scholarly journals Comprehensive mutagenesis of thefimSpromoter regulatory switch reveals novel regulation of type 1 pili in uropathogenicEscherichia coli

2016 ◽  
Vol 113 (15) ◽  
pp. 4182-4187 ◽  
Author(s):  
Huibin Zhang ◽  
Teodorus T. Susanto ◽  
Yue Wan ◽  
Swaine L. Chen
Keyword(s):  
Phase Variation ◽  
Inverted Repeats ◽  
Phase Variable ◽  
Genomic Context ◽  
Clonal Population ◽  
Control Phase ◽  
Additional Layer ◽  
Type 1 Pili ◽  
Tract Infections

Type 1 pili (T1P) are major virulence factors for uropathogenicEscherichia coli(UPEC), which cause both acute and recurrent urinary tract infections. T1P expression therefore is of direct relevance for disease. T1P are phase variable (both piliated and nonpiliated bacteria exist in a clonal population) and are controlled by an invertible DNA switch (fimS), which contains the promoter for thefimoperon encoding T1P. Inversion offimSis stochastic but may be biased by environmental conditions and other signals that ultimately converge atfimSitself. Previous studies offimSsequences important for T1P phase variation have focused on laboratory-adaptedE.colistrains and have been limited in the number of mutations or by alteration of thefimSgenomic context. We surmounted these limitations by using saturating genomic mutagenesis offimScoupled with accurate sequencing to detect both mutations and phase status simultaneously. In addition to the sequences known to be important for biasingfimSinversion, our method also identifies a previously unknown pair of 5′ UTR inverted repeats that act by altering the relativefimAlevels to control phase variation. Thus we have uncovered an additional layer of T1P regulation potentially impacting virulence and the coordinate expression of multiple pilus systems.

scholarly journals Human Urine Decreases Function and Expression of Type 1 Pili in Uropathogenic Escherichia coli

mBio ◽  
2015 ◽  
Vol 6 (4) ◽  
Author(s):  
Sarah E. Greene ◽  
Michael E. Hibbing ◽  
James Janetka ◽  
Swaine L. Chen ◽  
Scott J. Hultgren
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Urinary Tract Infections ◽  
Human Urine ◽  
Phase Variable ◽  
Bladder Epithelium ◽  
Content Type ◽  
Type 1 Pili ◽  
Tract Infections

ABSTRACTUropathogenicEscherichia coli(UPEC) is the primary cause of community-acquired urinary tract infections (UTIs). UPEC bind the bladder using type 1 pili, encoded by thefimoperon in nearly allE. coli. Assembled type 1 pili terminate in the FimH adhesin, which specifically binds to mannosylated glycoproteins on the bladder epithelium. Expression of type 1 pili is regulated in part by phase-variable inversion of the genomic element containing thefimSpromoter, resulting in phase ON (expressing) and OFF (nonexpressing) orientations. Type 1 pili are essential for virulence in murine models of UTI; however, studies of urine samples from human UTI patients demonstrate variable expression of type 1 pili. We provide insight into this paradox by showing that human urine specifically inhibits both expression and function of type 1 pili. Growth in urine induces thefimSphase OFF orientation, preventingfimexpression. Urine also contains inhibitors of FimH function, and this inhibition leads to a further bias infimSorientation toward the phase OFF state. The dual effect of urine onfimSregulation and FimH binding presents a potential barrier to type 1 pilus-mediated colonization and invasion of the bladder epithelium. However, FimH-mediated attachment to human bladder cells during growth in urine reverses these effects such thatfimexpression remains ON and/or turns ON. Interestingly, FimH inhibitors called mannosides also induce thefimSphase OFF orientation. Thus, the transduction of FimH protein attachment or inhibition into epigenetic regulation of type 1 pilus expression has important implications for the development of therapeutics targeting FimH function.IMPORTANCEUrinary tract infections (UTIs) are extremely common infections, frequently caused by uropathogenicEscherichia coli(UPEC), that are treated with antibiotics but often recur. Therefore, UTI treatment both is complicated by and contributes to bacterial antibiotic resistance. Thus, it is important to understand UTI pathogenesis to devise novel strategies and targets for prevention and treatment. Based on evidence from disease epidemiology and mouse models of infection, UPEC relies heavily on type 1 pili to attach to and invade the bladder epithelium during initial stages of UTI. Here, we demonstrate that the negative effect of planktonic growth in human urine on both the function and expression of type 1 pili is overcome by attachment to bladder epithelial cells, representing a strategy to subvert this alternative innate defense mechanism. Furthermore, this dually inhibitory action of urine is a mechanism shared with recently developed anti-type 1 pilus molecules, highlighting the idea that further development of antivirulence strategies targeting pili may be particularly effective for UPEC.

