A structure-function study of ZraP and ZraS provides new insights into the two-component system Zra

BackgroundZra belongs to the envelope stress response (ESR) two-component systems (TCS). It is atypical because of its third periplasmic repressor partner (ZraP), in addition to its histidine kinase sensor protein (ZraS) and its response regulator (ZraR) components. Furthermore, although it is activated by Zn²⁺, it is not involved in zinc homeostasis or protection against zinc toxicity. Here, we mainly focus on ZraS but also provide information on ZraP.MethodsThe purified periplasmic domain of ZraS and ZraP were characterized using biophysical and biochemical technics: multi-angle laser light scattering (MALLS), circular dichroism (CD), differential scanning fluorescence (DSF), inductively coupled plasma atomic emission spectroscopy (ICP-AES), cross-linking and small-angle X-ray scattering (SAXS). In-vivo experiments were carried out to determine the redox state of the cysteine residue in ZraP and the consequences for the cell of an over-activation of the Zra system.ResultsWe show that ZraS binds one Zn²⁺ molecule with high affinity resulting in conformational changes of the periplasmic domain, consistent with a triggering function of the metal ion. We also demonstrate that, in the periplasm, the only cysteine residue of ZraP is at least partially reduced. Using SAXS, we conclude that the previously determined X-ray structure is different from the structure in solution.ConclusionOur results allow us to propose a general mechanism for the Zra system activation and to compare it to the homologous Cpx system.General significanceWe bring new input on the so far poorly described Zra system and notably on ZraS.     

A Linkage between SmeIJK Efflux Pump,Cell Envelope Integrity,and σE-Mediated Envelope Stress Response in Stenotrophomonas maltophilia

Resistance nodulation division (RND) efflux pumps, such as the SmeIJK pump of Stenotrophomonas maltophilia, are known to contribute to the multidrug resistance in Gram-negative bacteria. However, some RND pumps are constitutively expressed even though no antimicrobial stresses occur, implying that there should be some physical implications for these RND pumps. In this study, the role of SmeIJK in antimicrobials resistance, envelope integrity, and σ^E-mediated envelope stress response (ESR) of S. maltophilia was assessed. SmeIJK was involved in the intrinsic resistance of S. maltophilia KJ to aminoglycosides and leucomycin. Compared with the wild-type KJ, the smeIJK deletion mutant exhibited growth retardation in the MH medium, an increased sensitivity to membrane-damaging agents (MDAs), as well as activation of an σ^E-mediated ESR. Moreover, the expression of smeIJK was further induced by sub-lethal concentrations of MDAs or surfactants in an σ^E-dependent manner. These data collectively suggested an alternative physiological role of smeIJK in cell envelope integrity maintenance and σ^E-mediated ESR beyond the efflux of antibiotics. Because of the necessity of the physiological role of SmeIJK in protecting S. maltophilia from the envelope stress, smeIJK is constitutively expressed, which, in turn, contributes the intrinsic resistance to aminoglycoside and leucomycin. This is the first demonstration of the linkage among RND-type efflux pump, cell envelope integrity, and σ^E-mediated ESR in S. maltophilia.     

Identification and analysis of novel small molecule inhibitors of RNase E: Implications for antibacterial targeting and regulation of RNase E

Increasing resistance of bacteria to antibiotics is a serious global challenge and there is a need to unlock the potential of novel antibacterial targets. One such target is the essential prokaryotic endoribonuclease RNase E. Using a combination of in silico high-throughput screening and in vitro validation we have identified three novel small molecule inhibitors of RNase E that are active against RNase E from Escherichia coli, Francisella tularensis and Acinetobacter baumannii. Two of the inhibitors are non-natural small molecules that could be suitable as lead compounds for the development of broad-spectrum antibiotics targeting RNase E. The third small molecule inhibitor is glucosamine-6-phosphate, a precursor of bacterial cell envelope peptidoglycans and lipopolysaccharides, hinting at a novel metabolite-mediated mechanism of regulation of RNase E.     

