scholarly journals Recognition of β-Strand Motifs by RseB Is Required for σEActivity in Escherichia coli

2011 ◽  
Vol 193 (22) ◽  
pp. 6179-6186 ◽  
Author(s):  
Adam Kulp ◽  
Meta J. Kuehn
Keyword(s):  
Stress Response ◽  
Stress Responses ◽  
Outer Membrane Proteins ◽  
Harsh Environments ◽  
Gram Negative Bacteria ◽  
Dependent Manner ◽  
Gram Negative ◽  
Stress Response Pathway ◽  
Envelope Stress ◽  
Different Types

Gram-negative bacteria react to misfolded proteins in the envelope through a myriad of different stress response pathways. This cohort of pathways allows the bacteria to specifically respond to different types of damage, and many of these have been discovered to have key roles in the virulence of bacterial pathogens. Misfolded outer membrane proteins (OMPs) are typically recognized by the σEpathway, a highly conserved envelope stress response pathway. We examined the features of misfolded OMPs with respect to their ability to generate envelope stress responses. We determined that the secondary structure, particularly the potential to form β strands, is critical to inducing the σEresponse in an RseB-dependent manner. The sequence of the potential β-strand motif modulates the strength of the σEresponse generated by the constructs. By understanding the details of how such stress response pathways are activated, we can gain a greater understanding of how bacteria survive in harsh environments.

scholarly journals Fine-Tuning of σEActivation Suppresses Multiple Assembly-Defective Mutations inEscherichia coli

2019 ◽  
Vol 201 (11) ◽  
Author(s):  
Elizabeth M. Hart ◽  
Aileen O’Connell ◽  
Kimberly Tang ◽  
Joseph S. Wzorek ◽  
Marcin Grabowicz ◽  
...  
Keyword(s):  
Stress Response ◽  
Cell Viability ◽  
Outer Membrane ◽  
Stress Responses ◽  
Expression Profiles ◽  
Outer Membrane Proteins ◽  
Fine Tuning ◽  
Permeability Barrier ◽  
Gram Negative ◽  
Envelope Stress

ABSTRACTThe Gram-negative outer membrane (OM) is a selectively permeable asymmetric bilayer that allows vital nutrients to diffuse into the cell but prevents toxins and hydrophobic molecules from entering. Functionally and structurally diverse β-barrel outer membrane proteins (OMPs) build and maintain the permeability barrier, making the assembly of OMPs crucial for cell viability. In this work, we characterize an assembly-defective mutant of the maltoporin LamB, LamBG439D. We show that the folding defect of LamBG439Dresults in an accumulation of unfolded substrate that is toxic to the cell when the periplasmic protease DegP is removed. Selection for suppressors of this toxicity identified the novel mutantdegSA323Eallele. The mutant DegSA323Eprotein contains an amino acid substitution at the PDZ/protease domain interface that results in a partially activated conformation of this protein. This activation increases basal levels of downstream σEstress response signaling. Furthermore, the enhanced σEactivity of DegSA323Esuppresses a number of other assembly-defective conditions without exhibiting the toxicity associated with high levels of σEactivity. We propose that the increased basal levels of σEsignaling primes the cell to respond to envelope stress before OMP assembly defects threaten cell viability. This finding addresses the importance of envelope stress responses in monitoring the OMP assembly process and underpins the critical balance between envelope defects and stress response activation.IMPORTANCEGram-negative bacteria, such asEscherichia coli, inhabit a natural environment that is prone to flux. In order to cope with shifting growth conditions and the changing availability of nutrients, cells must be capable of quickly responding to stress. Stress response pathways allow cells to rapidly shift gene expression profiles to ensure survival in this unpredictable environment. Here we describe a mutant that partially activates the σEstress response pathway. The elevated basal level of this stress response allows the cell to quickly respond to overwhelming stress to ensure cell survival.

scholarly journals The Rcs Phosphorelay Is a Cell Envelope Stress Response Activated by Peptidoglycan Stress and Contributes to Intrinsic Antibiotic Resistance

2008 ◽  
Vol 190 (6) ◽  
pp. 2065-2074 ◽  
Author(s):  
Mary E. Laubacher ◽  
Sarah E. Ades
Keyword(s):  
Outer Membrane ◽  
Single Molecule ◽  
Stress Responses ◽  
Structural Integrity ◽  
Cell Envelope ◽  
Gram Negative Bacteria ◽  
Intrinsic Resistance ◽  
Gram Negative ◽  
Envelope Stress ◽  
Peptidoglycan Layer