scholarly journals Phosphoethanolamine cellulose enhances curli-mediated adhesion of uropathogenicEscherichia colito bladder epithelial cells

2018 ◽  
Vol 115 (40) ◽  
pp. 10106-10111 ◽  
Author(s):  
Emily C. Hollenbeck ◽  
Alexandra Antonoplis ◽  
Chew Chai ◽  
Wiriya Thongsomboon ◽  
Gerald G. Fuller ◽  
...  
Keyword(s):  
Cell Surface ◽  
Bacterial Cell ◽  
Cell Monolayer ◽  
Surface Structures ◽  
Bacterial Cell Surface ◽  
Cell Surface Structures ◽  
Bladder Cell ◽  
Type 1 Pili ◽  
Tract Infections

UropathogenicEscherichia coli(UPEC) are the major causative agents of urinary tract infections, employing numerous molecular strategies to contribute to adhesion, colonization, and persistence in the bladder niche. Identifying strategies to prevent adhesion and colonization is a promising approach to inhibit bacterial pathogenesis and to help preserve the efficacy of available antibiotics. This approach requires an improved understanding of the molecular determinants of adhesion to the bladder urothelium. We designed experiments using a custom-built live cell monolayer rheometer (LCMR) to quantitatively measure individual and combined contributions of bacterial cell surface structures [type 1 pili, curli, and phosphoethanolamine (pEtN) cellulose] to bladder cell adhesion. Using the UPEC strain UTI89, isogenic mutants, and controlled conditions for the differential production of cell surface structures, we discovered that curli can promote stronger adhesive interactions with bladder cells than type 1 pili. Moreover, the coproduction of curli and pEtN cellulose enhanced adhesion. The LCMR enables the evaluation of adhesion under high-shear conditions to reveal this role for pEtN cellulose which escaped detection using conventional tissue culture adhesion assays. Together with complementary biochemical experiments, the results support a model wherein cellulose serves a mortar-like function to promote curli association with and around the bacterial cell surface, resulting in increased bacterial adhesion strength at the bladder cell surface.

scholarly journals Comprehensive Identification of Fim-Mediated Inversions in UropathogenicEscherichia coliwith Structural Variation Detection Using Relative Entropy

mSphere ◽  
2019 ◽  
Vol 4 (2) ◽  
Author(s):  
Colin W. Russell ◽  
Rashmi Sukumaran ◽  
Lu Ting Liow ◽  
Balamurugan Periaswamy ◽  
Shazmina Rafee ◽  
...  
Keyword(s):  
Relative Entropy ◽  
Structural Variation ◽  
Infection Type ◽  
Epigenetic Mechanism ◽  
Structural Variations ◽  
Entire Genome ◽  
Content Type ◽  
Type 1 Pili ◽  
Tract Infections