ZraP is a periplasmic molecular chaperone and a repressor of the zinc-responsive two-component regulator ZraSR

The bacterial envelope is the interface with the surrounding environment and is consequently subjected to a barrage of noxious agents including a range of compounds with antimicrobial activity. The ESR (envelope stress response) pathways of enteric bacteria are critical for maintenance of the envelope against these antimicrobial agents. In the present study, we demonstrate that the periplasmic protein ZraP contributes to envelope homoeostasis and assign both chaperone and regulatory function to ZraP from Salmonella Typhimurium. The ZraP chaperone mechanism is catalytic and independent of ATP; the chaperone activity is dependent on the presence of zinc, which is shown to be responsible for the stabilization of an oligomeric ZraP complex. Furthermore, ZraP can act to repress the two-component regulatory system ZraSR, which itself is responsive to zinc concentrations. Through structural homology, ZraP is a member of the bacterial CpxP family of periplasmic proteins, which also consists of CpxP and Spy. We demonstrate environmental co-expression of the CpxP family and identify an important role for these proteins in Salmonella's defence against the cationic antimicrobial peptide polymyxin B.     

Induction of the Cpx Envelope Stress Pathway Contributes to Escherichia coli Tolerance to Antimicrobial Peptides

Antimicrobial peptides produced by multicellular organisms as part of their innate system of defense against microorganisms are currently considered potential alternatives to conventional antibiotics in case of infection by multiresistant bacteria. However, while the mode of action of antimicrobial peptides is relatively well described, resistance mechanisms potentially induced or selected by these peptides are still poorly understood. In this work, we studied the mechanisms of action and resistance potentially induced by ApoEdpL-W, a new antimicrobial peptide derived from human apolipoprotein E. Investigation of the genetic response of Escherichia coli upon exposure to sublethal concentrations of ApoEdpL-W revealed that this antimicrobial peptide triggers activation of RcsCDB, CpxAR, and σ^E envelope stress pathways. This genetic response is not restricted to ApoEdpL-W, since several other antimicrobial peptides, including polymyxin B, melittin, LL-37, and modified S₄ dermaseptin, also activate several E. coli envelope stress pathways. Finally, we demonstrate that induction of the CpxAR two-component system directly contributes to E. coli tolerance toward ApoEdpL-W, polymyxin B, and melittin. These results therefore show that E. coli senses and responds to different antimicrobial peptides by activation of the CpxAR pathway. While this study further extends the understanding of the array of peptide-induced stress signaling systems, it also provides insight into the contribution of Cpx envelope stress pathway to E. coli tolerance to antimicrobial peptides.     

Everything old is new again: An update on current research on the Cpx envelope stress response

The Cpx envelope stress response (ESR) has been linked to proteins that are integrated into and secreted across the inner membrane for several decades. Initial studies of the cpx locus linked it to alterations in the protein content of both the inner and outer membrane, together with changes in proton motive driven transport and conjugation. Since the mid 1990s, the predominant view of the Cpx envelope stress response has been that it serves to detect and respond to secreted, misfolded proteins in the periplasm. Recent studies in Escherichia coli and other Gram negative organisms highlight a role for the Cpx ESR in specifically responding to perturbations that occur at the inner membrane (IM). It is clear that Cpx adaptation involves a broad suite of changes that encompass many functions in addition to protein folding. Interestingly, recent studies have refocused attention on Cpx-regulated phenotypes that were initially published over 30 years ago, including antibiotic resistance and transport across the IM. In this review I will focus on the insights and models that have arisen from recent studies and that may help explain some of the originally published Cpx phenotypes. Although the molecular nature of the inducing signal for the Cpx ESR remains enigmatic, recently solved structures of signaling proteins are yielding testable models concerning the molecular mechanisms behind signaling. The identification of connections between the Cpx ESR and other stress responses in the cell reveals a complex web of interactions that involves Cpx-regulated expression of other regulators as well as small proteins and sRNAs. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

