Peptides (v.22, #10)

Peptides: bacteria’s point of view by Naomi Balaban; Nir Koyfman (1517-1518).

A novel class of extracellular signaling peptides has been identified in Gram-positive bacteria that are actively transported into the cell to interact with intracellular receptors. The defining members of this novel class of signaling peptides are the Phr peptides of Bacillus subtilis and the mating pheromones of Enterococcus faecalis. These peptides are small and unmodified, gene encoded, and secreted by the bacterium. Most of these peptides diffuse into the extracellular medium, and when their concentration is sufficiently high, they are then actively transported into the cell by an oligopeptide permease (Opp). Once inside the cell, these peptides interact with an array of intracellular receptors. In B. subtilis, the Phr peptides regulate development of environmentally resistant spores and genetically competent cells (i.e. the natural ability to take up exogenous DNA). In E. faecalis, the mating pheromones regulate cell-cell transfer of plasmids, many of which encode antibiotic resistance or virulence factors. At least one component of the signaling pathway for these peptides is conserved in many bacteria, Opp. Opp is a non-specific transporter that transports peptides for use as carbon and nitrogen sources. The possibility that other bacteria could posses similar intracellularly functioning signaling peptides is discussed.

The tetracycline resistance plasmid pCF10 represents a class of unique mobile genetic elements of the bacterial genus Enterococcus, whose conjugative transfer functions are inducible by peptide sex pheromones excreted by potential recipient cells. These plasmids play a significant role in the dissemination of virulence and antibiotic resistance genes among the enterococci, which have become major nosocomial pathogens. Pheromone response by plasmid-carrying donor cells involves specific import of the peptide signal molecule, and subsequent interaction of the signal with one or more intracellular regulatory gene products. The pheromones are chromosomally encoded hydrophobic octa- or hepta-peptides, and different families of homologous plasmids encode the ability to respond to each pheromone. Among the four pheromone-responsive plasmids that have been characterized in some detail, there is considerable conservation in the genes encoding pheromone sensing and regulatory functions, and the peptides themselves show considerable similarity. In spite of this, there is extremely high specificity of response to each peptide, with virtually no “cross-induction” of transfer of non-cognate pheromone plasmids by the pheromones. This communication reviews the evidence for this specificity and discusses current molecular and genetic approaches to defining the basis for specificity.

Pentapeptide regulation of aspartyl-phosphate phosphatases by Marta Perego; James A. Brannigan (1541-1547).
Aspartyl-phosphate phosphatases are integral components of the phosphorelay signal transduction system for sporulation initiation in Bacillus subtilis. The Rap and Spo0E families of protein phosphatases specifically dephosphorylate the sporulation response regulators Spo0F and Spo0A, respectively. The phosphatases interpret regulatory signals antithetical to sporulation and the Rap phosphatases are subject to inactivation by specific pentapeptides generated from an inactive peptide precursor. Additional regulatory signals are brought about by the complex activation circuit that generates the Phr pentapeptide inhibitors of Rap phosphatases. Phr peptide’s recognition of the Rap phosphatase targets is remarkably specific. Specificity is dictated by the amino acid sequence of the pentapeptide. The identification of tetratricopeptide repeats in the Rap proteins may explain the mechanism by which Phr peptides bind to and inhibit the activity of Rap phosphatases.

In the Bacillus subtilis phosphorelay signal transduction system for sporulation initiation, signals competing with the differentiation process are interpreted by aspartyl-phosphate phosphatases that specifically dephosphorylate the Spo0F or Spo0A response regulators. The RapA phosphatase is regulated by the PhrA pentapeptide that directly and specifically inhibits its activity. PhrA specificity for RapA inhibition is dependent upon the amino acid sequence of the peptide. Here we show that the pentapeptide affinity for the phosphatase requires a free carboxylate group at the C-terminal amino acid. A free C-terminal carboxylic acid PhrA pentapeptide inhibits RapA phosphatase activity at a 1:1 ratio and it is approximately 200 fold more active than a C-terminal amide peptide. Therefore, coordination of the terminal carboxylate group appears to be critical for peptide binding to RapA.

Bacillus subtilis is known to produce an abundance of small polypeptides. Several of these have antimicrobial activity and others are pheromones or extracellular factors that affect internal signal transduction systems. The completion of the B. subtilis genomic nucleotide sequence has revealed 345 small polypeptide open-reading frames (of 85 codons or less), 81% of which are of unknown function. A significant number of these reside in prophage genomes or phage-like elements where they can be organized into large operons. It is likely that many more exist in the genome of B. subtilis but are “hidden” entirely or partially within other reading frames, or possess non-conventional translation start signals and have escaped detection. The discovery of so many small polypeptide orfs (SPORFs) and the likelihood of many more pose a challenging problem for those undertaking the complete functional analysis of genes that constitute prokaryotic genomes. A survey of known and potential peptide-encoding reading frames is presented herein as an attempt to classify those that are found in the B. subtilis genome according to function inferred from homology searches and to conservation among products of other microbial genomes.

