Adenylate Cyclase and the Cyclic AMP Receptor Protein Modulate Stress Resistance and Virulence Capacity of Uropathogenic Escherichia coli

In many bacteria, the second messenger cyclic AMP (cAMP) interacts with the transcription factor cAMP receptor protein (CRP), forming active cAMP-CRP complexes that can control a multitude of cellular activities, including expanded carbon source utilization, stress response pathways, and virulence. Here, we assessed the role of cAMP-CRP as a regulator of stress resistance and virulence in uropathogenicEscherichia coli(UPEC), the principal cause of urinary tract infections worldwide. Deletion of genes encoding either CRP or CyaA, the enzyme responsible for cAMP synthesis, attenuates the ability of UPEC to colonize the bladder in a mouse infection model, dependent on intact innate host defenses. UPEC mutants lacking cAMP-CRP grow normally in the presence of glucose but are unable to utilize alternate carbon sources like amino acids, the primary nutrients available to UPEC within the urinary tract. Relative to the wild-type UPEC isolate, thecyaAandcrpdeletion mutants are sensitive to nitrosative stress and the superoxide generator methyl viologen but remarkably resistant to hydrogen peroxide (H2O2) and acid stress. In the mutant strains, H2O2resistance correlates with elevated catalase activity attributable in part to enhanced translation of the alternate sigma factor RpoS. Acid resistance was promoted by both RpoS-independent and RpoS-dependent mechanisms, including expression of the RpoS-regulated DNA-binding ferritin-like protein Dps. We conclude that balanced input from many cAMP-CRP-responsive elements, including RpoS, is critical to the ability of UPEC to handle the nutrient limitations and severe environmental stresses present within the mammalian urinary tract.

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Characterization of Urinary Tract Infection-Associated Shiga Toxin-Producing Escherichia coli

Infection and Immunity ◽

10.1128/iai.01701-14 ◽

2014 ◽

Vol 82 (11) ◽

pp. 4631-4642 ◽

Cited By ~ 16

Author(s):

Francisco Toval ◽

Roswitha Schiller ◽

Iris Meisen ◽

Johannes Putze ◽

Ivan U. Kouzel ◽

...

Keyword(s):

Escherichia Coli ◽

Urinary Tract ◽

Urinary Bladder ◽

Shiga Toxin ◽

Infection Model ◽

Content Type ◽

E Coli ◽

Virulence Markers ◽

T24 Cells ◽

Tract Infections

ABSTRACTEnterohemorrhagicEscherichia coli(EHEC), a subgroup of Shiga toxin (Stx)-producingE. coli(STEC), is a leading cause of diarrhea and hemolytic-uremic syndrome (HUS) in humans. However, urinary tract infections (UTIs) caused by this microorganism but not associated with diarrhea have occasionally been reported. We geno- and phenotypically characterized three EHEC isolates obtained from the urine of hospitalized patients suffering from UTIs. These isolates carried typical EHEC virulence markers and belonged to HUS-associatedE. coli(HUSEC) clones, but they lacked virulence markers typical of uropathogenicE. coli. One isolate exhibited a localized adherence (LA)-like pattern on T24 urinary bladder epithelial cells. Since the glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer) are well-known receptors for Stx but also for P fimbriae, a major virulence factor of extraintestinal pathogenicE. coli(ExPEC), the expression of Gb3Cer and Gb4Cer by T24 cells and in murine urinary bladder tissue was examined by thin-layer chromatography and mass spectrometry. We provide data indicating that Stxs released by the EHEC isolates bind to Gb3Cer and Gb4Cer isolated from T24 cells, which were susceptible to Stx. All three EHEC isolates expressedstxgenes upon growth in urine. Two strains were able to cause UTI in a murine infection model and could not be outcompeted in urinein vitroby typical uropathogenicE. coliisolates. Our results indicate that despite the lack of ExPEC virulence markers, EHEC variants may exhibit in certain suitable hosts, e.g., in hospital patients, a uropathogenic potential. The contribution of EHEC virulence factors to uropathogenesis remains to be further investigated.

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Cyclic AMP (cAMP) Receptor Protein-cAMP Complex Regulates Heparosan Production in Escherichia coli Strain Nissle 1917

Applied and Environmental Microbiology ◽

10.1128/aem.01814-15 ◽

2015 ◽

Vol 81 (22) ◽

pp. 7687-7696 ◽

Cited By ~ 5

Author(s):

Huihui Yan ◽

Feifei Bao ◽

Liping Zhao ◽

Yanying Yu ◽

Jiaqin Tang ◽

...

