Though the bacterial opportunist Enterococcus faecalis causes a myriad of hospital-acquired infections (HAIs), including catheter-associated urinary tract infections (CAUTIs), little is known about the virulence mechanisms that it employs. However, the endocarditis- and biofilm-associated pilus (Ebp), a member of the sortase-assembled pilus family, was shown to play a role in a mouse model of E. faecalis ascending UTI. The Ebp pilus comprises the major EbpC shaft subunit and the EbpA and EbpB minor subunits. We investigated the biogenesis and function of Ebp pili in an experimental model of CAUTI using a panel of chromosomal pilin deletion mutants. A nonpiliated pilus knockout mutant (EbpABC− strain) was severely attenuated compared to its isogenic parent OG1RF in experimental CAUTI. In contrast, a nonpiliated ebpC deletion mutant (EbpC− strain) behaved similarly to OG1RF in vivo because it expressed EbpA and EbpB. Deletion of the minor pilin gene ebpA or ebpB perturbed pilus biogenesis and led to defects in experimental CAUTI. We discovered that the function of Ebp pili in vivo depended on a predicted metal ion-dependent adhesion site (MIDAS) motif in EbpA’s von Willebrand factor A domain, a common protein domain among the tip subunits of sortase-assembled pili. Thus, this study identified the Ebp pilus as a virulence factor in E. faecalis CAUTI and also defined the molecular basis of this function, critical knowledge for the rational development of targeted therapeutics.

Summary Uropathogenic Escherichia coli (UPEC), the causative agent of approximately 85% of urinary tract infections (UTI), is a major health concern primarily affecting women. During infection, neutrophils infiltrate the bladder, but the mechanism of recruitment is not well understood. Here, we investigated the role of UPEC-induced cytokine production in neutrophil recruitment and UTI progression. We first examined the kinetics of cytokine expression during UPEC infection of the bladder, and their contribution to neutrophil recruitment. We found that UPEC infection induces expression of several pro-inflammatory cytokines including granulocyte colony-stimulating factor (G-CSF, CSF-3), not previously known to be involved in the host response to UTI. G-CSF induces neutrophil emigration from the bone marrow; these cells are thought to be critical for bacterial clearance during infection. Upon neutralization of G-CSF during UPEC infection, we found fewer circulating neutrophils, decreased neutrophil infiltration into the bladder and, paradoxically, a decreased bacterial burden in the bladder. However, depletion of G-CSF resulted in a corresponding increase in macrophage-activating cytokines, such as monocyte chemotactic protein-1 (MCP-1, CCL-2) and Il-1β, which may be key in host response to UPEC infection, potentially resolving the paradoxical decreased bacterial burden. Thus, G-CSF acts in a previously unrecognized role to modulate the host inflammatory response during UPEC infection.

Urinary tract infections (UTI) are among the most common bacterial infections in humans, affecting millions of people every year. UTI cause significant morbidity in women throughout their lifespan, in infant boys, in older men, in individuals with underlying urinary tract abnormalities, and in those that require long-term urethral catheterization, such as patients with spinal cord injuries or incapacitated individuals living in nursing homes. Serious sequelae include frequent recurrences, pyelonephritis with sepsis, renal damage in young children, pre-term birth, and complications of frequent antimicrobial use including high-level antibiotic resistance and Clostridium difficile colitis. Uropathogenic E. coli (UPEC) cause the vast majority of UTI, but less common pathogens such as Enterococcus faecalis and other enterococci frequently take advantage of an abnormal or catheterized urinary tract to cause opportunistic infections. While antibiotic therapy has historically been very successful in controlling UTI, the high rate of recurrence remains a major problem, and many individuals suffer from chronically recurring UTI, requiring long-term prophylactic antibiotic regimens to prevent recurrent UTI. Furthermore, the global emergence of multi-drug resistant UPEC in the past ten years spotlights the need for alternative therapeutic and preventative strategies to combat UTI, including anti-infective drug therapies and vaccines. In this chapter, we review recent advances in the field of UTI pathogenesis, with an emphasis on the identification of promising drug and vaccine targets. We then discuss the development of new UTI drugs and vaccines, highlighting the challenges these approaches face and the need for a greater understanding of urinary tract mucosal immunity.

