Sheryl S. Justice

Morphological plasticity as a bacterial survival strategy

Bacteria have evolved complex systems to maintain consistent cell morphologies. Nevertheless, in certain circumstances, bacteria alter this highly regulated process to transform into filamentous organisms. Accumulating evidence attributes important biological roles to filamentation in stressful environments, including, but not limited to, sites of interaction between pathogenic bacteria and their hosts. Filamentation could represent an intended response to specific environmental cues that promote survival amidst the threats of consumption and killing.

Filamentation by Escherichia coli subverts innate defenses during urinary tract infection.

To establish disease, an infecting organism must overcome a vast array of host defenses. During cystitis, uropathogenic Escherichia coli (UPEC) subvert innate defenses by invading superficial umbrella cells and rapidly increasing in numbers to form intracellular bacterial communities (IBCs). In the late stages of the IBC pathway, filamentous and bacillary UPEC detach from the biofilm-like IBC, fluxing out of this safe haven to colonize the surrounding epithelium and initiate subsequent generations of IBCs, and eventually they establish a quiescent intracellular reservoir. Filamentous UPEC are not observed during acute infection in mice lacking functional Toll-like receptor 4 (TLR4), suggesting that the filamentous phenotype arises in response to host innate immunity. We investigated SulA, a cell division inhibitor associated with the SOS response, to gain insight into the role of filamentous UPEC in pathogenesis. A transcriptional reporter from PsulA revealed spatial and temporal differences in expression within IBCs, and it was active in the majority of filamentous UPEC. Although UTI89 and UTI89 ΔsulA both formed first-generation IBCs equally well, UTI89 ΔsulA was sharply attenuated in formation of second-generation IBCs and establishment of the quiescent intracellular reservoir. The virulence of UTI89 ΔsulA was restored in TLR4-deficient mice, suggesting that filamentation facilitates the transition to additional rounds of IBC formation by subverting innate immune responses. These findings demonstrate that transient SulA-mediated inhibition of cell division is essential for UPEC virulence in the murine model of cystitis.

CD14- and toll-like receptor-dependent activation of bladder epithelial cells by lipopolysaccharide and type 1 piliated Escherichia coli

ABSTRACT The gram-negative bacterium Escherichia coli is the leading cause of urinary tract infection. The interaction between type 1 piliated E. coli and bladder epithelial cells leads to the rapid production of inflammatory mediators, such as interleukin-6 (IL-6) and IL-8. Conflicting reports have been published in the literature regarding the mechanism by which uroepithelial cells are activated by type 1 piliated E. coli. In particular, the role of lipopolysaccharide (LPS) in these responses has been an area of significant debate. Much of the data arguing against LPS-mediated activation of bladder epithelial cells have come from studies using a renal epithelial cell line as an in vitro model of the urinary epithelium. In this report, we analyzed three bladder epithelial cell lines and demonstrated that they all respond to LPS. Furthermore, the LPS responsivity of the cell lines directly correlated with their ability to generate IL-6 after E. coli stimulation. The LPS receptor complex utilized by the bladder epithelial cell lines included CD14 and Toll-like receptors, and signaling involved the activation of NF-κB and p38 mitogen-activated protein kinase. Also, reverse transcription-PCR analysis demonstrated that bladder epithelial cells express CD14 mRNA. Thus, the molecular machinery utilized by bladder epithelial cells for the recognition of E. coli is very similar to that described for traditional innate immune cells, such as macrophages. In contrast, the A498 renal epithelial cell line did not express CD14, was hyporesponsive to LPS stimulation, and demonstrated poor IL-6 responses to E. coli.

Intracellular Lifestyles and Immune Evasion Strategies of Uropathogenic Escherichia coli

Paradigms in the pathogenesis of urinary tract infections have shifted dramatically as a result of recent scientific revelations. Beyond extracellular colonization of the bladder luminal surface, as traditional clinical thinking would hold, uropathogenic bacteria direct a complex, intracellular cascade that shelters bacteria from host defenses and leads to persistent bacterial residence within the epithelium. After epithelial invasion, many organisms are promptly expelled by bladder epithelial cells; a minority establish a niche in the cytoplasm that results in the development of biofilm-like intracellular bacterial communities and serves as the primary location for bacterial expansion. Exfoliation of the superficial epithelial layer acts to reduce the bacterial load but facilitates chronic residence of small nests of bacteria that later reemerge to cause some episodes of recurrent cystitis, a familiar clinical scenario in otherwise healthy women. Advances in both in vitro and animal models of cystitis promise to provide insights into the bacterial and host transcriptional and biochemical pathways that define these pathogenic stages.