Keyword(s):

Escherichia Coli ◽

Cyclic Amp ◽

Carbon Sources ◽

Coli Strain ◽

Site Directed Mutagenesis ◽

Receptor Protein ◽

Upstream Region ◽

Camp Receptor Protein ◽

Content Type ◽

Camp Receptor

ABSTRACTHeparosan serves as the starting carbon backbone for the chemoenzymatic synthesis of heparin, a widely used clinical anticoagulant drug. The availability of heparosan is a significant concern for the cost-effective synthesis of bioengineered heparin. The carbon source is known as the pivotal factor affecting heparosan production. However, the mechanism by which carbon sources control the biosynthesis of heparosan is unclear. In this study, we found that the biosynthesis of heparosan was influenced by different carbon sources. Glucose inhibits the biosynthesis of heparosan, while the addition of either fructose or mannose increases the yield of heparosan. Further study demonstrated that the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex binds to the upstream region of the region 3 promoter and stimulates the transcription of the gene cluster for heparosan biosynthesis. Site-directed mutagenesis of the CRP binding site abolished its capability of binding CRP and eliminated the stimulative effect on transcription.1H nuclear magnetic resonance (NMR) analysis was further performed to determine theEscherichia colistrain Nissle 1917 (EcN) heparosan structure and quantify extracellular heparosan production. Our results add to the understanding of the regulation of heparosan biosynthesis and may contribute to the study of other exopolysaccharide-producing strains.

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Iron Acquisition of Urinary Tract Infection Escherichia coli Involves Pathogenicity in Caenorhabditis elegans

Microorganisms ◽

10.3390/microorganisms9020310 ◽

2021 ◽

Vol 9 (2) ◽

pp. 310

Author(s):

Masayuki Hashimoto ◽

Yi-Fen Ma ◽

Sin-Tian Wang ◽

Chang-Shi Chen ◽

Ching-Hao Teng

Keyword(s):

Escherichia Coli ◽

Urinary Tract ◽

Caenorhabditis Elegans ◽

Large Scale ◽

Iron Acquisition ◽

Infection Model ◽

High Throughput Analysis ◽

E Coli ◽

C Elegans ◽

Tract Infections

Uropathogenic Escherichia coli (UPEC) is a major bacterial pathogen that causes urinary tract infections (UTIs). The mouse is an available UTI model for studying the pathogenicity; however, Caenorhabditis elegans represents as an alternative surrogate host with the capacity for high-throughput analysis. Then, we established a simple assay for a UPEC infection model with C. elegans for large-scale screening. A total of 133 clinically isolated E. coli strains, which included UTI-associated and fecal isolates, were applied to demonstrate the simple pathogenicity assay. From the screening, several virulence factors (VFs) involved with iron acquisition (chuA, fyuA, and irp2) were significantly associated with high pathogenicity. We then evaluated whether the VFs in UPEC were involved in the pathogenicity. Mutants of E. coli UTI89 with defective iron acquisition systems were applied to a solid killing assay with C. elegans. As a result, the survival rate of C. elegans fed with the mutants significantly increased compared to when fed with the parent strain. The results demonstrated, the simple assay with C. elegans was useful as a UPEC infectious model. To our knowledge, this is the first report of the involvement of iron acquisition in the pathogenicity of UPEC in a C. elegans model.

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Single Clinical Isolates from Acute Uncomplicated Urinary Tract Infections Are Representative of Dominant In Situ Populations

mBio ◽

10.1128/mbio.01064-13 ◽

2014 ◽

Vol 5 (2) ◽

Cited By ~ 18

Author(s):

Dana Willner ◽

Serene Low ◽

Jason A. Steen ◽

Narelle George ◽

Graeme R. Nimmo ◽

...

Keyword(s):

