Polysaccharide intercellular adhesin (PIA) protects Staphylococcus epidermidis against major components of the human innate immune system

The skin commensal and opportunistic pathogen Staphylococcus epidermidis is the leading cause of nosocomial and biofilm-associated infections. Little is known about the mechanisms by which S. epidermidis protects itself against the innate human immune system during colonization and infection. We used scanning electron microscopy to demonstrate that the exopolysaccharide intercellular adhesin (PIA) resides in fibrous strands on the bacterial cell surface, and that lack of PIA production results in complete loss of the extracellular matrix material that has been suggested to mediate immune evasion. Phagocytosis and killing by human polymorphonuclear leucocytes was significantly increased in a mutant strain lacking PIA production compared with the wild-type strain. The mutant strain was also significantly more susceptible to killing by major antibacterial peptides of human skin, cationic human beta-defensin 3 and LL-37, and anionic dermcidin. PIA represents the first defined factor of the staphylococcal biofilm matrix that protects against major components of human innate host defence.     

Staphylococcus epidermidis strategies to avoid killing by human neutrophils

Staphylococcus epidermidis is a leading nosocomial pathogen. In contrast to its more aggressive relative S. aureus, it causes chronic rather than acute infections. In highly virulent S. aureus, phenol-soluble modulins (PSMs) contribute significantly to immune evasion and aggressive virulence by their strong ability to lyse human neutrophils. Members of the PSM family are also produced by S. epidermidis, but their role in immune evasion is not known. Notably, strong cytolytic capacity of S. epidermidis PSMs would be at odds with the notion that S. epidermidis is a less aggressive pathogen than S. aureus, prompting us to examine the biological activities of S. epidermidis PSMs. Surprisingly, we found that S. epidermidis has the capacity to produce PSMδ, a potent leukocyte toxin, representing the first potent cytolysin to be identified in that pathogen. However, production of strongly cytolytic PSMs was low in S. epidermidis, explaining its low cytolytic potency. Interestingly, the different approaches of S. epidermidis and S. aureus to causing human disease are thus reflected by the adaptation of biological activities within one family of virulence determinants, the PSMs. Nevertheless, S. epidermidis has the capacity to evade neutrophil killing, a phenomenon we found is partly mediated by resistance mechanisms to antimicrobial peptides (AMPs), including the protease SepA, which degrades AMPs, and the AMP sensor/resistance regulator, Aps (GraRS). These findings establish a significant function of SepA and Aps in S. epidermidis immune evasion and explain in part why S. epidermidis may evade elimination by innate host defense despite the lack of cytolytic toxin expression. Our study shows that the strategy of S. epidermidis to evade elimination by human neutrophils is characterized by a passive defense approach and provides molecular evidence to support the notion that S. epidermidis is a less aggressive pathogen than S. aureus.     

The antimicrobial peptide-sensing system aps of Staphylococcus aureus

Staphylococcus aureus is a leading cause of hospital-associated and, more recently, community-associated infections caused by highly virulent methicillin-resistant strains (CA-MRSA). S. aureus survival in the human host is largely defined by the ability to evade attacks by antimicrobial peptides (AMPs) and other mechanisms of innate host defence. Here we show that AMPs induce resistance mechanisms in CA-MRSA via the aps AMP sensor/regulator system, including (i) the d-alanylation of teichoic acids, (ii) the incorporation of lysyl-phosphatidylglycerol in the bacterial membrane and a concomitant increase in lysine biosynthesis, and (iii) putative AMP transport systems such as the vraFG transporter, for which we demonstrate a function in AMP resistance. In contrast to the aps system of S. epidermidis, induction of the aps response in S. aureus was AMP-selective due to structural differences in the AMP binding loop of the ApsS sensor protein. Finally, using a murine infection model, we demonstrate the importance of the aps regulatory system in S. aureus infection. This study shows that while significant interspecies differences exist in the AMP-aps interaction, the AMP sensor system aps is functional and efficient in promoting resistance to a variety of AMPs in a clinically relevant strain of the important human pathogen S. aureus.     