Periplasmic Peptidyl Prolyl cis-trans Isomerases Are Not Essential for Viability, but SurA Is Required for Pilus Biogenesis in Escherichia coli

In Escherichia coli, FkpA, PpiA, PpiD, and SurA are the four known periplasmic cis-trans prolyl isomerases. These isomerases facilitate proper protein folding by increasing the rate of transition of proline residues between the cis and trans states. Genetic inactivation of all four periplasmic isomerases resulted in a viable strain that exhibited a decreased growth rate and increased susceptibility to certain antibiotics. Levels of the outer membrane proteins LamB and OmpA in the quadruple mutant were indistinguishable from those in the surA single mutant. In addition, expression of P and type 1 pili (adhesive organelles produced by uropathogenic strains of E. coli and assembled by the chaperone/usher pathway) were severely diminished in the absence of the four periplasmic isomerases. Maturation of the usher was significantly impaired in the outer membranes of strains devoid of all four periplasmic isomerases, resulting in a defect in pilus assembly. Moreover, this defect in pilus assembly and usher stability could be attributed to the absence of SurA. The data presented here suggest that the four periplasmic isomerases are not essential for growth under laboratory conditions but may have significant roles in survival in environmental and pathogenic niches, as indicated by the effect on pilus production.

Suppression of Bladder Epithelial Cytokine Responses by Uropathogenic Escherichia coli

ABSTRACT Urinary tract infections are most commonly caused by uropathogenic strains of Escherichia coli (UPEC), which invade superficial bladder epithelial cells via a type 1 pilus-dependent mechanism. Inside these epithelial cells, UPEC organisms multiply to high numbers to form intracellular bacterial communities, allowing them to avoid immune detection. Bladder epithelial cells produce interleukin-6 (IL-6) and IL-8 in response to laboratory strains of E. coli in vitro. We investigated the ability of UPEC to alter epithelial cytokine signaling by examining the in vitro responses of bladder epithelial cell lines to the cystitis strains UTI89 and NU14. The cystitis strains induced significantly less IL-6 than did the laboratory E. coli strain MG1655 from 5637 and T24 bladder epithelial cells. The cystitis strains also suppressed epithelial cytokine responses to exogenous lipopolysaccharide (LPS) and to laboratory E. coli. We found that insertional mutations in the rfa and rfb operons and in the surA gene all abolished the ability of UTI89 to suppress cytokine induction. The rfa and rfb operons encode LPS biosynthetic genes, while surA encodes a periplasmic cis-trans prolyl isomerase important in the biogenesis of outer membrane proteins. We conclude that, in this in vitro model system, cystitis strains of UPEC have genes encoding factors that suppress proinflammatory cytokine production by bladder epithelial cells.

Maturation of intracellular Escherichia coli communities requires SurA.

ABSTRACT Escherichia coli is the most common cause of community-acquired urinary tract infection (UTI). During murine cystitis, uropathogenic E. coli (UPEC) utilizes type 1 pili to bind and invade superficial bladder epithelial cells. UPEC then replicates within to form intracellular bacterial communities (IBCs), a process whose genetic determinants are as yet undefined. In this study, we investigated the role of SurA in the UPEC pathogenic cascade. SurA is a periplasmic prolyl isomerase/chaperone that facilitates outer membrane protein biogenesis and pilus assembly in E. coli. Invasion into bladder epithelial cells was disproportionately reduced when surA was genetically disrupted in the UPEC strain UTI89, demonstrating that binding alone is not sufficient for invasion. In a murine cystitis model, UTI89 surA::kan was unable to persist in the urinary tract. Complementation of UTI89 surA::kan with a plasmid (pDH15) containing surA under the control of an arabinose-inducible promoter restored in vivo binding and invasion events. However, the absence of arabinose within the mouse bladder resulted in depletion of SurA after invasion of the bacteria into the superficial epithelial cells. Under these conditions, invasion by UTI89/pDH15 surA::kan was normal, but in contrast to UTI89, UTI89/pDH15 surA::kan formed intracellular collections that contained fewer bacteria, were loosely organized, and lacked the normal transition to a densely packed, coccoid morphology. Our data argue that SurA is required within bladder epithelial cells for UPEC to undergo the morphological changes that underlie IBC maturation and completion of the UTI pathogenic cascade.

