研究生课程论文
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Bacterial Resistance to Antimicrobial Peptides
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2016年 1 月 15 日
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Abstract:Resistant bacterial infections are a major health problem in many parts of the world. The major commercial antibiotic classes often fail to combat common bacteria.Antimicrobial peptides (AMPs) are able to control bacterial infections by interfering with microbial metabolism and physiological processes in several ways.However, successful pathogens have developed mechanisms to resist AMPs.To gain a better understanding of the resistance process various technologies have been applied.we review progress on the use of in vivo infection models in AMP research and discuss the AMP resistance mechanisms that have been established by in vivo studies to contribute to microbial infection.we discuss multiple strategies by which bacteria could develop enhanced antimicrobial peptide resistance, focusing on sub-cellular regions from the surface to deep inside, evaluating bacterial membranes, cell walls and cytoplasmic metabolism.
Key word:Antimicrobial peptides; resistance; Membrane; cell wall
1 Introduction
In recent years, antibiotic resistance has increasingly become an uncontrollable health problem. Bacterial infections caused by resistant strains can be found in hospitals around the world, being extremely common in immune compromised patients [1]. Antibiotics are able to control bacterial infections, interfering with microbial metabolism and physiological processes, such as DNA replication and cell wall biosynthesis. Although multiple compounds are often used, cases of resistance to the majority of antibiotic classes used in hospitals have been reported. The last report from the American Centers for Disease Control estimated that over two million illnesses and 23,000 deaths were caused by drug-resistant microbes in the USA in 2013. These numbers have encouraged health organizations to establish stricter policies for antibiotic use in order to curtail the emergence of resistance. These policies are unquestionably helping to protect patients in many countries. If, on the one hand, a reliable policy for the use of antibiotics is necessary, the development of new drugs with potential activity against these pathogens is also essential.
Antimicrobial peptides (AMPs) are effective antibiotic agents found in plants, animals and microorganisms. These molecules have a broad spectrum of action, often being active against bacteria, fungi and protozoans. The amphipathic structure, common to AMPs, facilitates their interactions and insertion into the anionic cell wall and phospholipid membranes of microorganisms [2]. Frequently, AMP activity results from the disturbance of cell membrane integrity. However, AMPs can act in different cell targets including DNA , RNA , regulatory enzymes and other proteins, appearing as a promising alternative to classic antibiotics. Nevertheless, once AMPs have been put into current clinical use, the development of AMP-resistant strains will be inevitable [3]. Thus, the understanding of bacterial resistance against these
compounds is extremely necessary for a possible rational planning of the next antibiotic generation.
To shed some light on the bacterial resistance process, severaltechnologies including mass spectrometry and high-throughput techniques have been applied to analyses of bacterial physiology in response to antibiotic stress .A full understanding of how bacteria overcome AMP-mediated attack during infection requires a combination of in vitro and in vivo studies. In vitro work is critical to understanding the genes, proteins, and mechanisms involved in AMP resistance; however, the contribution of these in vitro-established mechanisms to bacterial disease can only be elucidated in vivo. Here, we review the literature on in vivo studies that examine the role of AMP resistance mechanisms in bacterial pathogenesis. We first describe the major in vivo models used in these studies; we then discuss the collective findings of in vitro and in vivo research that established AMP resistance mechanisms which contribute significantly to bacterial disease.we discuss different strategies by which bacteria can develop AMP resistance from the surface to deep inside, evaluating the bacterial resistance process layer by layer. Moreover, some technologies for detecting antimicrobial resistance are also discussed.
2 In vivo models of AMP resistance mechanisms in pathogenesis 2.1Human models of infection
To study human pathogens,an ideal in vivo model would be ahuman experimental infection model.Of course,concerns such as safetyto the human subjects and transmissibility to the public preclude theability to perform human infection experiments with most bacterialpathogens.However,within limitations imposed for medical andethical reasons,a few human experimental infection models arecurrently in use.These models provide the ability to accurately recapitulate the kinetics of natural,human disease.Importantly,humanmodels of infection allow the study of a human pathogen in the contextof the specific,host-derived pressures with which the pathogenevolved.One such host-derived pressure is attack by AMPs;as AMP resistance mechanisms are often specific for certain AMPs,it is beneficialto examine AMP resistance against the AMPs that pathogen encountersduring infection.Another advantage of human models is that they canbe adapted for testing new therapeutics for treatment of infections orvaccine candidates for disease prevention.
