Design, construction, and in vivo augmentation of a complex gut microbiome.

Efforts to model the human gut microbiome in mice have led to important insights into the mechanisms of host-microbe interactions. However, the model communities studied to date have been defined or complex, but not both, limiting their utility. Here, we construct and characterize in vitro a defined community of 104 bacterial species composed of the most common taxa from the human gut microbiota (hCom1). We then used an iterative experimental process to fill open niches: germ-free mice were colonized with hCom1 and then challenged with a human fecal sample. We identified new species that engrafted following fecal challenge and added them to hCom1, yielding hCom2. In gnotobiotic mice, hCom2 exhibited increased stability to fecal challenge and robust colonization resistance against pathogenic Escherichia coli. Mice colonized by either hCom2 or a human fecal community are phenotypically similar, suggesting that this consortium will enable a mechanistic interrogation of species and genes on microbiome-associated phenotypes.

Host-specific induction of Escherichia coli fitness genes during human urinary tract infection.

Uropathogenic Escherichia coli (UPEC) is the predominant etiological agent of uncomplicated urinary tract infection (UTI), manifested by inflammation of the urinary bladder, in humans and is a major global public health concern. Molecular pathogenesis of UPEC has been primarily examined using murine models of UTI. Translational research to develop novel therapeutics against this major pathogen, which is becoming increasingly antibiotic resistant, requires a thorough understanding of mechanisms involved in pathogenesis during human UTIs. Total RNA-sequencing (RNA-seq) and comparative transcriptional analysis of UTI samples to the UPEC isolates cultured in human urine and laboratory medium were used to identify novel fitness genes that were specifically expressed during human infection. Evidence for UPEC genes involved in ion transport, including copper efflux, nickel and potassium import systems, as key fitness factors in uropathogenesis were generated using an experimental model of UTI. Translational application of this study was investigated by targeting Cus, a bacterial copper efflux system. Copper supplementation in drinking water reduces E. coli colonization in the urinary bladder of mice. Additionally, our results suggest that anaerobic processes in UPEC are involved in promoting fitness during UTI in humans. In summary, RNA-seq was used to establish the transcriptional signature in UPEC during naturally occurring, community acquired UTI in women and multiple novel fitness genes used by UPEC during human infection were identified. The repertoire of UPEC genes involved in UTI presented here will facilitate further translational studies to develop innovative strategies against UTI caused by UPEC.

Blocking yersiniabactin import attenuates extraintestinal pathogenic Escherichia coli in cystitis and pyelonephritis and represents a novel target to prevent urinary tract infection.

The emergence and spread of extended-spectrum beta-lactamases and carbapenemases among common bacterial pathogens are threatening our ability to treat routine hospital- and community-acquired infections. With the pipeline for new antibiotics virtually empty, there is an urgent need to develop novel therapeutics. Bacteria require iron to establish infection, and specialized pathogen-associated iron acquisition systems like yersiniabactin, common among pathogenic species in the family Enterobacteriaceae, including multidrug-resistant Klebsiella pneumoniae and pathogenic Escherichia coli, represent potentially novel therapeutic targets. Although the yersiniabactin system was recently identified as a vaccine target for uropathogenic E. coli (UPEC)-mediated urinary tract infection (UTI), its contribution to UPEC pathogenesis is unknown. Using an E. coli mutant (strain 536ΔfyuA) unable to acquire yersiniabactin during infection, we established the yersiniabactin receptor as a UPEC virulence factor during cystitis and pyelonephritis, a fitness factor during bacteremia, and a surface-accessible target of the experimental FyuA vaccine. In addition, we determined through transcriptome sequencing (RNA-seq) analyses of RNA from E. coli causing cystitis in women that iron acquisition systems, including the yersiniabactin system, are highly expressed by bacteria during natural uncomplicated UTI. Given that yersiniabactin contributes to the virulence of several pathogenic species in the family Enterobacteriaceae, including UPEC, and is frequently associated with multidrug-resistant strains, it represents a promising novel target to combat antibiotic-resistant infections.

Immunization with the yersiniabactin receptor, FyuA, protects against pyelonephritis in a murine model of urinary tract infection.

