Uropathogenic E. coli (UPEC) causes the majority of urinary tract infections (UTI), a major global public health concern that is associated with significant morbidity and mortality. Antibiotic resistance in UPEC is prevalent, as exemplified by the rapid global dissemination of the multidrug-resistant E. coli sequence type 131 (ST131) clone. An understanding of interactions between UPEC and the innate immune system may ultimately lead to non-antibiotic-based therapeutic approaches for UTI. We recently demonstrated that some UPEC strains rapidly kill human macrophages, likely as a host evasion mechanism1. To identify the virulence factor/s responsible for killing human macrophages, we used an unbiased approach and generated a random transposon mutagenesis library in the reference strain CFT073. This approach revealed that the pore forming toxin hemolysin A (HlyA) mediated both cell death and inflammasome-dependent IL-1β release in human macrophages. The poorly characterized Cof phosphatase was also identified as a novel hemolysin regulator. In surveying a large panel of ST131 isolates, we found that only HlyA+ strains killed human macrophages. Interestingly, HlyA+ ST131 strains displayed heterogeneity in their ability to kill human macrophages, and this variation correlated with levels of secreted HlyA. Investigation into the biological significance of this phenomenon revealed that an ST131 strain producing low levels of HlyA initiated cell death that was partially NLRP3 inflammasome-dependent, with this response being associated with a host-protective role in a mouse UTI model. When the same ST131 strain was engineered to overexpress high HlyA levels, macrophage cell death occurred even when NLRP3 function was abrogated, and bladder colonisation was significantly increased. Thus, HlyA expression levels are variable between UPEC isolates, and this dictates how macrophages die, as well as host susceptibility versus resistance to colonization.
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