Murine models of urinary tract infection (UTI) have provided substantial data identifying uropathogenic E. coli (UPEC) virulence factors and assessing their expression in vivo. However, it is unclear how gene expression in these animal models compares to UPEC gene expression during UTI in humans. To address this, we used a UPEC strain CFT073-specific microarray to measure global gene expression in eight E. coli isolates monitored directly from the urine of eight women presenting at a clinic with bacteriuria. The resulting gene expression profiles were compared to those of the same E. coli isolates cultured statically to exponential phase in pooled, sterilized human urine ex vivo. Known fitness factors, including iron acquisition and peptide transport systems, were highly expressed during human UTI and support a model in which UPEC replicates rapidly in vivo. While these findings were often consistent with previous data obtained from the murine UTI model, host-specific differences were observed. Most strikingly, expression of type 1 fimbrial genes, which are among the most highly expressed genes during murine experimental UTI and encode an essential virulence factor for this experimental model, was undetectable in six of the eight E. coli strains from women with UTI. Despite the lack of type 1 fimbrial expression in the urine samples, these E. coli isolates were generally capable of expressing type 1 fimbriae in vitro and highly upregulated fimA upon experimental murine infection. The findings presented here provide insight into the metabolic and pathogenic profile of UPEC in urine from women with UTI and represent the first transcriptome analysis for any pathogenic E. coli during a naturally occurring infection in humans.

Uropathogenic Escherichia coli (UPEC) strains are responsible for the majority of uncomplicated urinary tract infections, which can present clinically as cystitis or pyelonephritis. UPEC strain CFT073, isolated from the blood of a patient with acute pyelonephritis, was most cytotoxic and most virulent in mice among our strain collection. Based on the genome sequence of CFT073, microarrays were utilized in comparative genomic hybridization (CGH) analysis of a panel of uropathogenic and fecal/commensal E. coli isolates. Genomic DNA from seven UPEC (three pyelonephritis and four cystitis) isolates and three fecal/commensal strains, including K-12 MG1655, was hybridized to the CFT073 microarray. The CFT073 genome contains 5,379 genes; CGH analysis revealed that 2,820 (52.4%) of these genes were common to all 11 E. coli strains, yet only 173 UPEC-specific genes were found by CGH to be present in all UPEC strains but in none of the fecal/commensal strains. When the sequences of three additional sequenced UPEC strains (UTI89, 536, and F11) and a commensal strain (HS) were added to the analysis, 131 genes present in all UPEC strains but in no fecal/commensal strains were identified. Seven previously unrecognized genomic islands (>30 kb) were delineated by CGH in addition to the three known pathogenicity islands. These genomic islands comprise 672 kb of the 5,231-kb (12.8%) genome, demonstrating the importance of horizontal transfer for UPEC and the mosaic structure of the genome. UPEC strains contain a greater number of iron acquisition systems than do fecal/commensal strains, which is reflective of the adaptation to the iron-limiting urinary tract environment. Each strain displayed distinct differences in the number and type of known virulence factors. The large number of hypothetical genes in the CFT073 genome, especially those shown to be UPEC specific, strongly suggests that many urovirulence factors remain uncharacterized.Escherichia coli strains capable of causing disease outside the gastrointestinal tract belong to a diverse group of isolates referred to as extraintestinal pathogenic E. coli (ExPEC) (50, 84). ExPEC strains are responsible for a variety of diseases, including urinary tract infections (UTIs), newborn meningitis, septicemia, nosocomial pneumonia, intra-abdominal infections, osteomyelitis and wound infections (22,27,49,84). Uropathogenic E. coli (UPEC), a prominent member of the ExPEC family, is responsible for up to 90% of uncomplicated UTIs in otherwise healthy individuals (108). An infection occurs primarily by the ascending route following the contamination of the periurethral area, presumably via a fecal reservoir. Bacteria ascend the urethra and colonize the bladder, resulting in cystitis, and in severe cases, infection may spread up the ureters to the kidneys, causing pyelonephritis (15). A serious and potentially life-threatening complication of pyelonephritis occurs when bacteria invade the bloodstream and produce a systemic infection. Due to anatomical differences, UTIs are significantly mo...