Escherichia Coli ◽

Urinary Tract ◽

Urinary Tract Infections ◽

Clinical Isolates ◽

Content Type ◽

E Coli ◽

Strain Diversity ◽

Amplicon Pyrosequencing ◽

Culture Independent ◽

Tract Infections

ABSTRACTUrinary tract infections (UTIs) are one of the most commonly acquired bacterial infections in humans, and uropathogenicEscherichia colistrains are responsible for over 80% of all cases. The standard method for identification of uropathogens in clinical laboratories is cultivation, primarily using solid growth media under aerobic conditions, coupled with morphological and biochemical tests of typically a single isolate colony. However, these methods detect only culturable microorganisms, and characterization is phenotypic in nature. Here, we explored the genotypic identity of communities in acute uncomplicated UTIs from 50 individuals by using culture-independent amplicon pyrosequencing and whole-genome and metagenomic shotgun sequencing. Genus-level characterization of the UTI communities was achieved using the 16S rRNA gene (V8 region). Overall UTI community richness was very low in comparison to other human microbiomes. We strain-typedEscherichia-dominated UTIs using amplicon pyrosequencing of the fimbrial adhesin gene,fimH. There were nine highly abundantfimHtypes, and each UTI sample was dominated by a single type. Molecular analysis of the corresponding clinical isolates revealed that in the majority of cases the isolate was representative of the dominant taxon in the community at both the genus and the strain level. Shotgun sequencing was performed on a subset of eightE. coliurine UTI and isolate pairs. The majority of UTI microbial metagenomic sequences mapped to isolate genomes, confirming the results obtained using phylogenetic markers. We conclude that for the majority of acute uncomplicatedE. coli-mediated UTIs, single cultured isolates are diagnostic of the infection.IMPORTANCEIn clinical practice, the diagnosis and treatment of acute uncomplicated urinary tract infection (UTI) are based on analysis of a single bacterial isolate cultured from urine, and it is assumed that this isolate represents the dominant UTI pathogen. However, these methods detect only culturable bacteria, and the existence of multiple pathogens as well as strain diversity within a single infection is not examined. Here, we explored bacteria present in acute uncomplicated UTIs using culture-independent sequence-based methods.Escherichia coliwas the most common organism identified, and analysis ofE. colidominant UTI samples and their paired clinical isolates revealed that in the majority of infections the cultured isolate was representative of the dominant taxon at both the genus and the strain level. Our data demonstrate that in most cases single cultured isolates are diagnostic of UTI and are consistent with the notion of bottlenecks that limit strain diversity during UTI pathogenesis.

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Biological Cost of Single and Multiple Norfloxacin Resistance Mutations in Escherichia coli Implicated in Urinary Tract Infections

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.6.2343-2351.2005 ◽

2005 ◽

Vol 49 (6) ◽

pp. 2343-2351 ◽

Cited By ~ 91

Author(s):

Patricia Komp Lindgren ◽

Linda L. Marcusson ◽

Dorthe Sandvang ◽

Niels Frimodt-Møller ◽

Diarmaid Hughes

Keyword(s):

Escherichia Coli ◽

Urinary Tract ◽

Infection Model ◽

Resistance Mutations ◽

Topoisomerase Iv ◽

E Coli ◽

Tract Infections ◽

Subsequent Selection

ABSTRACT Resistance to fluoroquinolones in urinary tract infection (UTIs) caused by Escherichia coli is associated with multiple mutations, typically those that alter DNA gyrase and DNA topoisomerase IV and those that regulate AcrAB-TolC-mediated efflux. We asked whether a fitness cost is associated with the accumulation of these multiple mutations. Mutants of the susceptible E. coli UTI isolate Nu14 were selected through three to five successive steps with norfloxacin. Each selection was performed with the MIC of the selected strain. After each selection the MIC was measured; and the regions of gyrA, gyrB, parC, and parE, previously associated with resistance mutations, and all of marOR and acrR were sequenced. The first selection step yielded mutations in gyrA, gyrB, and marOR. Subsequent selection steps yielded mutations in gyrA, parE, and marOR but not in gyrB, parC, or acrR. Resistance-associated mutations were identified in almost all isolates after selection steps 1 and 2 but in less than 50% of isolates after subsequent selection steps. Selected strains were competed in vitro, in urine, and in a mouse UTI infection model against the starting strain, Nu14. First-step mutations were not associated with significant fitness costs. However, the accumulation of three or more resistance-associated mutations was usually associated with a large reduction in biological fitness, both in vitro and in vivo. Interestingly, in some lineages a partial restoration of fitness was associated with the accumulation of additional mutations in late selection steps. We suggest that the relative biological costs of multiple mutations may influence the evolution of E. coli strains that develop resistance to fluoroquinolones.

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The Extracellular Domain of the β2Integrin β Subunit (CD18) Is Sufficient forEscherichia coliHemolysin andAggregatibacter actinomycetemcomitansLeukotoxin Cytotoxic Activity

mBio ◽

10.1128/mbio.01459-19 ◽

2019 ◽

Vol 10 (4) ◽

Cited By ~ 5

Author(s):

Laura C. Ristow ◽

Vy Tran ◽

Kevin J. Schwartz ◽

Lillie Pankratz ◽

Andrew Mehle ◽

...

