Activation of the Cpx-envelope stress response system promotes tolerance to antibacterials delivered by arginine-rich peptides and aminoglycosides in Escherichia coli

AbstractCell penetrating peptides (CPP) are increasingly used for cellular drug delivery in both pro- and eukaryotic cells, and oligoarginines have attracted special attention. However; their mechanism of action, particularly for prokaryotes is still unknown. Arginine-rich CPPs (R-CPP) efficiently delivers the antimicrobial peptide nucleic acid (PNA) into bacteria. Here, we show that resistance to an R-CPP PNA conjugate in Escherichia coli requires multiple genetic modifications and is specific to R-CPP and not to the PNA-part. An integral part of the resistance was the constitutively activated Cpx-envelope stress response system (cpx*), which decreased the cytoplasmic membrane potential and thereby indicates an indirectly energy dependent uptake mechanism. Interestingly, cpx* mutants also showed increased tolerance to aminoglycosides and R-CPP conjugated to a peptide targeting the DNA sliding clamp; i.e., similar uptake in E. coli for these antimicrobial compounds. We speculate that the cpx* phenotype could create an evolutionary opportunity to adapt and evolve in the presence of either compounds.Author summaryThe emergence of multidrug resistant bacteria is raising the need for new classes of antibiotics. Peptide nucleic acids (PNAs) may fill this requirement by their ability to block translation of essential mRNAs and hence inhibit growth. PNA needs conjugation to a delivery peptide (cell penetrating peptide; CPP) to enter the bacteria. Arginine-rich CPPs (CPPR) are receiving a lot of attention for use as delivery vessels. Here, we show, for the first time, CPPR-PNA resistance in Escherichia coli directed towards the delivery peptide. Consequently, resistance also applies to other antimicrobial compounds delivered by the same carrier. An integral part of CPPR resistance is due to a constitutive active Cpx-response system, which leads to a decreased electric potential (ΔΨ) across the inner membrane. The decreased ΔΨ is a result of down-regulation of two aerobic respiratory operons, namely NADH:ubiquinone oxidoreductase complex I and cytochrome bo3 ubiquinol oxidase. The decreased ΔΨ also led to increased tolerance to aminoglycosides. This shows that a (large) negative ΔΨ is important for providing sufficient free energy for membrane translocation of both CPPR and that the inner membrane is the main barrier for entry of both arginine-rich delivery peptides and aminoglycosides.

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The Escherichia coli CpxA-CpxR Envelope Stress Response System Regulates Expression of the Porins OmpF and OmpC

Journal of Bacteriology ◽

10.1128/jb.187.16.5723-5731.2005 ◽

2005 ◽

Vol 187 (16) ◽

pp. 5723-5731 ◽

Cited By ~ 99

Author(s):

Eric Batchelor ◽

Don Walthers ◽

Linda J. Kenney ◽

Mark Goulian

Keyword(s):

Escherichia Coli ◽

Stress Response ◽

Response Regulator ◽

Gene Encoding ◽

Fluorescent Reporter ◽

Response System ◽

Dnase I Footprinting ◽

Envelope Stress ◽

Two Component ◽

Stress Response System

ABSTRACT We performed transposon mutagenesis of a two-color fluorescent reporter strain to identify new regulators of the porin genes ompF and ompC in Escherichia coli. Screening of colonies by fluorescence microscopy revealed numerous mutants that exhibited interesting patterns of porin expression. One mutant harbored an insertion in the gene encoding the histidine kinase CpxA, the sensor for a two-component signaling system that responds to envelope stress. The cpxA mutant exhibited increased transcription of ompC and a very strong decrease in transcription of ompF under conditions in which acetyl phosphate levels were high. Subsequent genetic analysis revealed that this phenotype is dependent on phosphorylation of the response regulator CpxR and that activation of CpxA in wild-type cells results in similar regulation of porin expression. Using DNase I footprinting, we demonstrated that CpxR binds upstream of both the ompF and ompC promoters. It thus appears that two distinct two-component systems, CpxA-CpxR and EnvZ-OmpR, converge at the porin promoters. Within the context of envelope stress, outer membrane beta-barrel proteins have generally been associated with the sigma E pathway. However, at least for the classical porins OmpF and OmpC, our results show that the Cpx envelope stress response system plays a role in regulating their expression.

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The Cpx Stress Response System Potentiates the Fitness and Virulence of Uropathogenic Escherichia coli

Infection and Immunity ◽

10.1128/iai.01213-12 ◽

2013 ◽

Vol 81 (5) ◽

pp. 1450-1459 ◽

Cited By ~ 37

Author(s):

Irina Debnath ◽

J. Paul Norton ◽

Amelia E. Barber ◽

Elizabeth M. Ott ◽

Bijaya K. Dhakal ◽

...

