Abstract
Envelope-localized proteins, such as adhesins and secretion systems, play critical roles in host infection by Gram-negative pathogens. As such, their folding is monitored by envelope stress response systems. Previous studies demonstrated that the Cpx envelope stress response is required for virulence of Citrobacter rodentium, a murine pathogen used to model infections by the human pathogens enteropathogenic and enterohemorrhagic Escherichia coli; however, the mechanisms by which the Cpx response promotes host infection were previously unknown. Here, we characterized the C. rodentium Cpx regulon in order to identify genes required for host infection. Using transcriptomic and proteomic approaches, we found that the Cpx response upregulates envelope-localized protein folding and degrading factors but downregulates pilus genes and type III secretion effectors. Mouse infections with C. rodentium strains lacking individual Cpx-regulated genes showed that the chaperone/protease DegP and the disulfide bond oxidoreductase DsbA were essential for infection, but Cpx regulation of these genes did not fully account for attenuation of C. rodentium ΔcpxRA. Both deletion of dsbA and treatment with the reducing agent dithiothreitol activated the C. rodentium Cpx response, suggesting that it may sense disruption of disulfide bonding. Our results highlight the importance of envelope protein folding in host infection by Gram-negative pathogens.
Highlights
The Gram-negative envelope, which consists of the inner and outer membranes (IM and OM) and intervening periplasmic space, plays a critical role in the cell’s interactions with its environment
To characterize the C. rodentium Cpx regulon, we used a dual approach: RNA-Seq was used to identify transcripts whose abundance differs between C. rodentium DBS100 and ΔcpxRA
Secreted into the culture supernatant did not differ between wild-type and cpx mutant strains, nor did abundance of the other major secreted proteins, EspA and EspD (Figure S2). These results suggest that the C. rodentium Cpx response negatively regulates expression of T3S-related genes in two separate ways: the non-locus of enterocyte effacement (LEE) encoded effectors appear to be repressed primarily at the transcriptional level, while the LEE-encoded protein EspB may be repressed at the posttranscriptional or post-translational level
Summary
The Gram-negative envelope, which consists of the inner and outer membranes (IM and OM) and intervening periplasmic space, plays a critical role in the cell’s interactions with its environment. Envelope-localized proteins perform essential functions including nutrient uptake, extrusion of waste and toxic molecules, electron transport, adherence to surfaces, motility, and signal transduction, among others. Among the numerous envelope stress responses present in enterobacteria, the Cpx envelope stress response plays a important role in monitoring the folding of periplasmic and IM proteins (Vogt and Raivio, 2012; Raivio, 2014; Guest and Raivio, 2016). The Cpx response is mediated by a two-component system consisting of the IM-localized histidine kinase CpxA and the cytoplasmic response regulator CpxR.
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