Role of histone-like proteins H-NS and StpA in expression of virulence determinants of uropathogenic Escherichia coli

Abstract

The histone-like protein H-NS is a global regulator in Escherichia coli that has been intensively studied in nonpathogenic strains. However, no comprehensive study on the role of H-NS and its paralogue, StpA, in gene expression in pathogenic E. coli has been carried out so far. Here, we monitored the global effects of H-NS and StpA in a uropathogenic E. coli isolate by using DNA arrays. Expression profiling revealed that more than 500 genes were affected by an hns mutation, whereas no effect of StpA alone was observed. An hns stpA double mutant showed a distinct gene expression pattern that differed in large part from that of the hns single mutant. This suggests a direct interaction between the two paralogues and the existence of distinct regulons of H-NS and an H-NS/StpA heteromeric complex. hns mutation resulted in increased expression of alpha-hemolysin, fimbriae, and iron uptake systems as well as genes involved in stress adaptation. Furthermore, several other putative virulence genes were found to be part of the H-NS regulon. Although the lack of H-NS, either alone or in combination with StpA, has a huge impact on gene expression in pathogenic E. coli strains, its effect on virulence is ambiguous. At a high infection dose, hns mutants trigger more sudden lethality due to their increased acute toxicity in murine urinary tract infection and sepsis models. At a lower infectious dose, however, mutants lacking H-NS are attenuated through their impaired growth rate, which can only partially be compensated for by the higher expression of numerous virulence factors.

FIG. 1.

(A) Total numbers of altered genes in the hns mutant (shaded) and the hns stpA mutant (white) determined by expression profiling using a K-12-specific array (left) and an E. coli pathoarray (right). ORF, open reading frame. (B) Distribution of the deregulated K-12 genes into functional groups (upper panel, hns mutant; lower panel, hns stpA mutant). Upregulated genes are depicted by black bars; downregulated genes are shaded.

FIG. 2.

Expression levels of the major virulence factors of UPEC strain 536. Signal intensities derived from the array hybridizations for the three different fimbrial determinants fim (A), sfa (B), and prf (C) as well as for the alpha-hemolysin determinant hly (D) are depicted for wild-type (wt) strain 536 and its mutants.

FIG. 3.

Hyperfimbriated phenotype of hns mutants. (A) Protein levels of major subunits of S and P fimbriae in mid-log-phase cultures monitored by Western blotting. (B) Morphology of a wild-type cell (upper panel) and an hns mutant cell (lower panel) from agar plates visualized by atomic force microscopy.

FIG. 4.

H-NS-dependent siderophore production in E. coli strain 536. (A) Expression levels of the three different siderophore receptor genes of the H-NS-dependent siderophore systems determined by semiquantitative RT-PCR using RNA extracted from LB cultures and wild-type (WT) DNA as a positive control. (B) Chrome azurol S assay performed with supernatants from iron-depleted cultures grown in MM9 medium plus 2,2′-dipyridyl. Symbols: 536 wild type, ▴; ΔstpA, ×; Δhns, □; Δhns ΔstpA, ⋄.

FIG. 5.

Survival curves of mice infected intravenously with E. coli strain 536 or its isogenic mutants. Groups of 10 mice were infected with either 10⁹ CFU (A) or 10⁸ CFU (B) of washed bacteria through the tail veins.

FIG. 6.

Time course of lethality in the mouse lung toxicity assay. Groups of five mice were instilled intranasally with E. coli strain 536 or its isogenic mutants.