M. Chelsea Lane

Expression of flagella is coincident with uropathogenic Escherichia coli ascension to the upper urinary tract

Uropathogenic Escherichia coli (UPEC) cause most uncomplicated urinary tract infections (UTIs) in humans. Because UTIs are considered to occur in an ascending manner, flagellum-mediated motility has been suggested to contribute to virulence by enabling UPEC to disseminate to the upper urinary tract. Previous studies from our laboratory and others have demonstrated a modest yet important role for flagella during ascending UTI. To better understand the role of flagella in vivo, we used biophotonic imaging to monitor UPEC infection and temporospatial flagellin gene expression during ascending UTI. Using em7-lux (constitutive) and fliC-lux transcriptional fusions, we show that flagellin expression by UPEC coincides with ascension of the ureters and colonization of the kidney. The patterns of fliC luminescence observed in vitro and in vivo were also validated by comparative quantitative PCR. Because fliC expression appeared coincident during ascension, we reassessed the contribution of fliC to ascending UTI using a low-dose intraurethral model of ascending UTI. Although wild-type UPEC were able to establish infection in the bladder and kidneys by 6 hours postinoculation, fliC mutant bacteria were able to colonize the bladder but were significantly attenuated in the kidneys at this early time point. By 48 hours postinoculation, the fliC mutant bacteria were attenuated in the bladder and kidneys and were not detectable in the spleen. These data provide compelling evidence that wild-type UPEC express flagellin and presumably utilize flagellum-mediated motility during UTI to ascend to the upper urinary tract and disseminate within the host.

Role of motility in the colonization of uropathogenic Escherichia coli in the urinary tract

ABSTRACT Uropathogenic Escherichia coli (UPEC) causes most uncomplicated urinary tract infections (UTIs) in humans. Flagellum-mediated motility and chemotaxis have been suggested to contribute to virulence by enabling UPEC to escape host immune responses and disperse to new sites within the urinary tract. To evaluate their contribution to virulence, six separate flagellar mutations were constructed in UPEC strain CFT073. The mutants constructed were shown to have four different flagellar phenotypes: fliA and fliC mutants do not produce flagella; the flgM mutant has similar levels of extracellular flagellin as the wild type but exhibits less motility than the wild type; the motAB mutant is nonmotile; and the cheW and cheY mutants are motile but nonchemotactic. Virulence was assessed by transurethral independent challenges and cochallenges of CBA mice with the wild type and each mutant. CFU/ml of urine or CFU/g bladder or kidney was determined 3 days postinoculation for the independent challenges and at 6, 16, 48, 60, and 72 h postinoculation for the cochallenges. While these mutants colonized the urinary tract during independent challenge, each of the mutants was outcompeted by the wild-type strain to various degrees at specific time points during cochallenge. Altogether, these results suggest that flagella and flagellum-mediated motility/chemotaxis may not be absolutely required for virulence but that these traits contribute to the fitness of UPEC and therefore significantly enhance the pathogenesis of UTIs caused by UPEC.

Complex Interplay between Type 1 Fimbrial Expression and Flagellum-Mediated Motility of Uropathogenic Escherichia coli

Type 1 fimbriae and flagella have been previously shown to contribute to the virulence of uropathogenic Escherichia coli (UPEC) within the urinary tract. In this study, the relationship between motility and type 1 fimbrial expression was tested for UPEC strain CFT073 by examining the phenotypic effect of fimbrial expression on motility and the effect that induction of motility has on type 1 fimbrial expression. While constitutive expression of type 1 fimbriae resulted in a significant decrease in motility and flagellin expression (P < 0.0001), a loss of type 1 fimbrial expression did not result in increased motility. Additionally, hypermotility and flagellar gene over- and underexpression were not observed to affect the expression of type 1 fimbriae. Hence, it appeared that the relationship between type 1 fimbrial expression and motility is unidirectional, where the overexpression of type 1 fimbriae dramatically affects motility and flagellum expression but not vice versa. Moreover, the constitutive expression of type 1 fimbriae in UPEC cystitis isolate F11 and the laboratory strain E. coli K-12 MG1655 also resulted in decreased motility, suggesting that this phenomenon is not specific to CFT073 or UPEC in general. Lastly, by analyzing the repression of motility caused by constitutive type 1 fimbrial expression, it was concluded that the synthesis and presence of type 1 fimbriae at the bacterial surface is only partially responsible for the repression of motility, as evidenced by the partial restoration of motility in the CFT073 fim L-ON DeltafimAICDFGH mutant. Altogether, these data provide further insight into the complex interplay between type 1 fimbrial expression and flagellum-mediated motility.

