Differential and cell-type specific CDC42-regulated intestinal epithelial responses to pathogenic infection
Creators: name: Bandyopadhyay, Sheila
Publisher: name: No Publisher Supplied
Year: 2023

description: Enteric pathogens have evolved to hijack host cellular machinery, most notably Rho small GTPases, to modify the host cellular cytoskeleton for entry and invasion. Studies using non-intestinal epithelial cells suggested that Salmonella enterica serovar Typhimurium, an invasive enteric pathogen, manipulates host Cell-Division-Cycle-42 (CDC42) to remodel actin cytoskeletal network for its invasion. However, the role of CDC42 and its downstream intracellular protein effectors in intestinal epithelial cells during pathogen infection are relatively unclear. Additionally, the in vivo intestinal cell-type specific roles of CDC42 during Salmonella infections have not been explored. Understanding the molecular basis of pathogen-host cellular machinery, in particular involving CDC42, is imperative and will shed light on new targets and mechanisms for intervening infection and inflammation.First, a poorly-studied protein identified through a CDC42-dependent proteome, CDC42 effector protein (CDC42EP1) was found to rapidly redistribute and aggregate around invading Salmonella Typhimurium. To determine the contribution of CDC42 and CDC42EP1 to the cellular susceptibility to invasion, human enterocyte cell lines were used to perform S. Typhimurium infection assays. Salmonella-induced relocalization of CDC42EP1 was found to depend on CDC42 activity, as well as the lipid modification of the CDC42-lipid tail motif. Further analysis revealed that CDC42EP1 is also associated with VILLIN and SEPTIN-7, two cytoskeleton-regulating proteins, during early stages of infection. Unlike CDC42, CDC42EP1 is not required for S. Typhimurium’s initial cellular entry, but is found to associate with Salmonella-containing vacuoles and affect the pathogen’s intracellular growth and survival. These results uncovered a new host regulator of enteric Salmonella infections that may be targeted to restrict bacterial loads at the primary site of infection to prevent systemic spread.CDC42 is known to be a major regulator of actin cytoskeleton rearrangement during S. Typhimurium infections in vitro, but its role during a physiologically-relevant model has not been established in vivo. CDC42fl/fl; VillinCreER and CDC42-V2Tg mice were induced to ablate CDC42 expression or to overexpress CDC42-V2, a splicing variant of CDC42, respectively, in the mouse intestinal epithelial cells. The deletion of CDC42 impaired the host epithelial barrier and increased mortality, and affected the proper host defense against Salmonella infection. In contrast, an overexpression of CDC42 enhanced the defense response to pathogenic infection, strengthening the epithelial barrier. These findings, in a physiologically-relevant model, indicated significant function of intestinal epithelial cell-CDC42 in protection against Salmonella infection, an observation different from in vitro models. An intestinal epithelial deletion of CDC42 was previously found to impact Paneth cell localization; however, the Paneth cell-autonomous function of CDC42 has not been investigated, particularly in the context of pathogen infection. Transcriptomic studies revealed that CDC42 is highly expressed in Paneth cell progenitors, and the abundance of CDC42 in mature Paneth cells was reduced upon infection. Utilizing Paneth cell reporter mice (Cdc42fl/fl; Lyz13’UTR-CreER; Rosa26R-tdTomato), CDC42 was ablated specifically within Paneth cells. The deletion led to a reduction in Paneth cell-specific antimicrobial peptide expression and a changed localization of Paneth cells, a phenotype distinct from those observed in CDC42ΔIEC mice that lacked CDC42 in all intestinal epithelial cells. The deletion of CDC42 within Paneth cells exacerbated the intestinal inflammatory response to S. Typhimurium infection, suggesting that Paneth cell CDC42 facilitates mucosal defense against pathogen infection. The elevated reactive oxygen species (ROS) in CDC42ΔPC mouse Paneth cells may potentially contribute to the exacerbated disease progression in these mice. Taken together, these findings demonstrate differential and cell type-specific roles of CDC42 machinery in regulating host response to infection of enteric pathogens, such as Salmonella enterica Typhimurium.