Uropathogenic Escherichia coli (UPEC) strain CFT073 contains 13 large genomic islands ranging in size from 32 kb to 123 kb. Eleven of these genomic islands were individually deleted from the genome, and nine isogenic mutants were tested for their ability to colonize the CBA/J mouse model of ascending urinary tract infection. Three genomic island mutants (⌬PAI-aspV, ⌬PAI-metV, and ⌬PAI-asnT) were significantly outcompeted by wild-type CFT073 in the bladders and/or kidneys following transurethral cochallenge (P < 0.0139). The PAI-metV mutant also showed significant attenuation in the ability to independently colonize the kidneys (P ‫؍‬ 0.0011). Specific genes within these islands contributed to the observed phenotype, including a previously uncharacterized iron acquisition cluster, fbpABCD (c0294 to c0297 [c0294-97]), autotransporter, picU (c0350), and RTX family exoprotein, tosA (c0363) in the PAI-aspV island. The double deletion mutant with deletions in both copies of the fbp iron acquisition operon (⌬c0294-97 ⌬c2518-15) was significantly outcompeted by wild-type CFT073 in cochallenge. Strains with mutations in a type VI secretion system within the PAI-metV island did not show attenuation. The attenuation of the PAI-metV island was localized to genes c3405-10, encoding a putative phosphotransferase transport system, which is common to UPEC and avian pathogenic E. coli strains but absent from E. coli K-12. We have shown that, in addition to encoding virulence genes, genomic islands contribute to the overall fitness of UPEC strain CFT073 in vivo.Escherichia coli, a versatile microbe, can colonize the intestinal tract with no harmful effects to the host or can cause devastating and life-threatening disease (34). E. coli can be classified into one of three groups: commensal (nonpathogenic) E. coli strains that coexist with the host without causing overt disease, intestinal pathogenic (diarrheagenic) E. coli, and extraintestinal pathogenic E. coli (ExPEC). The latter category, ExPEC, was proposed in 2000 to classify E. coli isolates capable of causing disease outside of the intestinal tract, including uropathogenic E. coli (UPEC), sepsis-associated E. coli, and neonatal meningitis-associated E. coli (63). Within the human intestinal tract, ExPEC may colonize without causing disease. However, this subset of E. coli has the ability to disseminate to other sites of the body, including the urinary tract, bloodstream, and central nervous system, and elicit pathogenesis (77).Urinary tract infections (UTIs), the most common type of bacterial infection (16), affect 11% of adult women every year, with an estimated one-third of women requiring antibiotic therapy for a clinician-diagnosed UTI by 24 years of age (17). Approximately 60% of all women will experience a UTI during their lifetime (17). Nearly 7 million physician office visits, 1 million emergency room visits, and 100,000 hospitalizations per year are attributed to UTIs, with women twice as likely as men to seek medical treatment for infections of the urinary tract (6...

Three mutants of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (V106A, V179D, and Y181C), which occur in clinical isolates and confer resistance to nonnucleoside reverse transcriptase inhibitors (NNRTIs), were analyzed for RNA-and DNA-dependent DNA polymerization and RNase H cleavage. All mutants demonstrated processivities of polymerization that were indistinguishable from wild-type enzyme under conditions in which deoxynucleoside triphosphates were not limiting. The V106A reverse transcriptase demonstrated a three-to fourfold slowing of both DNA 3-end-directed and RNA 5-end-directed RNase H cleavage relative to both wild-type and V179D enzymes, similar to what was observed for P236L in a previously published study (P. Gerondelis et al., J. Virol. 73:5803-5813, 1999). In contrast, the Y181C reverse transcriptase demonstrated a selective acceleration of the secondary RNase H cleavage step during both modes of RNase H cleavage. The relative replication fitness of these mutants in H9 cells was assessed in parallel infections as well as in growth competition experiments. Of the NNRTI-resistant mutants, V179D was more fit than Y181C, and both of these mutants were more fit than V106A, which demonstrated the greatest reduction in RNase H cleavage. These findings, in combination with results from previous work, suggest that abnormalities in RNase H cleavage are a common characteristic of HIV-1 mutants resistant to NNRTIs and that combined reductions in the rates of DNA 3-end-and RNA 5-end-directed cleavages are associated with significant reductions in the replication fitness of HIV-1.Infection with human immunodeficiency virus (HIV) is the cause of AIDS and affects over 30 million people worldwide (64). The primary targets of therapy for HIV infection include the viral protease and reverse transcriptase (RT). HIV type 1 (HIV-1) RT is a heterodimer consisting of 66-and 51-kDa subunits (p66 and p51, respectively) (3). p66 contains both the polymerase and the RNase H active sites of the enzyme (34, 37, 39). The RNase H domain is present in the carboxy-terminal third of p66. Although p51 is derived from p66 by proteolytic cleavage, it assumes a very different tertiary structure and does not contain a catalytic site (37, 39). The function of p51 is not known, but it may play a role in binding the tRNA 3Lys -template complex (3, 39) and in maintaining the structural integrity of the heterodimer (1).RNase H cleavage is essential for HIV-1 replication (61; for a review see reference 11). Two modes of RNase H cleavage have been described (Fig. 1). "Polymerase-dependent" cleavage is thought to occur in concert with DNA polymerization to degrade the genomic RNA during minus strand DNA synthesis (26,46). The position of the primary DNA 3Ј-end-directed cleavage occurs 15 to 18 nucleotides (nt) from the recessed 3Ј end of the DNA (26, 33); we have referred to this mode of cleavage as DNA 3Ј-end-directed RNase H cleavage. A second mode of RNase H cleavage occurs independently of DNA polymerization. The position ...