A central metabolic circuit controlled by QseC in pathogenic Escherichia coli

Abstract

The QseC sensor kinase regulates virulence in multiple Gram-negative pathogens, by controlling the activity of the QseB response regulator. We have previously shown that qseC deletion interferes with dephosphorylation of QseB thus unleashing what appears to be an uncontrolled positive feedback loop stimulating increased QseB levels. Deletion of QseC downregulates virulence gene expression and attenuates enterohaemorrhagic and uropathogenic Escherichia coli (EHEC and UPEC), Salmonella typhimurium, and Francisella tularensis. Given that these pathogens employ different infection strategies and virulence factors, we used genome-wide approaches to better understand the role of the QseBC interplay in pathogenesis. We found that deletion of qseC results in misregulation of nucleotide, amino acid, and carbon metabolism. Comparable metabolic changes are seen in EHEC ΔqseC, suggesting that deletion of qseC confers similar pleiotropic effects in these two different pathogens. Disruption of representative metabolic enzymes phenocopied UPEC ΔqseC in vivo and resulted in virulence factor downregulation. We thus propose that in the absence of QseC, the constitutively active QseB leads to pleiotropic effects, impairing bacterial metabolism, and thereby attenuating virulence. These findings provide a basis for the development of antimicrobials targeting the phosphatase activity of QseC, as a means to attenuate a wide range of QseC-bearing pathogens.

Fig. 1

Deletion of qseC affects not only virulence factors, but primarily core metabolic processes. A) Classification of the 443 genes affected in UTI89ΔqseC, based on microarray analyses. Each gene is represented once, classified in the most relevant category. Percentage indicates the total number of affected factors in each category, relative to the total number of affected genes in the array. B) Average fold change of upregulated metabolic genes versus upregulated virulence genes. C) Graph showing the 53 altered proteins identified by GC/MS in UTI89ΔqseC. Functional classification was performed using KEGG and EcoCyc. D) The effects of qseC deletion on metabolism (Graph depicts the breakdown of all metabolic factors, including metabolite transporters, grouped in the “metabolism” and membrane transport” categories in 1A).

Fig. 2

QseC is implicated in the fimbrial regulatory networks. A) Expression of 7 CUP systems in UTI89ΔqseC, relative to UTI89, determined by microarray analysis. B) Relative fold change of yeh, F17-like, csgD and rstA in UTI89ΔqseC (red bar) compared to UTI89 (blue bar) by qRT-PCR. Values are normalized to the 16s rrsH gene.

Fig. 3

Effects of qseC deletion on nucleotide and amino acid metabolism. A) Nucleotide metabolism genes differentially expressed in UTI89ΔqseC, as determined by microarray analysis. B) Schematic of nucleotide metabolism. Upregulated genes, based on microarray analysis, are shown in green; downregulated genes in red; solid arrows indicate single steps; dashed arrows indicate more than one step. C) Differentially expressed amino acid metabolism genes. D) Schematic of the amino acid metabolic pathways affected upon qseC deletion. Color coding is same as in (B).

Fig. 4

Deletion of qseC impedes TCA cycle completion. A) Schematic of carbohydrate metabolism. Upregulated genes, based on microarray analysis, are shown in green; downregulated genes in red; solid arrows indicate single steps; dashed arrows indicate more than one step. B) Carbohydrate metabolism genes differentially expressed in the absence of QseC. C) Growth curves of UTI89ΔqseC (green) and wt UTI89 (red) in the presence of metabolites implicated in the glyoxylate shunt and the TCA cycle, generated by the Omnilog PM software. UTI89ΔqseC has a growth defect on metabolites that require completion of the TCA cycle (bottom 4 panels) and shows a preference for metabolites of the glyoxylate shunt (top 2 panels).

Fig. 5

TCA cycle mutants are attenuated exhibiting ΔqseC-like phenotypes. A) Bladder titers of UTI89ΔsdhB, UTI89Δmdh and UTI89ΔaceA compared to UTI89 and UTI89ΔqseC. Experiment was repeated 3 times. B) IBC production by UTI89, UTI89ΔsdhB, UTI89Δmdh, UTI89ΔaceA and UTI89ΔqseC. C) HA titers of UTI89ΔsdhB, UTI89Δmdh and UTI89ΔaceA compared to UTI89 and UTI89ΔqseC. Mannose addition inhibits type 1-dependent HA. D) Western Blot analysis probing for the CsgG curli subunit in UTI89ΔsdhB, UTI89Δmdh and UTI89ΔaceA compared to UTI89 and UTI89ΔqseC. UTI89ΔsdhB and UTI89Δmdh are defective in curli expression. E) Motility assays showing that UTI89ΔsdhB and UTI89Δmdh exhibit defective swimming motility similar to UTI89ΔqseC and UTI89ΔflhDC (negative for flagella). ***, P<0.0007, **, P<0.0099 and *, P<0.05