Keyword(s):

Escherichia Coli ◽

Urinary Tract ◽

Stress Response ◽

Aminoglycoside Antibiotic ◽

Deletion Mutants ◽

Content Type ◽

Response System ◽

Stress Response System ◽

Tract Infections ◽

Auxiliary Factor

ABSTRACTStrains of uropathogenicEscherichia coli(UPEC) are the primary cause of urinary tract infections, representing one of the most widespread and successful groups of pathogens on the planet. To colonize and persist within the urinary tract, UPEC must be able to sense and respond appropriately to environmental stresses, many of which can compromise the bacterial envelope. The Cpx two-component envelope stress response system is comprised of the inner membrane histidine kinase CpxA, the cytosolic response regulator CpxR, and the periplasmic auxiliary factor CpxP. Here, by using deletion mutants along with mouse and zebrafish infection models, we show that the Cpx system is critical to the fitness and virulence of two reference UPEC strains, the cystitis isolate UTI89 and the urosepsis isolate CFT073. Specifically, deletion of thecpxRAoperon impaired the ability of UTI89 to colonize the murine bladder and greatly reduced the virulence of CFT073 during both systemic and localized infections within zebrafish embryos. These defects coincided with diminished host cell invasion by UTI89 and increased sensitivity of both strains to complement-mediated killing and the aminoglycoside antibiotic amikacin. Results obtained with thecpxPdeletion mutants were more complicated, indicating variable strain-dependent and niche-specific requirements for this well-conserved auxiliary factor.

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The Cpx stress response system of Escherichia coli senses plasma membrane proteins and controls HtpX, a membrane protease with a cytosolic active site

Genes to Cells ◽

10.1046/j.1365-2443.2002.00554.x ◽

2002 ◽

Vol 7 (7) ◽

pp. 653-662 ◽

Cited By ~ 80

Author(s):

Nobuyuki Shimohata ◽

Shinobu Chiba ◽

Naoya Saikawa ◽

Koreaki Ito ◽

Yoshinori Akiyama

Keyword(s):

Escherichia Coli ◽

Plasma Membrane ◽

Stress Response ◽

Membrane Proteins ◽

Active Site ◽

Plasma Membrane Proteins ◽

Response System ◽

Stress Response System

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Phage-shock-protein (Psp) Envelope Stress Response: Evolutionary History & Discovery of Novel Players

10.1101/2020.09.24.301986 ◽

2020 ◽

Author(s):

Janani Ravi ◽

Vivek Anantharaman ◽

Samuel Zorn Chen ◽

Pratik Datta ◽

L Aravind ◽

...

Keyword(s):

Stress Response ◽

Web Application ◽

Cell Envelope ◽

Contextual Information ◽

Specific Cell ◽

Bacterial Phyla ◽

Response System ◽

Envelope Stress ◽

Stress Response System ◽

Conserved Gene

AbstractThe phage shock protein (Psp) stress-response system protects bacteria from envelope stress and stabilizes the cell membrane. Recent work from our group suggests that the psp systems have evolved independently in distinct Gram-positive and Gram-negative bacterial clades to effect similar stress response functions. Despite the prevalence of the key effector, PspA, and the functional Psp system, the various genomic contexts of Psp proteins, as well as their evolution across the kingdoms of life, have not yet been characterized. We have developed a computational pipeline for comparative genomics and protein sequence-structure-function analyses to identify sequence homologs, phyletic patterns, domain architectures, gene neighborhoods, and evolution of the candidates across the tree of life. This integrative pipeline enabled us to make several new discoveries, including the truly universal nature of PspA and the ancestry of the PspA/Snf7 dating all the way back to the Last Universal Common Ancestor. Using contextual information from conserved gene neighborhoods and their domain architectures, we delineated the phyletic patterns of all the Psp members. Next, we systematically identified all possible ‘flavors’ and genomic neighborhoods of the Psp systems. Finally, we traced their evolution, leading us to several interesting findings of their occurrence and co-migration that together point to the functions and roles of Psp in stress-response systems that are often lineage-specific. Conservation of the Psp systems across bacterial phyla emphasizes the established importance of this stress response system in prokaryotes, while the modularity in various lineages is indicative of adaptation to bacteria-specific cell-envelope structures, lifestyles, and adaptation strategies. We also developed an interactive web application that hosts all the data and results in this study that researchers can explore (https://jravilab.shinyapps.io/psp-evolution).

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Induction Kinetics of a Conditional pH Stress Response System in Escherichia coli

Journal of Molecular Biology ◽

10.1016/j.jmb.2009.08.037 ◽

2009 ◽

Vol 393 (2) ◽

pp. 272-286 ◽

Cited By ~ 48

Author(s):

Georg Fritz ◽

Christiane Koller ◽

Korinna Burdack ◽

Larissa Tetsch ◽

Ina Haneburger ◽

...