The peptidoglycan-associated protein NapA plays an important role in the envelope integrity and in the pathogenesis of the lyme disease spirochete

The bacterial pathogen responsible for causing Lyme disease, Borrelia burgdorferi, is an atypical Gram-negative spirochete that is transmitted to humans via the bite of an infected Ixodes tick. In diderms, peptidoglycan (PG) is sandwiched between the inner and outer membrane of the cell envelope. In many other Gram-negative bacteria, PG is bound by protein(s), which provide both structural integrity and continuity between envelope layers. Here, we present evidence of a peptidoglycan-associated protein (PAP) in B. burgdorferi. Using an unbiased proteomics approach, we identified Neutrophil Attracting Protein A (NapA) as a PAP. Interestingly, NapA is a Dps homologue, which typically functions to bind and protect cellular DNA from damage during times of stress. While B. burgdorferi NapA is known to be involved in the oxidative stress response, it lacks the critical residues necessary for DNA binding. Biochemical and cellular studies demonstrate that NapA is localized to the B. burgdorferi periplasm and is indeed a PAP. Cryo-electron microscopy indicates that mutant bacteria, unable to produce NapA, have structural abnormalities. Defects in cell-wall integrity impact growth rate and cause the napA mutant to be more susceptible to osmotic and PG-specific stresses. NapA-linked PG is secreted in outer membrane vesicles and augments IL-17 production, relative to PG alone. Using microfluidics, we demonstrate that NapA acts as a molecular beacon—exacerbating the pathogenic properties of B. burgdorferi PG. These studies further our understanding of the B. burgdorferi cell envelope, provide critical information that underlies its pathogenesis, and highlight how a highly conserved bacterial protein can evolve mechanistically, while maintaining biological function.     

Two-Component System Cross-Regulation Integrates Bacillus anthracis Response to Heme and Cell Envelope Stress

Two-component signaling systems (TCSs) are one of the mechanisms that bacteria employ to sense and adapt to changes in the environment. A prototypical TCS functions as a phosphorelay from a membrane-bound sensor histidine kinase (HK) to a cytoplasmic response regulator (RR) that controls target gene expression. Despite significant homology in the signaling domains of HKs and RRs, TCSs are thought to typically function as linear systems with little to no cross-talk between non-cognate HK-RR pairs. Here we have identified several cell envelope acting compounds that stimulate a previously uncharacterized Bacillus anthracis TCS. Furthermore, this TCS cross-signals with the heme sensing TCS HssRS; therefore, we have named it HssRS interfacing TCS (HitRS). HssRS reciprocates cross-talk to HitRS, suggesting a link between heme toxicity and cell envelope stress. The signaling between HssRS and HitRS occurs in the parental B. anthracis strain; therefore, we classify HssRS-HitRS interactions as cross-regulation. Cross-talk between HssRS and HitRS occurs at both HK-RR and post-RR signaling junctions. Finally, HitRS also regulates a previously unstudied ABC transporter implicating this transporter in the response to cell envelope stress. This chemical biology approach to probing TCS signaling provides a new model for understanding how bacterial signaling networks are integrated to enable adaptation to complex environments such as those encountered during colonization of the vertebrate host.     

Dynamic Interaction between the CpxA Sensor Kinase and the Periplasmic Accessory Protein CpxP Mediates Signal Recognition in E. coli

Two-component systems, consisting of an inner membrane sensor kinase and a cytosolic response regulator, allow bacteria to respond to changes in the environment. Some two-component systems are additionally orchestrated by an accessory protein that integrates additional signals. It is assumed that spatial and temporal interaction between an accessory protein and a sensor kinase modifies the activity of a two-component system. However, for most accessory proteins located in the bacterial envelope the mechanistic details remain unclear. Here, we analyzed the interaction between the periplasmic accessory protein CpxP and the sensor kinase CpxA in Escherichia coli in dependency of three specific stimuli. The Cpx two-component system responds to envelope stress and plays a pivotal role for the quality control of multisubunit envelope structures, including type three secretion systems and pili of different pathogens. In unstressed cells, CpxP shuts off the Cpx response by a yet unknown mechanism. We show for the first time the physical interaction between CpxP and CpxA in unstressed cells using bacterial two-hybrid system and membrane-Strep-tagged protein interaction experiments. In addition, we demonstrate that a high salt concentration and the misfolded pilus subunit PapE displace CpxP from the sensor kinase CpxA in vivo. Overall, this study provides clear evidence that CpxP modulates the activity of the Cpx system by dynamic interaction with CpxA in response to specific stresses.     

