Genome-wide mapping of cystitis due to Streptococcus agalactiae and Escherichia coli in mice identifies a unique bladder transcriptome that signifies pathogen-specific antimicrobial defense against urinary tract infection

Abstract

The most common causes of urinary tract infections (UTIs) are Gram-negative pathogens such as Escherichia coli; however, Gram-positive organisms, including Streptococcus agalactiae, or group B streptococcus (GBS), also cause UTI. In GBS infection, UTI progresses to cystitis once the bacteria colonize the bladder, but the host responses triggered in the bladder immediately following infection are largely unknown. Here, we used genome-wide expression profiling to map the bladder transcriptome of GBS UTI in mice infected transurethrally with uropathogenic GBS that was cultured from a 35-year-old women with cystitis. RNA from bladders was applied to Affymetrix Gene-1.0ST microarrays; quantitative reverse transcriptase PCR (qRT-PCR) was used to analyze selected gene responses identified in array data sets. A surprisingly small significant-gene list of 172 genes was identified at 24 h; this compared to 2,507 genes identified in a side-by-side comparison with uropathogenic E. coli (UPEC). No genes exhibited significantly altered expression at 2 h in GBS-infected mice according to arrays despite high bladder bacterial loads at this early time point. The absence of a marked early host response to GBS juxtaposed with broad-based bladder responses activated by UPEC at 2 h. Bioinformatics analyses, including integrative system-level network mapping, revealed multiple activated biological pathways in the GBS bladder transcriptome that regulate leukocyte activation, inflammation, apoptosis, and cytokine-chemokine biosynthesis. These findings define a novel, minimalistic type of bladder host response triggered by GBS UTI, which comprises collective antimicrobial pathways that differ dramatically from those activated by UPEC. Overall, this study emphasizes the unique nature of bladder immune activation mechanisms triggered by distinct uropathogens.

Fig 1

Attachment of uropathogenic serotype III GBS to human bladder uroepithelial cells in vitro (A, 30 min; B, 2 h; C, control) and colonization of the surface of bladder uroepithelium in murine GBS UTI as analyzed by fluorescence microscopy of whole tissue, illustrating focal areas of adhered GBS (D and E) alongside images of pockets of bound GBS between folds of uroepithelium (F and G) as analyzed with high-resolution SEM. Histological assessment of control (H to J) and GBS-infected (K to M) bladders illustrates a PMN infiltrate at 24 h (arrowheads), as demonstrated in serial sections stained using either H&E (H and K) or PMN-specific Gr-1 antibody and IHC techniques (I and J, L and M). Bars, 50 μM (small bars) and 180 μM (large bars).

Fig 2

Quantitation of adherence and intracellular survival of serotype III GBS in human bladder uroepithelial cells in vitro. The percentage of bacteria bound to 5637 cells after 30 min (representing initial adherence) is shown for GBS, alongside the percentage bound and invaded at 2 h and the percentage of intracellular bacteria recovered from bladder cells after 24 h after antibiotic protection. The equivalent data for UPEC CFT073 are shown for comparison.

Fig 3

Infectious dose and temporal colonization dynamics of uropathogenic serotype III GBS in a murine model of UTI. Data for escalating dose assays (A) and measures of early bladder adherence of GBS in mice challenged with the ID₉₀ after 30 min (B) are shown alongside persistence data for GBS in the bladder (C) and kidneys (D) and bacteriuria levels (E). Broken lines indicate the detection limit for each sample type based on the volume plated. Initial binding of GBS to bladder uroepithelium as represented in panel B was determined by comparing unwashed (U/W) bladders (bound GBS plus those present in bladder lumen) (n = 6) with washed (W) bladders (representing only adherent bacteria after 30 min), and the results are shown alongside data for UPEC for comparison. Temporal colonization data (C, D, and E) represent pooled data from mice challenged with the ID₉₀ in two separate experiments with at least 9 mice per group per experiment. Statistical comparisons between groups are based on Mann-Whitney U tests with P values set for significance at 0.05.

Fig 4

In vitro growth curve assays of uropathogenic serotype III GBS in pooled human urine (A) and in THB (B) as measured at OD₆₀₀. The equivalent growth curve data derived from control E. coli strains UPEC CFT073 and 83972 in parallel assays are shown for comparison.

Fig 5

Global gene expression in bladder during GBS cystitis in mice comprises a significant list of 172 genes (FDR < 0.1) derived from comparisons of infected and control (Ctrl) mice at 24 h (n = 5). Gene signal intensities are illustrated in the heat maps for individual arrays (A). The complete significant-gene list is provided in Table 2. The summary response is shown using a volcano scatterplot (B) where significant genes are displayed as shrinkage t test results [−log₁₀(P value)] (y axis) versus expression ratio [log₂(mean GBS infected/control)] (x axis), with vertical lines representing a 2-fold difference; red diamonds represent FDR < 0.1.

Fig 6

Immune activation in the GBS UTI transcriptome comprises prominent cytokine-cytokine receptor signaling (innateDB pathway number 604) (A) in which 12 genes were highly active (red circles; expression and significance cutoffs, 2.0 and 0.1). The pathway is illustrated according to subcellular localization and includes the total of 255 genes annotated in the pathway. The bladder transcriptome also includes chemokine signaling (innateDB pathway 4351) (B), with activation of the pathway occurring via 8 genes (red circles; Cxcl10, Cxcl9, and Stat2 are shown twice) among a total of 175 genes in the pathway.

Fig 7

Pathogen-specific defense against GBS in the bladder involves fewer gene expression responses, and these are slower to develop than responses elicited by UPEC. Volcano scatterplots (A and B) illustrate significant genes identified by shrinkage t test results at 2 h and 24 h for GBS, compared to those for UPEC (C and D). Red diamonds represent genes at FDR < 0.05 for both pathogens. Significant changes in gene expression for those genes identified as common (i.e., induced/repressed by both GBS and UPEC after 24 h) are denoted by black stars in panels B and D and are listed in detail in Table S4 in the supplemental material. These common responses illustrate the exact combination of shared and pathogen-specific responses in the bladder transcriptomes due to these two organisms.

Fig 8

Bladder uroepithelial cells contribute to defense against GBS through production of chemokines and cytokines. Human 5637 uroepithelial cells were infected with GBS at an MOI of 10 as described in Materials and Methods, and culture supernatants at 0.5 h, 2 h, and 24 h were used to measure a panel of 8 protein targets identified from array data and histopathology as part of the bladder response to GBS. Data derived from multiplex protein assay are shown for each target and represent the means ± standard errors of the means of the results determined for quadruplicate samples versus control (noninfected) bladder cells.