Unraveling CNF1-directed ExPEC colonization and reservoir formation to leverage novel therapies – ExPECtation

Urinary tract infections (UTI) are primarily caused by uropathogenic strains of E. coli (UPEC) that are genetically related to extra-intestinal pathogenic E. coli (ExPEC). UTI are common infections, impacting >150 million individuals annually, with a strong sex bias towards female incidence, and a propensity to recur frequently. This issue is compounded by the fact that although antibiotics are the primary therapy to eliminate acute infection, they have a limited impact against several recently emerged pandemic ExPEC sequence types (ST) that express multiple resistance mechanisms. The increase in antibiotic resistance among ExPEC, and the prospect of bacterial infections becoming unresponsive to antibiotic treatment, is considered one of the most serious threats to global health today.

The capacity of ExPEC to establish and persist in reservoirs in host tissue, in both the bladder (as pathogens) and the gastrointestinal tract (as commensals) is a key to the frequency and recurrence of UTI. We uncovered that CNF1, an AB-like toxin found in many ExPEC strains, potentially facilitates intestinal colonization. A large bioinformatic survey of genomes of pathogenic strains of E. coli found that cnf1-positive strains in the sequence type (ST)131 sublineage H30Rx/C2 have dramatically increased in proportion in recent years. E. coli ST131 is a pandemic multidrug resistant strain exhibiting unprecedented global expansion in the last decade and is the predominant E. coli isolated from extra-intestinal infections. E. coli ST131 also displays a remarkable capacity to colonize the gastrointestinal tract and dominates in this niche among E. coli species present. Together, this suggests that CNF1 imparts a fitness advantage to ExPEC, in vivo. Our preliminary data show that CNF1-expressing ExPEC outcompete isogenic strains lacking CNF1 in the intestine and the bladder, although, interestingly the kinetics of competition differ between these organs, suggesting that CNF1-mediated benefit is specific to each tissue. This would imply that CNF1 imparts advantages to both commensal and infectious lifestyles. To develop non-antibiotic based, host-directed therapeutics, we used a high throughput screen to discover novel chemical compounds that protect host cells against CNF1 cytotoxicity. We found that a specific compound can reduce the bacterial burden of CNF1-expressing E. coli ST131 H30Rx/C2 in the intestine.

Using a synergistic combination of cell biology and immunology approaches, and including sex as a biological variable, the aim of the proposal ExPECtation is to i) determine how CNF1 mediates bacterial interactions with host epithelial surfaces, ii) assess whether CNF1 specifically modulates host innate and adaptive immunity, and iii) define conditions in which our newly identified host-targeted anti-infective compound can specifically reduce or eliminate reservoirs in the intestine and/or during UTI in the bladder. With respect to future impact of this proposal, our results will provide foundational data to support potential application of a new therapeutic to treat recurrent UTIs. Given the extraordinary incidence of this infection, this would be expected to have broad and substantial benefits for health care systems and society. In addition, our compound has measured wide spectrum anti-infectious activity against a number of bacterial toxins and viruses, such as influenza A virus H1N1 and SARS-CoV-2. Thus, our study will provide essential knowledge for future development of a broad anti-infective therapy against multiple infections, including COVID-19.