ABSTRACTGram-negative bacteria possess stress responses to maintain the integrity of the cell envelope. Stress sensors monitor outer membrane permeability, envelope protein folding, and energization of the inner membrane. The systems used by gram-negative bacteria to sense and combat stress resulting from disruption of the peptidoglycan layer are not well characterized. The peptidoglycan layer is a single molecule that completely surrounds the cell and ensures its structural integrity. During cell growth, new peptidoglycan subunits are incorporated into the peptidoglycan layer by a series of enzymes called the penicillin-binding proteins (PBPs). To explore how gram-negative bacteria respond to peptidoglycan stress, global gene expression analysis was used to identifyEscherichia colistress responses activated following inhibition of specific PBPs by the β-lactam antibiotics amdinocillin (mecillinam) and cefsulodin. Inhibition of PBPs with different roles in peptidoglycan synthesis has different consequences for cell morphology and viability, suggesting that not all perturbations to the peptidoglycan layer generate equivalent stresses. We demonstrate that inhibition of different PBPs resulted in both shared and unique stress responses. The regulation of capsular synthesis (Rcs) phosphorelay was activated by inhibition of all PBPs tested. Furthermore, we show that activation of the Rcs phosphorelay increased survival in the presence of these antibiotics, independently of capsule synthesis. Both activation of the phosphorelay and survival required signal transduction via the outer membrane lipoprotein RcsF and the response regulator RcsB. We propose that the Rcs pathway responds to peptidoglycan damage and contributes to the intrinsic resistance ofE. colito β-lactam antibiotics.

scholarly journals Antiproliferative effects of porins extracted from Klebsiella pneumoniae on HL-60, NIH/3T3 and Human foreskin fibroblast cell (HFFC) lines measured by ELISA method

2010 ◽  
Vol 4 (1) ◽  
pp. 28-35
Author(s):  
Amir H. Al–Shammary ◽  
Essam F. Al-Jumaily ◽  
Nidhal Abdulmohymen
Keyword(s):  
Klebsiella Pneumoniae ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Primary Source ◽  
Gram Negative Bacteria ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Gram Negative ◽  
Isolation And Purification ◽  
Antiproliferative Effects

Approximately, 50% of the dry mass of the outer membrane of gram-negative bacteria consists of proteins, and more than 20 immunochemically distinct proteins (termed outer membrane proteins [OMPs]) have been identified. An identified local strain of Klebsiella pneumoniae was used as a primary source for the isolation and purification of porins. Multiple concentrations of purified porins (5, 10, 15, 20, 25) g/ml were incubated with three different cell lines for (24, 72 , 120) hrs, after the end of the incubation periods, the cells were treated with Cell proliferation ELISA, BrdU (colorimetric) kit to evaluate the antiproliferative effects of porins. The results revealed that porins are potent antiproliferative agent in a time and concentration dependent manner and thus could greatly affect prokaryote-eukaryote interaction as well as the whole inflammatory process resulted after infection with gram negative bacteria.

scholarly journals A Stress Response Monitoring Lipoprotein Trafficking to the Outer Membrane

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Kerrie L. May ◽  
Kelly M. Lehman ◽  
Angela M. Mitchell ◽  
Marcin Grabowicz
Keyword(s):  
Escherichia Coli ◽  
Stress Response ◽  
Outer Membrane ◽  
Stress Responses ◽  
Stress System ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Content Type ◽  
Essential Components ◽  
Trafficking Defects

