Stress responses as determinants of antimicrobial resistance in Gram-negative bacteria

Bacteria encounter a myriad of potentially growth-compromising conditions in nature and in hosts of pathogenic bacteria. These 'stresses' typically elicit protective and/or adaptive responses that serve to enhance bacterial survivability. Because they impact upon many of the same cellular components and processes that are targeted by antimicrobials, adaptive stress responses can influence antimicrobial susceptibility. In targeting and interfering with key cellular processes, antimicrobials themselves are 'stressors' to which protective stress responses have also evolved. Cellular responses to nutrient limitation (nutrient stress), oxidative and nitrosative stress, cell envelope damage (envelope stress), antimicrobial exposure and other growth-compromising stresses, have all been linked to the development of antimicrobial resistance in Gram-negative bacteria - resulting from the stimulation of protective changes to cell physiology, activation of resistance mechanisms, promotion of resistant lifestyles (biofilms), and induction of resistance mutations.     

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.     

Identification and functional analysis of antifungal immune response genes in Drosophila

Essential aspects of the innate immune response to microbial infection appear to be conserved between insects and mammals. Although signaling pathways that activate NF-kappaB during innate immune responses to various microorganisms have been studied in detail, regulatory mechanisms that control other immune responses to fungal infection require further investigation. To identify new Drosophila genes involved in antifungal immune responses, we selected genes known to be differentially regulated in SL2 cells by microbial cell wall components and tested their roles in antifungal defense using mutant flies. From 130 mutant lines, sixteen mutants exhibited increased sensitivity to fungal infection. Examination of their effects on defense against various types of bacteria and fungi revealed nine genes that are involved specifically in defense against fungal infection. All of these mutants displayed defects in phagocytosis or activation of antimicrobial peptide genes following infection. In some mutants, these immune deficiencies were attributed to defects in hemocyte development and differentiation, while other mutants showed specific defects in immune signaling required for humoral or cellular immune responses. Our results identify a new class of genes involved in antifungal immune responses in Drosophila.     

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.     

Pushing the envelope: extracytoplasmic stress responses in bacterial pathogens

Despite being nutrient rich, the tissues and fluids of vertebrates are hostile to microorganisms, and most bacteria that attempt to take advantage of this environment are rapidly eliminated by host defences. Pathogens have evolved various means to promote their survival in host tissues, including stress responses that enable bacteria to sense and adapt to adverse conditions. Many different stress responses have been described, some of which are responsive to one or a small number of cues, whereas others are activated by a broad range of insults. The surface layers of pathogenic bacteria directly interface with the host and can bear the brunt of the attack by the host armoury. Several stress systems that respond to perturbations in the microbial cell outside of the cytoplasm have been described and are known collectively as extracytoplasmic or envelope stress responses (ESRs). Here, we review the role of the ESRs in the pathogenesis of Gram-negative bacterial pathogens.     

Chemical modulation of physiological adaptation and cross-protective responses against oxidative stress in soil bacterium and phytopathogen, Xanthomonas

Soil bacteria need to adapt quickly to changes in the environmental conditions. Physiological adaptation plays an important role in microbial survival, especially under stressful conditions. Here the abilities of chemicals and pesticides to modulate physiological adaptive and cross-protective responses, that make the bacteria more resistant to oxidative stress, are examined in the soil bacterium and phytopathogen, Xanthomonas. The genetic basis for the observed stress resistance, as well as the regulatory mechanisms controlling gene expression during the process, has begun to be elucidated.     

Molecular mechanisms governing plant responses to high temperatures

The increased prevalence of high temperatures(HTs) around the world is a major global concern, as they dramatically affect agronomic productivity. Upon HT exposure, plants sense the temperature change and initiate cellular and metabolic responses that enable them to adapt to their new environmental conditions.Decoding the mechanisms by which plants cope with HT will facilitate the development of molecular markers to enable the production of plants with improved thermotolerance. In recent decades, genetic, physiological, molecular, and biochemical studies have revealed a number of vital cellular components and processes involved in thermoresponsive growth and the acquisition of thermotolerance in plants. This review summarizes the major mechanisms involved in plant HT responses, with a special focus on recent discoveries related to plant thermosensing, heat stress signaling, and HT-regulated gene expression networks that promote plant adaptation to elevated environmental temperatures.     