bEnterococci commonly cause hospital-acquired infections, such as infective endocarditis and catheter-associated urinary tract infections. In animal models of these infections, a long hairlike extracellular protein fiber known as the endocarditis-and biofilm-associated (Ebp) pilus is an important virulence factor for Enterococcus faecalis. For Ebp and other sortase-assembled pili, the pilus-associated sortases are essential for fiber formation as they create covalent isopeptide bonds between the sortase recognition motif and the pilin-like motif of the pilus subunits. However, the molecular requirements governing the incorporation of the three pilus subunits (EbpA, EbpB, and EbpC) have not been investigated in E. faecalis. Here, we show that a Lys residue within the pilin-like motif of the EbpC subunit was necessary for EbpC polymerization. However, incorporation of EbpA into the pilus fiber only required its sortase recognition motif (LPXTG), while incorporation of EbpB only required its pilin-like motif. Only the sortase recognition motif would be required for incorporation of the pilus tip subunit, while incorporation of the base subunit would only require the pilin recognition motif. Thus, these data support a model with EbpA at the tip and EbpB at the base of an EbpC polymer. In addition, the housekeeping sortase, SrtA, was found to process EbpB and its predicted catalytic Cys residue was required for efficient cell wall anchoring of mature Ebp pili. Thus, we have defined molecular interactions involved in fiber polymerization, minor subunit organization, and pilus subcellular compartmentalization in the E. faecalis Ebp pilus system. These studies advance our understanding of unique molecular mechanisms of sortase-assembled pilus biogenesis.

While many virulence factors promoting Streptococcus pyogenes invasive disease have been described, specific streptococcal factors and host properties influencing asymptomatic mucosal carriage remain uncertain. To address the need for a refined model of prolonged S. pyogenes asymptomatic mucosal colonization, we have adapted a preestrogenized murine vaginal colonization model for S. pyogenes. In this model, derivatives of strains HSC5, SF370, JRS4, NZ131, and MEW123 established a reproducible, asymptomatic colonization of the vaginal mucosa over a period of typically 3 to 4 weeks' duration at a relatively high colonization efficiency. Prior treatment with estradiol prolonged streptococcal colonization and was associated with reduced inflammation in the colonized vaginal epithelium as well as a decreased leukocyte presence in vaginal fluid compared to the levels of inflammation and leukocyte presence in non-estradiol-treated control mice. The utility of our model for investigating S. pyogenes factors contributing to mucosal carriage was verified, as a mutant with a mutation in the transcriptional regulator catabolite control protein A (CcpA) demonstrated significant impairment in vaginal colonization. An assessment of in vivo transcriptional activity in the CcpA ؊ strain for several known CcpA-regulated genes identified significantly elevated transcription of lactate oxidase (lctO) correlating with excessive generation of hydrogen peroxide to self-lethal levels. Deletion of lctO did not impair colonization, but deletion of lctO in a CcpA ؊ strain prolonged carriage, exceeding even that of the wild-type strain. Thus, while LctO is not essential for vaginal colonization, its dysregulation is deleterious, highlighting the critical role of CcpA in promoting mucosal colonization. The vaginal colonization model should prove effective for future analyses of S. pyogenes mucosal colonization. The Gram-positive pathogen Streptococcus pyogenes, or group A streptococcus, is responsible for a wide variety of clinical manifestations ranging from the relatively benign and superficial otitis media, impetigo, and pharyngitis to less common and more invasive conditions, including necrotizing fasciitis and toxic shock syndrome, in addition to the postinfectious complications rheumatic fever and glomerulonephritis (1). The ability to produce such a diversity of infections in so many different tissue compartments is testament to the extensive plasticity of the S. pyogenes transcriptome and an abundance of secreted virulence factors (2). While not considered normal human flora, S. pyogenes can be identified as a colonizer of mucosal surfaces of the oropharynx, rectum, and vaginal mucosa, and a prolonged asymptomatic carriage state can develop (3-7). How S. pyogenes carriage exists on a mucosal surface without inducing disease is poorly understood, but the issue is significant in terms of gaining an improved understanding of host-pathogen interactions and the regulation of mucosal immunity. Furthermore, asymptomatic oropharyngeal carri...