Type III secretion system confers enhanced virulence in clinical non-O1/non-O139 Vibrio cholerae.

Vibrio cholerae O1 infections mainly are responsible for significant mortality and morbidity amongst children, however, non-O1/non-O139 V. cholerae have also been reported to cause mild to severe infections because of their virulence potential. The pathogenic mechanisms of non-O1, non-O139 isolates are not as clearly understood as for that of O1 and O139 isolates. Type three secretion system (TTSS) is also considered one of the important virulent factors and during the current study, we investigated the role of TTSS in association with non-O1/non-O139 clinical isolates. We report that the presence of TTSS in non-O1/non-O139 V. cholerae clinical isolate (D13) from a child confers more virulence compared to the one lacking it (D15) in another clinical case during the small cholera epidemic. Moreover, the antibiotic susceptibility profiles of D13 and D15 indicate that they are multiple drug resistance (MDR) isolates. The sequence analysis for TTSS cluster was carried out for D13 and compared with the TTSS positive reference Vibrio parahaemolyticus RIMD2210633 and V. cholerae AM19226 non-O1/non-O139. Furthermore, the pathogenic potential of D13 & D15 was also explored in simple and economical invertebrate host model, Galleria mellonella and the results revealed that TTSS+ve isolate (D13) was more virulent compared to TTSS-ve isolate (D15). We suggest that this distinct genetic difference, seen in natural variants D13 and D15, is also reflected by the clinical picture of the former in contributing towards the severity of disease symptoms and this finding was further validated by assessing virulence potential of both isolates using inexpensive G. mellonella infection model.

Preliminary Work Towards Finding Proteins as Potential Vaccine Candidates for Vibrio cholerae Pakistani Isolates through Reverse Vaccinology.

Background and Objective: Vibrio cholerae continues to emerge as a dangerous pathogen because of increasing resistance to a number of antibiotics. This paper provides a solution to emerging antibiotic resistance by introducing novel proteins as vaccine candidates against cholera. Materials and Methods: Vibrio cholerae genome versatility is a hurdle for developing a vaccine to combat diarrhoeal infection, so its core gene information was used to determine a potential vaccine candidate. Whole genome sequence data of more than 100 Vibrio cholerae strains were used simultaneously to get core genome information. The VacSol pipeline based on reverse vaccinology was selected to address the problem of safe, cheap, temperature-stable, and effective vaccine candidates which can be used for vaccine development against Vibrio cholerae. VacSol screens vaccine candidates using integrated, well-known, and robust algorithms/tools for proteome analysis. The proteomes of the pathogens were initially screened to predict homology using BLASTp. Proteomes that are non-homologous to humans are then subjected to a predictor for localization. Helicer predicts transmembrane helices for the protein. Proteins failing to comply with the set parameters were filtered at each step, and finally, 11 proteins were filtered as vaccine candidates. Results: This selected group of vaccine candidates consists of proteins from almost all structural parts of Vibrio cholerae. Their blast results show that this filtered group includes flagellin A protein, a protein from the Zn transporter system, a lipocarrier outer membrane protein, a peptidoglycan-associated protein, a DNA-binding protein, a chemotaxis protein, a tRNA Pseuriudine synthase A, and two selected proteins, which were beta lactamases. The last two uncharacterized proteins possess 100% similarity to V. albensis and Enterobacter, respectively. Tertiary structure and active site determination show a large number of pockets on each protein. Conclusions: The most interesting finding of this study is that 10 proteins out of 11 filtered proteins are introduced as novel potential vaccine candidates. These novel vaccine candidates can result in the development of cost-effective and broad-spectrum vaccines which can be used in countries where cholera is a major contributor to diarrheal disease.

Comparative core/pan genome analysis of Vibrio cholerae isolates from Pakistan.

Cholera is an endemic disease in many regions of Asia including, Pakistan. Vibrio cholerae, the causative agent of cholera, is considered as one of the best adapted bacteria due to its ability to withstand severe environmental stresses. The V. cholerae genome is very plastic with many gene additions and deletions. In this study, we sought to understand the diversity of V. cholerae genes in two Pakistani subclades [e.g. Pakistani subclade I (PSC I) and Pakistani subclade II (PSC II)]. We have analyzed 44 PSC I and 56 PSC II strains, respectively. By analyzing our data, it was concluded that subclade group 2 (PSC II) has 2967 core genes repositories, while the PSC 1 group has just 1062 core genes. It was observed that the pangenome in the PSC II group is open while the pan-genome in PSC I are closed. It was also noted that the number of accessory genes (n = 2500) is higher in the PSC I group compared to the PSC II group (n = 550). Furthermore, analysis extended to the study of unique gene profiles suggested that all strains of the PSC II group have unique genes. One strain among the PSC II group had a high number of unique genes (n = 2612). However, in the PSC I group, only a few strains had unique genes with a maximum of 86 unique genes being found in a single strain. Core phylogeny of PSC I indicated that just three groups initially arose from a single common ancestor. At the same time, a complex pattern of evolution was found in the PSC II phylogenetic tree based on core gene information. This comparative genomic analysis has revealed 'waves' of V. cholerae evolution and information on its transmission and ability to modify its genetic content to survive in different environmental conditions. Here, we have investigated how the versatility of V. cholerae, a bacterium that persists across different habitats, is reflected in its genome. The data generated during the study should be extremely beneficial in defining the evolutionary relationship as well as diversity between V. cholerae subclades. It will also benefit epidemiological studies and the design of better treatment strategies for controlling epidemics.