A uropathogenic Escherichia coli strain CFT073-specific DNA microarray that includes each open reading frame was used to analyze the transcriptome of CFT073 bacteria isolated directly from the urine of infected CBA/J mice. The in vivo expression profiles were compared to that of E. coli CFT073 grown statically to exponential phase in rich medium, revealing the strategies this pathogen uses in vivo for colonization, growth, and survival in the urinary tract environment. The most highly expressed genes overall in vivo encoded translational machinery, indicating that the bacteria were in a rapid growth state despite specific nutrient limitations. Expression of type 1 fimbriae, a virulence factor involved in adherence, was highly upregulated in vivo. Five iron acquisition systems were all highly upregulated during urinary tract infection, as were genes responsible for capsular polysaccharide and lipopolysaccharide synthesis, drug resistance, and microcin secretion. Surprisingly, other fimbrial genes, such as pap and foc/sfa, and genes involved in motility and chemotaxis were downregulated in vivo. E. coli CFT073 grown in human urine resulted in the upregulation of iron acquisition, capsule, and microcin secretion genes, thus partially mimicking growth in vivo. On the basis of gene expression levels, the urinary tract appears to be nitrogen and iron limiting, of high osmolarity, and of moderate oxygenation. This study represents the first assessment of any E. coli pathotype's transcriptome in vivo and provides specific insights into the mechanisms necessary for urinary tract pathogenesis.Urinary tract infections (UTIs) are a serious health concern. Forty to 50% of women experience at least one UTI, leading to an estimated 8 million annual physician visits in the United States alone (39, 46). Uropathogenic Escherichia coli (UPEC) is by far the most common etiological agent of all UTIs. UPEC strain CFT073, derived from the clonal group O6:K2:H1 (26), was originally isolated from the blood and urine of a woman diagnosed with acute pyelonephritis (28). It is considered a prototype of the O6 serogroup, one of the most prevalent UPEC clonal lines (23,24). The virulence of this strain was reproduced in the well-established CBA mouse model of ascending UTI (28). In addition to numerous virulence studies, the genome of E. coli CFT073 has recently been sequenced and compared to that of enterohemorrhagic E. coli EDL933 and the nonpathogenic laboratory strain E. coli MG1655 (42).Mutations have been introduced into a number of candidate virulence genes in UPEC, leading to attenuated mutants in experimental UTI. These include fim, encoding type 1 fimbria (7, 13), sat, encoding secreted autotransporter toxin (14), cnf-1, encoding cytotoxic necrotizing factor (36), tonB, involved in iron transport (40), proP, involved in osmoprotectant transport (8), and degS (35). Large-scale screens for virulence factors of UPEC have also identified factors that aid UPEC during growth in urine (38) and have implicated capsule, lipopolysaccharide, ...

Uropathogenic Escherichia coli is the most common etiological agent of urinary tract infections. Bacteria can often express multiple adhesins during infection in order to favor attachment to specific niches within the urinary tract. We have recently demonstrated that type 1 fimbria, a phase-variable virulence factor involved in adherence, was the most highly expressed adhesin during urinary tract infection. Here, we examine whether the expression of type 1 fimbriae can affect the expression of other adhesins. Type 1 fimbrial phase-locked mutants of E. coli strain CFT073, which harbors genes for numerous adhesins, were employed in this study. CFT073-specific DNA microarray analysis of these strains demonstrates that the expression of type 1 fimbriae coordinately affects the expression of P fimbriae in an inverse manner. This represents evidence for direct communication between genes relating to pathogenesis, perhaps to aid the sequential occupation of different urinary tract tissues. While the role of type 1 fimbriae during infection has been clear, the role of P fimbriae must be further defined to assert the relevance of coordinated regulation in vivo. Therefore, we examined the ability of P fimbrial isogenic mutants, constructed in a type 1 fimbrial-negative background, to compete in the murine urinary tract over a period of 168 h. No differences in the colonization of these mutants were observed. However, comparison of these results with previous studies suggests that inversely coordinated expression of adhesin gene clusters does occur in vivo. Interestingly, the mutant that was incapable of expressing either type 1 or P fimbriae compensated by synthesizing F1C fimbriae.Uropathogenic Escherichia coli (UPEC) strains cause the majority of all urinary tract infections (UTIs). Forty to 50% of women experience at least one UTI during their lifetime, leading to an estimated 8 million physician visits annually in the United States (39, 55). Recent efforts to understand the mechanisms of virulence in this important pathogen include the sequencing of UPEC (52), complete transcriptome analysis (45), signature-tagged mutagenesis (4), and differential fluorescence induction (33). These studies collectively implicate adhesins, iron acquisition systems, capsules, lipopolysaccharides, and toxins in UPEC pathogenesis.Adherence to host tissues is often the first step towards colonization; thus, adhesins are essential for pathogenesis. The recent sequencing of UPEC strain CFT073, along with previous virulence studies, has predicted or demonstrated as many as 12 fimbrial gene clusters in this strain (5, 17, 52). Many fimbrial and afimbrial adhesins are phase variable (28, 34), including the most ubiquitous type 1 fimbriae encoded by the fim gene cluster. The expression of type 1 fimbriae is controlled by a promoter situated on an invertible element of DNA, also referred to as the fim switch (1). Bacteria are phase on, and type 1 fimbriae are expressed when the promoter faces the direction of fimA, which encodes the main structur...

