Comparative Genome Analysis of 19 Trueperella pyogenes Strains Originating from Different Animal Species Reveal a Genetically Diverse Open Pan-Genome.

Trueperella pyogenes is a Gram-positive opportunistic pathogen that causes severe cases of mastitis, metritis, and pneumonia in a wide range of animals, resulting in significant economic losses. Although little is known about the virulence factors involved in the disease pathogenesis, a comprehensive comparative genome analysis of T. pyogenes genomes has not been performed till date. Hence, present investigation was carried out to characterize and compare 19 T. pyogenes genomes originating in different geographical origins including the draftgenome of the first Indian origin strain T. pyogenes Bu5. Additionally, candidate virulence determinants that could be crucial for their pathogenesis were also detected and analyzed by using various bioinformatics tools. The pan-genome calculations revealed an open pan-genome of T. pyogenes. In addition, an inventory of virulence related genes, 190 genomic islands, 31 prophage sequences, and 40 antibiotic resistance genes that could play a significant role in organism's pathogenicity were detected. The core-genome based phylogeny of T. pyogenes demonstrates a polyphyletic, host-associated group with a high degree of genomic diversity. The identified core-genome can be further used for screening of drug and vaccine targets. The investigation has provided unique insights into pan-genome, virulome, mobiliome, and resistome of T. pyogenes genomes and laid the foundation for future investigations.

Comparative genome analysis of Salmonella enterica serovar Gallinarum biovars Pullorum and Gallinarum decodes strain specific genes.

Salmonella enterica serovar Gallinarum biovar Pullorum (bvP) and biovar Gallinarum (bvG) are the etiological agents of pullorum disease (PD) and fowl typhoid (FT) respectively, which cause huge economic losses to poultry industry especially in developing countries including India. Vaccination and biosecurity measures are currently being employed to control and reduce the S. Gallinarum infections. High endemicity, poor implementation of hygiene and lack of effective vaccines pose challenges in prevention and control of disease in intensively maintained poultry flocks. Comparative genome analysis unravels similarities and dissimilarities thus facilitating identification of genomic features that aids in pathogenesis, niche adaptation and in tracing of evolutionary history. The present investigation was carried out to assess the genotypic differences amongst S.enterica serovar Gallinarum strains including Indian strain S. Gallinarum Sal40 VTCCBAA614. The comparative genome analysis revealed an open pan-genome consisting of 5091 coding sequence (CDS) with 3270 CDS belonging to core-genome, 1254 CDS to dispensable genome and strain specific genes i.e. singletons ranging from 3 to 102 amongst the analyzed strains. Moreover, the investigated strains exhibited diversity in genomic features such as virulence factors, genomic islands, prophage regions, toxin-antitoxin cassettes, and acquired antimicrobial resistance genes. Core genome identified in the study can give important leads in the direction of design of rapid and reliable diagnostics, and vaccine design for effective infection control as well as eradication. Additionally, the identified genetic differences among the S. enterica serovar Gallinarum strains could be used for bacterial typing, structure based inhibitor development by future experimental investigations on the data generated.

Evaluation of in silico designed inhibitors targeting MelF (Rv1936) against Mycobacterium marinum within macrophages.

We recently identified inhibitors targeting Mycobacterium marinum MelF (Rv1936) by in silico analysis, which exhibited bacteriostatic/bactericidal activity against M. marinum and M. tuberculosis in vitro. Herein, we evaluated the effect of best four inhibitors (# 5175552, # 6513745, # 5255829, # 9125618) obtained from the ChemBridge compound libraries, on intracellular replication and persistence of bacteria within IFN-γ activated murine RAW264.7 and human THP-1 macrophages infected with M. marinum. Inhibitors # 5175552 and # 6513745 significantly reduced (p < 0.05) the intracellular replication of bacilli during day 7 post-infection (p.i.) within RAW264.7 and THP-1 macrophages infected at multiplicity of infection (MOI) of ~1.0. These observations were substantiated by electron microscopy, which revealed the protective effect of # 5175552 in clearing the bacilli inside murine macrophages. Strikingly, # 6513745 displayed synergism with isoniazid against M. marinum in murine macrophages, whereas # 5175552 significantly suppressed (p < 0.05) the persistent bacilli during day 10-14 p.i. in infected RAW264.7 and THP-1 macrophages (MOI of ~ 0.1). Moreover, # 5175552 and # 6513745 were non-cytotoxic to host macrophages at both 1X and 5X MIC. Further validation of these inhibitors against M. tuberculosis-infected macrophages and animal models has potential for development as novel anti-tubercular agents.

