Project Details

Inhibition of the RalGAP protein complex as a novel approach to broadly block enteric infections

Hinissan Kohio, PhD

Summary

BACKGROUND: Rotavirus, enterohemorrhagic Escherichia coli, and Cryptosporidium parvum are the principal viral, bacterial, and parasitic pathogens that cause severe gastroenteritis and life-threatening diarrhea in infants and young children. Rotavirus vaccine efficacy is low in endemic countries, and there are no effective therapeutics or vaccines against enterohemorrhagic Escherichia coli or Cryptosporidium parvum. GAP: Currently, there is a lack of knowledge regarding the role of host factors that support the infection of enteric pathogens, which will help design novel antiviral therapeutics. However, our previous CRISPR/Cas9 screens and published literature suggest that genetic deletion of the RalGAP complex suppresses infections caused by rotavirus, enterohemorrhagic Escherichia coli, and Cryptosporidium parvum. HYPOTHESIS: Given the role of RalGAP in endosomal vesicular trafficking, we speculate that these pathogens commonly hijack the RalGAP complex for endosomal entry and toxin release into the host intestinal epithelial cells. We hypothesize that inhibiting the host RalGAP complex will block the replication and pathogenesis of rotavirus, Escherichia coli, and Cryptosporidium parvum. METHODS: We genetically deleted the RALGAPA1 and RALGAPB subunits of the RALGAP protein complex via CRISPR/Cas9 in HT-29 cells. We further deleted the RALGAPA1 gene in a neonatal mouse model to assess its implication during infection in vivo. Additionally, we conducted a high-throughput drug screen of 17000 FDA-approved compounds to identify novel small molecules that can inhibit RALGAP activity. RESULTS: Our results showed a significant reduction in RV infection in the RALGAPA1 KO and RALGAPB KO cells, as determined by intracellular NSP5 levels measured by RT-qPCR. These findings indicate that both the RALGAPA1 and the RALGAPB subunit of the RALGAP protein complex might act as pro-viral factors during RV infection. Additionally, our results show 100% cell survival in the RALGAPB KO cells during enterohemorrhagic Escherichia coli infection. In contrast, only 66% of the wild-type cells survived. These data indicate that RALGAPB helps promote Escherichia coli infection, leading to increased cell death. Our preliminary findings show that Cryptosporidium parvum infection is not affected during RALGAP deletion. Deletion of the RALGAPA1 gene in neonatal mice did not affect rotavirus infection. The current results could be confounded by the host microbiome or mesenchymal and immune cells. Our drug screen identified a potential inhibitory compound of the RALGAP protein complex, which will require further evaluation in vivo. IMPACT: Our bench-to-bedside discovery pipeline has helped identify a potential cellular druggable target commonly usurped by these enteric pathogens and developed a pan-anti-microbial molecule to treat enteric infections, thereby reducing diarrhea-associated morbidity and mortality. Website Link: https://sdinglab.wustl.edu/people-page-page/