SummaryAlthough once thought to be unique to bacteria, Damino acids are also produced by mammals. For example, D -serine is excreted in human urine at concentrations ranging from 3.0 to 40 m m m m g ml ----1. An epidemiological survey demonstrated that urine isolates of E. coli are more likely to catabolise D -serine via expression of D -serine deaminase, DsdA than enteric disease isolates. The urosepsis strain, CFT073, and an isogenic dsdA mutant have similar growth kinetics in minimal or complex media. However, relative to the wild type, the dsdA mutant has a pleiomorphic cell shape and a prolonged, 4-6 h lag phase when grown in human urine. This suggests that D -serine catabolism provides a growth advantage in the urinary tract. Unexpectedly, in a direct competition model of urinary tract infection, the dsdA mutant was recovered 300-times more frequently than the wild type in the bladders of mice 48 h after infection. A new model of E. coli uropathogenesis is proposed where growth and gene expression are modulated in response to environmental D -serine levels. In support of this, the CFT073 dsdA mutant is hyperflagellated and more motile than the wild type indicating that intracellular levels of D -serine may directly or indirectly influence the expression of regulons associated with E. coli uropathogenesis.

In this study we show that deletion of the genes encoding L-serine deaminases SdaA and SdaB resulted in a mutant that accumulates higher intracellular levels of L-serine than CFT073. CFT073 sdaA sdaB has a mild competitive colonization defect whereas a CFT073 dsdA sdaA sdaB triple mutant shows a greater loss in competitive colonization ability. Thus, the inability to generate serine-specific catabolic products does not result in hypercolonization and the ability to catabolize serine represents a positive physiological trait during murine UTI. CFT073 dsdC and CFT073 dsdC dsdA mutants continue to outcompete the wild type in the UTI model. These results confirm that loss of DsdA activity results in the hypercolonization phenotype and that DsdC does not play a direct role in the elevated-colonization phenotype. Interestingly, a CFT073 dsdA mutant with deletions of D-serine transporter genes dsdX and cycA shows wild-type colonization levels of the bladder but is attenuated for kidney colonization. Thus, D-serine acts as a signal for hypercolonization and virulence gene expression by CFT073 dsdA, whereas overall catabolism of serine represents a positive Escherichia coli fitness trait during UTI.Urinary tract infections (UTIs) in adult women impose an estimated cost of $2.4 billion per year in the United States (12). Most women will experience at least one UTI in their lifetime, resulting in an estimated 6.8 million physician visits, 1.2 million emergency room visits, and nearly a quarter million hospitalizations each year. Escherichia coli remains by far the primary causative agent of community-acquired UTIs (11).The urinary tract is a normally sterile environment, and it poses daunting challenges to colonization by E. coli and other microorganisms. In addition to the cleansing flow of urine, numerous innate and acquired immune factors challenge the growth of uropathogenic Escherichia coli (UPEC) in the urinary tract. The host defense involves phagocytic attack, antimicrobial peptides, complement lytic and opsonizing factors, and reactive oxygen and nitrogen species. In addition, the urinary tract offers high-salt and high-osmolarity conditions while limiting E. coli nutrients common to the intestinal tract, especially neutral sugars and iron (4). Thus, we hypothesize that the ability of UPEC to import and metabolize the available carbon and nitrogen sources present in the urinary tract plays a special role in its ability to colonize and cause disease at that site.From a bacterial nutritional standpoint, urine is a dilute mixture of amino acids and small peptides, quite similar to tryptone broth, with the notable exception of the abundance of urea in urine (4). The growth of E. coli in tryptone broth is well characterized, where growing cells preferentially and sequentially utilize serine and then aspartate while secreting acetate. Once these amino acids are depleted, cells then import and use tryptophan and acetate, followed by alanine, glutamine, and threonine (31). This order of nutrient preference holds true for ...

