Vibrio cholerae is the causal organism of the cholera epidemic, which is mostly prevalent in developing and underdeveloped countries. However, incidences of cholera in developed countries are also alarming. Because of the emergence of new drug-resistant strains, even though several generic drugs and vaccines have been developed over time, Vibrio infections remain a global health problem that appeals for the development of novel drugs and vaccines against the pathogen. Here, applying comparative proteomic and reverse vaccinology approaches to the exoproteome and secretome of the pathogen, we have identified three candidate targets (ompU, uppP and yajC) for most of the pathogenic Vibrio strains. Two targets (uppP and yajC) are novel to Vibrio, and two targets (uppP and ompU) can be used to develop both drugs and vaccines (dual targets) against broad spectrum Vibrio serotypes. Using our novel computational approach, we have identified three peptide vaccine candidates that have high potential to induce both B- and T-cell-mediated immune responses from our identified two dual targets. These two targets were modeled and subjected to virtual screening against natural compounds derived from Piper betel. Seven compounds were identified first time from Piper betel to be highly effective to render the function of these targets to identify them as emerging potential drugs against Vibrio. Our preliminary validation suggests that these identified peptide vaccines and betel compounds are highly effective against Vibrio cholerae. Currently we are exhaustively validating these targets, candidate peptide vaccines, and betel derived lead compounds against a number of Vibrio species.

G-quadruplex structure forming motifs are among the most studied evolutionarily conserved drug targets that are present throughout the genome of different organisms and susceptible to influencing various biological processes. Here we report highly conserved potential G-quadruplex motifs (PGQs) in three essential genes (espK, espB, and cyp51) among 160 strains of the Mycobacterium tuberculosis genome. Products of these genes are involved in pathways that are responsible for virulence determination of bacteria inside the host cell and its survival by maintaining membrane fluidity. The espK and espB genes are essential players that prevent the formation of mature phagolysosome and antigen presentation by host macrophages. The cyp51 is another PGQ-possessing gene involved in sterol biosynthesis pathway and membrane formation. In the present study, we revealed the formation of stable intramolecular parallel G-quadruplex structures by Mycobacterium PGQs using a combination of techniques (NMR, circular dichroism [CD], and gel electrophoresis). Next, isothermal titration calorimetry (ITC) and CD melting analysis demonstrated that a well-known G-quadruplex ligand, TMPyP4, binds to and stabilizes these PGQ motifs. Finally, polymerase inhibition and qRT-PCR assays highlight the biological relevance of PGQ-possessing genes in this pathogen and demonstrate that G-quadruplexes are potential drug targets for the development of effective anti-tuberculosis therapeutics.

Several G-quadruplex forming motifs have been reported to be highly conserved in the regulatory regions of the genome of different organisms and influence various biological processes like DNA replication, recombination and gene expression. Here, we report the highly conserved and three potentially G-quadruplex forming motifs (SP-PGQs) in the essential genes (hsdS, recD, and pmrA) of the Streptococcus pneumoniae genome. These genes were previously observed to play a vital role in providing the virulence to the bacteria, by participating in the host-pathogen interaction, drug-efflux system and recombination- repair system. However, the presence and importance of highly conserved G-quadruplex motifs in these genes have not been previously recognized. We employed the CD spectroscopy, NMR spectroscopy, and electrophoretic mobility shift assay to confirm the adaptation of the G-quadruplex structure by the SP-PGQs. Further, ITC and CD melting analysis revealed the energetically favorable and thermodynamically stable interaction between a candidate G4 binding small molecule TMPyP4 and SP-PGQs. Next, TFP reporter based assay confirmed the regulatory role of SP-PGQs in the expression of PGQ harboring genes. All these experiments together characterized the SP-PGQs as a promising drug target site for combating the Streptococcus pneumoniae infection.

Due to next-generation sequence technologies, sequencing of bacterial genomes is no longer one of the main bottlenecks in bacterial research and the number of new genomes deposited in public databases continues to increase at an accelerating rate. Among these new genomes, several belong to the same species and were generated for pan-genomic studies. A pan-genomic study allows investigation of strain phenotypic differences based on genotypic differences. Along with a need for good assembly quality, it is also fundamental to guarantee good functional genome annotation of the different strains. In order to ensure quality and standards for functional genome annotation among different strains, we developed and made available PANNOTATOR (http://bnet.egr.vcu.edu/iioab/agenote.php), a web-based automated pipeline for the annotation of closely related and well-suited genomes for pan-genome studies, aiming at reducing the manual work to generate reports and corrections of various genome strains. PANNOTATOR achieved 98 and 76% of correctness for gene name and function, respectively, as result of an annotation transfer, with a similarity cut-off of 70%, compared with a gold standard annotation for the same species. These results surpassed the RAST and BASys softwares by 41 and 21% and 66 and 17% for gene name and function annotation, respectively, when there were reliable genome annotations of closely related species. PANNOTATOR provides fast and reliable pan-genome annotation; thereby allowing us to maintain the research focus on the main genotype differences between strains.

Nipah virus (NPV) is one of the most notorious viruses with a very high fatality rate. Because of the recurrent advent of this virus and its severe neurological implications, often leading to high mortality, the WHO R&D Blueprint, 2018 has listed the Nipah virus as one of the emerging infectious diseases requiring urgent research and development effort. Yet there is a major layback in the development of effective vaccines or drugs against NPV. In this study, we have designed a stable multivalent vaccine combining several T-cell and B-cell epitopes of the essential Nipah viral proteins with the help of different ligands and adjuvants which can effectively induce both humoral and cellular immune responses in human. Different advanced immune-informatic tools confirm the stability, high immunogenicity and least allergenicity of the vaccine candidate. The standard molecular dynamic cascade analysis validates the stable interaction of the vaccine construct with the human Toll-like receptor 3 (TLR3) complex. Later, codon optimization and in silico cloning in a known pET28a vector system shows the possibility for the expression of this vaccine in a simple organism like E.coli. It is believed that with further in vitro and in vivo validation, this vaccine construct can pose to be a better prophylactic solution to the Nipah viral disease.