Quorum sensing enables unicellular organisms to behave in a multicellular way by allowing population-wide synchronized adaptive responses that involve modulation of a wide range of physiological responses in a cell density-, cell proximity- or growth phase-dependent manner. Examples of processes modulated by quorum sensing are the development of genetic competence, conjugative plasmid transfer, sporulation and cell differentiation, biofilm formation, virulence response, production of antibiotics, antimicrobial peptides and toxins, and bioluminescence (for reviews see ). The cell-to-cell communication strategies involved in these processes are based on the utilization of small signal molecules produced and released into the environment by the microorganisms. These communication molecules are referred to as pheromones and act as chemical messengers that transmit information across space. The extracellular pheromones accumulate in the environment and trigger a response in the target cells when its concentration reaches a certain threshold value. Elucidation of the chemical nature of the pheromones modulating the processes mentioned above reveals that most of them are unmodified peptides, post-translationally modified peptides, N-acyl homoserine lactones, or butyrolactones. Lactone-based pheromones are the preferred communication signals in Gram-negative bacteria (for review see ), whereas peptide-based pheromones are the predominant extracellular signals among Gram-positive bacteria (for review see ). However, lactone-based pheromones are utilized as signals that modulate differentiation and secondary metabolism production in Streptomyces (for review see ).This review focuses on the major advances and current views of the peptide-pheromone dependent regulatory circuits involved in production of antimicrobial peptides in Gram-positive bacteria.
Keywords: Antimicrobial peptide; Signal transduction; Peptide pheromone; Regulation; Quorum sensing;

A two-component signal-transduction cascade in Carnobacterium piscicola LV17B: two signaling peptides and one sensor-transmitter by Michiel Kleerebezem; Oscar P. Kuipers; Willem M. de Vos; Michael E. Stiles; Luis E.N. Quadri (1597-1601).
In the lactic acid bacterium Carnobacterium piscicola LV17B a peptide-pheromone dependent quorum-sensing mode is involved in the regulation of bacteriocin production. Bacteriocin CB2 was identified as an environmental signal that induces bacteriocin production. Here, we demonstrate that a second 24 amino acid peptide (CS) also induces bacteriocin production. Transcription activation of several carnobacteriocin operons is triggered by CB2 or CS via a two-component signal transduction system composed of CbnK and CbnR.
Keywords: Bacteriocin; Signal transduction; Peptide pheromone; Regulation; Quorum sensing;

The accessory gene regulator (agr) system of staphylococci regulates the expression of virulence factors in response to cell density. The extracellular signaling molecule encoded by this system is a thiolactone-containing pheromone peptide whose primary sequence varies among staphylococcal strains. A post-translational modification of the peptide is believed to be carried out by an enzyme with a novel function, AgrB. Staphylococcal pheromones show cross-inhibiting properties: Pheromones of self and pheromones of non-self induce and suppress the agr response, respectively, and have therefore been proposed as novel anti-staphylococcal drugs. As inhibition of agr leads to diminished expression of toxins, but to increased expression of colonization factors and biofilm formation, their therapeutic potential remains yet to be evaluated in depth.
Keywords: Staphylococcus; agr; Pheromone;

RNAIII inhibiting peptide (RIP), a global inhibitor of Staphylococcus aureus pathogenesis: structure and function analysis by Yael Gov; Arkady Bitler; Giorgo Dell’Acqua; José V Torres; Naomi Balaban (1609-1620).
Staphylococcus aureus are gram-positive bacteria that can cause serious diseases in humans and animals. S. aureus infections can be prevented by the heptapeptide RNAIII inhibiting peptide (RIP). RIP was originally isolated from culture supernatants of coagulase negative staphylococci presumed to be S. xylosus. The sequence of RIP was identified as YSPXTNF. Native RIP and its synthetic analogue YSPWTNF have been shown to be effective inhibitors of diseases caused by various strains of S. aureus, including, cellulitis, keratitis, septic arthritis, osteomylitis and mastitis. RIP is therefore considered to be a global inhibitor of S. aureus. We show here that: 1) the amide form of RIP (YSPWTNF-NH2) is highly stable and is therefore the one recommended for use. 2) RIP inhibits S. aureus pathogenesis by inhibiting the synthesis of both agr transcripts RNAII and RNAIII. 3) Although RIP inhibits agr, it also reduces bacterial adherence to mammalian cells and to plastic (tested on HEp2 cells and on polystyrene by fluorescence and atomic force microscopy), suggesting that RIP can be used safely as a therapeutic molecule. 4) RIP derivatives were designed and tested for their ability to inhibit RNAIII in vitro and cellulitis in vivo. Not all peptides that inhibited RNAIII also inhibited an infection in vivo, indicating that studies must be carried out in vivo before considering a peptide to be of therapeutic potential. 5) The RIP derivative containing Lysine and Isoleucine at positions 2 and 4, respectively, inhibited S. aureus infections in vivo (tested on cellulitis), suggesting that both RIP YSPWTNF and its derivative YKPITNF are effective inhibitors of infections caused by S. aureus.
Keywords: Staphylococcus aureus; Bacterial virulence; Quorum sensing; Synthetic peptide inhibitors; Atomic force microscopy; Adhesion; RIP; RAP; agr;