Deletional inactivation of the gene encoding D-serine deaminase, dsdA, in uropathogenic Escherichia coli strain CFT073 results in a hypermotile strain with a hypercolonization phenotype in the bladder and kidneys of mice in a model of urinary tract infection (UTI). The in vivo gene expression profiles of CFT073 and CFT073 dsdA were compared by isolating RNA directly from the urine of mice challenged with each strain individually. Hybridization of cDNAs derived from these samples to CFT073-specific microarrays allowed identification of genes that were up-or down-regulated in the dsdA deletion strain during UTI. Up-regulated genes included the known D-serine-responsive gene dsdX, suggesting in vivo intracellular accumulation of D-serine by CFT073 dsdA. Genes encoding F1C fimbriae, both copies of P fimbriae, hemolysin, OmpF, a dipeptide transporter DppA, a heat shock chaperone IbpB, and clusters of open reading frames with unknown functions were also up-regulated. To determine the role of these genes as well as motility in the hypercolonization phenotype, mutants were constructed in the CFT073 dsdA background and tested in competition against the wild type in the murine model of UTI. Strains with deletions of one or both of the two P fimbrial operons, hlyA, fliC, ibpB, c0468, locus c3566 to c3568, or c2485 to c2490 colonized mouse bladders and kidneys at levels indistinguishable from wild type. CFT073 dsdA c2398 and CFT073 dsdA focA maintained a hypercolonization phenotype. A CFT073 dsdA dppA mutant was attenuated 10-to 50-fold in its colonization ability compared to CFT073. Our results support a role for D-serine catabolism and signaling in global virulence gene regulation of uropathogenic E. coli.Urinary tract infections (UTI) in adult women impose an estimated cost of $2.4 billion per year in the United States (30). Most women will experience at least one UTI in their lifetimes, resulting in an estimated 6.8 million physician visits, 1.2 million emergency room visits, and nearly a quarter million hospitalizations each year. Escherichia coli remains, by far, the primary causative agent of community-acquired UTIs.The urinary tract is a normally sterile environment, and it poses several challenges to colonization by E. coli and other microorganisms. From its natural reservoir in the colon, uropathogenic E. coli (UPEC) must colonize the perineum and the periurethral areas, ascend through the urethra to the bladder, and then continue to ascend via the ureter to the kidneys to cause pyelonephritis. The ascent of UPEC is thwarted by the cleansing flow of urine, free iron limitation, exfoliation of host cells to which UPEC attach, and attack by phagocytic cells and inflammatory mediators. A subset of UTIs progresses to septicemia, exposing the microbe to complement lytic factors. UPEC must be able to bind a variety of differentiated cell surfaces during the ascent and colonization process. At the same time, UPEC must obtain nutrients that may be limited, invade urinary tract epithelial cells, defend against the host res...

In vivo accumulation of D-serine by Escherichia coli CFT073 leads to elevated expression of PAP fimbriae and hemolysin by an unknown mechanism. Loss of D-serine catabolism by CFT073 leads to a competitive advantage during murine urinary tract infection (UTI), but loss of both D-and L-serine catabolism results in attenuation. Serine is the first amino acid to be consumed in closed tryptone broth cultures and precedes the production of acetyl phosphate, a high-energy molecule involved in intracellular signaling, and the eventual secretion of acetate. We propose that the colonization defect associated with the loss of serine catabolism is due to perturbations of acetate metabolism. CFT073 grows more rapidly on acetogenic substrates than does E. coli K-12 isolate MG1655. As shown by transcription microarray results, D-serine is catabolized into acetate via the phosphotransacetylase (pta) and acetate kinase (ackA) genes while downregulating expression of acetyl coenzyme A synthase (acs). CFT073 acs, which is unable to reclaim secreted acetate, colonized mouse bladders and kidneys in the murine model of UTI indistinguishably from the wild type. Both pta and ackA are involved in the maintenance of intracellular acetyl phosphate. CFT073 pta and ackA mutants were screened to investigate the role of acetyl phosphate in UTI pathogenesis. Both single mutants are at a competitive disadvantage relative to the wild type in the kidneys but normally colonize the bladder. CFT073 ackA pta was attenuated in both the bladder and the kidneys. Thus, we demonstrate that CFT073 is adapted to acetate metabolism as a result of requiring a proper cycling of the acetyl phosphate pathway for colonization of the upper urinary tract.Urinary tract infections (UTIs) place a significant burden on the United States healthcare system, costing upwards of $2.4 billion per year (28). The majority of women will experience a UTI in their lifetime, and every year there are approximately 6.8 million physician visits, 1.2 million emergency room visits, a quarter million hospitalizations, and thousands of deaths due to complications of UTIs, most often sepsis (10,18,28,47). Escherichia coli is the most commonly isolated causative agent of community-acquired UTIs (10).The transition from residence in the gastrointestinal tract, where uropathogenic Escherichia coli (UPEC) transiently resides, to the urinary tract represents a significant change in environment. While the gastrointestinal tract is densely populated with many different species of bacteria, the bladder is normally a sterile environment yet one that presents significant challenges to bacterial growth. In addition to the cleansing flow of urine, numerous innate and acquired immune factors challenge the growth of UPEC in the urinary tract. The host defense involves phagocytic attack, antimicrobial peptides, complement lytic and opsonizing factors, and reactive oxygen and nitrogen species. In addition, the urinary tract as reflected in urine is limited in nutrients common to the intestinal tract, esp...

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