ABSTRACTMost urinary tract infections (UTIs) are caused by uropathogenicEscherichia coli(UPEC), which depends on an extracellular organelle (type 1 pili) for adherence to bladder cells during infection. Type 1 pilus expression is partially regulated by inversion of a piece of DNA referred to asfimS, which contains the promoter for thefimoperon encoding type 1 pili.fimSinversion is regulated by up to five recombinases collectively known as Fim recombinases. These Fim recombinases are currently known to regulate two other switches: theipuSandhyxSswitches. A long-standing question has been whether the Fim recombinases regulate the inversion of other switches, perhaps to coordinate expression for adhesion or virulence. We answered this question using whole-genome sequencing with a newly developed algorithm (structural variation detection using relative entropy [SVRE]) for calling structural variations using paired-end short-read sequencing. SVRE identified all of the previously known switches, refining the specificity of which recombinases act at which switches. Strikingly, we found no new inversions that were mediated by the Fim recombinases. We conclude that the Fim recombinases are each highly specific for a small number of switches. We hypothesize that the unlinked Fim recombinases have been recruited to regulatefimS, andfimSonly, as a secondary locus; this further implies that regulation of type 1 pilus expression (and its role in gastrointestinal and/or genitourinary colonization) is important enough, on its own, to influence the evolution and maintenance of multiple additional genes within the accessory genome ofE. coli.IMPORTANCEUTI is a common ailment that affects more than half of all women during their lifetime. The leading cause of UTIs is UPEC, which relies on type 1 pili to colonize and persist within the bladder during infection. The regulation of type 1 pili is remarkable for an epigenetic mechanism in which a section of DNA containing a promoter is inverted. The inversion mechanism relies on what are thought to be dedicated recombinase genes; however, the full repertoire for these recombinases is not known. We show here that there are no additional targets beyond those already identified for the recombinases in the entire genome of two UPEC strains, arguing that type 1 pilus expression itself is the driving evolutionary force for the presence of these recombinase genes. This further suggests that targeting the type 1 pilus is a rational alternative nonantibiotic strategy for the treatment of UTI.

scholarly journals The UbiI (VisC) Aerobic Ubiquinone Synthase Is Required for Expression of Type 1 Pili, Biofilm Formation, and Pathogenesis in Uropathogenic Escherichia coli

2016 ◽  
Vol 198 (19) ◽  
pp. 2662-2672 ◽  
Author(s):  
Kyle A. Floyd ◽  
Courtney A. Mitchell ◽  
Allison R. Eberly ◽  
Spencer J. Colling ◽  
Ellisa W. Zhang ◽  
...  
Keyword(s):  
Urinary Tract ◽  
Biofilm Formation ◽  
Energy Deficit ◽  
Wild Type ◽  
Content Type ◽  
Anoxic Conditions ◽  
E Coli ◽  
Type 1 Pili ◽  
Tract Infections

ABSTRACTUropathogenicEscherichia coli(UPEC), which causes the majority of urinary tract infections (UTI), uses pilus-mediated adherence to initiate biofilm formation in the urinary tract. Oxygen gradients withinE. colibiofilms regulate expression and localization of adhesive type 1 pili. A transposon mutant screen for strains defective in biofilm formation identified theubiI(formerlyvisC) aerobic ubiquinone synthase gene as critical for UPEC biofilm formation. In this study, we characterized a nonpolarubiIdeletion mutant and compared its behavior to that of wild-type bacteria grown under aerobic and anoxic conditions. Consistent with its function as an aerobic ubiquinone-8 synthase, deletion ofubiIin UPEC resulted in reduced membrane potential, diminished motility, and reduced expression of chaperone-usher pathway pili. Loss of aerobic respiration was previously shown to negatively impact expression of type 1 pili. To determine whether this reduction in type 1 pili was due to an energy deficit, wild-type UPEC and theubiImutant were compared for energy-dependent phenotypes under anoxic conditions, in which quinone synthesis is undertaken by anaerobic quinone synthases. Under anoxic conditions, the two strains exhibited wild-type levels of motility but produced diminished numbers of type 1 pili, suggesting that the reduction of type 1 pilus expression in the absence of oxygen is not due to a cellular energy deficit. Acute- and chronic-infection studies in a mouse model of UTI revealed a significant virulence deficit in theubiImutant, indicating that UPEC encounters enough oxygen in the bladder to induce aerobic ubiquinone synthesis during infection.IMPORTANCEThe majority of urinary tract infections are caused by uropathogenicE. coli, a bacterium that can respire in the presence and absence of oxygen. The bladder environment is hypoxic, with oxygen concentrations ranging from 4% to 7%, compared to 21% atmospheric oxygen. This work provides evidence that aerobic ubiquinone synthesis must be engaged during bladder infection, indicating that UPEC bacteria sense and use oxygen as a terminal electron acceptor in the bladder and that this ability drives infection potential despite the fact that UPEC is a facultative anaerobe.

scholarly journals Comprehensive Identification of Fim-Mediated Inversions in Uropathogenic Escherichia coli with Structural Variation Detection Using Relative Entropy