RNAIII inhibiting peptide (RIP) inhibits agr-regulated toxin production by Olney Vieira-da-Motta; Patrı́cia Damasceno Ribeiro; Wilmar Dias da Silva; Enrique Medina-Acosta (1621-1627).
Staphylococcal enterotoxins (SEs) are emetic toxins that cause food poisoning. SEs also function as powerful pyrogenic toxin superantigens that stimulate non-specific T-cell proliferation. Together with the hemolysins, SEs have been largely implicated as virulence factors in multiple infection models. Recent biochemical and genetic analyses have demonstrated that production of some of these toxins is partially regulated by quorum sensing mechanisms where proteins and peptides activate the accessory gene regulator (agr). Because toxin production is central to bacterial pathogenesis, therapeutic strategies alternative to antibiotics, and based on rational interference of the quorum sensing systems involved, are currently being developed. This approach would lead to repression of toxin production and, thus, to disease prevention. Here we provide evidence to conclude that synthetic analogs of the RNAIII inhibiting peptide (RIP) and antibodies to its target molecule TRAP function in vitro as efficient suppressors of agr-regulated exotoxin production by Staphylococcus aureus.
Keywords: Enterotoxin; Hemolysin; IgY; Quorum sensing; Staphylococcus;

Living organisms of all types produce a large repertoire of gene-encoded, net positively charged, antimicrobial peptides as part of their innate immunity to microbial invasion. Despite significant variations in composition, length and secondary structure most antimicrobial peptides are active in micromolar concentrations, suggesting a common general mechanism for their mode of action. Many antimicrobial peptides bind bacterial phospholipid membranes up to a threshold concentration, followed by membrane permeation/disintegration (the “carpet” mechanism). Recent data suggest that the details of the permeation pathways may vary for different peptides and are assigned to different modes of action. Accumulating data reveal that the molecular basis for cell selectivity is the ability of peptides to specifically bind the negatively charged bacterial membrane, as well as their oligomeric state in solution and in the membrane. Based on the “carpet” mechanism and the role of the peptide oligomeric state, a novel group of diastereomeric (containing D- and L-amino acids) antimicrobial peptides were developed. These peptides may serve as promising templates for the future designs of antimicrobial peptides.
Keywords: Antibacterial peptides; Diastereomers; Lysis; Lytic peptide; Innate immunity;

Processing site and gene structure for the murine antimicrobial peptide CRAMP by Vasumati K. Pestonjamasp; Kenneth H. Huttner; Richard L. Gallo (1643-1650).
Cathelicidins are a mammalian gene family notable for the presence of an antibiotic peptide encoded at the carboxy-terminal domain of the nascent pre-pro-protein. Following proteolytic release, this peptide has direct antimicrobial activity. To understand the function and regulation of cathelicidin we investigated the peptide processing site and gene structure of the mouse cathelicidin CRAMP. Amino acid sequencing of the purified native 5 kDa peptide identified the functionally critical amino terminal sequence of mature CRAMP. Characterization of the CRAMP gene (Cnlp) showed homology in structure and sequence identity in several potential transcription factors binding sites found in the human cathelicidin LL-37. Overall, CRAMP shows striking similarities with LL-37, making it a useful model for study of human cathelicidin function and regulation.
Keywords: Innate immunity; Neutrophils; Antibiotic; Mouse;

Antimicrobial host defense peptides, such as defensins, protegrins, and platelet microbicidal proteins are deployed by mammalian skin, epithelia, phagocytes, and platelets in response to Staphylococcus aureus infection. In addition, staphylococcal products with similar structures and activities, called bacteriocins, inhibit competing microorganisms. Staphylococci have developed resistance mechanisms, which are either highly specific for certain host defense peptides or bacteriocins or which broadly protect against a range of cationic antimicrobial peptides. Experimental infection models can be used to study the molecular mechanisms of antimicrobial peptides, the peptide resistance strategies of S. aureus, and the therapeutic potential of peptides in staphylococcal diseases.
Keywords: Staphylococcus aureus; Host defense peptides; Bacteriocins; Innate immunity; Animal studies; Defensins; Protegrins; Platelet microbicidal proteins; Thrombocidins; Lantibiotics; Teichoic acids; ABC transporters; Secondary transporters; Immunomodulation;

