Lipid complexes with cationic peptides and OAKs; their role in antimicrobial action and in the delivery of antimicrobial agents

Antimicrobial agents are toxic to bacteria by a variety of mechanisms. One mechanism that is very dependent on the lipid composition of the bacterial membrane is the clustering of anionic lipid by cationic antimicrobial agents. Certain species of oligo-acyl-lysine (OAK) antimicrobial agents are particularly effective in clustering anionic lipids in mixtures mimicking the composition of bacterial membranes. The clustering of anionic lipids by certain cationic antimicrobial agents contributes to the anti-bacterial action of these agents. Bacterial membrane lipids are a determining factor, resulting in some species of bacteria being more susceptible than others. In addition, lipids can be used to increase the effectiveness of antimicrobial agents when administered in vivo. Therefore, we review some of the structures in which lipid mixtures can assemble, to more effectively be utilized as antimicrobial delivery systems. We describe in more detail the complexes formed between mixtures of lipids mimicking bacterial membranes and an OAK and their usefulness in synergizing with antibiotics to overcome bacterial multidrug resistance.     

The Sushi peptides: structural characterization and mode of action against Gram-negative bacteria

The compositional difference in microbial and human cell membranes allows antimicrobial peptides to preferentially bind microbes. Peptides which specifically target lipopolysaccharide (LPS) and palmitoyl-oleoyl-phosphatidylglycerol (POPG) are efficient antibiotics. From the core LPS-binding region of Factor C, two 34-mer Sushi peptides, S1 and S3, were derived. S1 functions as a monomer, while S3 is active as a dimer. Both S1 and S3 display detergent-like properties in disrupting LPS aggregates, with specificity for POPG resulting from electrostatic and hydrophobic forces between the peptides and the bacterial lipids. During interaction with POPG, the S1 transitioned from a random coil to an α-helix, while S3 resumed a mixture of α-helix and β-sheet structures. The unsaturated nature of POPG confers fluidity and enhances insertion of the peptides into the lipid bilayer, causing maximal disruption of the bacterial membrane. These parameters should be considered in designing and developing new generations of peptide antibiotics with LPS-neutralizing capability. Received 2 October 2007; received after revision 2 November 2007; accepted 4 December 2007 J. L. Ding, B. Ho: Co-senior authors.     

Multifunctional cationic host defence peptides and their clinical applications

With the rapid rise in the emergence of bacterial strains resistant to multiple classes of antimicrobial agents, there is an urgent need to develop novel antimicrobial therapies to combat these pathogens. Cationic host defence peptides (HDPs) and synthetic derivatives termed innate defence regulators (IDRs) represent a promising alternative approach in the treatment of microbial-related diseases. Cationic HDPs (also termed antimicrobial peptides) have emerged from their origins as nature’s antibiotics and are widely distributed in organisms from insects to plants to mammals and non-mammalian vertebrates. Although their original and primary function was proposed to be direct antimicrobial activity against bacteria, fungi, parasites and/or viruses, cationic HDPs are becoming increasingly recognized as multifunctional mediators, with both antimicrobial activity and diverse immunomodulatory properties. Here we provide an overview of the antimicrobial and immunomodulatory activities of cationic HDPs, and discuss their potential application as beneficial therapeutics in overcoming infectious diseases.     

β-Lactam resistance in Staphylococcus aureus: the adaptive resistance of a plastic genome

Staphylococci have two mechanisms for resistance to β-lactam antibiotics. One is the production of β-lactamases, enzymes that hydrolytically destroy β-lactams. The other is the expression of penicillin-binding protein 2a (PBP 2a), which is not susceptible to inhibition by β-lactam antibiotics. Strains of S. aureus exhibiting either β-lactamase or PBP 2a-directed resistance (or both) have established a considerable ecological niche among human pathogens. The emergence and subsequent spread of bacterial strains designated as methicillin-resistant S. aureus (MRSA), from the 1960s to the present, has created clinical difficulties for nosocomial treatment on a global scale. The recent variants of MRSA that are resistant to glycopeptide antibiotics (such as vancomycin) have ushered in a new and disconcerting chapter in the evolution of this organism. Received 2 April 2005; received after revision 15 July 2005; accepted 25 July 2005     

Autonomous translocation and intracellular trafficking of the cell-penetrating and immune-suppressive effector protein YopM