Antimicrobial peptide resistance mechanisms of human bacterial pathogens

The critical role played by antimicrobial peptides (AMPs) in mammalian innate immunity is increasingly recognized. Bacteria differ in their intrinsic susceptibility to AMPs, and the relative resistance of some important human pathogens to these defense molecules is now appreciated as an important virulence phenotype. Experimental analysis has identified diverse mechanisms of bacterial AMP resistance including altered cell surface charge, active efflux, production of proteases or trapping proteins, and modification of host cellular processes. The contribution of these resistance mechanisms to pathogenesis is confirmed through direct comparison of wild-type bacteria and AMP-sensitive mutants using in vivo infection models. Knowledge of the molecular basis of bacterial AMP resistance may provide new targets for antimicrobial therapy of human infectious diseases.     

Bacterial resistance to antimicrobial peptides

Antimicrobial peptides (AMPs) or host defense peptides (HDPs) are vital components of human innate defense system targeting human‐related bacteria. Many bacteria have various mechanisms interfering with AMP activity, causing resistance to AMPs. Since AMPs are considered as potential novel antimicrobial drugs, understanding the mechanisms of bacterial resistance to direct killing of AMPs is of great significance. In this review, a comparative overview of bacterial strategies for resistance to direct killing of various AMPs is presented. Such strategies include bacterial cell envelope modification, AMP degradation, sequestration, expelling, and capsule.     

The expanding scope of antimicrobial peptide structures and their modes of action

Antimicrobial peptides (AMPs) are an integral part of the innate immune system that protect a host from invading pathogenic bacteria. To help overcome the problem of antimicrobial resistance, cationic AMPs are currently being considered as potential alternatives for antibiotics. Although extremely variable in length, amino acid composition and secondary structure, all peptides can adopt a distinct membrane-bound amphipathic conformation. Recent studies demonstrate that they achieve their antimicrobial activity by disrupting various key cellular processes. Some peptides can even use multiple mechanisms. Moreover, several intact proteins or protein fragments are now being shown to have inherent antimicrobial activity. A better understanding of the structure-activity relationships of AMPs is required to facilitate the rational design of novel antimicrobial agents.     

Evolution of defence cocktails: Antimicrobial peptide combinations reduce mortality and persistent infection

The simultaneous expression of costly immune effectors such as multiple antimicrobial peptides is a hallmark of innate immunity of multicellular organisms, yet the adaptive advantage remains unresolved. Here, we test current hypotheses on the evolution of such defence cocktails. We use RNAi gene knock‐down to explore, the effects of three highly expressed antimicrobial peptides, displaying different degrees of activity in vitro against Staphylococcus aureus, during an infection in the beetle Tenebrio molitor. We find that a defensin confers no survival benefit but reduces bacterial loads. A coleoptericin contributes to host survival without affecting bacterial loads. An attacin has no individual effect. Simultaneous knock‐down of the defensin with the other AMPs results in increased mortality and elevated bacterial loads. Contrary to common expectations, the effects on host survival and bacterial load can be independent. The expression of multiple AMPs increases host survival and contributes to the control of persisting infections and tolerance. This is an emerging property that explains the adaptive benefit of defence cocktails.     

Meningococcal resistance to antimicrobial peptides is mediated by bacterial adhesion and host cell RhoA and Cdc42 signalling

Antimicrobial peptides (AMPs) constitute an essential part of the innate immune defence. Pathogenic bacteria have evolved numerous strategies to withstand AMP‐mediated killing. The influence of host epithelia on bacterial AMP resistance is, however, still largely unknown. We found that adhesion to pharyngeal epithelial cells protected Neisseria meningitidis, a leading cause of meningitis and sepsis, from the human cathelicidin LL‐37, the cationic model amphipathic peptide (MAP) and the peptaibol alamethicin, but not from polymyxin B. Adhesion to primary airway epithelia resulted in a similar increase in LL‐37 resistance. The inhibition of selective host cell signalling mediated by RhoA and Cdc42 was found to abolish the adhesion‐induced LL‐37 resistance by a mechanism unrelated to the actin cytoskeleton. Moreover, N. meningitidis triggered the formation of cholesterol‐rich membrane microdomains in pharyngeal epithelial cells, and host cell cholesterol proved to be essential for adhesion‐induced resistance. Our data highlight the importance of Rho GTPase‐dependent host cell signalling for meningococcal AMP resistance. These results indicate that N. meningitidis selectively exploits the epithelial microenvironment in order to protect itself from LL‐37.     

