SummaryMany respiratory pathogens establish persistent infection or a carrier state in the human nasopharynx without overt disease symptoms but the presence of these in the lungs usually results in disease. Although the anatomy and microenvironments between nasopharynx and lungs are different, a virulence factor with an organ-specific function in the colonization of the nasopharynx is unknown. In contrast to the severity of pertussis and mortality in non-vaccinated young children, Bordetella pertussis results in milder and prolonged cough in vaccinated adolescents and adults. Individuals harbouring bacteria in the nasopharynx serve as reservoirs for intrafamilial and nosocomial transmission. We show that the Bps polysaccharide of B. pertussis is critical for initial colonization of the mouse nose and the trachea but not of the lungs. Our data reveal a biofilm lifestyle for B. pertussis in the nose and the requirement of Bps in this developmental process. Bps functions as an adhesin by promoting adherence of B. pertussis and Escherichia coli to human nasal but not to human lung epithelia. Patient serum specifically recognized Bps suggesting its expression during natural human infections. We describe the first bacterial factor that exhibits a differential role in colonization and adherence between the nasopharynx and the lungs.

Urinary tract infections (UTIs) are caused by uropathogenic Escherichia coli (UPEC) strains. In contrast to many enteric E. coli pathogroups, no genetic signature has been identified for UPEC strains. We conducted a high-resolution comparative genomic study using E. coli isolates collected from the urine of women suffering from frequent recurrent UTIs. These isolates were genetically diverse and varied in urovirulence, or the ability to infect the bladder of a mouse model of cystitis. Importantly, we found no set of genes, including previously defined putative urovirulence factors (PUFs), that were predictive of urovirulence. In addition, in some patients, the E. coli strain causing a recurrent UTI had fewer PUFs than the supplanted strain. In competitive experimental infections in mice, the supplanting strain was more efficient at colonizing the mouse bladder than the supplanted strain. Despite the lack of a clear genomic signature for urovirulence, comparative transcriptomic and phenotypic analyses revealed that the expression of key conserved functions during culture, such as motility and sugar metabolism, could be used to predict subsequent mouse bladder colonization. Taken together, our findings suggest that UTI risk and outcome may be determined by complex interactions between host susceptibility and the urovirulence potential of diverse bacterial strains.

Bordetella spp. form biofilms in the mouse nasopharynx, thereby providing a potential mechanism for establishing chronic infections in humans and animals. Filamentous hemagglutinin (FHA) is a major virulence factor of B. pertussis, the causative agent of the highly transmissible and infectious disease, pertussis. In this study, we dissected the role of FHA in the distinct biofilm developmental stages of B. pertussis on abiotic substrates and in the respiratory tract by employing a murine model of respiratory biofilms. Our results show that the lack of FHA reduced attachment and decreased accumulation of biofilm biomass on artificial surfaces. FHA contributes to biofilm development by promoting the formation of microcolonies. Absence of FHA from B. pertussis or antibody-mediated blockade of surface-associated FHA impaired the attachment of bacteria to the biofilm community. Exogenous addition of FHA resulted in a dose-dependent inhibitory effect on bacterial association with the biofilms. Furthermore, we show that FHA is important for the structural integrity of biofilms formed on the mouse nose and trachea. Together, these results strongly support the hypothesis that FHA promotes the formation and maintenance of biofilms by mediating cell-substrate and inter-bacterial adhesions. These discoveries highlight FHA as a key factor in establishing structured biofilm communities in the respiratory tract.

SignificanceThe emergence of multidrug-resistant bacteria, including uropathogenic Escherichia coli (UPEC), makes the development of targeted antivirulence therapeutics a critical focus of research. During urinary tract infections (UTIs), UPEC uses chaperone–usher pathway pili tipped with an array of adhesins that recognize distinct receptors with sterochemical specificity to facilitate persistence in various tissues and habitats. We used an interdisciplinary approach driven by structural biology and synthetic glycoside chemistry to design and optimize glycomimetic inhibitors of the UPEC adhesin FmlH. These inhibitors competitively blocked FmlH in vitro, in in vivo mouse UTI models, and in ex vivo healthy human kidney tissue. This work demonstrates the utility of structure-driven drug design in the effort to develop antivirulence therapeutic compounds.

Uropathogenic Escherichia coli (UPEC) is the primary etiological agent of over 85% of community-acquired urinary tract infections (UTIs). Mouse models of infection have shown that UPEC can invade bladder epithelial cells in a type 1 pilus-dependent mechanism, avoid a TLR4-mediated exocytic process, and escape into the host cell cytoplasm. The internalized UPEC can clonally replicate into biofilm-like intracellular bacterial communities (IBCs) of thousands of bacteria while avoiding many host clearance mechanisms. Importantly, IBCs have been documented in urine from women and children suffering acute UTI. To understand this protected bacterial niche, we elucidated the transcriptional profile of bacteria within IBCs using microarrays. We delineated the upregulation within the IBC of genes involved in iron acquisition, metabolism, and transport. Interestingly, lacZ was highly upregulated, suggesting that bacteria were sensing and/or utilizing a galactoside for metabolism in the IBC. A ΔlacZ strain displayed significantly smaller IBCs than the wild-type strain and was attenuated during competitive infection with a wild-type strain. Similarly, a galK mutant resulted in smaller IBCs and attenuated infection. Further, analysis of the highly upregulated gene yeaR revealed that this gene contributes to oxidative stress resistance and type 1 pilus production. These results suggest that bacteria within the IBC are under oxidative stress and, consistent with previous reports, utilize nonglucose carbon metabolites. Better understanding of the bacterial mechanisms used for IBC development and establishment of infection may give insights into development of novel anti-virulence strategies.