Background and Aims: Neural Wiskott-Aldrich Syndrome protein (N-WASP) is a key regulator of the actin cytoskeleton in epithelial tissues and is poised to mediate cytoskeletal-dependent aspects of apical junction complex (AJC) homeostasis. Attaching-and-effacing (AE) pathogens disrupt this homeostasis through translocation of the effector molecule early secreted antigenic target-6 (ESX)-1 secretion-associated protein F (EspF). Although the mechanisms underlying AJC disruption by EspF are unknown, EspF contains putative binding sites for N-WASP and the endocytic regulator sorting nexin 9 (SNX9). We hypothesized that N-WASP regulates AJC integrity and AE pathogens use EspF to induce junction disassembly through an N-WASP- and SNX9-dependent pathway. Methods: We analyzed mice with intestine-specific N-WASP deletion and generated cell lines with N-WASP and SNX9 depletion for dynamic functional assays. We generated EPEC and Citrobacter rodentium strains complemented with EspF bearing point mutations abolishing N-WASP and SNX9 binding to investigate the requirement for these interactions. Results: Mice lacking N-WASP in the intestinal epithelium showed spontaneously increased permeability, abnormal AJC morphology, and mislocalization of occludin. N-WASP depletion in epithelial cell lines led to impaired assembly and disassembly of tight junctions in response to changes in extracellular calcium. Cells lacking N-WASP or SNX9 supported actin pedestals and type III secretion, but were resistant to EPEC-induced AJC disassembly and loss of transepithelial resistance. We found that during in vivo infection with AE pathogens, EspF must bind both N-WASP and SNX9 to disrupt AJCs and induce intestinal barrier dysfunction. Conclusions: Overall, these studies show that N-WASP critically regulates AJC homeostasis, and the AE pathogen effector EspF specifically exploits both N-WASP and SNX9 to disrupt intestinal barrier integrity during infection.

It is breathtaking to consider how the response to pandemic viral pathogens has been transformed over the past century by greater knowledge of fundamental biology and technological innovations including PCR and next-generation sequencing. In striking contrast to the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, the pathogen responsible for the 1918 influenza pandemic was not identified until years after the outbreak. The definitive text in 1927 described influenza as “an epidemiologic conception” likely caused by the bacterium Haemophilus influenzae. Six decades later, HIV-1 was discovered within a few years of the first report of AIDS, although it took another decade before HIV-1 RNA detection methods were sensitive enough to correlate viral load during clinical latency with rate of progression to AIDS. Four decades later, the genomic sequence of SARS-CoV-2 was publicly available on the internet within weeks of the unexplained outbreak of fatal pneumonia that is now known as COVID-19. This critical information enabled academic researchers, vaccine manufacturers, diagnostic laboratories, and some governments to spring into action. In the midst of COVID-19 lockdown, despite collapse of reagent supply chains, independent investigators around the world shared expertise and reagents in order to establish desperately needed local screening programs for SARS-CoV-2. A paper by Yang et al. in PNAS describes the analysis of viral load data from one local screening program, the results of which have important implications for efforts to control the spread of SARS-CoV-2 and for understanding the pathogenesis of SARS-CoV-2 infection.