Regulation of fimbrial phase transition frequencies in uropathogenic Escherichia coli

Uropathogenic Escherichia coli (UPEC) are the etiologic agent of more than 80% of uncomplicated urinary tract infections (UTIs) in humans. Pyelonephritis-associated pili (Pap) are fimbrial adhesins that facilitate binding of UPEC to Gala-(l,4)-Gal/i moieties contained in membrane glycolipids on human uroepithelial cells and are associated with acute kidney infection (pyelonephritis). Pap expression is phase variable and the frequency with which phase transition occurs determines the proportion of P-fimbriate bacteria in the population. In this study, pap phase transition frequencies were measured in clinical isolates for the first time and were shown to be markedly higher than the frequencies displayed by the same pap operons measured in E. coli K-12. In this relevant regulatory context, phase variation frequencies of homologous pap operons were found to be differentially affected by culture conditions, indicating a hierarchy of expression depending on environmental signals. Cross-talk between pap operons was also found to be dependent on culture conditions. The molecular mechanism leading to different phase variation frequencies between homologous pap operons was investigated by sequencing 82 pap regulatory regions and their regulators (papl and papB) from 54 UPEC isolates of different clinical origin (asymptomatic vs. symptomatic UTI). One variable region identified was a high affinity binding site for the pap autoregulatory protein PapB. The site contained a variable number of 9 bp repeats with (T/A)3 sequences, which affected PapB binding and the frequency of off-to-on phase transition, under particular environmental conditions. Sequence diversification via point mutation was also observed among papl genes, encoding for the pap transcriptional activator Papl, and were shown to be under positive selection (dN/dS > 1) for functionally adaptive amino acid replacements. Certain Papl variants correlated with symptomatic disease and differed in their ability to activate pap operons. This study provided different lines of evidence supporting the hypothesis that UPEC isolates have evolved mechanisms to regulate the phase variation frequencies of homologous fimbrial operons, potentially to achieve their differential activation and sequential expression. The ability of UPEC to coordinate expression of multiple surface factors is critical for the successful colonisation of the varied complex micro-environments encountered in the human urinary tract

Predictability studies of coastal marine ecosystem behavior

The study presented in this thesis is principally meant to analyze the genericity of a deterministic, comprehensive marine ecosystem model in combination with various refined representations of hydrodynamical processes, and to evaluate the potential predictability skills of this combined modelling system with specific applications in two rather different coastal basins. This objective has been realized by first developing a modular coupling interface between the Princeton Ocean Model (POM) and the European Regional Seas Ecosystem Model (ERSEM), called High-Resolution OpenSESAME POM ERSEM (HiROPE). Secondly, this model framework, embedding a composite of 'complex' conceptual principles of the functioning of the main biogeochemical processes, has been applied to substantially different marine systems, the Baltic proper and the northern Adriatic Sea. The generic biological first principles of the ERSEM ecosystem model have been throughly controlled for consistency, and a suitable mathematical syntax has been defined in order to accomodate the various biogeochemical cycles of the resolved elements. The model has been specifically applied in the chosen basins with different temporal and spatial resolutions: a one-dimensional (vertical, 1D-V), climatological implementation in the northern Adriatic Sea; a 1D-V implementation in the Baltic proper with realistic forcing functions in the period 1979-1991 and a fully three-dimensional, high-frequency realistic implementation in the northern Adriatic Sea (October 1995). General conclusions are that the representation of hydrodynamical variability, the definition and resolution of boundary processes, the introduction of new source terms or the implementation of new biological state variables, affect the predictability of the system behavior more than the utilization of incomplete initial conditions of biological variables in a complex comprehensive ecosystem model

Uropathogenic Escherichia coli virulence and innate immune responses during urinary tract infection

Urinary tract infections (UTI) are among the most common infectious diseases of humans and are the most common nosocomial infections in the developed world. It is estimated that 40-50% of women and 5% of men will develop a UTI in their lifetime, and UTI accounts for more than 1 million hospitalizations and $1.6 billion in medical expenses each year in the USA. Uropathogenic Escherichia coli (UPEC) is the primary cause of UTI. This review presents an overview of recent discoveries related to the primary virulence factors of UPEC and major innate immune responses to infection of the lower urinary tract. New and emerging themes in UPEC research are discussed in the context of the interface between host and pathogen

Functional heterogeneity of the UpaH autotransporter protein from uropathogenic Escherichia coli