Development of an Intranasal Vaccine To Prevent Urinary Tract Infection by Proteus mirabilis

ABSTRACT Proteus mirabilis commonly infects the complicated urinary tract and is associated with urolithiasis. Stone formation is caused by bacterial urease, which hydrolyzes urea to ammonia, causing local pH to rise, and leads to the subsequent precipitation of magnesium ammonium phosphate (struvite) and calcium phosphate (apatite) crystals. To prevent these infections, we vaccinated CBA mice with formalin-killed bacteria or purified mannose-resistant, Proteus-like (MR/P) fimbriae, a surface antigen expressed by P. mirabilis during experimental urinary tract infection, via four routes of immunization: subcutaneous, intranasal, transurethral, and oral. We assessed the efficacy of vaccination using the CBA mouse model of ascending urinary tract infection. Subcutaneous or intranasal immunization with formalin-killed bacteria and intranasal or transurethral immunization with purified MR/P fimbriae significantly protected CBA mice from ascending urinary tract infection by P. mirabilis (P < 0.05). To investigate the potential of MrpH, the MR/P fimbrial tip adhesin, as a vaccine, the mature MrpH peptide (residues 23 to 275, excluding the signal peptide), and the N-terminal receptor-binding domain of MrpH (residues 23 to 157) were overexpressed as C-terminal fusions to maltose-binding protein (MBP) and purified on amylose resins. Intranasal immunization of CBA mice with MBP-MrpH (residues 23 to 157) conferred effective protection against urinary tract infection by P. mirabilis (P < 0.002).

Role of the K2 Capsule in Escherichia coli Urinary Tract Infection and Serum Resistance

BACKGROUND Capsule expression may be important during ascending Escherichia coli urinary tract infections (UTIs). METHODS An isogenic ksl(k2)ABCDE mutant of extraintestinal pathogenic E. coli (ExPEC) strain CFT073 that could not synthesize the K2 capsule was compared with wild-type CFT073, to determine virulence in a murine model of ascending UTI and in vitro killing assays. RESULTS No significant differences were observed regarding the abilities of the mutant and the wild-type CFT073 strains to colonize the murine urinary tract in single-challenge infection experiments. However, in competitive-colonization experiments, the mutant was significantly outcompeted by the wild-type strain in urine and the kidneys. The mutant strain was also more susceptible to human serum. Complementation of the mutant with a plasmid containing the ksl(k2)ABCDE genes restored capsule expression, enhanced survival in the murine urinary tract, and restored serum resistance. CONCLUSION These results indicate that expression of the K2 capsule is important for the pathogenesis of UTI and provides protection against complement-mediated killing. To our knowledge, this is the first study in which the E. coli capsule has been proven to play a role in infection by use of isogenic mutants and genetic complementation.

Dissemination and Systemic Colonization of Uropathogenic Escherichia coli in a Murine Model of Bacteremia

ABSTRACT Infection with uropathogenic Escherichia coli (UPEC), the causative agent of most uncomplicated urinary tract infections, proceeds in an ascending manner and, if left untreated, may result in bacteremia and urosepsis. To examine the fate of UPEC after its entry into the bloodstream, we developed a murine model of sublethal bacteremia. CBA/J mice were inoculated intravenously with 1 × 106 CFU of pyelonephritis strain E. coli CFT073 carrying a bioluminescent reporter. Biophotonic imaging, used to monitor the infection over 48 h, demonstrated that the bacteria disseminated systemically and appeared to localize at discrete sites. UPEC was recovered from the spleen, liver, kidneys, lungs, heart, brain, and intestines as early as 20 min postinoculation, peaking at 24 h postinoculation. A nonpathogenic E. coli K-12 strain, however, disseminated at significantly lower levels (P < 0.01) and was cleared from the liver and cecum by 24 h postinoculation. Isogenic mutants lacking type 1 fimbriae, P fimbriae, capsule, TonB, the heme receptors Hma and ChuA, or particularly the sialic acid catabolism enzyme NanA were significantly outcompeted by wild-type CFT073 during bacteremia (P < 0.05), while flagellin and hemolysin mutants were not. IMPORTANCE E. coli is the primary cause of urinary tract infections. In severe cases of kidney infection, bacteria can enter the bloodstream and cause systemic disease. While the ability of E. coli to cause urinary tract infection has been extensively studied, the fate of these bacteria once they enter the bloodstream is largely unknown. Here we used an imaging technique to develop a mouse model of E. coli bloodstream infection and identify bacterial genes that are important for the bacteria to spread to and infect various organs. Understanding how urinary tract pathogens like E. coli cause disease after they enter the bloodstream may aid in the development of protective and therapeutic treatments. E. coli is the primary cause of urinary tract infections. In severe cases of kidney infection, bacteria can enter the bloodstream and cause systemic disease. While the ability of E. coli to cause urinary tract infection has been extensively studied, the fate of these bacteria once they enter the bloodstream is largely unknown. Here we used an imaging technique to develop a mouse model of E. coli bloodstream infection and identify bacterial genes that are important for the bacteria to spread to and infect various organs. Understanding how urinary tract pathogens like E. coli cause disease after they enter the bloodstream may aid in the development of protective and therapeutic treatments.

