Tracy L. Raivio

Envelope stress responses and Gram-negative bacterial pathogenesis.

The σE, Cpx and Bae envelope stress responses of Escherichia coli are involved in the maintenance, adaptation and protection of the bacterial envelope in response to a variety of stressors. Recent studies indicate that the Cpx and σE stress responses exist in many Gram‐negative bacterial pathogens. The envelope is of particular importance to these organisms because most virulence determinants reside in, or must transit through, this cellular compartment. The Cpx system has been implicated in expression of pili, type IV secretion systems and key virulence regulators, while the σE pathway has been shown to be critical for protection from oxidative stress and intracellular survival. Homologues of the σE– and Cpx‐regulated protease DegP are essential for full virulence in numerous pathogens, and, like σE, DegP appears to confer resistance to oxidative stress and intracellular survival capacity. Some pathogens contain multiple homologues of the Cpx‐regulated, disulphide bond catalyst DsbA protein, which has been demonstrated to play roles in the expression of secreted virulence determinants, type III secretion systems and pili. This review highlights recent studies that indicate roles for the σE, Cpx and Bae envelope stress responses in Gram‐negative bacterial pathogenesis.

The σE and Cpx regulatory pathways: Overlapping but distinct envelope stress responses

The Cpx and sigmaE extracytoplasmic stress responses sense and respond to misfolded proteins in the bacterial envelope. Recent studies have highlighted differences between these regulatory pathways in terms of activating signals, mechanisms of signal transduction and the nature of the responses. Cumulatively, the findings suggest distinct physiological roles for these partially overlapping envelope stress responses. The sigmaE pathway is essential for survival and is primarily responsible for monitoring and responding to alterations in outer membrane protein folding. Mounting evidence suggests that the Cpx regulon may have been adapted to ensure properly timed expression and assembly of adhesive organelles.

MicroReview: Envelope stress responses and Gram‐negative bacterial pathogenesis

The σE, Cpx and Bae envelope stress responses of Escherichia coli are involved in the maintenance, adaptation and protection of the bacterial envelope in response to a variety of stressors. Recent studies indicate that the Cpx and σE stress responses exist in many Gram‐negative bacterial pathogens. The envelope is of particular importance to these organisms because most virulence determinants reside in, or must transit through, this cellular compartment. The Cpx system has been implicated in expression of pili, type IV secretion systems and key virulence regulators, while the σE pathway has been shown to be critical for protection from oxidative stress and intracellular survival. Homologues of the σE– and Cpx‐regulated protease DegP are essential for full virulence in numerous pathogens, and, like σE, DegP appears to confer resistance to oxidative stress and intracellular survival capacity. Some pathogens contain multiple homologues of the Cpx‐regulated, disulphide bond catalyst DsbA protein, which has been demonstrated to play roles in the expression of secreted virulence determinants, type III secretion systems and pili. This review highlights recent studies that indicate roles for the σE, Cpx and Bae envelope stress responses in Gram‐negative bacterial pathogenesis.

Characterization of the Cpx Regulon in Escherichia coli Strain MC4100

The Cpx two-component signal transduction pathway of Escherichia coli mediates adaptation to envelope protein misfolding. However, there is experimental evidence that at least 50 genes in 34 operons are part of the Cpx regulon and many have functions that are undefined or unrelated to envelope protein maintenance. No comprehensive analysis of the Cpx regulon has been presented to date. In order to identify strongly Cpx-regulated genes that might play an important role(s) in envelope protein folding and/or to further define the role of the Cpx response and to gain insight into what makes a gene subject to strong Cpx regulation, we have carried out a uniform characterization of a Cpx-regulated lux reporter library in a single-strain background. Strongly Cpx-regulated genes encode proteins that are directly linked to envelope protein folding, localized to the envelope but uncharacterized, or involved in limiting the cellular concentration of noxious molecules. Moderately Cpx-regulated gene clusters encode products implicated in biofilm formation. An analysis of CpxR binding sites in strongly regulated genes indicates that while neither a consensus match nor their orientation predicts the strength of Cpx regulation, most genes contain a CpxR binding site within 100 bp of the transcriptional start site. Strikingly, we found that while there appears to be little overlap between the Cpx and Bae envelope stress responses, the sigma(E) and Cpx responses reciprocally regulate a large group of strongly Cpx-regulated genes, most of which are uncharacterized.

Cpx signaling pathway monitors biogenesis and affects assembly and expression of P pili

P pili are important virulence factors in uropathogenic Escherichia coli. The Cpx two‐component signal transduction system controls a stress response and is activated by misfolded proteins in the periplasm. We have discovered new functions for the Cpx pathway, indicating that it may play a critical role in pathogenesis. P pili are assembled via the chaperone/usher pathway. Subunits that go ‘OFF‐pathway’ during pilus biogenesis generate a signal. This signal is derived from the misfolding and aggregation of subunits that failed to come into contact with the chaperone in the periplasm. In response, Cpx not only controls the stress response, but also controls genes necessary for pilus biogenesis, and is involved in regulating the phase variation of pap expression and, potentially, the expression of a panoply of other virulence factors. This study demonstrates how the prototypic chaperone/usher pathway is intricately linked and dependent upon a signal transduction system.