Uropathogenic Escherichia coli (UPEC) are responsible for the majority of urinary tract infections(UTI). To cause UTI, UPEC must adhere to epithelial cells of the urinary tract and overcome the shear flow forces of urine. This function is primarily mediated by fimbrial adhesins, which mediate specific attachment to host cell receptors. Another group of adhesins that contribute to UPEC mediated UTI are autotransporter (AT) proteins. AT proteins possess a range of virulence properties such as adherence, aggregation, invasion and biofilm formation. One recently characterized AT protein of UPEC is UpaH, a large AIDA-I type AT protein that contributes to biofilm formation and bladder colonization. In this study, we have characterized a series of naturally occurring variants of UpaH. We demonstrate that extensive sequence variation exists within the passenger-encoding domain of UpaH variants from different UPEC strains. This sequence variation is associated with functional heterogeneity with respect to the ability of UpaH to mediate biofilm formation. In contrast, all of the UpaH variants examined retained a conserved ability to mediate binding to extracellular matrix (ECM) proteins. Bioinformatic analysis of the UpaH passenger domain identified a conserved region (UpaHCR) and hydrophobic region (UpaHHR). Deletion of these domains reduced biofilm formation but not binding to ECM proteins. Despite variation in upaH sequence, the transcription of upaH was repressed by a conserved mechanism involving the global regulator H-NS, and mutation of the hns gene relieved this repression. Overall, our findings shed new light on the regulation and function of the UpaH AT protein

Identification of genes important for growth of asymptomatic Bacteriuria Escherichia coli in urine

Escherichia coli is the most important etiological agent of urinary tract infections (UTIs). Unlike uropathogenic E. coli, which causes symptomatic infections, asymptomatic bacteriuria (ABU) E. coli strains typically lack essential virulence factors and colonize the bladder in the absence of symptoms. While ABU E. coli can persist in the bladder for long periods of time, little is known about the genetic determinants required for its growth and fitness in urine. To identify such genes, we have employed a transposon mutagenesis approach using the prototypic ABU E. coli strain 83972 and the clinical ABU E. coli strain VR89. Six genes involved in the biosynthesis of various amino acids and nucleobases were identified (carB, argE, argC, purA, metE, and ilvC), and site-specific mutants were subsequently constructed in E. coli 83972 and E. coli VR89 for each of these genes. In all cases, these mutants exhibited reduced growth rates and final cell densities in human urine. The growth defects could be complemented in trans as well as by supplementation with the appropriate amino acid or nucleobase. When assessed in vivo in a mouse model, E. coli 83972carAB and 83972argC showed a significantly reduced competitive advantage in the bladder and/or kidney during coinoculation experiments with the parent strain, whereas 83972metE and 83972ilvC did not. Taken together, our data have identified several biosynthesis pathways as new important fitness factors associated with the growth of ABU E. coli in human urine

Molecular analysis of type 3 fimbrial genes from Escherichia coli, Klebsiella and Citrobacter species

Background: Catheter-associated urinary tract infection (CAUTI) is the most common nosocomial infection in the United States and is caused by a range of uropathogens. Biofilm formation by uropathogens that cause CAUTI is often mediated by cell surface structures such as fimbriae. In this study, we characterised the genes encoding type 3 fimbriae from CAUTI strains of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Citrobacter koseri and Citrobacter freundii

Contribution of siderophore systems to growth and urinary tract colonization of asymptomatic bacteriuria Escherichia coli

The molecular mechanisms that define asymptomatic bacteriuria (ABU) E. coli colonization of the human urinary tract remain to be properly elucidated. Here we utilize ABU E. coli strain 83972 as a model to dissect the contribution of siderophores to iron acquisition, growth, fitness and colonization of the urinary tract. We show that E. coli 83972 produces enterobactin, salmochelin, aerobactin and yersiniabactin, and examine the role of these systems using mutants defective in siderophore biosynthesis and uptake. Enterobactin and aerobactin contributed most to total siderophore activity and growth in defined iron-deficient media. No siderophores were detected in an 83972 quadruple mutant deficient in all four siderophore biosynthesis pathways; this mutant did not grow in defined iron-deficient media but grew in iron-limited pooled human urine due to iron uptake via the FecA ferric citrate receptor. In a mixed 1:1 growth assay with 83972 there was no fitness disadvantage of the 83972 quadruple biosynthetic mutant, demonstrating its capacity to act as a ‘cheater’ and utilize siderophores produced by the wild-type strain for iron uptake. An 83972 enterobactin/salmochelin double receptor mutant was outcompeted by 83972 in human urine and the mouse urinary tract, indicating a role for catecholate receptors in urinary tract colonization