Proteomic analysis of the adaptation to warming in the Antarctic bacteria Shewanella frigidimarina

Antarctica is subjected to extremely variable conditions, but the importance of the temperature increase in cold adapted bacteria is still unknown. To study the molecular adaptation to warming of Antarctic bacteria, cultures of Shewanella frigidimarina were incubated at temperatures ranging from 0 °C to 30 °C, emulating the most extreme conditions that this strain could tolerate. A proteomic approach was developed to identify the soluble proteins obtained from cells growing at 4 °C, 20 °C and 28 °C. The most drastic effect when bacteria were grown at 28 °C was the accumulation of heat shock proteins as well as other proteins related to stress, redox homeostasis or protein synthesis and degradation, and the decrease of enzymes and components of the cell envelope. Furthermore, two main responses in the adaptation to warm temperature were detected: the presence of diverse isoforms in some differentially expressed proteins, and the composition of chaperone interaction networks at the limits of growth temperature. The abundance changes of proteins suggest that warming induces a stress situation in S. frigidimarina forcing cells to reorganize their molecular networks as an adaptive response to these environmental conditions.     

Adapting with Microbial Help: Microbiome Flexibility Facilitates Rapid Responses to Environmental Change

Animals and plants are metaorganisms and associate with microbes that affect their physiology, stress tolerance, and fitness. Here the hypothesis that alteration of the microbiome may constitute a fast-response mechanism to environmental change is examined. This is supported by recent reciprocal transplant experiments with reef corals, which have shown that their microbiome adapts to thermally variable habitats and changes over time when transplanted into different environments. Further, inoculation of corals with beneficial bacteria increases their stress tolerance. But corals differ in their ability to flexibly associate with different bacteria. How scales of microbiome flexibility may reflect different metaorganism adaptation mechanisms is discussed and future directions for research are pinpointed. It is posited that microbiome flexibility is a broad phenomenon that contributes to the ability of organisms to respond to environmental change. Importantly, adapting with microbial help may provide an alternate route to organismal adaptation that facilitates rapid responses.     

Chemical modulation of physiological adaptation and cross-protective responses against oxidative stress in soil bacterium and phytopathogen,Xanthomonas

Soil bacteria need to adapt quickly to changes in the environmental conditions. Physiological adaptation plays an important role in microbial survival, especially under stressful conditions. Here the abilities of chemicals and pesticides to modulate physiological adaptive and cross-protective responses, that make the bacteria more resistant to oxidative stress, are examined in the soil bacterium and phytopathogen, Xanthomonas. The genetic basis for the observed stress resistance, as well as the regulatory mechanisms controlling gene expression during the process, has begun to be elucidated.

     

Signal transduction during cold, salt, and drought stresses in plants

Abiotic stresses, especially cold, salinity and drought, are the primary causes of crop loss worldwide. Plant adaptation to environmental stresses is dependent upon the activation of cascades of molecular networks involved in stress perception, signal transduction, and the expression of specific stress-related genes and metabolites. Plants have stress-specific adaptive responses as well as responses which protect the plants from more than one environmental stress. There are multiple stress perception and signaling pathways, some of which are specific, but others may cross-talk at various steps. In this review article, we first expound the general stress signal transduction pathways, and then highlight various aspects of biotic stresses signal transduction networks. On the genetic analysis, many cold induced pathways are activated to protect plants from deleterious effects of cold stress, but till date, most studied pathway is ICE-CBF-COR signaling pathway. The Salt-Overly-Sensitive (SOS) pathway, identified through isolation and study of the sos1, sos2, and sos3 mutants, is essential for maintaining favorable ion ratios in the cytoplasm and for tolerance of salt stress. Both ABA-dependent and -independent signaling pathways appear to be involved in osmotic stress tolerance. ROS play a dual role in the response of plants to abiotic stresses functioning as toxic by-products of stress metabolism, as well as important signal transduction molecules and the ROS signaling networks can control growth, development, and stress response. Finally, we talk about the common regulatory system and cross-talk among biotic stresses, with particular emphasis on the MAPK cascades and the cross-talk between ABA signaling and biotic signaling.     

Oligopolyphenylenevinylene-Conjugated Oligoelectrolyte Membrane Insertion Molecules Selectively Disrupt Cell Envelopes of Gram-Positive Bacteria

The modification of microbial membranes to achieve biotechnological strain improvement with exogenous small molecules, such as oligopolyphenylenevinylene-conjugated oligoelectrolyte (OPV-COE) membrane insertion molecules (MIMs), is an emerging biotechnological field. Little is known about the interactions of OPV-COEs with their target, the bacterial envelope. We studied the toxicity of three previously reported OPV-COEs with a selection of Gram-negative and Gram-positive organisms and demonstrated that Gram-positive bacteria are more sensitive to OPV-COEs than Gram-negative bacteria. Transmission electron microscopy demonstrated that these MIMs disrupt microbial membranes and that this occurred to a much greater degree in Gram-positive organisms. We used a number of mutants to probe the nature of MIM interactions with the microbial envelope but were unable to align the membrane perturbation effects of these compounds to previously reported membrane disruption mechanisms of, for example, cationic antimicrobial peptides. Instead, the data support the notion that OPV-COEs disrupt microbial membranes through a suspected interaction with diphosphatidylglycerol (DPG), a major component of Gram-positive membranes. The integrity of model membranes containing elevated amounts of DPG was disrupted to a greater extent by MIMs than those prepared from Escherichia coli total lipid extracts alone.     