Beyond electrostatics: Antimicrobial peptide selectivity and the influence of cholesterol-mediated fluidity and lipid chain length on protegrin-1 activity

Antimicrobial peptides (AMPs) are a promising class of innate host defense molecules for next-generation antibiotics, as they uniquely target and permeabilize membranes of pathogens. This selectivity has been explained by the electrostatic attraction between these predominantly cationic peptides and the bacterial membrane, which is heavily populated with anionic lipids. However, AMP-resistant bacteria have non-electrostatic countermeasures that modulate membrane rigidity and thickness. We explore how variations in physical properties affect the membrane affinity and disruption process of protegrin-1 (PG-1) in phosphatidylcholine (PC) membranes with altered lipid packing densities and thicknesses. From isothermal titration calorimetry and atomic force microscopy, our results showed that PG-1 could no longer insert into membranes of increasing cholesterol amounts nor into monounsaturated PC membranes of increasing thicknesses with similar fluidities. Prevention of PG-1’s incorporation consequently made the membranes more resistant to peptide-induced structural transformations like pore formation. Our study provides evidence that AMP affinity and activity are strongly correlated with the fluidity and thickness of the membrane. A basic understanding of how physical mechanisms can regulate cell selectivity and resistance towards AMPs will aid in the development of new antimicrobial agents.     

Interplay between Candida albicans and the Antimicrobial Peptide Armory

Antimicrobial peptides (AMPs) are key elements of innate immunity, which can directly kill multiple bacterial, viral, and fungal pathogens. The medically important fungus Candida albicans colonizes different host niches as part of the normal human microbiota. Proliferation of C. albicans is regulated through a complex balance of host immune defense mechanisms and fungal responses. Expression of AMPs against pathogenic fungi is differentially regulated and initiated by interactions of a variety of fungal pathogen-associated molecular patterns (PAMPs) with pattern recognition receptors (PRRs) on human cells. Inflammatory signaling and other environmental stimuli are also essential to control fungal proliferation and to prevent parasitism. To persist in the host, C. albicans has developed a three-phase AMP evasion strategy, including secretion of peptide effectors, AMP efflux pumps, and regulation of signaling pathways. These mechanisms prevent C. albicans from the antifungal activity of the major AMP classes, including cathelicidins, histatins, and defensins leading to a basal resistance. This minireview summarizes human AMP attack and C. albicans resistance mechanisms and current developments in the use of AMPs as antifungal agents.     

Physico-chemical properties of staphylococci of different species in resistance to human thrombodefensins

Testing 54 strains of staphylococci (Staphylococcus aureus, S. epidermidis, S. haemolyticus, S. warneri, S. hominis, S. capitis) revealed that S. aureus in contrast to coagulase-negative staphylococci (CNS) is more resistant to bactoriocidal action of human thrombodefensins (resistance index: 60.3 vs 25.6%), less hydrophilicolipophilic balance-HLB: -0.42 vs -0.64) and less charged (x-potential: -32.4 vs -35.6 mV). In groups of staphylococci (S. aureus and CNS) correlation links of bacterial resistance to human thrombodefensins with their HLB and x-potential (r=-0.32...-0.36). By In vitro experiments, it was shown that 5 passages of staphylococci in meat-peptonic broth with human thrombodefensins (50 mkg protein/ml) lead to adaptation of bacteria followed by the formation of resistance to cationic peptides from thrombocytes, a decrease of hydrophobicity and x-potencial. The role of physico-chemical properties in providing thrombodefensin-resistance of staphylococci as a developmental factor of infectious-and-inflammatory process and persistence of bacteria was confirmed with Salmonella infection.     

Biofilms of clinical strains of Staphylococcus that do not contain polysaccharide intercellular adhesin

Staphylococcus aureus and coagulase-negative staphylococci, primarily Staphylococcus epidermidis, are recognized as a major cause of nosocomial infections associated with the use of implanted medical devices. The capacity of S. epidermidis to form biofilms, allowing it to evade host immune defence mechanisms and antibiotic therapy, is considered to be crucial in colonizing the surfaces of medical implants and dissemination of infection. It has previously been demonstrated that the biofilm of a model strain S. epidermidis RP62A comprises two carbohydrate-containing moieties, a polysaccharide having a structure of a linear poly-N-acetyl-(1-->6)-beta-D-glucosamine and teichoic acid. In the present paper we show that, unlike this model strain, certain clinical isolates of coagulase-negative staphylococci produce biofilms that do not contain detectable amounts of poly-N-acetyl-(1-->6)-beta-D-glucosamine. In contrast to that of S. epidermidis RP62A, these biofilms are not detached with metaperiodate, while proteinase K causes their partial dispersal.     