Urinary tract infections (UTI) are common and represent a substantial economic and public health burden. Roughly 80% of these infections are caused by a heterogeneous group of uropathogenic Escherichia coli (UPEC) strains. Antibiotics are standard therapy for UTI, but a rise in antibiotic resistance has complicated treatment, making the development of a UTI vaccine more urgent. Iron receptors are a promising new class of vaccine targets for UTI, as UPEC require iron to colonize the iron-limited host urinary tract and genes encoding iron acquisition systems are highly expressed during infection. Previously, three of six UPEC siderophore and heme receptors were identified as vaccine candidates by intranasal immunization in a murine model of ascending UTI. To complete the assessment of iron receptors as vaccine candidates, an additional six UPEC iron receptors were evaluated. Of the six vaccine candidates tested in this study (FyuA, FitA, IroN, the gene product of the CFT073 locus c0294, and two truncated derivatives of ChuA), only FyuA provided significant protection (P = 0.0018) against UPEC colonization. Intranasal immunization induced a robust and long-lived humoral immune response. In addition, the levels of FyuA-specific serum IgG correlated with bacterial loads in the kidneys [Spearman's rank correlation coefficient ρ(14) = -0.72, P = 0.0018], providing a surrogate of protection. FyuA is the fourth UPEC iron receptor to be identified from our screens, in addition to IutA, Hma, and IreA, which were previously demonstrated to elicit protection against UPEC challenge. Together, these iron receptor antigens will facilitate the development of a broadly protective, multivalent UTI vaccine to effectively target diverse strains of UPEC.

Redundancy and specificity of Escherichia coli iron acquisition systems during urinary tract infection.

Uropathogenic Escherichia coli (UPEC), the predominant cause of uncomplicated urinary tract infection (UTI), utilizes an array of outer membrane iron receptors to facilitate siderophore and heme import from within the iron-limited urinary tract. While these systems are required for UPEC in vivo fitness and are assumed to be functionally redundant, the relative contributions of specific receptors to pathogenesis are unknown. To delineate the relative roles of distinct UPEC iron acquisition systems in UTI, isogenic mutants in UPEC strain CFT073 or 536 lacking individual receptors were competed against one another in vivo in a series of mixed infections. When combinations of up to four mutants were coinoculated using a CBA/J mouse model of ascending UTI, catecholate receptor mutants (ΔfepA, Δiha, and ΔiroN mutants) were equally fit, suggesting redundant function. However, noncatecholate siderophore receptor mutants, including the ΔiutA aerobactin receptor mutant and the ΔfyuA yersiniabactin receptor mutant, were frequently outcompeted by coinoculated mutants, indicating that these systems contribute more significantly to UPEC iron acquisition in vivo. A tissue-specific preference for heme acquisition was also observed, as a heme uptake-deficient Δhma ΔchuA double mutant was outcompeted by siderophore receptor mutants specifically during kidney colonization. The relative contribution of each receptor to UTI only partially correlated with in vivo levels of receptor gene expression, indicating that other factors likely contributed to the observed fitness differences. Overall, our results suggest that UPEC iron receptors provide both functional redundancy and niche specificity for this pathogen as it colonizes distinct sites within the urinary tract.

Preventing urinary tract infection: progress toward an effective Escherichia coli vaccine.

Uncomplicated urinary tract infections (UTIs) are common, with nearly half of all women experiencing at least one UTI in their lifetime. This high frequency of infection results in huge annual economic costs, decreased workforce productivity and high patient morbidity. At least 80% of these infections are caused by uropathogenic Escherichia coli (UPEC). UPEC can reside side by side with commensal strains in the gastrointestinal tract and gain access to the bladder via colonization of the urethra. Antibiotics represent the current standard treatment for UTI; however, even after treatment, patients frequently suffer from recurrent infection with the same or different strains. In addition, successful long-term treatment has been complicated by a rise in both the number of antibiotic-resistant strains and the prevalence of antibiotic-resistance mechanisms. As a result, preventative approaches to UTI, such as vaccination, have been sought. This review summarizes recent advances in UPEC vaccine development and outlines future directions for the field.