Uropathogenic Escherichia coli (UPEC) is a major cause of urinary tract and bloodstream infections and possesses an array of virulence factors for colonization, survival, and persistence. One such factor is the polysaccharide K capsule. Among the different K capsule types, the K1 serotype is strongly associated with UPEC infection. In this study, we completely sequenced the K1 UPEC urosepsis strain PA45B and employed a novel combination of a lytic K1 capsule-specific phage, saturated Tn 5 transposon mutagenesis, and high-throughput transposon-directed insertion site sequencing (TraDIS) to identify the complement of genes required for capsule production. Our analysis identified known genes involved in capsule biosynthesis, as well as two additional regulatory genes ( mprA and lrhA ) that we characterized at the molecular level. Mutation of mprA resulted in protection against K1 phage-mediated killing, a phenotype restored by complementation. We also identified a significantly increased unidirectional Tn 5 insertion frequency upstream of the lrhA gene and showed that strong expression of LrhA induced by a constitutive Pcl promoter led to loss of capsule production. Further analysis revealed loss of MprA or overexpression of LrhA affected the transcription of capsule biosynthesis genes in PA45B and increased sensitivity to killing in whole blood. Similar phenotypes were also observed in UPEC strains UTI89 (K1) and CFT073 (K2), demonstrating that the effects were neither strain nor capsule type specific. Overall, this study defined the genome of a UPEC urosepsis isolate and identified and characterized two new regulatory factors that affect UPEC capsule production. IMPORTANCE Urinary tract infections (UTIs) are among the most common bacterial infections in humans and are primarily caused by uropathogenic Escherichia coli (UPEC). Many UPEC strains express a polysaccharide K capsule that provides protection against host innate immune factors and contributes to survival and persistence during infection. The K1 serotype is one example of a polysaccharide capsule type and is strongly associated with UPEC strains that cause UTIs, bloodstream infections, and meningitis. The number of UTIs caused by antibiotic-resistant UPEC is steadily increasing, highlighting the need to better understand factors (e.g., the capsule) that contribute to UPEC pathogenesis. This study describes the original and novel application of lytic capsule-specific phage killing, saturated Tn 5 transposon mutagenesis, and high-throughput transposon-directed insertion site sequencing to define the entire complement of genes required for capsule production in UPEC. Our comprehensive approach uncovered new genes involved in the regulation of this key virulence determinant.

UropathogenicEscherichia coli(UPEC) is the most common cause of urinary tract infection, which in some patients can develop into life-threatening urosepsis. Serum resistance is a key virulence trait of strains that cause urosepsis. Recently, we identified a novel method of serum resistance in patients withPseudomonas aeruginosalung infections, where patients possessed antibodies that inhibited complement-mediated killing (instead of protecting against infection). These inhibitory antibodies were of the IgG2 subtype, specific to the O-antigen component of lipopolysaccharide (LPS) and coated the bacterial surface, preventing bacterial lysis by complement. As this mechanism could apply to any Gram-negative bacterial infection, we hypothesized that inhibitory antibodies may represent an uncharacterized mechanism of serum resistance in UPEC. To test this, 45 urosepsis patients with paired blood culture UPEC isolates were screened for serum titers of IgG2 specific for their cognate strain’s LPS. Eleven patients had sufficiently high titers of the antibody to inhibit serum-mediated killing of UPEC isolates by pooled healthy control sera. Depletion of IgG or removal of O-antigen restored sensitivity of the isolates to the cognate patient serum. Importantly, the isolates from these 11 patients were more sensitive to killing by serum than isolates from patients with no inhibitory antibodies. This suggests the presence of inhibitory antibodies may have allowed these strains to infect the bloodstream. The high prevalence of patients with inhibitory antibodies (24%) suggests that this phenomenon is an important mechanism of UPEC serum resistance. LPS-specific inhibitory antibodies have now been identified against three Gram-negative pathogens that cause disparate diseases.IMPORTANCEDespite improvements in the early detection and management of sepsis, morbidity and mortality are still high. Infections of the urinary tract are one of the most frequent sources of sepsis withEscherichia colithe main causative agent. Serum resistance is vital for bacteria to infect the bloodstream. Here we report a novel method of serum resistance found in patients with UPEC-mediated sepsis. Antibodies in sera usually protect against infection, but here we found that 24% of patients expressed “inhibitory antibodies” capable of preventing serum-mediated killing of their infecting isolate. Our data suggest that these antibodies would allow otherwise serum-sensitive UPEC strains to cause sepsis. The high prevalence of patients with inhibitory antibodies in this cohort suggests that this is a widespread mechanism of resistance to complement-mediated killing in urosepsis patients, invoking the potential for the application of new methods to prevent and treat sepsis.

E. coli is a multifaceted pathogen of major significance to global human health and an important contributor to increasing antibiotic resistance. Given the paucity of therapies still effective against multidrug-resistant pathogenic E. coli strains, novel treatment and prevention strategies are urgently required. In this study, we defined the core and accessory components of the E. coli genome by examining a large collection of draft and completely sequenced strains available from public databases. This data set was mined by employing a reverse-vaccinology approach in combination with proteomics to identify putative broadly protective vaccine antigens. One such antigen was identified that was highly immunogenic and induced protection in a mouse model of bacteremia. Overall, our study provides a genomic and proteomic framework for the selection of novel vaccine antigens that could mediate broad protection against pathogenic E. coli .

