Pangenome analyses of bovine rectoanal junction squamous epithelial cell–specific adherence factors in E. coli

Authors: NAWROCKI, ERIN - Pennsylvania State University; Kudva, Indira; DUDLEY, EDWARD - Pennsylvania State University

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 1/30/2023
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Shiga toxin–producing Escherichia coli (STEC) are major foodborne pathogens that cause thousands of hospitalizations each year in the United States. Cattle are the natural reservoir of STEC and are colonized asymptomatically at the recto-anal junction (RAJ). The molecular mechanisms that allow E. coli to adhere to the RAJ are not fully understood, in part because most studies of STEC adherence focus on human cell culture models. To identify a set of bovine-specific E. coli adherence factors, we isolated primary RAJ squamous epithelial (RSE) cells directly from healthy Holstein cattle and co-cultured them with diverse E. coli strains from bovine and nonbovine sources. Briefly, RSE cells and E. coli were incubated together in suspension for 4 hours, following which the RSEs were repeatedly centrifuged and washed to isolate adherent E. coli. Bacterial CFU/ml in the total suspension and the attached portion were enumerated by serial dilution. Adherence was calculated by taking the ratio of RSE-attached CFU/ml to total CFU/ml. In parallel, we performed a pangenome analysis of the E. coli strain collection with the bioinformatic tool Roary. From a set of 60 E. coli genomes, we categorized 3159 gene clusters as core or soft-core genes (present in > 95% of isolates) and 18,655 gene clusters as accessory genes. We hypothesized that the accessory genes in bovine E. coli include factors that contribute to the RSE adherence phenotype. Using Scoary to correlate empirical adherence data with the presence and absence of genes in the pangenome, we compiled a list of accessory genes associated with RSE adherence. These include members of the marRAB operon, which regulates dozens of genes involved in antibiotic resistance and other stress responses; the pga operon, which is a known contributor to biofilm formation; and several genes that encode putative fimbrial adhesins such as yadN, yfcV, and ygiL. To supplement our pangenome study, we also constructed a transposon library of EDL933, a model STEC serotype O157:H7 strain, and adapted the RSE co-culture assay to screen mutants that were either over- or underrepresented in the adherent fraction. High-throughput sequencing of the total and RSE-attached populations of EDL933 revealed additional candidate adherence factors. A mutant defective for bcsA, a main component of the bacterial cellulose synthesis system, was impaired for RSE adherence when compared to wild-type EDL933. Further molecular characterization of RSE-specific adherence factors is in progress. Relative to those involved in human infection, E. coli genes that promote bovine colonization are poorly characterized. In this work, we have explored RSE adherence in E. coli strains of various pathotypes and serotypes to search for adherence factors in diverse genomes. Surveying the species in this way may lead to new candidates for drugs, vaccines, or probiotic interventions that reduce carriage of STEC in cattle and prevent contamination of the food chain.