Antimicrobials,Stress and Mutagenesis

Cationic antimicrobial peptides are ancient and ubiquitous immune effectors that multicellular organisms use to kill and police microbes whereas antibiotics are mostly employed by microorganisms. As antimicrobial peptides (AMPs) mostly target the cell wall, a microbial ‘Achilles heel’, it has been proposed that bacterial resistance evolution is very unlikely and hence AMPs are ancient ‘weapons’ of multicellular organisms. Here we provide a new hypothesis to explain the widespread distribution of AMPs amongst multicellular organism. Studying five antimicrobial peptides from vertebrates and insects, we show, using a classic Luria-Delbrück fluctuation assay, that cationic antimicrobial peptides (AMPs) do not increase bacterial mutation rates. Moreover, using rtPCR and disc diffusion assays we find that AMPs do not elicit SOS or rpoS bacterial stress pathways. This is in contrast to the main classes of antibiotics that elevate mutagenesis via eliciting the SOS and rpoS pathways. The notion of the ‘Achilles heel’ has been challenged by experimental selection for AMP-resistance, but our findings offer a new perspective on the evolutionary success of AMPs. Employing AMPs seems advantageous for multicellular organisms, as it does not fuel the adaptation of bacteria to their immune defenses. This has important consequences for our understanding of host-microbe interactions, the evolution of innate immune defenses, and also sheds new light on antimicrobial resistance evolution and the use of AMPs as drugs.     

Molecular Adaptation Mechanisms Employed by Ethanologenic Bacteria in Response to Lignocellulose-derived Inhibitory Compounds

Current international interest in finding alternative sources of energy to the diminishing supplies of fossil fuels has encouraged research efforts in improving biofuel production technologies. In countries which lack sufficient food, the use of sustainable lignocellulosic feedstocks, for the production of bioethanol, is an attractive option. In the pre-treatment of lignocellulosic feedstocks for ethanol production, various chemicals and/or enzymatic processes are employed. These methods generally result in a range of fermentable sugars, which are subjected to microbial fermentation and distillation to produce bioethanol. However, these methods also produce compounds that are inhibitory to the microbial fermentation process. These compounds include products of sugar dehydration and lignin depolymerisation, such as organic acids, derivatised furaldehydes and phenolic acids. These compounds are known to have a severe negative impact on the ethanologenic microorganisms involved in the fermentation process by compromising the integrity of their cell membranes, inhibiting essential enzymes and negatively interact with their DNA/RNA. It is therefore important to understand the molecular mechanisms of these inhibitions, and the mechanisms by which these microorganisms show increased adaptation to such inhibitors. Presented here is a concise overview of the molecular adaptation mechanisms of ethanologenic bacteria in response to lignocellulose-derived inhibitory compounds. These include general stress response and tolerance mechanisms, which are typically those that maintain intracellular pH homeostasis and cell membrane integrity, activation/regulation of global stress responses and inhibitor substrate-specific degradation pathways. We anticipate that understanding these adaptation responses will be essential in the design of ''intelligent'' metabolic engineering strategies for the generation of hyper-tolerant fermentation bacteria strains.     

根除细菌生物被膜的新视角：分散

细菌生物被膜(biofilm,BF)是细菌为对抗外界压力形成的一种自我保护结构,对于抗菌药物具有极高的耐受性,在临床上极易引发难治性慢性感染。BF分散是指在BF形成周期中,膜内细胞主动逸出,恢复浮游生长模式,寻找新定植位点的过程。由于细菌在浮游状态下,更易受到抗菌药与免疫反应的作用,诱导BF分散是控制BF相关感染(biofilm-associated infections, BAI)的一条富有前景的策略。本文从BF分散的方式和信号分子等角度,对BF分散的调控机制进行分析;归纳能影响BF分散的物质,并对BF分散后可能带来的危害及未来的研究思路进行简述,以期为研发新型分散剂和深入研究药物作用的靶点提供理论参考。     

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.