The Human Cathelicidin LL-37 Has Antiviral Activity against Respiratory Syncytial Virus

Respiratory syncytial virus is a leading cause of lower respiratory tract illness among infants, the elderly and immunocompromised individuals. Currently, there is no effective vaccine or disease modifying treatment available and novel interventions are urgently required. Cathelicidins are cationic host defence peptides expressed in the inflamed lung, with key roles in innate host defence against infection. We demonstrate that the human cathelicidin LL-37 has effective antiviral activity against RSV in vitro, retained by a truncated central peptide fragment. LL-37 prevented virus-induced cell death in epithelial cultures, significantly inhibited the production of new infectious particles and diminished the spread of infection, with antiviral effects directed both against the viral particles and the epithelial cells. LL-37 may represent an important targetable component of innate host defence against RSV infection. Prophylactic modulation of LL-37 expression and/or use of synthetic analogues post-infection may represent future novel strategies against RSV infection.     

The antimicrobial peptide cathelicidin interacts with airway mucus

Antimicrobial peptides (AMPs) and mucins are components of airway secretions and both contribute to the innate host defense system. At neutral pH, AMPs are positively charged, mucins negatively. It was the aim of the study to test whether these opposite charges result in interactions between AMPs and mucins. We measured binding of mucins isolated from porcine gastric mucosa to the cathelicidin LL-37 coated to multiwell plates and found that LL-37 electrostatically interacts with mucins. Circular dichroism spectra of the peptide revealed the induction of -helical conformation by mucins. Addition of mucins to solutions of LL-37 significantly decreased the antimicrobial activity of the peptide against Pseudomonas aeruginosa and Streptococcus pneumoniae. We then tested whether LL-37 is bound to mucins in airway secretions from human subjects and found that a significant proportion of the peptide and its propeptide are bound to high molecular weight components. Together these data show that cationic AMPs interact with anionic mucins in airway secretions. Functions of AMPs are modulated by this interaction.     

Biological characterization and modes of action of temporins and bombinins H, multiple forms of short and mildly cationic anti-microbial peptides from amphibian skin.

Genetically encoded cationic anti-microbial peptides (AMPs) are essential components of the ancient and non-specific innate immune system, which is the principal defence mechanism of all species of life, with the primary role to kill infectious microorganisms. Amphibian skin is one of the richest natural sources of such molecules, which are produced by holocrine-type dermal glands and released upon stimulation. This review highlights the attractive and unique structural/functional properties of temporins and bombinins H, two families of short and mildly cationic peptides, isolated from the skin of frogs belonging to Rana and Bombina genera, respectively. Beside improving our knowledge on the role of AMPs in the regulation of the innate immunity, the biological significance of the existence of multiple forms of a prototypic peptide sequence within the same organism and the implication of short peptides in the endotoxin neutralization, these two classes of AMPs can be also considered as valid candidates for the design of novel anti-infective and anti-sepsis drugs.     

Antimicrobial peptides: biochemical determinants of activity and biophysical techniques of elucidating their functionality

Antimicrobial peptides (AMPs) have been established over millennia as powerful components of the innate immune system of many organisms. Due to their broad spectrum of activity and the development of host resistance against them being unlikely, AMPs are strong candidates for controlling drug-resistant pathogenic microbial pathogens. AMPs cause cell death through several independent or cooperative mechanisms involving membrane lysis, non-lytic activity, and/or intracellular mechanisms. Biochemical determinants such as peptide length, primary sequence, charge, secondary structure, hydrophobicity, amphipathicity and host cell membrane composition together influence the biological activities of peptides. A number of biophysical techniques have been used in recent years to study the mechanisms of action of AMPs. This work appraises the molecular parameters that determine the biocidal activity of AMPs and overviews their mechanisms of actions and the diverse biochemical, biophysical and microscopy techniques utilised to elucidate these.     