The RpoE Stress Response Pathway Mediates Reduction of the Virulence of Enteropathogenic Escherichia coli by Zinc

Zinc supplements are an effective clinical treatment for infantile diarrheal disease caused by enteric pathogens. Previous studies demonstrated that zinc acts on enteropathogenic Escherichia coli (EPEC) bacteria directly to suppress several virulence-related genes at a concentration that can be achieved by oral delivery of dietary zinc supplements. Our in vitro studies showed that a micromolar concentration of zinc induced the envelope stress response and suppressed virulence in EPEC, providing a possible mechanistic explanation for zinc''s therapeutic action. In this report, we investigated the molecular and physiological changes in EPEC induced by zinc. We found that micromolar concentrations of zinc reduced the bacterial growth rate without affecting viability. We observed increased membrane permeability caused by zinc. Zinc upregulated the RpoE-dependent envelope stress response pathway and suppressed EPEC virulence gene expression. RpoE alone was sufficient to inhibit virulence factor expression and to attenuate attaching and effacing lesion formation on human host cells. By mutational analysis we demonstrate that the DNA-binding motif of RpoE is necessary for suppression of the LEE1, but not the LEE4, operon. Predictably, inhibition of the RpoE-mediated envelope stress response in combination with micromolar concentrations of zinc reduced EPEC viability. In conclusion, zinc induces the RpoE and stress response pathways in EPEC, and the alternate sigma factor RpoE downregulates EPEC LEE and non-LEE virulence genes by multiple mechanisms.     

Similarities between Exogenously- and Endogenously-Induced Envelope Stress: The Effects of a New Antibacterial Molecule,TPI1609-10

Antibiotics with novel and/or multiple targets are highly desirable in the face of the steady rise of clinical antibiotic resistance. We have screened and identified small molecules, typified by the compound TPI1609-10 (aka SM10), with antibiotic activity against both gram-positive and gram-negative bacteria. SM10 was screened in vitro to bind branched Holliday junction intermediates of homologous recombination and tyrosine recombinase-mediated recombination; thus, the cellular targets of the small molecules were expected to include the RuvABC Holliday junction resolvasome and the XerCD complex involved in proper segregation of replicated chromosomes to daughter cells. SM10 indeed induces DNA damage and filamentation in E. coli. However, SM10 also induces envelope stress and causes increased production of intracellular reactive oxygen species. In addition, SM10 has similar effects to endogenously-induced envelope stress via overproducing outer membrane proteins (OmpC and OmpF), which also induces the SOS response, chromosome fragmentation, and production of reactive oxygen species. The synergy between SM10, and cerulenin, a fatty acid synthesis inhibitor, together with the SM10 hypersensitivity of cpx and rpoE mutants, further support that SM10''s mode of action damages membrane damage. The lethality of SM10 treatment and of OmpC overproduction are observed in both aerobically- and anaerobically-grown cells, and is accompanied by substantial DNA damage even anaerobically. Thus, only some DNA damage is due to reactive oxygen. We propose that membrane depolarization and the potential reduction in intracellular pH, leading to abasic site formation, cause a substantial amount of the DNA damage associated with both SM10 treatment and endogenous envelope stress. While it is difficult to completely exclude effects related to envelope damage as the sources of DNA damage, trapping intermediates associated with DNA repair and chromosome segregation pathways remains very likely. Thus SM10 may have distinct but synergistic modes of action.     