Keyword(s):

Escherichia Coli ◽

Stress Response ◽

Response System ◽

Induction Kinetics ◽

Ph Stress ◽

Stress Response System ◽

Kinetics Of

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Toward a Semisynthetic Stress Response System To Engineer Microbial Solvent Tolerance

mBio ◽

10.1128/mbio.00308-12 ◽

2012 ◽

Vol 3 (5) ◽

Cited By ~ 57

Author(s):

Kyle A. Zingaro ◽

Eleftherios Terry Papoutsakis

Keyword(s):

Escherichia Coli ◽

Stress Response ◽

Heat Shock ◽

Heat Shock Proteins ◽

Solvent Tolerance ◽

Content Type ◽

Response System ◽

Wide Range ◽

Stress Response System ◽

Shock Proteins

ABSTRACT Strain tolerance to toxic metabolites is an important trait for many biotechnological applications, such as the production of solvents as biofuels or commodity chemicals. Engineering a complex cellular phenotype, such as solvent tolerance, requires the coordinated and tuned expression of several genes. Using combinations of heat shock proteins (HSPs), we engineered a semisynthetic stress response system in Escherichia coli capable of tolerating high levels of toxic solvents. Simultaneous overexpression of the HSPs GrpE and GroESL resulted in a 2-fold increase in viable cells (CFU) after exposure to 5% (vol/vol) ethanol for 24 h. Co-overexpression of GroESL and ClpB on coexisting plasmids resulted in 1,130%, 78%, and 25% increases in CFU after 24 h in 5% ethanol, 1% n-butanol, and 1% i-butanol, respectively. Co-overexpression of GrpE, GroESL, and ClpB on a single plasmid produced 200%, 390%, and 78% increases in CFU after 24 h in 7% ethanol, 1% n-butanol, or 25% 1,2,4-butanetriol, respectively. Overexpression of other autologous HSPs (DnaK, DnaJ, IbpA, and IbpB) alone or in combinations failed to improve tolerance. Expression levels of HSP genes, tuned through inducible promoters and the plasmid copy number, affected the effectiveness of the engineered stress response system. Taken together, these data demonstrate that tuned co-overexpression of GroES, GroEL, ClpB, and GrpE can be engaged to engineer a semisynthetic stress response system capable of greatly increasing the tolerance of E. coli to solvents and provides a starting platform for engineering customized tolerance to a wide variety of toxic chemicals. IMPORTANCE Microbial production of useful chemicals is often limited by the toxicity of desired products, feedstock impurities, and undesired side products. Improving tolerance is an essential step in the development of practical platform organisms for production of a wide range of chemicals. By overexpressing autologous heat shock proteins in Escherichia coli, we have developed a modular semisynthetic stress response system capable of improving tolerance to ethanol, n-butanol, and potentially other toxic solvents. Using this system, we demonstrate that a practical stress response system requires both tuning of individual gene components and a reliable framework for gene expression. This system can be used to seek out new interacting partners to improve the tolerance phenotype and can be used in the development of more robust solvent production strains.

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A Bacterial Stress Response Regulates Respiratory Protein Complexes To Control Envelope Stress Adaptation

Journal of Bacteriology ◽

10.1128/jb.00153-17 ◽

2017 ◽

Vol 199 (20) ◽

Cited By ~ 21

Author(s):

Randi L. Guest ◽

Junshu Wang ◽

Julia L. Wong ◽

Tracy L. Raivio

Keyword(s):

Escherichia Coli ◽

Antibiotic Resistance ◽

Stress Response ◽

Stress Responses ◽

Nadh Dehydrogenase ◽

Cytoplasmic Membrane ◽

Protein Complexes ◽

Inner Membrane ◽

Content Type ◽

Envelope Stress

ABSTRACT The Cpx envelope stress response mediates adaptation to stresses that affect protein folding within the envelope of Gram-negative bacteria. Recent transcriptome analyses revealed that the Cpx response impacts genes that affect multiple cellular functions predominantly associated with the cytoplasmic membrane. In this study, we examined the connection between the Cpx response and the respiratory complexes NADH dehydrogenase I and cytochrome bo 3 in enteropathogenic Escherichia coli. We found that the Cpx response directly represses the transcription of the nuo and cyo operons and that Cpx-mediated repression of these complexes confers adaptation to stresses that compromise envelope integrity. Furthermore, we found that the activity of the aerobic electron transport chain is reduced in E. coli lacking a functional Cpx response despite no change in the transcription of either the nuo or the cyo operon. Finally, we show that expression of NADH dehydrogenase I and cytochrome bo 3 contributes to basal Cpx pathway activity and that overproduction of individual subunits can influence pathway activation. Our results demonstrate that the Cpx response gauges and adjusts the expression, and possibly the function, of inner membrane protein complexes to enable adaptation to envelope stress. IMPORTANCE Bacterial stress responses allow microbes to survive environmental transitions and conditions, such as those encountered during infection and colonization, that would otherwise kill them. Enteric microbes that inhabit or infect the gut are exposed to a plethora of stresses, including changes in pH, nutrient composition, and the presence of other bacteria and toxic compounds. Bacteria detect and adapt to many of these conditions by using envelope stress responses that measure the presence of stressors in the outermost compartment of the bacterium by monitoring its physiology. The Cpx envelope stress response plays a role in antibiotic resistance and host colonization, and we have shown that it regulates many functions at the bacterial inner membrane. In this report, we describe a novel role for the Cpx response in sensing and controlling the expression of large, multiprotein respiratory complexes at the cytoplasmic membrane of Escherichia coli. The significance of our research is that it will increase our understanding of how these stress responses are involved in antibiotic resistance and the mechanisms used by bacteria to colonize the gut.

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