Computational insights into promoter architecture of toxin-antitoxin systems of Mycobacterium tuberculosis.

Toxin-antitoxin (TA) systems are two component genetic modules widespread in many bacterial genomes, including Mycobacterium tuberculosis (Mtb). The TA systems play a significant role in biofilm formation, antibiotic tolerance and persistence of pathogen inside the host cells. Deciphering regulatory motifs of Mtb TA systems is the first essential step to understand their transcriptional regulation. In this study, in silico approaches, that is, the knowledge based motif discovery and de novo motif discovery were used to identify the regulatory motifs of 79 Mtb TA systems. The knowledge based motif discovery approach was used to design a Perl based bio-tool Mtb-sig-miner available at (https://github.com/zoozeal/Mtb-sig-miner), which could successfully detect sigma (σ) factor specific regulatory motifs in the promoter region of Mtb TA modules. The manual curation of Mtb-sig-miner output hits revealed that the majority of them possessed σB regulatory motif in their promoter region. On the other hand, de novo approach resulted in the identification of a novel conserved motif [(T/A)(G/T)NTA(G/C)(C/A)AT(C/A)] within the promoter region of 14 Mtb TA systems. The identified conserved motif was also validated for its activity as conserved core region of operator sequence of corresponding TA system by molecular docking studies. The strong binding of respective antitoxin/toxin with the identified novel conserved motif reflected the validation of identified motif as the core region of operator sequence of respective TA systems. These findings provide computational insight to understand the transcriptional regulation of Mtb TA systems.

Insights from the protein-protein interaction network analysis of Mycobacterium tuberculosis toxin-antitoxin systems.

Protein-protein interaction (PPI) network analysis is a powerful strategy to understand M. tuberculosis (Mtb) system level physiology in the identification of hub proteins. In the present study, the PPI network of 79 Mtb toxin-antitoxin (TA) systems comprising of 167 nodes and 234 edges was investigated. The topological properties of PPI network were examined by 'Network analyzer' a cytoscape plugin app and STRING database. The key enriched biological processes and the molecular functions of Mtb TA systems were analyzed by STRING. Manual curation of the PPI data identified four proteins (i.e. Rv2762c, VapB14, VapB42 and VapC42) to possess the highest number of interacting partners. The top 15% hub proteins were identified in the PPI network by employing two statistical measures, i.e. betweenness and radiality by employing cytohubba. Insights gained from the molecular protein models of VapC9 and VapC10 are also documented.

Immuno-PCR, a new technique for the serodiagnosis of tuberculosis.

Rapid and accurate diagnosis of tuberculosis (TB) is essential to control the disease. The conventional microbiological tests have limitations and there is an urgent need to devise a simple, rapid and reliable point-of-care (POC) test. The failure of TB diagnostic tests based on antibody detection due to inconsistent and imprecise results has stimulated renewed interest in the development of rapid antigen detection methods. However, the World Health Organization (WHO) has emphasized to continue research for designing new antibody-based detection tests with improved accuracy. Immuno-polymerase chain reaction (I-PCR) combines the simplicity and versatility of enzyme-linked immunosorbent assay (ELISA) with the exponential amplification capacity and sensitivity of PCR thus leading to several-fold increase in sensitivity in comparison to analogous ELISA. In this review, we have described the serodiagnostic potential of I-PCR assays for an early diagnosis of TB based on the detection of potential mycobacterial antigens and circulating antibodies in body fluids of TB patients.

Homology modeling and structural validation of tissue factor pathway inhibitor.

Blood coagulation is a cascade of complex enzymatic reactions which involves specific proteins and cellular components to interact and prevent blood loss. The coagulation process begins by either "Tissue Dependent Pathway" (also known as extrinsic pathway) or by "contact activation pathway" (also known as intrinsic pathway). TFPI is an endogenous multivalent Kunitz type protease inhibitor which inhibits Tissue factor dependent pathway by inhibiting Tissue Factor:Factor VIIa (TF:FVIIa) complex and Factor Xa. TFPI is one of the most studied coagulation pathway inhibitor which has various clinical and potential therapeutic applications, however, its exact mechanism of inhibition is still unknown. Structure based mechanism elucidation is commonly employed technique in such cases. Therefore, in the current study the generated a complete TFPI structural model so as to understand the mechanistic details of it's functioning. The model was checked for stereochemical quality by PROCHECK-NMR, WHATIF, ProSA, and QMEAN servers. The model was selected, energy minimized and simulated for 1.5ns. The result of the study may be a guiding point for further investigations on TFPI and its role in coagulation mechanism.