Keyword(s):

Escherichia Coli ◽

Urinary Tract ◽

Urinary Tract Infections ◽

Integrin Signaling ◽

Β Subunit ◽

Wild Type ◽

Α Subunit ◽

Content Type ◽

Animal Pathogens ◽

Tract Infections

ABSTRACTTheEscherichia colihemolysin (HlyA) is a pore-forming exotoxin associated with severe complications of human urinary tract infections. HlyA is the prototype of the repeats-in-toxin (RTX) family, which includes LtxA fromAggregatibacter actinomycetemcomitans, a periodontal pathogen. The existence and requirement for a host cell receptor for these toxins are controversial. We performed an unbiased forward genetic selection in a mutant library of human monocytic cells, U-937, for host factors involved in HlyA cytotoxicity. The top candidate was the β2integrin β subunit. Δβ2cell lines are approximately 100-fold more resistant than wild-type U-937 cells to HlyA, but remain sensitive to HlyA at high concentrations. Similarly, Δβ2cells are more resistant than wild-type U-937 cells to LtxA, as Δβ2cells remain LtxA resistant even at >1,000-fold-higher concentrations of the toxin. Loss of any single β2integrin α subunit, or even all four α subunits together, does not confer resistance to HlyA. HlyA and LtxA bind to the β2subunit, but not to αL, αM, or αXin far-Western blots. Genetic complementation of Δβ2cells with either β2or β2with a cytoplasmic tail deletion restores HlyA and LtxA sensitivity, suggesting that β2integrin signaling is not required for cytotoxicity. Finally, β2mutations do not alter sensitivity to unrelated pore-forming toxins, as wild-type or Δβ2cells are equally sensitive toStaphylococcus aureusα-toxin andProteus mirabilisHpmA. Our studies show two RTX toxins use the β2integrin β subunit alone to facilitate cytotoxicity, but downstream integrin signaling is dispensable.IMPORTANCEUrinary tract infections are one of the most common bacterial infections worldwide. UropathogenicEscherichia colistrains are responsible for more than 80% of community-acquired urinary tract infections. Although we have known for nearly a century that severe infections stemming from urinary tract infections, including kidney or bloodstream infections are associated with expression of a toxin, hemolysin, from uropathogenicEscherichia coli, how hemolysin functions to enhance virulence is unknown. Our research defines the interaction of hemolysin with the β2integrin, a human white cell adhesion molecule, as a potential therapeutic target during urinary tract infections. TheE. colihemolysin is the prototype for a toxin family (RTX family) produced by a wide array of human and animal pathogens. Our work extends to the identification and characterization of the receptor for an additional member of the RTX family, suggesting that this interaction may be broadly conserved throughout the RTX toxin family.

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The Cyclic AMP Receptor Protein Regulates Quorum Sensing and Global Gene Expression in Yersinia pestis during Planktonic Growth and Growth in Biofilms

mBio ◽

10.1128/mbio.02613-19 ◽

2019 ◽

Vol 10 (6) ◽

Cited By ~ 6

Author(s):

Jeremy T. Ritzert ◽

George Minasov ◽

Ryan Embry ◽

Matthew J. Schipma ◽

Karla J. F. Satchell

Keyword(s):