Molecular characterization of endocarditis-associated Staphylococcus aureus

Infective endocarditis (IE) is a life-threatening infection of the heart endothelium and valves. Staphylococcus aureus is a predominant cause of severe IE and is frequently associated with infections in health care settings and device-related infections. Multilocus sequence typing (MLST), spa typing, and virulence gene microarrays are frequently used to classify S. aureus clinical isolates. This study examined the utility of these typing tools to investigate S. aureus epidemiology associated with IE. Ninety-seven S. aureus isolates were collected from patients diagnosed with (i) IE, (ii) bloodstream infection related to medical devices, (iii) bloodstream infection not related to medical devices, and (iv) skin or soft-tissue infections. The MLST clonal complex (CC) for each isolate was determined and compared to the CCs of members of the S. aureus population by eBURST analysis. The spa type of all isolates was also determined. A null model was used to determine correlations of IE with CC and spa type. DNA microarray analysis was performed, and a permutational analysis of multivariate variance (PERMANOVA) and principal coordinates analysis were conducted to identify genotypic differences between IE and non-IE strains. CC12, CC20, and spa type t160 were significantly associated with IE S. aureus. A subset of virulence-associated genes and alleles, including genes encoding staphylococcal superantigen-like proteins, fibrinogen-binding protein, and a leukocidin subunit, also significantly correlated with IE isolates. MLST, spa typing, and microarray analysis are promising tools for monitoring S. aureus epidemiology associated with IE. Further research to determine a role for the S. aureus IE-associated virulence genes identified in this study is warranted

Insights into a Multidrug Resistant Escherichia coli Pathogen of the Globally Disseminated ST131 Lineage: Genome Analysis and Virulence Mechanisms

Escherichia coli strains causing urinary tract infection (UTI) are increasingly recognized as belonging to specific clones. E. coli clone O25b:H4-ST131 has recently emerged globally as a leading multi-drug resistant pathogen causing urinary tract and bloodstream infections in hospitals and the community. While most molecular studies to date examine the mechanisms conferring multi-drug resistance in E. coli ST131, relatively little is known about their virulence potential. Here we examined E. coli ST131 clinical isolates from two geographically diverse collections, one representing the major pathogenic lineages causing UTI across the United Kingdom and a second representing UTI isolates from patients presenting at two large hospitals in Australia. We determined a draft genome sequence for one representative isolate, E. coli EC958, which produced CTX-M-15 extended-spectrum β-lactamase, CMY-23 type AmpC cephalosporinase and was resistant to ciprofloxacin. Comparative genome analysis indicated that EC958 encodes virulence genes commonly associated with uropathogenic E. coli (UPEC). The genome sequence of EC958 revealed a transposon insertion in the fimB gene encoding the activator of type 1 fimbriae, an important UPEC bladder colonization factor. We identified the same fimB transposon insertion in 59% of the ST131 UK isolates, as well as 71% of ST131 isolates from Australia, suggesting this mutation is common among E. coli ST131 strains. Insertional inactivation of fimB resulted in a phenotype resembling a slower off-to-on switching for type 1 fimbriae. Type 1 fimbriae expression could still be induced in fimB-null isolates; this correlated strongly with adherence to and invasion of human bladder cells and bladder colonisation in a mouse UTI model. We conclude that E. coli ST131 is a geographically widespread, antibiotic resistant clone that has the capacity to produce numerous virulence factors associated with UTI

Intestinal Colonization Traits of Pandemic Multidrug-Resistant Escherichia coli ST131

Background. Epidemiological studies point to the gut as a key reservoir of multidrug resistant Escherichia coli multilocus sequence type 131 (ST131), a globally dominant pathogenic clone causing urinary tract and bloodstream infections. Here we report a detailed investigation of its intestinal lifestyle. Methods. Clinical ST131 isolates and type 1 fimbriae null mutants were assessed for colonization of human intestinal epithelia and in mouse intestinal colonization models. Mouse gut tissue underwent histologic analysis for pathology and ST131 localization. Key findings were corroborated in mucus-producing human cell lines and intestinal biopsy specimens. Results. ST131 strains adhered to and invaded human intestinal epithelial cells more than probiotic and commensal strains. The reference ST131 strain EC958 established persistent intestinal colonization in mice, and expression of type 1 fimbriae mediated higher colonization levels. Bacterial loads were highest in the distal parts of the mouse intestine and did not cause any obvious pathology. Further analysis revealed that EC958 could bind to both mucus and underlying human intestinal epithelia. Conclusions. ST131 strains can efficiently colonize the mammalian gut and persist long term. Type 1 fimbriae enhance ST131 intestinal colonization, suggesting that mannosides, currently developed as therapeutics for bladder infections and Crohn’s disease, could also be used to limit intestinal ST131 reservoirs