Extracellular Gram-negative pathogenic bacteria target essential cytoplasmic processes of eukaryotic cells by using effector protein delivery systems such as the type III secretion system (T3SS). These secretion systems directly inject effector proteins into the host cell cytoplasm. Among the T3SS-dependent Yop proteins of pathogenic Yersinia, the function of the effector protein YopM remains enigmatic. In a recent study, we demonstrated that recombinant YopM from Yersinia enterocolitica enters host cells autonomously without the presence of bacteria and thus identified YopM as a novel bacterial cell-penetrating protein. Following entry YopM down-regulates expression of pro-inflammatory cytokines such as tumor necrosis factor α. These properties earmark YopM for further development as a novel anti-inflammatory therapeutic. To elucidate the uptake and intracellular targeting mechanisms of this bacterial cell-penetrating protein, we analyzed possible routes of internalization employing ultra-cryo electron microscopy. Our results reveal that under physiological conditions, YopM enters cells predominantly by exploiting endocytic pathways. Interestingly, YopM was detected free in the cytosol and inside the nucleus. We could not observe any colocalization of YopM with secretory membranes, which excludes retrograde transport as the mechanism for cytosolic release. However, our findings indicate that direct membrane penetration and/or an endosomal escape of YopM contribute to the cytosolic and nuclear localization of the protein. Surprisingly, even when endocytosis is blocked, YopM was found to be associated with endosomes. This suggests an intracellular endosome-associated transport of YopM.     

Mobile genetic elements of Staphylococcus aureus

Bacteria such as Staphylococcus aureus are successful as commensal organisms or pathogens in part because they adapt rapidly to selective pressures imparted by the human host. Mobile genetic elements (MGEs) play a central role in this adaptation process and are a means to transfer genetic information (DNA) among and within bacterial species. Importantly, MGEs encode putative virulence factors and molecules that confer resistance to antibiotics, including the gene that confers resistance to beta-lactam antibiotics in methicillin-resistant S. aureus (MRSA). Inasmuch as MRSA infections are a significant problem worldwide and continue to emerge in epidemic waves, there has been significant effort to improve diagnostic assays and to develop new antimicrobial agents for treatment of disease. Our understanding of S. aureus MGEs and the molecules they encode has played an important role toward these ends and has provided detailed insight into the evolution of antimicrobial resistance mechanisms and virulence.     

Ornithine decarboxylase, S-adenosyl-L-methionine decarboxylase and arginine decarboxylase from Mycobacterium bovis (BCG)

Ornithine decarboxylase (ODC), S-adenosyl-L-methionine decarboxylase (AMDC) and arginine decarboxylase (ADC) activities were detected for the first time in extracts of Mycobacterium bovis (BCG). All the decarboxylases differed from corresponding known bacterial decarboxylases in that: a) ODC did not require GTP for activity; b) ODC was not inhibited by any known inhibitor of bacterial ODCs; c) AMDC and ADC did not require Mg2+-ion for activity and were not markedly inhibited by any known inhibitor of the decarboxylases of other bacteria.     

Ornithine decarboxylase,S-adenosyl-L-methionine decarboxylase and arginine decarboxylase fromMycobacterium bovis (BCG)

Summary Ornithine decarboxylase (ODC), S-adenosyl-L-methionine decarboxylase (AMDC) and arginine decarboxylase (ADC) activities were detected for the first time in extracts ofMycobacterium bovis (BCG). All the decarboxylases differed from corresponding known bacterial decarboxylases in that: a) ODC did not require GTP for activity; b) ODC was not inhibited by any known inhibitor of bacterial ODCs; c) AMDC and ADC did not require Mg²⁺-ion for activity and were not markedly inhibited by any known inhibitor of the decarboxylases of other bacteria.     