WWP-1 Is a Novel Modulator of the DAF-2 Insulin-Like Signaling Network Involved in Pore-Forming Toxin Cellular Defenses in Caenorhabditis elegans

Pore-forming toxins (PFTs) are the single largest class of bacterial virulence factors. The DAF-2 insulin/insulin-like growth factor-1 signaling pathway, which regulates lifespan and stress resistance in Caenorhabditis elegans, is known to mutate to resistance to pathogenic bacteria. However, its role in responses against bacterial toxins and PFTs is as yet unexplored. Here we reveal that reduction of the DAF-2 insulin-like pathway confers the resistance of Caenorhabditis elegans to cytolitic crystal (Cry) PFTs produced by Bacillus thuringiensis. In contrast to the canonical DAF-2 insulin-like signaling pathway previously defined for aging and pathogenesis, the PFT response pathway diverges at 3-phosphoinositide-dependent kinase 1 (PDK-1) and appears to feed into a novel insulin-like pathway signal arm defined by the WW domain Protein 1 (WWP-1). In addition, we also find that WWP-1 not only plays an important role in the intrinsic cellular defense (INCED) against PFTs but also is involved in innate immunity against pathogenic bacteria Pseudomonas aeruginosa and in lifespan regulation. Taken together, our data suggest that WWP-1 and DAF-16 function in parallel within the fundamental DAF-2 insulin/IGF-1 signaling network to regulate fundamental cellular responses in C. elegans.     

Outer membrane permeabilization by the membrane attack complex sensitizes Gram-negative bacteria to antimicrobial proteins in serum and phagocytes

Infections with Gram-negative bacteria form an increasing risk for human health due to antibiotic resistance. Our immune system contains various antimicrobial proteins that can degrade the bacterial cell envelope. However, many of these proteins do not function on Gram-negative bacteria, because the impermeable outer membrane of these bacteria prevents such components from reaching their targets. Here we show that complement-dependent formation of Membrane Attack Complex (MAC) pores permeabilizes this barrier, allowing antimicrobial proteins to cross the outer membrane and exert their antimicrobial function. Specifically, we demonstrate that MAC-dependent outer membrane damage enables human lysozyme to degrade the cell wall of E. coli. Using flow cytometry and confocal microscopy, we show that the combination of MAC pores and lysozyme triggers effective E. coli cell wall degradation in human serum, thereby altering the bacterial cell morphology from rod-shaped to spherical. Completely assembled MAC pores are required to sensitize E. coli to the antimicrobial actions of lysozyme and other immune factors, such as Human Group IIA-secreted Phospholipase A2. Next to these effects in a serum environment, we observed that the MAC also sensitizes E. coli to more efficient degradation and killing inside human neutrophils. Altogether, this study serves as a proof of principle on how different players of the human immune system can work together to degrade the complex cell envelope of Gram-negative bacteria. This knowledge may facilitate the development of new antimicrobials that could stimulate or work synergistically with the immune system.     

Cell envelope stress response in Gram-positive bacteria

The bacterial cell envelope is the first and major line of defence against threats from the environment. It is an essential and yet vulnerable structure that gives the cell its shape and counteracts the high internal osmotic pressure. It also provides an important sensory interface and molecular sieve, mediating both information flow and the controlled transport of solutes. The cell envelope is also the target for numerous antibiotics. Therefore, the monitoring and maintenance of cell envelope integrity in the presence of envelope perturbating agents and conditions is crucial for survival. The underlying signal transduction is mediated by two regulatory principles, two-component systems and extracytoplasmic function sigma factors, in both the Firmicutes (low-GC) and Actinobacteria (high-GC) branches of Gram-positive bacteria. This study presents a comprehensive overview of cell envelope stress-sensing regulatory systems. This knowledge will then be applied for in-depth comparative genomics analyses to emphasize the distribution and conservation of cell envelope stress-sensing systems. Finally, the cell envelope stress response will be placed in the context of the overall cellular physiology, demonstrating that its regulatory systems are linked not only to other stress responses but also to the overall homeostasis and lifestyle of Gram-positive bacteria.