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Corynebacterium pseudotuberculosis (Cp) é o agente etiológico de diversas doenças infecciosas e contagiosas crônicas, incluindo linfadenite caseosa (LC), linfangite ulcerativa, mastite e doença edematosa da pele e afeta um amplo número de hospedeiros. Neste trabalho, primeiramente, foi realizada a anotação gênica manual em duas linhagens de C. pseudotuberculosis utilizando a ferramenta Artêmis. A linhagem Cp162, isolado de camelo no Reino Unido e a P54B96, isolada de antílope na África do Sul, as quais tem genomas de 2.293.464 pb e 2.337.657 pb, 52,17% e 52,2% de conteúdo GC, 2002 e 2084 sequências codificadoras, 87 e 62 pseudogenes respectivamente. Ambas possuem 49 genes de tRNA e 4 operons de rRNA. Os genomas foram depositadas no banco de dados do NCBI GenBank sob os números de acesso CP003652 e CP003385, respectivamente. No segundo momento foram usados 15 genomas de C. pseudotuberculosis para um estudo em larga escala das proteínas preditas a partir destas sequências genômicas, através de técnicas de bioinformática e modelagem comparativa, usando o workflow MHOLline. Somente as sequências de qualidade, categorizadas em muito alta, alta, boa e média a boa foram usadas a partir dos grupos classificados por MHOLline. Usando alguns scripts desenvolvido pela equipe do LGCM, foram selecionadas um conjunto de 331 proteínas conservadas, que apresentavam estruturas 3D e considerando 95-100% de similaridade entre sequências intra-espécies. Visando identificar proteínas essenciais homólogas e não homólogas aos hospedeiros humano, equino, bovino e ovino, o core-modeloma foi filtrado resultando em um conjunto final de 10 proteínas. Apenas quatro proteínas foram identificadas como essenciais e não homólogas e foram consideradas como alvos putativos para drogas e vacinas, submetidos a triagem virtual e análises de docking. Adicionalmente, entre outros, um parâmetro para avaliar a drogabilidade de todas as proteínas, também foi calculado, permitindo a predição de cavidades das proteínas. Pesquisamos as outras 6 proteínas essenciais e homólogas realizando uma análise profunda das suas estruturas (Cp e dos hospedeiros) em nível de resíduos confirmando alguns resíduos conservados nos sítios ativos com conformações diferentes ou resíduos completamente diferentes. A conservação desses resíduos foi validada através de um alinhamento múltiplo de sequências. Além disso, o papel destes alvos em diferentes vias metabólicas bacterianas, patogenicidade e virulência também foram determinados. Baseado nesses estudos, as 4 proteínas essenciais e não homólogas foram propostas como potenciais alvos para tratamento e as 6 proteínas essenciais e homólogas também podem ser utilizadas como possíveis alvos terapêuticos devido às diferenças observadas em resíduos conservados. Espera-se que os nossos dados possam ajudar na seleção de proteínas de C. pseudotuberculosis para o desenvolvimento bem sucedido de novas drogas e vacinas para uma ampla classe de hospedeiros. Corynebacterium pseudotuberculosis (Cp) is the etiological agent of several infectious and contagious chronic diseases, including caseous lymphadenitis (CLA), ulcerative lymphangitis, mastitis, and edematous skin disease thus affecting a broad spectrum of animal hosts, including ruminants that cause a huge decrease in wool production and carcass quality, thus threatening economic and dairy industries worldwide. In this work, first, manual annotation was performed using Artemis: the annotation tool, for two C. pseudotuberculosis strains. For Cp162, isolated from a camel in UK and completely sequenced using the SOLiD v3 Plus NGS platform, a genome of 2,293,464 bp in size, having 52.17% GC content, 2,002 coding sequences, 4 rRNA operons, 49 tRNA genes, and 87 pseudogenes, was obtained. For strain P54B96, isolated from an antelope in South Africa and sequenced using the Ion Torrent PGM NGS platform, a genome of 2,337,657 bp in size, having 52.2% GC content, 2,084 coding sequences, 4 rRNA operons, 49 tRNA genes, and 62 pseudogenes, was obtained. They were deposited in the NCBI GenBank database under accession numbers CP003652 and CP003385, respectively. Motivated by an increasing demand for new drug and/or vaccine targets, secondly, a novel strategy using a highthroughput workflow, called MHOLline, was followed to construct the modelome (3D models) of 15 C. pseudotuberculosis strains from various hosts and countries. Only Very High, High, Good and Medium to Good quality sequences were used from MHOLline classified groups(G2). Using a locally installed BALST NCBI tool, a set of 331 conserved proteins with 3D structures was selected showing 95-100% intra-species sequence similarity. The host proteomes considered in this study were human, horse, cow and sheep. Further filtering this core-modelome for essential and non-host homologous proteins, resulted in a final set of 10 proteins. Among these, only 4 proteins were identified as essential and non-host homologous and were considered as putative drug and vaccine targets, subjected to virtual screening and docking analyses. The druggability score, a drug target prioritization parameter, among others, was also calculated for all these proteins, allowing the prediction of druggable protein cavities. We further extrapolated our research to the other 6 essential host homologous proteins. A deep structure analysis at their residue level confirmed some conserved active site residues either exhibiting different conformations or even completely different residues. A multiple sequence alignment proved the residues conservation in these targets. Further, the role of these targets in different bacterial metabolic pathways, pathogenicity and virulence were also determined. It was proposed that the 6 essential host homologous proteins might also provide an extended choice for therapeutic targets. It is expected that our data will facilitate selection of C. pseudotuberculosis proteins for successful designing of new drugs and vaccines for a broad range of hosts.

In the last decade, robust sequencing technologies have revolutionized the genomic science. As a result, comparative genomics is now recognized as a new discipline. Comparative microbial genomics exploits both similarities and differences in the genomes, proteome, trascriptome, and regulatory regions of different organisms to infer the evolutionary relations, along with conserved and unique characteristics of species. These analyses have resulted in some surprising biological discoveries in the recent past. This study presents comparative genomic analysis of multiple pathogenic and non-pathogenic bacteria from related species, to dissect the genomic information and to get insights into evolutionary relationships, conserved information and mechanisms of pathogenicity. Starting from genus Corynebacterium, 11 representative species are analysed and compared, resulting in 741 conserved Gene Families (GFs) in all of them, and significant intra-species proteome similarities (98-99%) were observed. Subsequently, the pan- (7059 GFs) and core genome (552 GFs) of genus Campylobacter is estimated. A detailed comparative pathogenomic study of Campylobacter fetus subspecies resulted in identification of common and novel regions associated with pathogenicity; and species specific virulence factors and vaccine candidates have been characterized. Furthermore, comparative genomics and pathogenomics analysis of the genus Helicobacter (46 genomes) is accomplished. 38 Helicobacter pylori were found to share 1,185 core gene families representing ~77% of the average genome size. The core essential genes families (EGFs) are ascertained, and explored for potential therapeutics against H. pylori. In conclusion, we propose that, these observed genomic variations, species specific features and core virulence factors will enhance understanding of the lifestyle of the organisms, and will contribute to the development of antibiotics, drugs and vaccines.