A simple assay to quantify mycobacterial lipid antigen-specific T cell receptors in human tissues and blood

T cell receptors (TCRs) encode the history of antigenic challenge within an individual and have the potential to serve as molecular markers of infection. In addition to peptide antigens bound to highly polymorphic MHC molecules, T cells have also evolved to recognize bacterial lipids when bound to non-polymorphic CD1 molecules. One such subset, germline-encoded, mycolyl lipid-reactive (GEM) T cells, recognizes mycobacterial cell wall lipids and expresses a conserved TCR-ɑ chain that is shared among genetically unrelated individuals. We developed a quantitative PCR assay to determine expression of the GEM TCR-ɑ nucleotide sequence in human tissues and blood. This assay was validated on plasmids and T cell lines. We tested blood samples from South African subjects with or without tuberculin reactivity or with active tuberculosis disease. We were able to detect GEM TCR-ɑ above the limit of detection in 92% of donors but found no difference in GEM TCR-ɑ expression among the three groups after normalizing for total TCR-ɑ expression. In a cohort of leprosy patients from Nepal, we successfully detected GEM TCR-ɑ in 100% of skin biopsies with histologically confirmed tuberculoid and lepromatous leprosy. However, GEM TCR-ɑ expression was not different between leprosy patients and control subjects after normalization. Thus, GEM T cells constitute part of the T cell repertoire in the skin. Further, these results reveal the feasibility of developing a simple, field deployable molecular diagnostic based on mycobacterial lipid antigen-specific TCR sequences that are readily detectable in human tissues and blood independent of genetic background.

Design and Synthesis of Highly Active Antimycobacterial Mutual Esters of 2-(2-Isonicotinoylhydrazineylidene)propanoic Acid

The combination of two active scaffolds into one molecule represents a proven approach in drug design to overcome microbial drug resistance. We designed and synthesized more lipophilic esters of 2-(2-isonicotinoylhydrazineylidene)propanoic acid, obtained from antitubercular drug isoniazid, with various alcohols, phenols and thiols, including several drugs, using carbodiimide-mediated coupling. Nineteen new esters were evaluated as potential antimycobacterial agents against drug-sensitive Mycobacterium tuberculosis (Mtb.) H37Rv, Mycobacterium avium and Mycobacterium kansasii. Selected derivatives were also tested for inhibition of multidrug-resistant (MDR) Mtb., and their mechanism of action was investigated. The esters exhibited high activity against Mtb. (minimum inhibitory concentrations, MIC, from ≤0.125 μM), M. kansasii, M. avium as well as MDR strains (MIC from 0.25, 32 and 8 µM, respectively). The most active mutual derivatives were derived from 4-chloro/phenoxy-phenols, triclosan, quinolin-8-ol, naphthols and terpene alcohols. The experiments identified enoyl-acyl carrier protein reductase (InhA), and thus mycobacterial cell wall biosynthesis, as the main target of the molecules that are activated by KatG, but for some compounds can also be expected adjunctive mechanism(s). Generally, the mutual esters have also avoided cytotoxicity and are promising hits for the discovery of antimycobacterial drugs with improved properties compared to parent isoniazid.

In silico Approach to Identify Potent Bioactive Compounds as Inhibitors against the Enoyl-acyl Carrier Protein (acp) Reductase Enzyme of Mycobacterium tuberculosis

The enoyl-acyl carrier protein (ACP) reductase (InhA) of Mycobacterium tuberculosis elongates acyl fatty acids, which are progenitors of mycolic acids and that are mycobacterial cell wall parts. The aim is to discover potent therapeutic novel bioactive compounds as enoyl-acyl carrier protein (ACP) reductase (InhA, PDB ID: 4U0J) antagonists using an in silico drug design scheme. Structure-based computerized prediction of drug-receptor interactions. PyRx virtual screening tool was used to conduct molecular docking investigations on enoyl-ACP reductase. A target-based ligand selection strategy to choose ligand compounds was employed. The ligand structure was chosen using LEA3D-CNRS. Medication data set that was approved by the FDA: 2028 molecule (s) were used in the study. Around 27 bioactive molecules can bind to the 4U0J, with docking scores ranging from -6.2 to -11.2 Kcal/mol. Compound CHEMBL441373 was shown to have the highest acceptable docking energy (-11.1Kcal/mol), making it a good candidate for a cell wall protein inhibitor (4U0J) that should be investigated further in vivo and in vitro. The anti-mycobacterial ability of triazole scaffolding in a new therapeutic was determined. Compound CHEMBL441373 is located to possess high docking energy (-11.1Kcal/mol) and is shown as a suitable molecule of cell wall protein inhibitor (4U0J).