ABSTRACTGram-negative bacteria produce lipid-anchored lipoproteins that are trafficked to their outer membrane (OM). These lipoproteins are essential components in each of the molecular machines that build the OM, including the Bam machine that assembles β-barrel proteins and the Lpt pathway that transports lipopolysaccharide. Stress responses are known to monitor Bam and Lpt function, yet no stress system has been found that oversees the fundamental process of lipoprotein trafficking. We used genetic and chemical biology approaches to induce several different lipoprotein trafficking stresses inEscherichia coli. Our results identified the Cpx two-component system as a stress response for monitoring trafficking. Cpx is activated by trafficking defects and is required to protect the cell against the consequence of the resulting stress. The OM-targeted lipoprotein NlpE acts as a sensor that allows Cpx to gauge trafficking efficiency. We reveal that NlpE signals to Cpx while it is transiting the inner membrane (IM)en routeto the OM and that only a small highly conserved N-terminal domain is required for signaling. We propose that defective trafficking causes NlpE to accumulate in the IM, activating Cpx to mount a transcriptional response that protects cells. Furthermore, we reconcile this new role of NlpE in signaling trafficking defects with its previously proposed role in sensing copper (Cu) stress by demonstrating that Cu impairs acylation of lipoproteins and, consequently, their trafficking to the OM.IMPORTANCEThe outer membrane built by Gram-negative bacteria such asEscherichia coliforms a barrier that prevents antibiotics from entering the cell, limiting clinical options at a time of prevalent antibiotic resistance. Stress responses ensure that barrier integrity is continuously maintained. We have identified the Cpx signal transduction system as a stress response that monitors the trafficking of lipid-anchored lipoproteins to the outer membrane. These lipoproteins are needed by every machine that builds the outer membrane. Cpx monitors just one lipoprotein, NlpE, to detect the efficiency of lipoprotein trafficking in the cell. NlpE and Cpx were previously shown to play a role in resistance to copper. We show that copper blocks lipoprotein trafficking, reconciling old and new observations. Copper is an important element in innate immunity against pathogens, and our findings suggest that NlpE and Cpx helpE. colisurvive the assault of copper on a key outer membrane assembly pathway.

scholarly journals Characterization of theVibrio choleraePhage Shock Protein Response

2019 ◽  
Vol 201 (14) ◽  
Author(s):  
Cara M. DeAngelis ◽  
Dhrubajyoti Nag ◽  
Jeffrey H. Withey ◽  
Jyl S. Matson
Keyword(s):  
Stress Response ◽  
Vibrio Cholerae ◽  
Membrane Damage ◽  
Regulatory Elements ◽  
Life Cycles ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Content Type ◽  
Genetic Components ◽  
Stress Response Pathway

ABSTRACTThe phage shock protein (Psp) system is a stress response pathway that senses and responds to inner membrane damage. The genetic components of the Psp system are present in several clinically relevant Gram-negative bacteria, includingVibrio cholerae. However, most of the current knowledge about the Psp response stems fromin vitrostudies inEscherichia coliandYersinia enterocolitica. In fact, the Psp response inV. choleraehas remained completely uncharacterized. In this study, we demonstrate thatV. choleraedoes have a functional Psp response system. We found that overexpression of GspD (EpsD), the type II secretion system secretin, induces the Psp response, whereas otherV. choleraesecretins do not. In addition, we have identified several environmental conditions that induce this stress response. Our studies on the genetic regulation and induction of the Psp system inV. choleraesuggest that the key regulatory elements are conserved with those of other Gram-negative bacteria. While apspnull strain is fully capable of colonizing the infant mouse intestine, it exhibits a colonization defect in a zebrafish model, indicating that this response may be important for disease transmission in the environment. Overall, these studies provide an initial understanding of a stress response pathway that has not been previously investigated inV. cholerae.IMPORTANCEVibrio choleraeleads a dual life cycle, as it can exist in the aquatic environment and colonize the human small intestine. In both life cycles,V. choleraeencounters a variety of stressful conditions, including fluctuating pH and temperature and exposure to other agents that may negatively affect cell envelope homeostasis. The phage shock protein (Psp) response is required to sense and respond to such insults in other bacteria but has remained unstudied inV. cholerae. Interestingly, the Psp system has protein homologs, principally, PspA, in a number of bacterial clades as well as in archaea and plants. Therefore, our findings not only fill a gap in knowledge about an unstudied extracytoplasmic stress response inV. cholerae, but also may have far-reaching implications.

scholarly journals Fine-Tuning of the Cpx Envelope Stress Response Is Required for Cell Wall Homeostasis inEscherichia coli

mBio ◽  
2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Antoine Delhaye ◽  
Jean-François Collet ◽  
Géraldine Laloux
Keyword(s):  
Quality Control ◽  
Cell Wall ◽  
Control System ◽  
Stress Response ◽  
Cell Shape ◽  
Protein Quality ◽  
Fine Tuning ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Envelope Stress