Gene Expression ◽

Quorum Sensing ◽

Carbon Source ◽

Cyclic Amp ◽

Yersinia Pestis ◽

Carbon Sources ◽

Transcriptional Regulator ◽

Receptor Protein ◽

Content Type ◽

Bacterial Physiology

ABSTRACT Cyclic AMP (cAMP) receptor protein (Crp) is an important transcriptional regulator of Yersinia pestis. Expression of crp increases during pneumonic plague as the pathogen depletes glucose and forms large biofilms within lungs. To better understand control of Y. pestis Crp, we determined a 1.8-Å crystal structure of the protein-cAMP complex. We found that compared to Escherichia coli Crp, C helix amino acid substitutions in Y. pestis Crp did not impact the cAMP dependency of Crp to bind DNA promoters. To investigate Y. pestis Crp-regulated genes during plague pneumonia, we performed RNA sequencing on both wild-type and Δcrp mutant bacteria growing in planktonic and biofilm states in minimal media with glucose or glycerol. Y. pestis Crp was found to dramatically alter expression of hundreds of genes in a manner dependent upon carbon source and growth state. Gel shift assays confirmed direct regulation of the malT and ptsG promoters, and Crp was then linked to Y. pestis growth on maltose as a sole carbon source. Iron regulation genes ybtA and fyuA were found to be indirectly regulated by Crp. A new connection between carbon source and quorum sensing was revealed as Crp was found to regulate production of acyl-homoserine lactones (AHLs) through direct and indirect regulation of genes for AHL synthetases and receptors. AHLs were subsequently identified in the lungs of Y. pestis-infected mice when crp expression was highest in Y. pestis biofilms. Thus, in addition to the well-studied pla gene, other Crp-regulated genes likely have important functions during plague infection. IMPORTANCE Bacterial pathogens have evolved extensive signaling pathways to translate environmental signals into changes in gene expression. While Crp has long been appreciated for its role in regulating metabolism of carbon sources in many bacterial species, transcriptional profiling has revealed that this protein regulates many other aspects of bacterial physiology. The plague pathogen Y. pestis requires this global regulator to survive in blood, skin, and lungs. During disease progression, this organism adapts to changes within these niches. In addition to regulating genes for metabolism of nonglucose sugars, we found that Crp regulates genes for virulence, metal acquisition, and quorum sensing by direct or indirect mechanisms. Thus, this single transcriptional regulator, which responds to changes in available carbon sources, can regulate multiple critical behaviors for causing disease.

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The Repeat-In-Toxin Family Member TosA Mediates Adherence of Uropathogenic Escherichia coli and Survival during Bacteremia

Infection and Immunity ◽

10.1128/iai.05713-11 ◽

2011 ◽

Vol 80 (2) ◽

pp. 493-505 ◽

Cited By ~ 31

Author(s):

Patrick D. Vigil ◽

Travis J. Wiles ◽

Michael D. Engstrom ◽

Lev Prasov ◽

Matthew A. Mulvey ◽

...

Keyword(s):

Escherichia Coli ◽

Urinary Tract ◽

Virulence Factors ◽

Upper Urinary Tract ◽

Host Cells ◽

Content Type ◽

E Coli ◽

Wide Range ◽

Novel Target ◽

Tract Infections

ABSTRACTUropathogenicEscherichia coli(UPEC) is responsible for the majority of uncomplicated urinary tract infections (UTI) and represents the most common bacterial infection in adults. UPEC utilizes a wide range of virulence factors to colonize the host, including the novel repeat-in-toxin (RTX) protein TosA, which is specifically expressed in the host urinary tract and contributes significantly to the virulence and survival of UPEC.tosA, found in strains within the B2 phylogenetic subgroup ofE. coli, serves as a marker for strains that also contain a large number of well-characterized UPEC virulence factors. The presence oftosAin anE. coliisolate predicts successful colonization of the murine model of ascending UTI, regardless of the source of the isolate. Here, a detailed analysis of the function oftosArevealed that this gene is transcriptionally linked to genes encoding a conserved type 1 secretion system similar to other RTX family members. TosA localized to the cell surface and was found to mediate (i) adherence to host cells derived from the upper urinary tract and (ii) survival in disseminated infections and (iii) to enhance lethality during sepsis (as assessed in two different animal models of infection). An experimental vaccine, using purified TosA, protected vaccinated animals against urosepsis. From this work, it was concluded that TosA belongs to a novel group of RTX proteins that mediate adherence and host damage during UTI and urosepsis and could be a novel target for the development of therapeutics to treat ascending UTIs.

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Peptidoglycan Sensing Prevents Quiescence and Promotes Quorum-Independent Colony Growth of Uropathogenic Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.00157-20 ◽

2020 ◽

Vol 202 (20) ◽

Author(s):

Eric C. DiBiasio ◽

Hilary J. Ranson ◽

James R. Johnson ◽

David C. Rowley ◽

Paul S. Cohen ◽

...

Keyword(s):