Lipid mediated inhibition of Niemann-Pick C1 protein is an evolutionary conserved feature of multiple Mycobacterium lineages and non-tubercular mycobacteria.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB) and is a major cause of human morbidity and mortality. Crucially, Mtb can persist and replicate within host macrophages (MF) and subvert multiple antimicrobial defense mechanisms. How this is achieved is incompletely understood. Previously, we reported that lipids shed by persistent mycobacteria inhibit NPC1, the lysosomal protein deficient in most cases of the rare, inherited lysosomal storage disorder Niemann-Pick disease type C (NPC). Inhibition of NPC1 leads to a drop in lysosomal calcium levels blocking phagosome-lysosome fusion and thereby leads to mycobacterial persistence. Studies of mycobacterial lineages have identified events during mycobacterial evolution that result in the acquisition of persistence. We speculated that the production of specific cell wall lipid(s) capable of inhibiting NPC1 activities could have been a critical step in the evolution of pathogenicity. In this study, we have therefore investigated whether lipid extracts from clinical Mtb strains representative of multiple Mtb lineages, members of the Mtb complex (MTBC) and selected non-tubercular mycobacteria (NTM) inhibit the NPC pathway. We have found that the ability to inhibit the NPC pathway was present in all clinical isolates studied from Mtb lineages 1, 2 and 4. We also found that lipids from MTBC member, Mycobacterium bovis and the NTM, Mycobacterium abscessus and Mycobacterium avium also inhibited the NPC pathway. However, when lipids were assayed from Mycobacterium canettii (M. canettii), a smooth tubercle mycobacterium, which is considered to resemble the common ancestor of the MTBC no inhibition of the NPC1 pathway was detected. We therefore conclude that the evolution of mycobacterial cell wall lipids that inhibit the NPC pathway evolved early and post divergence from M. canettii related mycobacteria and NPC1 inhibition significantly contributes to the ability of these pathogens to persist and cause disease.

AN EXHAUSTIVE REVIEW ON EMERGING DRUG TARGETS OF TUBERCULOSIS WITH SPECIAL EMPHASIS ON CELL WALL SYNTHESIS

Tuberculosis (TB) is one of the top 10 causes of mortality and morbidity. Worldwide, yet, it has been over 60 years since a novel drug was introduced in market to treat the disease exclusively. Increased number of drug resistant TB cases has prompted the search for novel potent anti-TB drug. Mycobacterial cell wall has unique structure which provides integrity to the cell. The future development of new potent anti-TB drug targets is associated with the synthesis of various cell wall constituents; the structural and genetic information about mycobacterial cell wall envelope is now available. In the present review, we have focused on prospective drug targets that can be optimum triumph for successful drug candidate.

IL-4 and helminth infection downregulate MINCLE-dependent macrophage response to mycobacteria and Th17 adjuvanticity

