RESUMO
Uropathogenic Escherichia coli (UPEC) modulates aspects of the innate immune response during urinary tract infection to facilitate bacterial invasion of the bladder epithelium, a requirement for the propagation of infection. For example, UPEC-encoded YbcL suppresses the traversal of bladder epithelia by neutrophils in both an in vitro model and an in vivo murine cystitis model. The suppressive activity of YbcL requires liberation from the bacterial periplasm, though the mechanism of release is undefined. Here we present findings on the site of action of YbcL and demonstrate a novel mode of secretion for a UPEC exoprotein. Suppression of neutrophil migration by purified YbcL(UTI), encoded by cystitis isolate UTI89, required the presence of a uroepithelial layer; YbcL(UTI) did not inhibit neutrophil chemotaxis directly. YbcL(UTI) was released to a greater extent during UPEC infection of uroepithelial cells than during that of neutrophils. Release of YbcL(UTI) was maximal when UPEC and bladder epithelial cells were in close proximity. Established modes of secretion, including outer membrane vesicles, the type II secretion system, and the type IV pilus, were dispensable for YbcL(UTI) release from UPEC. Instead, YbcL(UTI) was liberated during bacterial death, which was augmented upon exposure to bladder epithelial cells, as confirmed by detection of bacterial cytoplasmic proteins and DNA in the supernatant and enumeration of bacteria with compromised membranes. As YbcL(UTI) acts on the uroepithelium to attenuate neutrophil migration, this mode of release may represent a type of altruistic cooperation within a UPEC population during colonization of the urinary tract.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli/metabolismo , Doenças do Sistema Imunitário , Imunossupressores/metabolismo , Transtornos Leucocíticos , Neutrófilos/efeitos dos fármacos , Escherichia coli Uropatogênica/imunologia , Adulto , Células Epiteliais/microbiologia , Humanos , Neutrófilos/fisiologia , Proteínas Periplásmicas/metabolismoRESUMO
The recruitment of immune cells from the periphery to the site of inflammation is an essential step in the innate immune response at any mucosal surface. During infection of the urinary bladder, polymorphonuclear leukocytes (PMN; neutrophils) migrate from the bloodstream and traverse the bladder epithelium. Failure to resolve infection in the absence of a neutrophilic response demonstrates the importance of PMN in bladder defense. To facilitate colonization of the bladder epithelium, uropathogenic Escherichia coli (UPEC), the causative agent of the majority of urinary tract infections (UTIs), dampen the acute inflammatory response using a variety of partially defined mechanisms. To further investigate the interplay between host and bacterial pathogen, we developed an in vitro model of this aspect of the innate immune response to UPEC. In the transuroepithelial neutrophil migration assay, a variation on the Boyden chamber, cultured bladder epithelial cells are grown to confluence on the underside of a permeable support. PMN are isolated from human venous blood and are applied to the basolateral side of the bladder epithelial cell layers. PMN migration representing the physiologically relevant basolateral-to-apical direction in response to bacterial infection or chemoattractant molecules is enumerated using a hemocytometer. This model can be used to investigate interactions between UPEC and eukaryotic cells as well as to interrogate the molecular requirements for the traversal of bladder epithelia by PMN. The transuroepithelial neutrophil migration model will further our understanding of the initial inflammatory response to UPEC in the bladder.
Assuntos
Movimento Celular/fisiologia , Técnicas Citológicas/métodos , Células Epiteliais/citologia , Neutrófilos/citologia , Bexiga Urinária/citologia , Movimento Celular/imunologia , Células Epiteliais/imunologia , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/patologia , Humanos , Imunidade Inata/imunologia , Neutrófilos/imunologia , Bexiga Urinária/imunologia , Escherichia coli Uropatogênica/imunologiaRESUMO
Uropathogenic Escherichia coli (UPEC) strains suppress the acute inflammatory response in the urinary tract to ensure access to the intracellular uroepithelial niche that supports the propagation of infection. Our understanding of this initial cross talk between host and pathogen is incomplete. Here we report the identification of a previously uncharacterized periplasmic protein, YbcL, encoded by UPEC that contributes to immune modulation in the urinary tract by suppressing acute neutrophil migration. In contrast to wild-type UPEC, an isogenic strain lacking ybcL expression (UTI89 ΔybcL) failed to suppress transepithelial polymorphonuclear leukocyte (PMN) migration in vitro, a defect complemented by expressing ybcL episomally. YbcL homologs are present in many E. coli genomes; expression of the YbcL variant encoded by nonpathogenic E. coli K-12 strain MG1655 (YbcL(MG)) failed to complement the UTI89 ΔybcL defect, whereas expression of the UPEC YbcL variant (YbcL(UTI)) in MG1655 conferred the capacity for suppressing PMN migration. This phenotypic difference was due to a single amino acid difference (V78T) between the two YbcL homologs, and a majority of clinical UPEC strains examined were found to encode the suppressive YbcL variant. Purified YbcL(UTI) protein suppressed PMN migration in response to live or killed MG1655, and YbcL(UTI) was detected in the supernatant during UPEC infection of bladder epithelial cells or PMNs. Lastly, early PMN influx to murine bladder tissue was augmented upon in vivo infection with UTI89 ΔybcL compared with wild-type UPEC. Our findings demonstrate a role for UPEC YbcL in suppression of the innate immune response during urinary tract infection.