Uropathogenic E. coli (UPEC) causes the majority of urinary tract infections (UTI), which is associated with significant morbidity and mortality. An understanding of the interactions between UPEC and the innate immune system may lead to new therapeutic approaches for UTI. We recently demonstrated that some UPEC strains rapidly kill human macrophages, likely as a host evasion mechanism. Using a random transposon mutagenesis library in the reference strain CFT073, we identified eight clones that were impaired in their ability to kill primary human macrophages. These hits were independently validated and sequenced to identify mutated genes, and independent knock-outs were generated to further validate the findings. This approach revealed that the pore forming toxin, hemolysin A (HlyA) is essential for triggering 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 isolates of the clinically-relevant globally disseminated E. coli sequence type 131 (ST131) clone, we also found that only HlyA+ strains kill human macrophages. Further characterization of these strains revealed variation in the levels of HlyA secretion, and consequent heterogeneity in their capacity to trigger human macrophage cell death. This impacted on mechanisms of cell death; in an ST131 strain that secretes low HlyA levels, cell death was partially dependent on the NLRP3 inflammasome. In contrast, in a UPEC strain secreting high amounts of HlyA, cell death was NLRP3 independent. To understand the functional relevance of this, the effect of HlyA levels on bladder colonization was assessed in a mouse UTI model. These studies revealed that fine-tuning of HlyA production likely dictates host evasion verses host protection. Overall, this study highlights that HlyA mediates UPEC-initiated inflammasome activation and cell death in human macrophages, and that an NLRP3-independent cell death pathway exists in these cells. We are currently investigating the alternative cell death pathway, as this likely has important implications for understanding UTI.