Cytotoxic necrotizing factor 1 and hemolysin from uropathogenic Escherichia coli elicit different host responses in the murine bladder - PubMed (original) (raw)

Cytotoxic necrotizing factor 1 and hemolysin from uropathogenic Escherichia coli elicit different host responses in the murine bladder

Tamako A Garcia et al. Infect Immun. 2013 Jan.

Abstract

Cytotoxic necrotizing factor 1 (CNF1) and hemolysin (HlyA1) are toxins produced by uropathogenic Escherichia coli (UPEC). We previously showed that these toxins contribute to the inflammation and tissue damage seen in a mouse model of ascending urinary tract infection. CNF1 constitutively activates small Rho GTPases by deamidation of a conserved glutamine residue, and HlyA1 forms pores in eukaryotic cell membranes. In this study, we used cDNA microarrays of bladder tissue isolated from mice infected intraurethrally with wild-type CP9, CP9cnf1, or CP9ΔhlyA to further evaluate the role that each toxin plays in the host response to UPEC. Regardless of the strain used, we found that UPEC itself elicited a significant change in host gene expression 24 h after inoculation. The largest numbers of upregulated genes were in the cytokine and chemokine signaling and Toll-like receptor signaling pathways. CNF1 exerted a strong positive influence on expression of genes involved in innate immunity and signal transduction and a negative impact on metabolism- and transport-associated genes. HlyA1 evoked an increase in expression of genes that encode innate immunity factors and a decrease in expression of genes involved in cytoskeletal and metabolic processes. Multiplex cytokine and myeloperoxidase assays corroborated our finding that a strong proinflammatory response was elicited by all strains tested. Bladders challenged intraurethrally with purified CNF1 displayed pathology similar to but significantly less intense than the pathology that we observed in CP9-challenged mice. Our data demonstrate substantial roles for CNF1 and HlyA1 in initiation of a strong proinflammatory response to UPEC in the bladder.

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Figures

Fig 1

Fig 1

UPEC elicits significant host transcriptional changes in the bladder. Complete linkage cluster (A) and heat map (B) of all of the 2,200 unique BioIDs that were significantly different in any of the five comparisons in Table S1 in the supplemental material. Red, an increase in gene expression relative to the control; green, a decrease in gene expression relative to the control. (C) Complete linkage cluster of 783 BioIDs from CP9-versus-CP9_cnf1_ and/or CP9-versus-CP9Δ_hlyA1_ comparisons. For complete linkage clusters, gene expression relative to the reference control (normal bladder RNA) is shown for RNA isolated from the bladder of each mouse challenged with CP9 (CP9), CP9_cnf1_ (cnf1), CP9Δ_hlyA1_ (hlyA), or PBS (PBS).

Fig 2

Fig 2

CNF1 alters the expression of more genes than HlyA1. Genes whose expression changed 3-fold or more in the CP9-versus-CP9_cnf1_ and/or CP9-versus-CP9Δ_hlyA1_ comparisons were separated into GO biological process categories. The total numbers of genes altered in each comparison are as follows: 91 genes upregulated in CP9 versus CP9_cnf1_, 93 genes downregulated in CP9 versus CP9_cnf1_, 49 genes upregulated in CP9 versus CP9Δ_hlyA1_, and 37 genes downregulated in CP9 versus CP9Δ_hlyA1_.

Fig 3

Fig 3

Proinflammatory cytokines (A to E) and myeloperoxidase (F) are present in the bladders or urine by 24 h after challenge with UPEC. The proinflammatory cytokines IL-6 (A), MIP-2 (CXCL-2) (B), KC (CXCL-1) (C), IFN-γ (D), and TNF-α (E) were measured by use of the Milliplex MAG mouse cytokine/chemokine panel and Luminex 100 MultiAnalyte platform in a magnetic bead format. The limit of detection was 2.2 pg/ml for each cytokine. (F) The level of myeloperoxidase in mouse urine was detected by ELISA. Prechallenge urine samples within an experiment were pooled. Horizontal black bars represent the median. Data were analyzed by the Kruskal-Wallis test with Dunn's multiple-comparison posttest. *, P < 0.05; **, P < 0.005.

Fig 4

Fig 4

Bladders from CP9- and CNF1-inoculated mice exhibit significant pathology. Female C3H/HeOuJ mice were challenged with CP9 (A and B), 2 μg CNF1 (C and D), or PBS (E and F). Each panel is a representative sample from its respective treatment group. Magnifications, ×40 (A, C, and E) and ×400 (B, D, and F). The rectangles in panels A, C, and E correspond to the selection shown at ×400. Symbols: ●, edema; ▼, epithelial damage. (G) Histological scores of mouse bladders challenged with CP9, CNF1, or PBS. The four histological parameters scored were epithelial damage, edema, hemorrhage, and neutrophil infiltration, and each was assigned a score of 0 to 5. The scoring scale is as follows: 0, no lesion; 1, minimal; 2, mild; 3, moderate; 4, marked; 5, severe.

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