As with any model,human infection models have limitations.For subject safety reasons,these models are generally limited to local infections and to the early stages of disease,with treatment at the onset of symptoms or discomfort;long-term infections,systemic infections, and sequelae cannot be safely examined in human volunteers.To ensure control of the infection,human models are typically restricted to one or two well-characterized wild-type bacterial strains,and isogenic derivatives thereof,that are readily treatable and do not harbor plasmids or phages that could transmit genetic material to the host’s microbial flora.Many other aspects of human infection models,such as route of inoculation and dosage,are far less flexible than in animal models.Working within these limitations,however,human experimental
infection models accurately reproduce naturally acquired disease and provide important information about the host-pathogen relationship that is directly relevant to humans.Two such models have been used to examine the role of AMP resistance mechanisms in human infectious disease[4]. 2.2. Nonhuman in vivo models of infection
For most pathogens, no option exists for human experimentation; researchers rely instead on various nonhuman vertebrate and invertebrate models to study pathogenic mechanisms, including the importance of AMP-resistance systems, in vivo. These models, which we will refer to collectively as animal models, offer several advantages, including the generally lower cost of non-primate animal studies compared with human studies, the ability to study later stages of infection, and the ability to choose and even manipulate the genetic background of the host. Disadvantages of animal infection models include the inability to study host-restricted aspects of the disease or host response, and the difficulty in establishing the relevance to humans of results obtained in animals. Within these limitations, however, most bacterial pathogenesis studies have been performed in animals and have provided a wealth of information on host-pathogen interactions[5].
3 Membrane alterations that cause antimicrobial peptide resistance
Most AMPs are capable of permeabilizing microbial membranes causing an osmotic cellular imbalance. In general terms, the electrostatic forces start the interaction between the negatively charged cell surface and the positively charged peptide. This initial interaction leads to a second step in which the peptide with hydrophobic patches binds to the lipidic membrane, resulting in membrane disruption. Barrel-stave, toroidal, and carpet are the main models of this concept[6]. In barrel-stave, peptides seem to oligomerize and form transmembrane pores. Otherwise, toroidal pores could be formed by monomer peptides, which induce a local membrane curvature that also results in membrane disruption[6]. Finally, a carpet mechanism occurs when the AMPs cover the membrane surface, causing a detergent-like effect that is able to disintegrate the membranes. Although these three classical mechanisms of action have been extensively discussed in the last two decades, some authors have proposed several other possibilities.
The mechanisms of bacterial resistance to AMPs are still not fully established,but the modifications in the physical–chemical interaction between the bacterial cell membrane and the AMP molecule seem to be the first step commonly involved in the resistance process. In general,bacterial resistance can be achieved by bacteria changing the AMP target to make it less susceptible to AMP action or even by mechanisms related to the removal of AMPs from their site of action in the bacterial membrane.Often,fluidity and permeability of the bacterial cell membranes decrease due to alterations in the architecture of the outer and inner membranes[7].Reduced levels of specific membrane proteins and ions(such as Mg2+and Ca2+),and changes in membrane lipid composition afford protection to the site of action of various AMPs.This was observed in bacterial resistance to polymyxins,defensins and
cathelicidins,where these compounds main target is the cytoplasmic bacterial membrane.
Included in the main constituents of bacterial membranes are various types of phospholipids, such as phosphatidylethanolamine (PE),phosphatidyglycerol (PG) and cardiolipin (CL) [8]. The bacterial membrane structure fulfils several vital tasks, while the phospholipid membrane composition prevents cell damage in unfavorable growth conditions, and this modulation seems to be crucial for bacterial survival. The roles of the various phospholipids, their biosynthesis, turnover and regulation are very important in bacterial membrane structure and function, showing a close relation to antibiotic resistance. One example is about the expression of postulated flippase proteins that are required for translocation of lipids from the inner to the outer leaflet of cytoplasmic membranes, resulting in changes in the membrane composition and giving it a stable profile against antimicrobial action.
Under the control of the TCS systems, the bacteria perceive environmental stimulus, activate signal transduction and respond to the presence of AMPs, resulting in increased AMP resistance . Along with this, the resistance is often also increased by the activity of transmembrane transporters, the efflux pumps that transduce electrochemical energy to displace the drugs out of the periplasm . Active efflux plays a major role in this resistance, and multidrug efflux pumps decrease the accumulation of drugs within cells.
The tripartite-transport systems composed of inner and outer membrane proteins, connected by a periplasmic membrane fusion protein(i.e. AcrABTolC in E. coli), are mainly an important efflux system to pump cytotoxic compounds and antimicrobial drugs. Their overexpression in bacteria is really correlated with their antibiotic resistance [9]. Thus, the bacteria can adapt to a wide range of environmental conditions, including the presence of antimicrobial compounds, antibiotics and AMPs, which constitute environmental chemical stresses for bacterial cells, through appropriate developed mechanisms that confer protection against this external attack, such as the mechanism involving the cell membrane that seems to be pivotal in bacterial survival and resistance.
4. Modifications in cell wall of AMP-resistant bacterial strains
Cationic antimicrobial peptides(AMPs)are produced by all living organisms including bacteria and come in many different chemical structures including ribosomal peptides(e.g.linear helical or disulphide-stabilized),ribosomal peptides with extensive posttranslational modifications(so-called RIPPs),such as lantibiotics as well as peptides with unusual amino acids of non-ribosomal origin such as glyco-and lipopeptide antibiotics.The unifying structural and biophysical theme of AMPs is the cationic and amphiphilic nature which enables the interaction with the negatively charged cell envelope components and phospholipids.Based on these features,it was a longheld view that AMPs kill microbes by forming more or less defined pores in the lipid bilayer of microbial membranes.However,more recently it became that the antibiotic activity even of unmodified ribosomal peptides can be much more specific
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