Escherichia coli global gene expression in urine from women with urinary tract infection

Abstract

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.

Figure 1. E. coli gene expression in voided urine from cystitis patients and culture in urine ex vivo.

(A) Correlation of gene expression levels obtained by microarray and qPCR. Ct values determined by qPCR are plotted for 13 genes (see text) versus normalized microarray signal intensity for in vivo (left two panels) and in vitro (right two panels) cDNA samples from patient isolates 371 (first and third panel) and 151 (second and last panel). Correlation coefficient (r) values are shown and P<0.001 for all correlations. (B) Genes differentially expressed during UTI in women and culture in urine ex vivo. Log₂ fold changes in vivo, compared to in vitro expression, were measured by qPCR and are normalized to gapA transcript. (C) Confirmation of in vivo gene expression by clinical isolates as shown by PCR using genomic DNA or cDNA template.

Figure 2. E. coli virulence factor expression in urines from UTI patients.

Heat maps indicate normalized microarray signal intensities for genes encoding (A) UPEC fitness factors and (B) genes in the fim locus in eight E. coli isolates in urine collected from women with cystitis. For reference, the overall most (rpsK, +) and least (pheM, −) expressed genes are shown in the bottom panels, representing average signal intensities of 15.821 and 3.881, respectively.

Figure 3. Host-specific virulence gene expression by E. coli.

Relative virulence and fitness gene expression by E. coli isolates in urine from human UTI plotted versus relative expression by E. coli CFT073 in urine from experimental murine UTI. Line shows the theoretical perfect correlation. Panels highlight fitness genes encoding adherence and motility factors (top left), toxins (top right), iron acquisition systems (bottom left), and metabolic and transport proteins (bottom right). Relative expression is based on normalized microarray signal intensity rank (1, gene with lowest signal; 5379, gene with highest signal). Relative human expression is the median rank of all eight isolates, except for fimA, papA, papA_2, hlyA, sat, pic, tsh, tosA, chuA, iutA, iroN, fyuA, hma, iha, and sitA, which are medians from isolates positive for given gene by PCR (P. Vigil and H. Mobley, in preparation). Highlighted genes are colored to indicate the expression rank difference of each gene between human and murine UTI: red, less than 10% difference in expression rank; orange, 10–30% difference; green, 30–50% difference; blue, greater than 50% difference. Spearman rank correlation coefficient for all genes is r = 0.5890 (P<0.0001). Murine data were derived from our previously-published transcriptome study .

Figure 4. In vitro expression of type 1 fimbriae by clinical E. coli isolates.

(A) Invertible element (IE) assay for type 1 fimbriae . IE is shown in gray; inverted repeats (IR) located within the IE; orientation of the fim promoter (P) indicated by arrow in phase-on (top panel) and phase-off (bottom panel) positions. Expected fragment sizes following PCR (half arrows show primer sites) and asymmetrical SnaBI digest are indicated by black bars. (B) IE orientation determined as described in (A) for strains cultured statically under type 1 fimbriae-enriching conditions or with aeration. Arrows indicate expected sizes for phase-on and phase-off. CFT073 strains with mutations preventing IE switching and locking the “phase-on” (L-ON) or “phase-off” (L-OFF) orientation are also included. M, molecular mass standards; sizes (in kb) are indicated. (C) Western blot using anti-FimA polyclonal antibody. Strains were cultured as in (B) and acid-treated whole cell lysate separated on 15% SDS-PAGE gels. Top panel, type 1 fimbriae-enriching static culture; bottom panel, aerated culture. Arrows indicate the ∼12 kDa FimA band. (D) Mannose-sensitive hemagglutination. Strains were cultured as in (B) (statically, top two rows; aeration, bottom two rows) and their ability to agglutinate guinea pig erythrocytes in the presence (+) or absence (−) of alpha-methyl-mannoside (α mm) was assessed. −, no agglutination; +/−, weak agglutination; +, agglutination with undiluted or 1∶2 dilution of bacterial suspension; ++, agglutination with 1∶4 or 1∶8 dilution; +++, agglutination with 1∶16 or 1∶32 dilution; ND, not determined. Data represent median agglutination reactions of three independent experiments.

Figure 5. Murine colonization and type 1 fimbriae expression by clinical isolates.

CBA/J mice were transurethrally inoculated with 10⁸ CFU E. coli EFC4 (fecal strain), CFT073 (model pyelonephritis strain), or clinical isolates AL371 or AL151. (A) Bladder colonization at 48 hpi. Symbols represent data from individual animals and bars indicate the median. (B) fimA expression by isolates AL371 (gray bars) and AL151 (black bars) during murine UTI. Urine was collected from infected mice 6–48 hpi and transcript levels from expelled bacteria measured by qPCR. For each strain, urines were pooled from five mice (n = 10) and represent two biological replicates. Expression fold change following expulsion from the murine bladder relative to expression following expulsion from the human bladder is shown.