2018 ◽  
Author(s):  
Colin W. Russell ◽  
Rashmi Sukumaran ◽  
Lu Ting Liow ◽  
Balamurugan Periaswamy ◽  
Shazmina Rafee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Relative Entropy ◽  
Structural Variation ◽  
Infection Type ◽  
Uropathogenic Escherichia Coli ◽  
Structural Variations ◽  
Type 1 Pili ◽  
Bladder Cells ◽  
Tract Infections

Most urinary tract infections (UTIs) are caused by uropathogenic Escherichia coli (UPEC), which depend on an extracellular organelle (Type 1 pili) for adherence to bladder cells during infection. Type 1 pilus expression is partially regulated by inversion of a piece of DNA referred to as fimS, which contains the promoter for the fim operon encoding Type 1 pili. fimS inversion is regulated by up to five recombinases collectively known as Fim recombinases. These Fim recombinases are currently known to regulate two other switches: the ipuS and hyxS switches. A long-standing question has been whether the Fim recombinases regulate the inversion of other switches, perhaps to coordinate expression for adhesion or virulence. We answered this question using whole genome sequencing with a newly developed algorithm (Structural Variation detection using Relative Entropy, SVRE) for calling structural variations using paired-end short read sequencing. SVRE identified all of the previously known switches, refining the specificity of which recombinases act at which switches. Strikingly, we found no new inversions that were mediated by the Fim recombinases. We conclude that the Fim recombinases are each highly specific for a small number of switches. We hypothesize that the unlinked Fim recombinases have been recruited to regulate fimS, and fimS only, as a secondary locus; this further implies that regulation of Type 1 pilus expression (and its role in gastrointestinal and/or genitourinary colonization) is important enough, on its own, to influence the evolution and maintenance of multiple additional genes within the accessory genome of E. coli.

scholarly journals Systematic Analysis of REBASE Identifies Numerous Type I Restriction-Modification Systems with Duplicated, Distinct hsdS Specificity Genes That Can Switch System Specificity by Recombination

mSystems ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
John M. Atack ◽  
Chengying Guo ◽  
Thomas Litfin ◽  
Long Yang ◽  
Patrick J. Blackall ◽  
...  
Keyword(s):  
Vaccine Development ◽  
Phase Variation ◽  
Dna Methyltransferases ◽  
Inverted Repeats ◽  
Global Changes ◽  
Type I ◽  
Phase Variable ◽  
Global Methylation ◽  
Content Type ◽  
Restriction Modification

ABSTRACT N6-Adenine DNA methyltransferases associated with some Type I and Type III restriction-modification (R-M) systems are able to undergo phase variation, randomly switching expression ON or OFF by varying the length of locus-encoded simple sequence repeats (SSRs). This variation of methyltransferase expression results in genome-wide methylation differences and global changes in gene expression. These epigenetic regulatory systems are called phasevarions, phase-variable regulons, and are widespread in bacteria. A distinct switching system has also been described in Type I R-M systems, based on recombination-driven changes in hsdS genes, which dictate the DNA target site. In order to determine the prevalence of recombination-driven phasevarions, we generated a program called RecombinationRepeatSearch to interrogate REBASE and identify the presence and number of inverted repeats of hsdS downstream of Type I R-M loci. We report that 3.9% of Type I R-M systems have duplicated variable hsdS genes containing inverted repeats capable of phase variation. We report the presence of these systems in the major pathogens Enterococcus faecalis and Listeria monocytogenes, which could have important implications for pathogenesis and vaccine development. These data suggest that in addition to SSR-driven phasevarions, many bacteria have independently evolved phase-variable Type I R-M systems via recombination between multiple, variable hsdS genes. IMPORTANCE Many bacterial species contain DNA methyltransferases that have random on/off switching of expression. These systems, called phasevarions (phase-variable regulons), control the expression of multiple genes by global methylation changes. In every previously characterized phasevarion, genes involved in pathobiology, antibiotic resistance, and potential vaccine candidates are randomly varied in their expression, commensurate with methyltransferase switching. Our systematic study to determine the extent of phasevarions controlled by invertible Type I R-M systems will provide valuable information for understanding how bacteria regulate genes and is key to the study of physiology, virulence, and vaccine development; therefore, it is critical to identify and characterize phase-variable methyltransferases controlling phasevarions.

scholarly journals Phase variation of a signal transduction system controlsClostridioides difficilecolony morphology, motility, and virulence