Oxygen-Limiting Conditions Enrich for Fimbriate Cells of Uropathogenic Proteus mirabilis and Escherichia coli

MR/P fimbriae of uropathogenic Proteus mirabilis undergo invertible element-mediated phase variation whereby an individual bacterium switches between expressing fimbriae (phase ON) and not expressing fimbriae (phase OFF). Under different conditions, the percentage of fimbriate bacteria within a population varies and could be dictated by either selection (growth advantage of one phase) or signaling (preferentially converting one phase to the other in response to external signals). Expression of MR/P fimbriae increases in a cell-density dependent manner in vitro and in vivo. However, rather than the increased cell density itself, this increase in fimbrial expression is due to an enrichment of fimbriate bacteria under oxygen limitation resulting from increased cell density. Our data also indicate that the persistence of MR/P fimbriate bacteria under oxygen-limiting conditions is a result of both selection (of MR/P fimbrial phase variants) and signaling (via modulation of expression of the MrpI recombinase). Furthermore, the mrpJ transcriptional regulator encoded within the mrp operon contributes to phase switching. Type 1 fimbriae of Escherichia coli, which are likewise subject to phase variation via an invertible element, also increase in expression during reduced oxygenation. These findings provide evidence to support a mechanism for persistence of fimbriate bacteria under oxygen limitation, which is relevant to disease progression within the oxygen-restricted urinary tract.

Dissemination of a Highly Virulent Pathogen: Tracking The Early Events That Define Infection

The series of events that occurs immediately after pathogen entrance into the body is largely speculative. Key aspects of these events are pathogen dissemination and pathogen interactions with the immune response as the invader moves into deeper tissues. We sought to define major events that occur early during infection of a highly virulent pathogen. To this end, we tracked early dissemination of Yersinia pestis, a highly pathogenic bacterium that causes bubonic plague in mammals. Specifically, we addressed two fundamental questions: (1) do the bacteria encounter barriers in disseminating to draining lymph nodes (LN), and (2) what mechanism does this nonmotile bacterium use to reach the LN compartment, as the prevailing model predicts trafficking in association with host cells. Infection was followed through microscopy imaging in addition to assessing bacterial population dynamics during dissemination from the skin. We found and characterized an unexpected bottleneck that severely restricts bacterial dissemination to LNs. The bacteria that do not pass through this bottleneck are confined to the skin, where large numbers of neutrophils arrive and efficiently control bacterial proliferation. Notably, bottleneck formation is route dependent, as it is abrogated after subcutaneous inoculation. Using a combination of approaches, including microscopy imaging, we tested the prevailing model of bacterial dissemination from the skin into LNs and found no evidence of involvement of migrating phagocytes in dissemination. Thus, early stages of infection are defined by a bottleneck that restricts bacterial dissemination and by neutrophil-dependent control of bacterial proliferation in the skin. Furthermore, and as opposed to current models, our data indicate an intracellular stage is not required by Y. pestis to disseminate from the skin to draining LNs. Because our findings address events that occur during early encounters of pathogen with the immune response, this work can inform efforts to prevent or control infection.

Uropathogenic Escherichia coli Strains Generally Lack Functional Trg and Tap Chemoreceptors Found in the Majority of E. coli Strains Strictly Residing in the Gut

The prevalence and function of four chemoreceptors, Tsr, Tar, Trg, and Tap, were determined for a collection of uropathogenic, fecal-commensal, and diarrheagenic Escherichia coli strains. tar and tsr were present or functional in nearly all isolates. However, trg and tap were significantly less prevalent or functional among the uropathogenic E. coli strains (both in 6% of strains) than among fecal-commensal strains (both in > or =50% of strains) or diarrheal strains (both in > or =75% of strains) (P < 0.02).

Expression during Host Infection and Localization of Yersinia pestis Autotransporter Proteins

Yersinia pestis CO92 has 12 open reading frames encoding putative conventional autotransporters (yaps), nine of which appear to produce functional proteins. Here, we demonstrate the ability of the Yap proteins to localize to the cell surface of both Escherichia coli and Yersinia pestis and show that a subset of these proteins undergoes processing by bacterial surface omptins to be released into the supernatant. Numerous autotransporters have been implicated in pathogenesis, suggesting a role for the Yaps as virulence factors in Y. pestis. Using the C57BL/6 mouse models of bubonic and pneumonic plague, we determined that all of these genes are transcribed in the lymph nodes during bubonic infection and in the lungs during pneumonic infection, suggesting a role for the Yaps during mammalian infection. In vitro transcription studies did not identify a particular environmental stimulus responsible for transcriptional induction. The primary sequences of the Yaps reveal little similarity to any characterized autotransporters; however, two of the genes are present in operons, suggesting that the proteins encoded in these operons may function together. Further work aims to elucidate the specific functions of the Yaps and clarify the contributions of these proteins to Y. pestis pathogenesis.