Abstract
Uropathogenic Escherichia coli (UPEC) is the major cause of urinary tract infections. Nearly half of all UPEC strains secrete hemolysin, a cytotoxic pore-forming toxin. Here, we show that the prevalence of the hemolysin toxin gene (hlyA) is highly variable among the most common 83 E. coli sequence types (STs) represented on the EnteroBase genome database. To explore this diversity in the context of a defined monophyletic lineage, we contextualized sequence variation of the hlyCABD operon within the genealogy of the globally disseminated multidrug-resistant ST131 clone. We show that sequence changes in hlyCABD and its newly defined 1.616-kb-long leader sequence correspond to phylogenetic designation, and that ST131 strains with the strongest hemolytic activity belong to the most extensive multidrug-resistant sublineage (clade C2). To define the set of genes involved in hemolysin production, the clade C2 strain S65EC was completely sequenced and subjected to a genome-wide screen by combining saturated transposon mutagenesis and transposon-directed insertion site sequencing with the capacity to lyse red blood cells. Using this approach, and subsequent targeted mutagenesis and complementation, 13 genes were confirmed to be specifically required for production of active hemolysin. New hemolysin-controlling elements included discrete sets of genes involved in lipopolysaccharide (LPS) inner core biosynthesis (waaC, waaF, waaG, and rfaE) and cytoplasmic chaperone activity (dnaK and dnaJ), and we show these are required for hemolysin secretion. Overall, this work provides a unique description of hemolysin sequence diversity in a single clonal lineage and describes a complex multilevel system of regulatory control for this important toxin.IMPORTANCE Uropathogenic E. coli (UPEC) is the major cause of urinary tract infections and a frequent cause of sepsis. Nearly half of all UPEC strains produce the potent cytotoxin hemolysin, and its expression is associated with enhanced virulence. In this study, we explored hemolysin variation within the globally dominant UPEC ST131 clone, finding that strains from the ST131 sublineage with the greatest multidrug resistance also possess the strongest hemolytic activity. We also employed an innovative forward genetic screen to define the set of genes required for hemolysin production. Using this approach, and subsequent targeted mutagenesis and complementation, we identified new hemolysin-controlling elements involved in LPS inner core biosynthesis and cytoplasmic chaperone activity, and we show that mechanistically they are required for hemolysin secretion. These original discoveries substantially enhance our understanding of hemolysin regulation, secretion and function.
Highlights
Uropathogenic Escherichia coli (UPEC) is the major cause of urinary tract infections
We found that variation in hemolysin expression by UPEC can have profound effects on biological outcomes; low-level hemolysin expression triggers NOD-like receptor pyrin domain-containing 3 (NLRP3)-mediated macrophage cell death that is associated with host protection in a mouse model of experimental urinary tract infection (UTI), whereas
Epidemiological studies show that hlyA prevalence is associated with UPEC strains that cause severe UTI [16, 17]
Summary
Uropathogenic Escherichia coli (UPEC) is the major cause of urinary tract infections. We show that the prevalence of the hemolysin toxin gene (hlyA) is highly variable among the most common 83 E. coli sequence types (STs) represented on the EnteroBase genome database To explore this diversity in the context of a defined monophyletic lineage, we contextualized sequence variation of the hlyCABD operon within the genealogy of the globally disseminated multidrug-resistant ST131 clone. Despite the presence of unique features that define ST131 and other clones, strains within these phylogenetically related lineages exhibit extensive diversity in their accessory genome This occurs primarily through the possession of multiple large genomic islands that contain different combinations of genes encoding virulence factors such as adhesins (e.g., fimbriae and autotransporters), surface polysaccharides (e.g., capsule and O antigen), iron acquisition systems (e.g., siderophores and heme scavenging systems), and toxins (e.g., hemolysin and cytotoxic necrotizing factor-1) that are associated with the capacity to cause disease [10, 12, 13]. We found that variation in hemolysin expression by UPEC can have profound effects on biological outcomes; low-level hemolysin expression triggers NLRP3-mediated macrophage cell death that is associated with host protection in a mouse model of experimental UTI, whereas
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