Short peptides conferring resistance to macrolide antibiotics by Tanel Tenson; Alexander S Mankin (1661-1668).
Translation of specific short peptides can render the ribosome resistant to macrolide antibiotics such as erythromycin. Peptides act in cis upon the ribosome on which they have been translated. Amino acid sequence and size are critical for peptide activity. Pentapeptides with different consensus sequences confer resistance to structurally different macrolide antibiotics, suggesting direct interaction between the peptide and the drug on the ribosome. Translation of resistance peptides may result in expulsion of the macrolide antibiotics from the ribosome. The consensus sequence of peptides conferring erythromycin resistance is similar to the sequence of the leader peptide involved in translational attenuation of erythromycin resistance genes, indicating that a similar type of interaction between the nascent peptide and antibiotics can occur in both cases.

The effect of charge increase on the specificity and activity of a short antimicrobial peptide by Sung Yu Hong; Tae Gwan Park; Keun-Hyeung Lee (1669-1674).
By using short linear antimicrobial peptides as a model system, the effect of peptide charge on the specificity between Candida albicans (fungi) and Gram-positive bacteria was investigated. In a present study, we added and/or deleted lysine residue(s) at the C-terminal and/or N-terminal end(s) of an antimicrobial peptide (KKVVFKVKFK-NH2) and synthesized the peptides that had similar α helical structures in a lipid membrane mimic condition. The increase of peptide charge improved antifungal activity without the change of antibacterial activity. Structure-activity relationship study about the peptides revealed that the net positive charge must play an important role in the specificity between C. albicans and Gram-positive bacteria and the increase of the net positive charge without the moderate change of secondary structure could improve activity for C. albicans rather than Gram-positive bacteria.
Keywords: Antibacterial peptide; α-helical structure; Hydrophobicity; Net positive charge;

N-terminal modifications of Polymyxin B nonapeptide and their effect on antibacterial activity by Haim Tsubery; Itzhak Ofek; Sofia Cohen; Mati Fridkin (1675-1681).
Polymyxin B (PMB) is a potent antibacterial lipopeptide composed of a positively charged cyclic peptide ring and a fatty acid containing tail. Polymyxin B nonapeptide (PMBN), the deacylated amino derivative of polymyxin B, is much less bactericidal but able to permeabilize the outer membrane of Gram-negative bacteria and to neutralize the toxic effects of lipopolysaccharide (LPS). In this study, we synthesized and evaluated the antibacterial and LPS neutralizing activities of four PMBN analogs modified at their N-terminal. Our results suggest that oligoalanyl substitutions of PMBN do not effect most of PMBN activities. However, a hydrophobic aromatic substitution generated a PMB-like molecule with high antibacterial activity and significant reduced toxicity.
Keywords: Antibacterial peptide; Lipopolysaccharide; Polymyxin B; Polymyxin B nonapeptide;

Affinity driven molecular transfer from erythrocyte membrane to target cells by Rina Feder; Rachel Nehushtai; Amram Mor (1683-1690).
A wide variety of antimicrobial peptides are known to bind to - and disrupt microbial plasma membranes. Recently, derivatives of the antimicrobial peptide dermaseptin S4 were shown to selectively disrupt the plasma membrane of the intracellular parasite Plasmodium falciparum without harming that of the mammalian host cell. The resulting antimalarial activity is allegedly exerted after the harmless peptide binding to the membrane of the host cell, followed by peptide translocation across a number of intracellular membrane systems and interaction with that of the intraerythrocyte parasite. In this study, we present evidence in support of the ability of a membrane-bound peptide, the dermaseptin S4 derivative K4-S4(1–13)a, to transfer from red blood cells (RBCs) to another distant membrane. Binding of K4-S4(1–13)a to the plasma membrane of RBCs was assessed in vitro and in vivo, and found to be rapid, spontaneous and receptor independent, as was the transfer of the RBC-bound peptide to the plasma membrane of microorganisms. The present study further provides a basis for the possible use of RBCs as a transport vehicle to deliver drugs to distant targets. This drug delivery system involves the transient “loading” of RBCs with a lipophilic “hook” peptide. Such a peptide has enough affinity for the RBC’s plasma membrane to bind to the membrane, but given the opportunity, the peptide will exit its position and transfer to another (target) cell for which it has a greater affinity. The efficacy of such an affinity driven transfer system was demonstrated experimentally by the transfer of K4-S4(1–13)a from pre-loaded RBCs to bacteria, yeast and protozoan target cells.