Integrated metabolic modeling, culturing and transcriptomics explains enhanced virulence ofV. choleraeduring co-infection with ETEC
Authors: Abdel-Haleem Alyaa M. , Ravikumar Vaishnavi , Ji Boyang , Mineta Katsuhiko , Gao Xin , Nielsen Jens , Gojobori Takashi , Mijakovic Ivan 
Published: June 2020

AbstractGene essentiality is altered during polymicrobial infections. Nevertheless, most studies rely on single-species infections to assess pathogen gene essentiality. Here, we use genome-scale metabolic models to explore the effect of co-infection of the diarrheagenic pathogenVibrio cholerae(V. cholerae) with another enteric pathogen, enterotoxigenicE. coli(ETEC). Model predictions showed thatV. choleraemetabolic capabilities were increased due to ample cross-feeding opportunities enabled by ETEC. This is in line with increased severity of cholera symptoms known to occur in patients with dual-infections by the two pathogens.In vitroco-culture systems confirmed thatV. choleraegrowth is enhanced in co-cultures relative to single-cultures. Further, expression levels of severalV. choleraemetabolic genes were significantly perturbed as shown by dual RNAseq analysis of its co-cultures with different ETEC strains. A decrease in ETEC growth was also observed, probably mediated by non-metabolic factors. Single gene essentiality analysis predicted conditionally-independent genes that are essential for the pathogen’s growth in both single- and co-infection scenarios. Our results reveal growth differences that are of relevance to drug targeting and efficiency in polymicrobial infections.ImportanceMost studies proposing new strategies to manage and treat infections have been largely focused on identifying druggable targets that can inhibit a pathogen’s growth when it is the single cause of infection.In vivo, however, infections can be caused by multiple species. This is important to take into account when attempting to develop or use current antibacterials since their efficacy can change significantly between single and co-infections. In this study, we used genome-scale metabolic models (GEMs) to interrogate the growth capabilities ofVibrio cholerae(V. cholerae) in single and co-infections with enterotoxigenicE. coli(ETEC), which co-occur in large fraction of diarrheagenic patients. Co-infection model predictions showed thatV. choleraegrowth capabilities are enhanced in presence of ETEC relative toV. choleraesingle-infection, through cross-fed metabolites made available toV. choleraeby ETEC.In vitro, co-cultures of the two enteric pathogens further confirmed model predictions showing an increased growth ofV. choleraein co-culture relative toV. choleraesingle-cultures while ETEC growth was suppressed. Dual RNAseq analysis of the co-cultures also confirmed that the transcriptome ofV. choleraeis distinct during co-infection compared to single infection scenarios where processes related to metabolism were significantly perturbed. Further,in silicogene-knock out simulations uncovered discrepancies in gene essentiality forV. choleraegrowth between single and co-infections. Integrative model-guided analysis thus identified druggable targets that would be critical forV. choleraegrowth in both single and co-infections, thus designing inhibitors against those targets would provide a broader spectrum coverage against cholera infections.