Antibodies against Hemolysin and Cytotoxic Necrotizing Factor Type 1 (CNF1) Reduce Bladder Inflammation in a Mouse Model of Urinary Tract Infection with Toxigenic Uropathogenic Escherichia coli

Abstract

Uropathogenic Escherichia coli (UPEC) is the leading cause of cystitis. Cytotoxic necrotizing factor 1 (CNF1) and hemolysin (Hly) are toxins made by approximately 50% of UPEC isolates. CNF1 and Hly contribute to the robust inflammatory response in the bladders of mice challenged with UPEC strain CP9. We hypothesized that antibodies against CNF1 and/or Hly would reduce cystitis caused by CP9. To test this theory, we immunized female C3H/HeOuJ mice subcutaneously with a genetically derived Hly toxoid or genetically derived CNF1 toxoid plus sublethal doses of CNF1. We collected serum and observed increasing titers of specific and neutralizing antibodies against Hly or CNF1 over time. We challenged the mice intraurethrally with CP9 and euthanized them 24 h later. We observed 10-fold lower bacterial titers in the urine of Hly-immunized mice than in that of shamimmunized mice but no difference in kidney bacterial titers. Immunized mice also exhibited significantly less cystitis than shamimmunized mice. In CNF1-vaccinated mice, we detected neither a difference in urine or kidney bacterial titers nor a reduction in the severity of cystitis versus that of sham-immunized mice. We then passively administered an anti-CNF1 monoclonal antibody intraperitoneally to female C3H/HeOuJ mice prior to intraurethral challenge with CP9. Upon challenge, we noted no difference in colonization of the urine or kidney; however, cystitis was reduced significantly in mice treated with the anti-CNF1 antibody versus that in the bladders of mice given an isotype control antibody. Taken together, our data demonstrate that antibodies against CNF1 or Hly reduce the bladder pathology caused by UPEC. E scherichia coli is responsible for 70 to 80% of uncomplicated urinary tract infections (UTIs), which most commonly occur in women (1). Indeed, in 2007, UTIs accounted for 10.5 million ambulatory patient visits (2). The causal E. coli strains are called uropathogenic E. coli (UPEC), and they express an array of virulence factors that enable them to colonize the urinary tract, invade urothelial cells, and overcome host defenses. These microbial products include specialized adhesive structures (pili and fimbriae) and toxins (3-6). One such toxin, hemolysin (Hly), is expressed by 60% of UPEC strains, while another UPEC toxin, cytotoxic necrotizing factor 1 (CNF1), is made by about 40% of such isolates (7-9). The genes that encode these toxins and other virulence factors are often found clustered on pathogenicity islands (10).E. coli Hly can form pores in the membranes of a variety of mammalian cell types with subsequent lysis of these cells (11-13). Even at sublytic concentrations, Hly can modulate host cell signaling pathways, modify the host immune response, and cause cell death (14). An operon composed of four genes, hlyCABD, is required for the production and secretion of active Hly (15, 16). The hlyA gene encodes a 107-kDa heat-labile inactive Hly precursor or protoxin. HlyC is an acyltransferase that activates Hly by fatty acid acylation of two lysi...

Hemolysin of uropathogenic Escherichia coli: A cloak or a dagger?

“…A super-saturating transposon library generated to assess genes of importance in UPEC bacterial fitness, inoculated intravenously, did not recover transposon mutants of HlyA (100) . Although the authors do not specifically report each (1,101,102) . Characterization of the mechanism of HlyA pore formation or identification of a receptor for the toxin could not only lead to the development of therapeutic treatments for severe UPEC infections, but would benefit a much larger field, as members of the RTX toxin family span a variety of human pathogens, including those that infect the urinary tract, blood stream, intestinal tract and respiratory tract.…”

The Bacterial Toxin CNF1 Induces Activation and Maturation of Human Monocyte-Derived Dendritic Cells

“…In recent years, pioneering studies have provided evidence that CNF1 has the capacity to promote inflammation, stimulate host immunity and function as a potent immunoadjuvant [ 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 ]. Nevertheless, the immunostimulatory properties of the toxin are not fully understood and little is known about its direct effects on different immune cell subsets.…”

“…Infection studies have further reported that CNF1 has the capacity to promote inflammation in vivo, indicating that the toxin also possesses immunostimulatory properties [ 10 , 11 , 12 , 13 ]. In line with this conclusion, CNF1 has been shown to be a potent immunoadjuvant that can augment antigen-specific adaptive immune responses and host protection [ 14 , 15 , 16 ].…”

Genomic, Physiologic, and Symbiotic Characterization of Serratia marcescens Strains Isolated from the Mosquito Anopheles stephensi

“…Direct contact between S. marcescens Db11 and infected C. elegans was required for killing effect, indicating that the function of hemolysins synergized other components in S. marcescens Db11 for pathogenicity (Kurz and Ewbank, 2000 ; Kurz et al, 2003 ). Moreover, hemolysins attacked various immune cells as an immune evasion strategy (Smith et al, 2015 ).…”