The myeloid C-type lectin receptor (CLR) MINCLE senses the mycobacterial cell wall component trehalose-6,6-dimycolate (TDM). Recently, we found that IL-4 down-regulates MINCLE expression in macrophages. IL-4 is a hallmark cytokine in helminth infections, which appear to increase the risk for mycobacterial infection and active tuberculosis. Here, we investigated functional consequences of IL-4 and helminth infection on MINCLE-driven macrophage activation and Th1/Th17 adjuvanticity. IL-4 inhibited MINCLE and cytokine induction after macrophage infection with Mycobacterium bovis Bacille Calmette-Guerin (BCG). Infection of mice with BCG upregulated MINCLE on myeloid cells, which was inhibited by IL-4 plasmid injection and by infection with the nematode Nippostrongylus brasiliensis in monocytes. To determine the impact of helminth infection on MINCLE dependent immune responses, we vaccinated mice with a recombinant protein together with the MINCLE ligand Trehalose-6,6-dibehenate (TDB) as adjuvant. Concurrent infection with N. brasiliensis or with Schistosoma mansoni promoted T cell-derived IL-4 production and suppressed Th1/Th17 differentiation in the spleen. In contrast, helminth infection did not reduce Th1/Th17 induction by TDB in draining peripheral lymph nodes, where IL-4 levels were unaltered. Upon use of the TLR4-dependent adjuvant G3D6A, N. brasiliensis infection impaired selectively the induction of splenic antigen-specific Th1 but not of Th17 cells. Thus, helminth infection attenuated the Th17 response to MINCLE-dependent immunization in an organ-specific manner. Taken together, our results demonstrate down-regulation of MINCLE expression on monocytes and macrophages by IL-4 as a possible mechanism of thwarted Th17 vaccination responses by underlying helminth infection.

Structures of the mycobacterial membrane protein MmpL3 reveal its mechanism of lipid transport

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 Cell Wall Stimulant in the Treatment of Squamous Cell Carcinoma: A Case Series Regarding Treatment in Equine, Bovine and Caprine Patients

Squamous cell carcinoma (SCC) is a common dermatological neoplasia found in large animal species. Treatment options, such as surgery and cryotherapy may be difficult or not feasible. Alternative therapies, such as immunomodulating drugs, can potentially be used for companion large animals. The hypothesis of the following retrospective study is: following multiple intravenous and intralesional injections of a mycobacterial cell wall stimulant (MCW) regression of SCC in equine, bovine and caprine patients will be observed. In this observational-retrospective case series, patients included are 2 bovine, 2 caprine and 3 equine patients. The medical records at two different teaching veterinary hospitals were searched for cases with a positive histopathological diagnosis of squamous cell carcinoma that were subsequently treated with MCW, as either the sole therapy, or in conjunction with other therapies. Seven cases were included in this retrospective study. The median duration of therapy was 56.5 days, with 3 of the 7 patients being euthanized. Significant complications were seen in 3/7 patients. Repeated injections of a MCW may lead to reduction in lesion size of SCC in some cases, but long-term resolution is unlikely and the risk of significant complications is high; due to limited sample size and the variety in species, it is difficult to conclude if MCW is an effective therapy for SCC.

Exploring targets of cell wall protein synthesis and overexpression mediated drug resistance for the discovery of potential M. tb inhibitors

: Tuberculosis is an infectious disease engulfing millions of lives worldwide; it is caused by mammalian tubercle bacilli, Mycobacterium tuberculosis complex which may consist of strains viz. M. tuberculosis hominis (human strain), M. microti, M. pinnipedii and M. canettii. The other pathogenic strain is M.africanum which belongs to the M. tuberculosis complex and it is fully virulent for humans. The non-pathogenic strains in the complex may include, M. fortuitum and M.smegmatis. Extensive research has been carried out to combat this dangerous disease. World Health Organization proposed Directly Observed Treatment Short-course regimen (DOTS) for the eradication of the TB. In addition, the compounds such as TBA-7371, TBI-166, AZD5847 and PBTZ-169 are under clinical trials whereas the recently FDA approved anti tubercular drugs are Pretomanid (PA-824), Bedaquiline (TMC207), Linezolid (PNU-100480) and Delamanid (OPC-67683). The early detection of mycobacterium tuberculosis can be permanently cured by DOTS comprising of Pyrazinamide (Z), Isoniazid (H), Rifampin (R) and Ethambutol (E). Duration of the treatment depends on viability of the disease. DOTS can target to disrupt the biosynthesis of mycobacterial cell wall proteins expressed by various genes. Overexpression of these genes may produce drug-resistant due to dose misuse or the intake of quality compromised anti tubercular drug regimen. Therefore, in the present review there has been a necessity to report the second line antitubercular chemotherapeutics to target various proteins which are the building block of M. tb cell wall, overexpression of which may produce drug resistance.