribution of these routes and precise transmission mechanisms needs to be better understood.

Effects of benzothiazinone and ethambutol on the integrity of the corynebacterial cell envelope

The mycomembrane (MM) is a mycolic acid layer covering the surface of Mycobacteria and related species. This group includes important pathogens such as Mycobacterium tuberculosis, Corynebacterium diphtheriae, but also the biotechnologically important strain Corynebacterium glutamicum. Biosynthesis of the MM is an attractive target for antibiotic intervention. The first line anti-tuberculosis drug ethambutol (EMB) and the new drug candidate, benzothiazinone 043 (BTZ) interfere with the synthesis of the arabinogalactan (AG), which is a structural scaffold for covalently attached mycolic acids that form the inner leaflet of the MM. We previously showed that C. glutamicum cells treated with a sublethal concentration of EMB lose the integrity of the MM. In this study we examined the effects of BTZ on the cell envelope. Our work shows that BTZ efficiently blocks the apical growth machinery, however effects in combinatorial treatment with β-lactam antibiotics are only additive, not synergistic. Transmission electron microscopy (TEM) analysis revealed a distinct middle layer in the septum of control cells considered to be the inner leaflet of the MM covalently attached to the AG. This layer was not detectable in the septa of BTZ or EMB treated cells. In addition, we observed that EMB treated cells have a thicker and less electron dense peptidoglycan (PG). While EMB and BTZ both effectively block elongation growth, BTZ also strongly reduces septal cell wall synthesis, slowing down growth effectively. This renders BTZ treated cells likely more tolerant to antibiotics that act on growing bacteria.

3, 5-disubstituted 1,2,4- oxadiazole: A potent inhibitor of Mycolic acid synthesis

Oxadiazoles are 5-membered, unsaturated, unstable, heteroaromatic ring containing two carbon atom, one oxygen atom and two pyridine type nitrogen atom linked in continuity. They fall under non beta lactam class of antibiotics. Oxadiazoles are potent inhibitors of mycolic acid synthesis. Mycolic acids are long fatty acids important for cell wall formation in class of bacterias called Mycolata taxon. The article focuses on various scaffolds of 1, 2, and 4- oxadiazole having inhibitory action on mycolic acid synthesis and can be applied for anti-tuberculosis treatment.

Corynebacterium suedekumii sp. nov and Corynebacterium breve sp. nov., isolated from raw cow's milk.

Four Gram-positive, rod-shaped, none-sporeforming, non-motile isolates were obtained from various raw milk samples taken from the cooling tank on a research farm in Königswinter, Germany. Based on phylogenetic analysis of the 16S rRNA genes and whole genome sequences, all isolates were assigned to the genus Corynebacterium, but were divided in two different groups. All isolates contained C18 : 1 cis 9 and C16 : 0 as predominant fatty acids, as well as traces of C18 : 0. They all contained menaquinones MK-8 (H2) and MK-9 (H2) and produced mycolic acids characteristic for the majority of species belonging to the genus Corynebacterium. 16S rRNA gene sequence similarity values to the closest related type strains Corynebacterium humireducens DSM 45392T and Corynebacterium pilosum DSM 20521T were below 98.7 %, average nucleotide identity values were below 86 % and digital DNA-DNA-hybridization values were below 25 %, indicating that the isolates represent two novel species. The names Corynebacterium suedekumii sp. nov. and Corynebacterium breve sp. nov. are proposed, represented by the type strains LM112T (=DSM 116216T=HAMBI 3782T) and R4T (=DSM 116183T=HAMBI 3785T), respectively.

Vaccination with mycobacterial lipid loaded nanoparticle leads to lipid antigen persistence and memory differentiation of antigen-specific T cells

Mycobacterium tuberculosis (Mtb) infection elicits both protein and lipid antigen-specific T cell responses. However, the incorporation of lipid antigens into subunit vaccine strategies and formulations has been underexplored, and the characteristics of vaccine-induced Mtb lipid-specific memory T cells have remained elusive. Mycolic acid (MA), a major lipid component of the Mtb cell wall, is presented by human CD1b molecules to unconventional T cell subsets. These MA-specific CD1b-restricted T cells have been detected in the blood and disease sites of Mtb-infected individuals, suggesting that MA is a promising lipid antigen for incorporation into multicomponent subunit vaccines. In this study, we utilized the enhanced stability of bicontinuous nanospheres (BCN) to efficiently encapsulate MA for in vivo delivery to MA-specific T cells, both alone and in combination with an immunodominant Mtb protein antigen (Ag85B). Pulmonary administration of MA-loaded BCN (MA-BCN) elicited MA-specific T cell responses in humanized CD1 transgenic mice. Simultaneous delivery of MA and Ag85B within BCN activated both MA- and Ag85B-specific T cells. Notably, pulmonary vaccination with MA-Ag85B-BCN resulted in the persistence of MA, but not Ag85B, within alveolar macrophages in the lung. Vaccination of MA-BCN through intravenous or subcutaneous route, or with attenuated Mtb likewise reproduced MA persistence. Moreover, MA-specific T cells in MA-BCN-vaccinated mice differentiated into a T follicular helper-like phenotype. Overall, the BCN platform allows for the dual encapsulation and in vivo activation of lipid and protein antigen-specific T cells and leads to persistent lipid depots that could offer long-lasting immune responses.