2019 ◽  
Author(s):  
Elizabeth M. Garrett ◽  
Ognjen Sekulovic ◽  
Daniela Wetzel ◽  
Joshua B. Jones ◽  
Adrianne N. Edwards ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Recent Work ◽  
Phase Variation ◽  
Phenotypic Heterogeneity ◽  
Colony Morphology ◽  
Disease Development ◽  
Phase Variable ◽  
Signal Transduction System ◽  
Clonal Population ◽  
Swimming Motility

AbstractRecent work has revealed thatClostridioides difficile, a major cause of nosocomial diarrheal disease, exhibits phenotypic heterogeneity within a clonal population as a result of phase variation. ManyC. difficilestrains representing multiple ribotypes develop two colony morphotypes, termed rough and smooth, but the biological implications of this phenomenon have not been explored. Here, we examine the molecular basis and physiological relevance of the distinct colony morphotypes produced by this bacterium. We show thatC. difficilereversibly differentiates into rough and smooth colony morphologies, and that bacteria derived from the isolates display opposing surface and swimming motility behaviors. We identified an atypical phase-variable signal transduction system consisting of a histidine kinase and two response regulators, named herein CmrRST, which mediates the switch in colony morphology and motility behaviors. The CmrRST-regulated surface motility is independent of Type IV pili, suggesting a novel mechanism of surface expansion inC. difficile. Microscopic analysis of cell and colony structure indicates that CmrRST promotes the formation of elongated bacteria arranged in bundled chains, which may contribute to bacterial migration. In a hamster model of acuteC. difficiledisease, colony morphology correlates with virulence, and the CmrRST system is required for disease development. Furthermore, we provide evidence that CmrRST phase varies during infection, suggesting that the intestinal environment impacts the proportion of CmrRST-expressingC. difficile. Our findings indicate thatC. difficileemploys phase variation of the CmrRST signal transduction system to generate phenotypic heterogeneity during infection, with concomitant effects on bacterial physiology and pathogenesis.Significance StatementPhenotypic heterogeneity within a genetically clonal population allows many mucosal pathogens to survive within their hosts, balancing the need to produce factors that promote colonization and persistence with the need to avoid the recognition of those factors by the host immune system. Recent work suggests that the human intestinal pathogenClostridium difficileemploys phase variation during infection to generate a heterogeneous population differing in swimming motility, toxin production, and more. This study identifies a signal transduction system that broadly impactsC. difficilephysiology and behaviorin vitroand in an animal model. Phase variation of this system is therefore poised to modulate the coordinated expression of multiple mechanisms influencingC. difficiledisease development.

scholarly journals What Flips the Switch? Signals and Stress Regulating Extraintestinal Pathogenic Escherichia coli Type 1 Fimbriae (Pili)

2021 ◽  
Vol 10 (1) ◽  
pp. 5
Author(s):  
Hicham Bessaiah ◽  
Carole Anamalé ◽  
Jacqueline Sung ◽  
Charles M. Dozois
Keyword(s):  
Stress Response ◽  
Stress Responses ◽  
Bacterial Colonization ◽  
Phase Variation ◽  
Neonatal Meningitis ◽  
E Coli ◽  
Type 1 Fimbriae ◽  
Colonization Factor ◽  
Tract Infections

Pathogens are exposed to a multitude of harmful conditions imposed by the environment of the host. Bacterial responses against these stresses are pivotal for successful host colonization and pathogenesis. In the case of many E. coli strains, type 1 fimbriae (pili) are an important colonization factor that can contribute to diseases such as urinary tract infections and neonatal meningitis. Production of type 1 fimbriae in E. coli is dependent on an invertible promoter element, fimS, which serves as a phase variation switch determining whether or not a bacterial cell will produce type 1 fimbriae. In this review, we present aspects of signaling and stress involved in mediating regulation of type 1 fimbriae in extraintestinal E. coli; in particular, how certain regulatory mechanisms, some of which are linked to stress response, can influence production of fimbriae and influence bacterial colonization and infection. We suggest that regulation of type 1 fimbriae is potentially linked to environmental stress responses, providing a perspective for how environmental cues in the host and bacterial stress response during infection both play an important role in regulating extraintestinal pathogenic E. coli colonization and virulence.

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