ABSTRACTThe envelope of Gram-negative bacteria is an essential compartment that constitutes a protective and permeability barrier between the cell and its environment. The envelope also hosts the cell wall, a mesh-like structure made of peptidoglycan (PG) that determines cell shape and provides osmotic protection. Since the PG must grow and divide in a cell-cycle-synchronized manner, its synthesis and remodeling are tightly regulated. Here, we discovered that PG homeostasis is intimately linked to the levels of activation of the Cpx system, an envelope stress response system traditionally viewed as being involved in protein quality control in the envelope. We first show that Cpx is activated when PG integrity is challenged and that this activation provides protection to cells exposed to antibiotics inhibiting PG synthesis. By rerouting the outer membrane lipoprotein NlpE, a known Cpx activator, to a different envelope subcompartment, we managed to manipulate Cpx activation levels. We found that Cpx overactivation leads to aberrant cellular morphologies, to an increased sensitivity to β-lactams, and to dramatic division and growth defects, consistent with a loss of PG homeostasis. Remarkably, these phenotypes were largely abrogated by the deletion ofldtD, a Cpx-induced gene involved in noncanonical PG cross-linkage, suggesting that this transpeptidase is an important link between PG homeostasis and the Cpx system.Altogether our data show that fine-tuning of an envelope quality control system constitutes an important layer of regulation of the highly organized cell wall structure.IMPORTANCEThe envelope of Gram-negative bacteria is essential for viability. First, it includes the cell wall, a continuous polymer of peptidoglycan (PG) that determines cell morphology and protects against osmotic stress. Moreover, the envelope constitutes a protective barrier between the cell interior and the environment. Therefore, mechanisms called envelope stress response systems (ESRS) exist to monitor and defend envelope integrity against harmful conditions. Cpx is a major ESRS that detects and manages the accumulation of misfolded proteins in the envelope ofEscherichia coli. We found that this protein quality control system also plays a fundamental role in the regulation of PG assembly. Strikingly, the level of Cpx response is critical, as an excessive activation leads to phenotypes associated with a loss of cell wall integrity. Thus, by contributing to PG homeostasis, the Cpx system lies at the crossroads between key processes of bacterial life, including cell shape, growth, division, and antibiotic resistance.

scholarly journals Metabolism of long-chain fatty acids affects disulfide bond formation in Escherichia coli and activates envelope stress response pathways as a combat strategy

2020 ◽  
Author(s):  
Kanchan Jaswal ◽  
Megha Shrivastava ◽  
Deeptodeep Roy ◽  
Shashank Agrawal ◽  
Rachna Chaba
Keyword(s):  
Escherichia Coli ◽  
Stress Response ◽  
Disulfide Bond ◽  
Bond Formation ◽  
Redox Status ◽  
Disulfide Bond Formation ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Envelope Stress ◽  
Long Chain

AbstractThe envelope of gram-negative bacteria serves as the first line of defense against environmental insults. Therefore, its integrity is continuously monitored and maintained by several envelope stress response (ESR) systems. Due to its oxidizing environment, the envelope represents an important site for disulfide bond formation. In Escherichia coli, the periplasmic oxidoreductase, DsbA introduces disulfide bonds in substrate proteins and transfers electrons to the inner membrane oxidoreductase, DsbB. Under aerobic conditions, the reduced form of DsbB is re-oxidized by ubiquinone, an electron carrier in the electron transport chain (ETC). Given the critical role of ubiquinone in transferring electrons derived from the oxidation of reduced cofactors, we were intrigued whether metabolic conditions that generate a large number of reduced cofactors render ubiquinone unavailable for disulfide bond formation. To test this, here we investigated the influence of metabolism of long-chain fatty acid (LCFA), an energy-rich carbon source, on the redox state of the envelope. We show that LCFA degradation increases electron flow in the ETC. Further, we find that whereas cells metabolizing LCFAs exhibit several characteristics of insufficient disulfide bond formation, these hallmarks are averted in cells exogenously provided with ubiquinone. Importantly, the ESR pathways, Cpx and σE, are activated by envelope signals generated during LCFA metabolism, and these systems maintain proper disulfide bond formation. We find that σE downregulation hampers disulfide bond formation only in the absence of Cpx, and amongst the two ESR systems, only Cpx senses redox-dependent signal and is induced to a greater extent by LCFAs. Therefore, we argue that Cpx is the primary ESR that senses and maintains envelope redox homeostasis. Taken together, our results demonstrate an intricate relationship between cellular metabolism and disulfide bond formation dictated by ETC and ESR, and provide the basis for examining whether similar mechanisms control envelope redox status in other gram-negative bacteria.Author summaryDisulfide bonds contribute to the folding and stability of many extracytoplasmic proteins in all domains of life. In gram-negative bacteria, including Escherichia coli, disulfide bond formation occurs in the oxidizing environment of the periplasmic space enclosed within the outer and inner membrane layers of the envelope. Because disulfide-bonded proteins are involved in diverse biological processes, bacteria must monitor the envelope redox status and elicit an appropriate response when perturbations occur; however, these mechanisms are not well elucidated. Here, we demonstrated that the metabolism of an energy-rich carbon source, long-chain fatty acid (LCFA) hampers disulfide bond formation in E. coli. An envelope stress response (ESR) system, Cpx, senses this redox imbalance and maintains proper disulfide bond formation. The σE pathway, another ESR system, plays an ancillary role in maintaining redox homeostasis. LCFA metabolism, disulfide bond formation, and ESR systems have independently been implicated in the pathogenesis of several gram-negative bacteria. The present study sets the basis to explore whether LCFA metabolism impacts the virulence of these bacteria by influencing the redox status of their envelope and activation of ESR pathways.