Polysaccharide Capsule and Sialic Acid-Mediated Regulation Promote Biofilm-Like Intracellular Bacterial Communities during Cystitis

ABSTRACT Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs). A murine UTI model has revealed an infection cascade whereby UPEC undergoes cycles of invasion of the bladder epithelium, intracellular proliferation in polysaccharide-containing biofilm-like masses called intracellular bacterial communities (IBC), and then dispersal into the bladder lumen to initiate further rounds of epithelial colonization and invasion. We predicted that the UPEC K1 polysaccharide capsule is a key constituent of the IBC matrix. Compared to prototypic E. coli K1 strain UTI89, a capsule assembly mutant had a fitness defect in functionally TLR4+ and TLR4− mice, suggesting a protective role of capsule in inflamed and noninflamed hosts. K1 capsule assembly and synthesis mutants had dramatically reduced IBC formation, demonstrating the common requirement for K1 polysaccharide in IBC development. The capsule assembly mutant appeared dispersed in the cytoplasm of the bladder epithelial cells and failed to undergo high-density intracellular replication during later stages of infection, when the wild-type strain continued to form serial generations of IBC. Deletion of the sialic acid regulator gene nanR partially restored IBC formation in the capsule assembly mutant. These data suggest that capsule is necessary for efficient IBC formation and that aberrant sialic acid accumulation, resulting from disruption of K1 capsule assembly, produces a NanR-mediated defect in intracellular proliferation and IBC development. Together, these data demonstrate the complex but important roles of UPEC polysaccharide encapsulation and sialic acid signaling in multiple stages of UTI pathogenesis.

Ribonuclease 7 is a potent antimicrobial peptide within the human urinary tract

Although the urinary tract is constantly challenged by microbial invasion, it remains free from colonization. Although little is known about how the urinary tract maintains sterility, the presence of antimicrobial peptides (AMPs) in the urine suggests that they may play a role in its protection from infection. Ribonuclease 7 (RNase 7) is a potent AMP that was first identified in the skin. Here, we characterize the expression and relevance of RNase 7 in the human kidney and urinary tract. Using RNA isolated from healthy human tissue, we performed quantitative real-time PCR and found basal RNASE7 expression in kidney and bladder tissue. Immunohistochemical and immunofluorescent analysis localized RNase 7 to the urothelium of the bladder, ureter, and the intercalated cells of the collecting tubules. In control urine samples from healthy individuals, the concentration of RNase 7 was found to be in the low micromolar range; very abundant for an AMP. Antibacterial neutralization assays showed that urinary RNase 7 has potent antimicrobial properties against Gram-negative and Gram-positive uropathogenic bacteria. Thus, RNase 7 is expressed in the human kidney and urinary tract and it may have an important antimicrobial role in maintaining tract sterility.

Morphological plasticity promotes resistance to phagocyte killing of uropathogenic Escherichia coli

Uropathogenic Escherichia coli proceed through a complex intracellular developmental pathway that includes multiple morphological changes. During intracellular growth within Toll-like receptor 4-activated superficial bladder epithelial cells, a subpopulation of uropathogenic E. coli initiates SulA-mediated filamentation. In this study, we directly investigated the role of bacterial morphology in the survival of uropathogenic E. coli from killing by phagocytes. We initially determined that both polymorphonuclear neutrophils and macrophages are recruited to murine bladder epithelium at times coincident with extracellular bacillary and filamentous uropathogenic E. coli. We further determined that bacillary uropathogenic E. coli were preferentially destroyed when mixed uropathogenic E. coli populations were challenged with cultured murine macrophages in vitro. Consistent with studies using elliptical-shaped polymers, the initial point of contact between the phagocyte and filamentous uropathogenic E. coli influenced the efficacy of internalization. These findings demonstrate that filamentous morphology provides a selective advantage for uropathogenic E. coli evasion of killing by phagocytes and defines a mechanism for the essential role for SulA during bacterial cystitis. Thus, morphological plasticity can be viewed as a distinct class of mechanism used by bacterial pathogens to subvert host immunity.