Induction of Staphylococcus epidermidis biofilm formation via proteolytic processing of the accumulation-associated protein by staphylococcal and host proteases

Because of its biofilm forming potential Staphylococcus epidermidis has evolved as a leading cause of device-related infections. The polysaccharide intercellular adhesin (PIA) is significantly involved in biofilm accumulation. However, infections because of PIA-negative strains are not uncommon, suggesting the existence of PIA-independent biofilm accumulation mechanisms. Here we found that biofilm formation in the clinically significant S. epidermidis 5179 depended on the expression of a truncated 140 kDa isoform of the 220 kDa accumulation-associated protein Aap. As expression of the truncated Aap isoform leads to biofilm formation in aap-negative S. epidermidis 1585, this domain mediates intercellular adhesion in a polysaccharide-independent manner. In contrast, expression of full-length Aap did not lead to a biofilm-positive phenotype. Obviously, to gain adhesive function, full-length Aap has to be proteolytically processed through staphylococcal proteases as demonstrated by inhibition of biofilm formation by alpha(2)-macroglobulin. Importantly, also exogenously added granulocyte proteases activated Aap, thereby inducing biofilm formation in S. epidermidis 5179 and four additional, independent clinical S. epidermidis strains. It is therefore reasonable to assume that in vivo effector mechanisms of the innate immunity can directly induce protein-dependent S. epidermidis cell aggregation and biofilm formation, thereby enabling the pathogen to evade clearance by phagocytes.     

Differential activity of lytic α-helical peptides on lactobacilli and lactobacilli-derived liposomes

Eukaryotic antimicrobial peptides (AMPs) interact with plasma membrane of bacteria, fungi and eukaryotic parasites. Noteworthy, Lactobacillus delbrueckii subsp. lactis (CIDCA 133) and L. delbrueckii subsp. bulgaricus (CIDCA 331) show different susceptibility to human beta-defensins (β-sheet peptides). In the present work we extended the study to α-helical peptides from anuran amphibian (Aurein 1.2, Citropin 1.1 and Maculatin 1.1). We studied the effect on whole bacteria and liposomes formulated with bacterial lipids through growth kinetics, flow cytometry, leakage of liposome content and studies of peptide insertion in lipid monolayers.Growth of strain CIDCA 331 was dramatically inhibited in the presence of all three peptides and minimal inhibitory concentrations were lower than those for strain CIDCA 133. Flow cytometry revealed that AMPs lead to the permeabilization of bacteria.In addition, CIDCA 331-derived liposomes showed high susceptibility, leading to content leakage and structural disruption. Accordingly, peptide insertion in lipid monolayers demonstrated spontaneous interaction of AMPs with CIDCA 331 lipids. In contrast, lipids monolayers from strain CIDCA 133 were less susceptible.Summarizing we demonstrate that the high resistance of the probiotic strain CIDCA 133 to AMPs extends to α helix peptides Aurein, Citropin and Maculatin. This behavior could be ascribed in part to differences in membrane composition. These findings, along with the previously demonstrated resistance to β defensins from human origin, suggest that strain CIDCA 133 is well adapted to host innate immune effectors from both mammals and amphibians thus indicating conserved mechanisms of interaction with key components of the innate immune system.     

Proteomics assisted profiling of antimicrobial peptide signatures from black pepper (<Emphasis Type="Italic">Piper nigrum</Emphasis> L.)

Plant antimicrobial peptides are the interesting source of studies in defense response as they are essential components of innate immunity which exert rapid defense response. In spite of abundant reports on the isolation of antimicrobial peptides (AMPs) from many sources, the profile of AMPs expressed/identified from single crop species under certain stress/physiological condition is still unknown. This work describes the AMP signature profile of black pepper and their expression upon Phytophthora infection using label-free quantitative proteomics strategy. The differential expression of 24 AMPs suggests that a combinatorial strategy is working in the defense network. The 24 AMP signatures belonged to the cationic, anionic, cysteine-rich and cysteine-free group. As the first report on the possible involvement of AMP signature in Phytophthora infection, our results offer a platform for further study on regulation, evolutionary importance and exploitation of theses AMPs as next generation molecules against pathogens.