A third envelope stress signal transduction pathway in Escherichia coli

Escherichia coli uses overlapping envelope stress responses to adapt to insults to the bacterial envelope that cause protein misfolding. The σ E and Cpx envelope stress responses are activated by both common and distinct envelope stresses and respond by increasing the expression of the periplasmic protease DegP as well as target genes unique to each response. The σ E pathway is involved in outer membrane protein (OMP) folding quality control whereas the Cpx pathway plays an important role in the assembly of at least one pilus. Previously, we identified the spy gene as a new Cpx regulon member of unknown function. Interestingly, induction of spy expression by severe envelope stresses such as spheroplasting is only partially dependent on an intact Cpx signalling pathway, unlike other Cpx‐regulated genes. Here we show that the BaeS sensor kinase and BaeR response regulator also control expression of spy in response to envelope stress. BaeS and BaeR do not affect expression of other known Cpx‐regulated genes, however, baeR cpxR double mutants show increased sensitivity to envelope stresses relative to either single mutant alone. We propose that the Bae signal transduction pathway controls a third envelope stress response in E. coli that induces expression of a distinct set of adaptive genes.

Just scratching the surface: an expanding view of the Cpx envelope stress response

To detect and effectively respond to damage to the cell envelope, Gram-negative bacteria possess multiple envelope stress responses. Among these, the CpxAR two-component system has been shown to sense the presence of misfolded periplasmic proteins and increase the production of envelope-localized protein folding and degrading factors in response. However, recent studies have revealed that additional parameters, such as adhesion and central metabolism, can also be sensed by the Cpx signalling system. The discovery that the Cpx regulon contains dozens to hundreds of genes indicates that the cellular functions of the Cpx response are also likely much broader than previously realized. These newly recognized functions include other aspects of envelope maintenance, communication with other regulatory pathways, and pathogenesis. A new model is emerging in which the Cpx response integrates diverse signals and promotes cell survival by protecting the envelope in multiple ways.

Three (and more) component regulatory systems - auxiliary regulators of bacterial histidine kinases.

Two‐component signal transduction (TCST) is the most prevalent mechanism employed by microbes to sense and respond to environmental changes. It is characterized by the signal‐induced transfer of phosphate from a sensor histidine kinase (HK) to a response regulator (RR), resulting in a cellular response. An emerging theme in the field of TCST signalling is the discovery of auxiliary factors, distinct from the HK and RR, which are capable of influencing phosphotransfer. One group of TCST auxiliary proteins accomplishes this task by acting on HKs. Auxiliary regulators of HKs are widespread and have been identified in all cellular compartments, where they can influence HK activity through interactions with the sensing, transmembrane or enzymatic domains of the HK. The effects of an auxiliary regulator are controlled by its regulated expression, modification and/or through ligand binding. Ultimately, auxiliary regulators can connect a given TCST system to other regulatory networks in the cell or result in regulation of the TCST system in response to an expanded range of stimuli. The studies highlighted in this review draw attention to an emerging view of bacterial TCST systems as core signalling units upon which auxiliary factors act.

Tethering of CpxP to the inner membrane prevents spheroplast induction of the Cpx envelope stress response

The Cpx envelope stress response of Escherichia coli is controlled by a two‐component regulatory system that senses misfolded proteins in extracytoplasmic compartments and responds by inducing the expression of envelope protein folding and degrading factors. We have proposed that in the absence of envelope stress the pathway is maintained in a downregulated state, in part through interactions between the periplasmic inhibitor molecule CpxP and the sensing domain of the histidine kinase CpxA. In this study, we show that depletion of the periplasmic contents of the cell by spheroplast formation does indeed lead to induction of the Cpx envelope stress response. Further, removal of CpxP is an important component of this induction because tethering an MBP–CpxP fusion protein to the spheroplast inner membranes prevents full activation by this treatment. Spheroplast formation has previously been demonstrated to induce the expression of a periplasmic protein of unknown function, Spy. Analysis of spy expression in response to spheroplast formation by Western blot analysis and by lacZ operon fusion in various cpx mutant backgrounds demonstrated that spy is a member of the Cpx regulon. Interestingly, although the only known spy homologue is cpxP, Spy does not appear to perform the same function as CpxP as it is not involved in inhibiting the Cpx envelope stress response. Rather, deletion of spy leads to activation of the σE stress response. Because the σE response is specifically affected by alterations in outer membrane protein biogenesis, we think it possible that Spy may be involved in this process.

The Cpx Envelope Stress Response Affects Expression of the Type IV Bundle-Forming Pili of Enteropathogenic Escherichia coli

The Cpx envelope stress response mediates adaptation to potentially lethal envelope stresses in Escherichia coli. The two-component regulatory system consisting of the sensor kinase CpxA and the response regulator CpxR senses and mediates adaptation to envelope insults believed to result in protein misfolding in this compartment. Recently, a role was demonstrated for the Cpx response in the biogenesis of P pili, attachment organelles expressed by uropathogenic E. coli. CpxA senses misfolded P pilus assembly intermediates and initiates increased expression of both assembly and regulatory factors required for P pilus elaboration. In this report, we demonstrate that the Cpx response is also involved in the expression of the type IV bundle-forming pili of enteropathogenic E. coli (EPEC). Bundle-forming pili were not elaborated from an exogenous promoter in E. coli laboratory strain MC4100 unless the Cpx pathway was constitutively activated. Further, an EPEC cpxR mutant synthesized diminished levels of bundle-forming pili and was significantly affected in adherence to epithelial cells. Since type IV bundle-forming pili are very different from chaperone-usher-type P pili in both form and biogenesis, our results suggest that the Cpx envelope stress response plays a general role in the expression of envelope-localized organelles with diverse structures and assembly pathways.