Escherichia Coli ◽

Urinary Tract ◽

Urinary Tract Infections ◽

Recurrent Infection ◽

Colony Growth ◽

Quiescent State ◽

Content Type ◽

Tract Infections

ABSTRACT Uropathogenic Escherichia coli (UPEC) is the leading cause of human urinary tract infections (UTIs), and many patients experience recurrent infection after successful antibiotic treatment. The source of recurrent infections may be persistent bacterial reservoirs in vivo that are in a quiescent state and thus are not susceptible to antibiotics. Here, we show that multiple UPEC strains require a quorum to proliferate in vitro with glucose as the carbon source. At low cell density, the bacteria remain viable but enter a quiescent, nonproliferative state. Of the clinical UPEC isolates tested to date, 35% (51/145) enter this quiescent state, including isolates from the recently emerged, multidrug-resistant pandemic lineage ST131 (i.e., strain JJ1886) and isolates from the classic endemic lineage ST73 (i.e., strain CFT073). Moreover, quorum-dependent UPEC quiescence is prevented and reversed by small-molecule proliferants that stimulate colony formation. These proliferation cues include d-amino acid-containing peptidoglycan (PG) tetra- and pentapeptides, as well as high local concentrations of l-lysine and l-methionine. Peptidoglycan fragments originate from the peptidoglycan layer that supports the bacterial cell wall but are released as bacteria grow. These fragments are detected by a variety of organisms, including human cells, other diverse bacteria, and, as we show here for the first time, UPEC. Together, these results show that for UPEC, (i) sensing of PG stem peptide and uptake of l-lysine modulate the quorum-regulated decision to proliferate and (ii) quiescence can be prevented by both intra- and interspecies PG peptide signaling. IMPORTANCE Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs). During pathogenesis, UPEC cells adhere to and infiltrate bladder epithelial cells, where they may form intracellular bacterial communities (IBCs) or enter a nongrowing or slowly growing quiescent state. Here, we show in vitro that UPEC strains at low population density enter a reversible, quiescent state by halting division. Quiescent cells resume proliferation in response to sensing a quorum and detecting external signals, or cues, including peptidoglycan tetra- and pentapeptides.

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Flexible Metabolism and Suppression of Latent Enzymes Are Important forEscherichia coliAdaptation to Diverse Environments within the Host

Journal of Bacteriology ◽

10.1128/jb.00181-19 ◽

2019 ◽

Vol 201 (16) ◽

Cited By ~ 2

Author(s):

Christopher J. Alteri ◽

Stephanie D. Himpsl ◽

Allyson E. Shea ◽

Harry L. T. Mobley

Keyword(s):

Escherichia Coli ◽

Urinary Tract ◽

Pyruvate Kinase ◽

Tricarboxylic Acid Cycle ◽

Financial Burden ◽

Content Type ◽

Bacterial Physiology ◽

E Coli ◽

Growth Requirements ◽

Tract Infections

ABSTRACTBacterial metabolism is necessary for adaptation to the host microenvironment. Flexible metabolic pathways allow uropathogenicEscherichia coli(UPEC) to harmlessly reside in the human intestinal tract and cause disease upon extraintestinal colonization.E. coliintestinal colonization requires carbohydrates as a carbon source, while UPEC extraintestinal colonization requires gluconeogenesis and the tricarboxylic acid cycle. UPEC containing disruptions in two irreversible glycolytic steps involving 6-carbon (6-phosphofructokinase;pfkA) and 3-carbon (pyruvate kinase;pykA) substrates have no fitness defect during urinary tract infection (UTI); however, both reactions are catalyzed by isozymes: 6-phosphofructokinases Pfk1 and Pfk2, encoded bypfkAandpfkB, and pyruvate kinases Pyk II and Pyk I, encoded bypykAandpykF. UPEC strains lacking one or both phosphofructokinase-encoding genes (pfkBandpfkA pfkB) and strains lacking one or both pyruvate kinase genes (pykFandpykA pykF) were investigated to determine their regulatory roles in carbon flow during glycolysis by examining their fitness during UTI andin vitrogrowth requirements. Loss of a single phosphofructokinase-encoding gene has no effect on fitness, while thepfkA pfkBdouble mutant outcompeted the parental strain in the bladder. A defect in bladder and kidney colonization was observed with loss ofpykF, while loss ofpykAresulted in a fitness advantage. ThepykA pykFmutant was indistinguishable from wild-typein vivo, suggesting that the presence of Pyk II rather than the loss of Pyk I itself is responsible for the fitness defect in thepykFmutant. These findings suggest thatE. colisuppresses latent enzymes to survive in the host urinary tract.IMPORTANCEUrinary tract infections are the most frequently diagnosed urologic disease, with uropathogenicEscherichia coli(UPEC) infections placing a significant financial burden on the health care system by generating more than two billion dollars in annual costs. This, in combination with steadily increasing antibiotic resistances to present day treatments, necessitates the discovery of new antimicrobial agents to combat these infections. By broadening our scope beyond the study of virulence properties and investigating bacterial physiology and metabolism, we gain a better understanding of how pathogens use nutrients and compete within host microenvironments, enabling us to cultivate new therapeutics to exploit and target pathogen growth requirements in a specific host environment.

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