scholarly journals Inhibitor of intramembrane protease RseP blocks the σE response causing lethal accumulation of unfolded outer membrane proteins

2018 ◽  
Vol 115 (28) ◽  
pp. E6614-E6621 ◽  
Author(s):  
Anna Konovalova ◽  
Marcin Grabowicz ◽  
Carl J. Balibar ◽  
Juliana C. Malinverni ◽  
Ronald E. Painter ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Stress Response ◽  
Nutrient Uptake ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Selective Inhibitor ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Underlying Basis

The outer membrane (OM) of Gram-negative bacteria forms a robust permeability barrier that blocks entry of toxins and antibiotics. Most OM proteins (OMPs) assume a β-barrel fold, and some form aqueous channels for nutrient uptake and efflux of intracellular toxins. The Bam machine catalyzes rapid folding and assembly of OMPs. Fidelity of OMP biogenesis is monitored by the σE stress response. When OMP folding defects arise, the proteases DegS and RseP act sequentially to liberate σE into the cytosol, enabling it to activate transcription of the stress regulon. Here, we identify batimastat as a selective inhibitor of RseP that causes a lethal decrease in σE activity in Escherichia coli, and we further identify RseP mutants that are insensitive to inhibition and confer resistance. Remarkably, batimastat treatment allows the capture of elusive intermediates in the OMP biogenesis pathway and offers opportunities to better understand the underlying basis for σE essentiality.

scholarly journals BING, a novel antimicrobial peptide isolated from Japanese medaka plasma, targets bacterial envelope stress response by suppressing cpxR expression

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Miao Dong ◽  
Shu Hin Kwok ◽  
Joseph L. Humble ◽  
Yimin Liang ◽  
Sze Wing Tang ◽  
...  
Keyword(s):  
Stress Responses ◽  
Pathogenic Bacteria ◽  
Efflux Pump ◽  
Gram Negative Bacteria ◽  
Promising Alternative ◽  
Gram Negative ◽  
Upstream Regulator ◽  
Mammalian Cell Lines ◽  
Envelope Stress ◽  
Peptidyl Prolyl Isomerases

AbstractAntimicrobial peptides (AMPs) have emerged as a promising alternative to small molecule antibiotics. Although AMPs have previously been isolated in many organisms, efforts on the systematic identification of AMPs in fish have been lagging. Here, we collected peptides from the plasma of medaka (Oryzias latipes) fish. By using mass spectrometry, 6399 unique sequences were identified from the isolated peptides, among which 430 peptides were bioinformatically predicted to be potential AMPs. One of them, a thermostable 13-residue peptide named BING, shows a broad-spectrum toxicity against pathogenic bacteria including drug-resistant strains, at concentrations that presented relatively low toxicity to mammalian cell lines and medaka. Proteomic analysis indicated that BING treatment induced a deregulation of periplasmic peptidyl-prolyl isomerases in gram-negative bacteria. We observed that BING reduced the RNA level of cpxR, an upstream regulator of envelope stress responses. cpxR is known to play a crucial role in the development of antimicrobial resistance, including the regulation of genes involved in drug efflux. BING downregulated the expression of efflux pump components mexB, mexY and oprM in P. aeruginosa and significantly synergised the toxicity of antibiotics towards these bacteria. In addition, exposure to sublethal doses of BING delayed the development of antibiotic resistance. To our knowledge, BING is the first AMP shown to suppress cpxR expression in Gram-negative bacteria. This discovery highlights the cpxR pathway as a potential antimicrobial target.

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