Escherichia coli is a commensal or pathogenic bacterium that can survive in diverse environments. Adhesion to surfaces is essential for E. coli colonization, and thus it is important to understand the molecular mechanisms that promote this process in different niches. Autotransporter proteins are a class of cell-surface factor used by E. coli for adherence. Here we characterized the regulation and function of YeeJ, a poorly studied but widespread representative from an emerging class of autotransporter proteins, the inverse autotransporters (IAT). We showed that the yeeJ gene is present in ~40% of 96 completely sequenced E. coli genomes and that YeeJ exists as two length variants, albeit with no detectable functional differences. We demonstrated that YeeJ promotes biofilm formation in different settings through exposition at the cell-surface. We also showed that YeeJ contains a LysM domain that interacts with peptidoglycan and thus assists its localization into the outer membrane. Additionally, we identified the Polynucleotide Phosphorylase PNPase as a repressor of yeeJ transcription. Overall, our work provides new insight into YeeJ as a member of the recently defined IAT class, and contributes to our understanding of how commensal and pathogenic E. coli colonise their environments.

The bladder is innervated by primary afferent nerve fibres that detect bladder distension and, through projections into the spinal cord, provide sensory input to the central nervous system circuits regulating bladder sensation and function. Uropathogenic E. coli (UPEC) bacteria are the primary cause of urinary tract infection (UTI) in adults, inducing clinical symptoms characterised by exaggerated bladder sensation, including urgency, frequency, and pelvic pain. However, the mechanisms underlying UTI-induced modulation of bladder afferent function are yet to be explored. Here, we isolated supernatants from the bladders of female mice acutely infected with UPEC (strain CFT073), or those sham-treated with phosphate buffered saline. Supernatants were then applied into the bladder lumen of healthy donor mice, and multiunit bladder afferent nerve responses to distension measured ex-vivo. Supernatant constituents from UPEC or sham-treated mice were analysed using a mouse cytokine multiplex assay. Supernatants from UPEC-infected mice significantly enhanced bladder afferent firing to distension in the absence of changes in muscle compliance. Further evaluation revealed that UPEC supernatants exclusively sensitised high-threshold bladder mechanoreceptors to graded bladder distension and also recruited a population of “silent nociceptors” to become mechanosensitive, thereby amplifying bladder afferent responses to physiological stimuli. UPEC supernatants contained significantly elevated concentrations of a range of cytokines released from innate immune cells, including but not limited to TNF-α, IL-1β, IL-6, IL-17, IFN-gamma, and MCP-1. These data provide novel mechanistic insight into how UPEC-mediated UTI induces bladder hypersensitivity and the symptoms of frequency, urgency, and pelvic pain.

Urinary tract infection (UTI) caused by uropathogenic Escherichia coli (UPEC) engages interleukin-10 (IL-10) as an early innate immune response to regulate inflammation and promote the control of bladder infection. However, the mechanism of engagement of innate immunity by UPEC that leads to elicitation of IL-10 in the bladder is unknown. Here, we identify the major UPEC flagellar filament, FliC, as a key bacterial component sensed by the bladder innate immune system responsible for the induction of IL-10 synthesis. IL-10 responses of human as well as mouse bladder epithelial cell-monocyte cocultures were triggered by flagella of three major UPEC representative strains, CFT073, UTI89, and EC958. FliC purified to homogeneity induced IL-10 in vitro and in vivo as well as other functionally related cytokines, including IL-6. The genome-wide innate immunological context of FliC-induced IL-10 in the bladder was defined using RNA sequencing that revealed a network of transcriptional and antibacterial defenses comprising 1,400 genes that were induced by FliC. Of the FliC-responsive bladder transcriptome, altered expression of il10 and 808 additional genes were dependent on Toll-like receptor 5 (TLR5), according to analysis of TLR5-deficient mice. Examination of the potential of FliC and associated innate immune signature in the bladder to boost host defense, based on prophylactic or therapeutic administration to mice, revealed significant benefits for the control of UPEC. We conclude that detection of FliC through TLR5 triggers rapid IL-10 synthesis in the bladder, and FliC represents a potential immune modulator that might offer benefit for the treatment or prevention of UPEC UTI. IMPORTANCE Interleukin-10 is part of the immune response to urinary tract infection (UTI) due to E. coli, and it is important in the early control of infection in the bladder. Defining the mechanism of engagement of the immune system by the bacteria that enables the protective IL-10 response is critical to exploring how we might exploit this mechanism for new infection control strategies. In this study, we reveal part of the bacterial flagellar apparatus (FliC) is an important component that is sensed by and responsible for induction of IL-10 in the response to UPEC. We show this response occurs in a TLR5-dependent manner. Using infection prevention and control trials in mice infected with E. coli, this study also provides evidence that purified FliC might be of value in novel approaches for the treatment of UTI or in preventing infection by exploiting the FliC-triggered bladder transcriptome.