Adenylate Cyclase and the Cyclic AMP Receptor Protein Modulate Stress Resistance and Virulence Capacity of Uropathogenic Escherichia coli

Abstract

In many bacteria, the second messenger cyclic AMP (cAMP) interacts with the transcription factor cAMP receptor protein (CRP), forming active cAMP-CRP complexes that can control a multitude of cellular activities, including expanded carbon source utilization, stress response pathways, and virulence. Here, we assessed the role of cAMP-CRP as a regulator of stress resistance and virulence in uropathogenic Escherichia coli (UPEC), the principal cause of urinary tract infections worldwide. Deletion of genes encoding either CRP or CyaA, the enzyme responsible for cAMP synthesis, attenuates the ability of UPEC to colonize the bladder in a mouse infection model, dependent on intact innate host defenses. UPEC mutants lacking cAMP-CRP grow normally in the presence of glucose but are unable to utilize alternate carbon sources like amino acids, the primary nutrients available to UPEC within the urinary tract. Relative to the wild-type UPEC isolate, the cyaA and crp deletion mutants are sensitive to nitrosative stress and the superoxide generator methyl viologen but remarkably resistant to hydrogen peroxide (H 2 O 2 ) and acid stress. In the mutant strains, H 2 O 2 resistance correlates with elevated catalase activity attributable in part to enhanced translation of the alternate sigma factor RpoS. Acid resistance was promoted by both RpoS-independent and RpoS-dependent mechanisms, including expression of the RpoS-regulated DNA-binding ferritin-like protein Dps. We conclude that balanced input from many cAMP-CRP-responsive elements, including RpoS, is critical to the ability of UPEC to handle the nutrient limitations and severe environmental stresses present within the mammalian urinary tract. U nder homeostatic conditions, the mammalian urinary tract is maintained as a sterile environment through the production of antimicrobial peptides and other toxic compounds, the bulk flow of urine, innate immune cell surveillance mechanisms, and nutrient limitations (1-5). However, select microbial pathogens are capable of colonizing and infecting this normally inhospitable niche. Uropathogenic Escherichia coli (UPEC) is the major cause of urinary tract infections (UTI) worldwide, affecting millions and requiring billions of dollars for diagnosis and treatment annually (6). To overcome host defenses and effectively colonize the urinary tract, UPEC employs a variety of virulence factors and stress resistance mechanisms, including adhesive and motility organelles that mediate attachment to and invasion of host cells, toxins that disarm innate immune responses, and multiple iron-scavenging proteins (1, 7-9). The ability to sense and prioritize the use of limited carbon sources within the nutrient-poor environment of the urinary tract is also likely critical to the success of UPEC, but our understanding of the impact that bacterial metabolic pathways have on the establishment and progression of a UTI is incomplete.Within the urinary tract, UPEC relies largely on the catabolism of small peptides and amino acids for survival and growth (4). U...

Fitness, Stress Resistance, and Extraintestinal Virulence in Escherichia coli

“…Extensive, rapid diversification of isolates has been reported to occur during extraintestinal infection in humans, with RpoS levels varying as a result of the trade-off between protection against stress and the preservation of metabolic properties (27). Such a balance between carbon source use and oxidative stress has also recently been reported to be critical in urinary tract infection (28).…”

Strengths and Limitations of Model Systems for the Study of Urinary Tract Infections and Related Pathologies

“…Due to mutations in the pattern recognition receptor TLR4 and possibly other immune regulators, C3H/HeJ mice are hyporesponsive to lipopolysaccharide and therefore have very limited inflammatory responses during experimental UTI (196,(199)(200)(201). Together, these observations indicate that TLR4-dependent innate host defenses within wild-type mice, and not nutrient availability per se, are the major factors that restrict colonization of the bladder by the cya and crp UPEC mutants (197).…”

“…Consequently, the use of surrogate hosts like C. elegans and zebrafish, or even larvae of the wax moth Galleria mellonella (250)(251)(252), is not so tangential to UTI research and has the potential to drive important discoveries in human patients. Findings made by using animal model systems can be complemented and extended in broth culture or on agar plates using genetic and biochemical assays that are designed to examine how UPEC mutants and wild-type strains handle specific stresses such as reactive oxygen species or serum components like complement (197,243). Mathematical and computer modeling of infectious processes and gene regulation in UPEC provides additional insight into the pathogenesis of UTIs and can suggest new hypotheses to be tested in vivo (46,253,254).…”

“…Mouse lines that vary in their susceptibility to UTI have helped identify host susceptibility factors and provide a platform for assessing the functionality of bacterial fitness determinants. Consider, for example, a recent analysis of isogenic UPEC mutants that lack the cya or crp gene (197). These genes enable UPEC to synthesize and respond to the second messenger cyclic AMP (cAMP).…”

“…In vitro, the UPEC cystitis isolate UTI89 requires both cya and crp to effectively utilize alternate carbon sources like amino acids, the principal nutrients available to UPEC within the urinary tract (96,197,198). In broth culture assays, the cya and crp mutants also have increased sensitivities to the damaging effects of reactive nitrogen species and superoxide radicals, akin to those generated by host inflammatory cells (197). In wild-type mice, the cya and crp mutants are each impaired in their ability to colonize the bladder, whereas in C3H/HeJ mice, the mutants are no longer defective.…”