Flagellin glycosylation with pseudaminic acid in <Emphasis Type="Italic">Campylobacter</Emphasis> and <Emphasis Type="Italic">Helicobacter</Emphasis>: prospects for development of novel therapeutics

Many pathogenic bacteria require flagella-mediated motility to colonise and persist in their hosts. Helicobacter pylori and Campylobacter jejuni are flagellated epsilonproteobacteria associated with several human pathologies, including gastritis, acute diarrhea, gastric carcinoma and neurological disorders. In both species, glycosylation of flagellin with an unusual sugar pseudaminic acid (Pse) plays a crucial role in the biosynthesis of functional flagella, and thereby in bacterial motility and pathogenesis. Pse is found only in pathogenic bacteria. Its biosynthesis via six consecutive enzymatic steps has been extensively studied in H. pylori and C. jejuni. This review highlights the importance of flagella glycosylation and details structural insights into the enzymes in the Pse pathway obtained via a combination of biochemical, crystallographic, and mutagenesis studies of the enzyme–substrate and –inhibitor complexes. It is anticipated that understanding the underlying structural and molecular basis of the catalytic mechanisms of the Pse-synthesising enzymes will pave the way for the development of novel antimicrobials.     

Two classes of metabolites fromTheonella swinhoei are localized in distinct populations of bacterial symbionts

The marine spongeTheonella swinhoei (lithistid Family Theonellidae, Order Astrophorida) has yielded many important, bioactive natural products, most of which share structural features with bacterial natural products. The presence of microbial symbionts inT. swinhoei has been reported, and it was originally suggested that the cytotoxic macrolide swinholide A and many of the bioactive cyclic peptides fromT. swinhoei were all produced by symbiotic cyanobacteria. By transmission electron microscopy, we found four distinct cell populations to be consistently present inT. swinhoei: eukaryotic sponge cells, unicellular heterotrophic bacteria, unicellular cyanobacteria and filamentous heterotrophic bacteria. Purification and chemical analyses of each cell type showed the macrolide swinholide A to be limited to the mixed population of unicellular heterotrophic bacteria, and an anti-fungal cyclic peptide occurred only in the filamentous heterotrophic bacteria. Contrary to prior speculation, no major metabolites were located in the cyanobacteria or sponge cells.     

The short proline-rich antibacterial peptide family

From the many peptide families that are induced upon bacterial infection and can be isolated from all classes of animals, the short, proline-rich antibacterial peptides enjoy particular interest. These molecules were shown to inactivate an intracellular biopolymer in bacteria without destroying or remaining attached to the bacterial cell membrane, and as such emerged as viable candidates for the treatment of mammalian infections. These peptides were originally isolated from insects, they kill mostly Gram-negative bacteria with high efficiency and they show structural similarities with longer insect- and mammal-derived antimicrobial peptides. However, while the distant relatives appear to carry multiple functional domains, apidaecin, drosocin, formaecin and pyrrhocoricin consist of only minimal determinants needed to penetrate across the cell membrane and bind to the target biopolymer. These peptides appear to inhibit metabolic processes, such as protein synthesis or chaperone-assisted protein folding. Pyrrhocoricin derivatives protect mice from experimental infections in vivo, suggesting the utility of modified analogs in the clinical setting. Sequence variations of the target protein at the peptide-binding site may allow the development of new peptide variants that kill currently unresponsive strains or species. Received 12 December 2001; received after revision 11 February 2002; accepted 19 February 2002     

Evolution of bacterial pathogenesis

The evolution of bacteria is associated with continuous generation of novel genetic variants. The major driving forces in this process are point mutations, genetic rearrangements, and horizontal gene transfer. A large number of human and animal bacterial pathogens have evolved the capacity to produce virulence factors that are directly involved in infection and disease. Additionally, many bacteria express resistance traits against antibiotics. Both virulence factors and resistance determinants are subject to intrastrain genetic and phenotypic variation. They are often encoded on unstable DNA regions. Thus, they can be readily transferred to bacteria of the same species or even to non-related prokaryotes. This review article focuses on the main mechanisms of bacterial microevolution responsible for the rapid emergence of variants with novel virulence and resistance properties. In addition, processes of macroevolution are described with special emphasis on gene transfer and fixation of adaptive mutations in the genome of pathogens.     

Lactoferrin

The first function attributed to lactoferrin (Lf), an iron binding protein belonging to the non-immune natural defences, was antimicrobial activity that depended on its capacity to sequester iron. Iron-independent microbicidal activities, requiring direct interaction between this cationic protein and microbial surface components, were later demonstrated. Many other anti-microbial and anti-viral functions have since been ascribed to Lf. In mucosal secretions, iron and Lf modulate the motility and aggregation of pathogenic bacteria. Lf inhibits bacterial adhesion on abiotic surfaces through ionic binding to biomaterials, or specific binding to bacterial structures or both. Lf inhibition of bacterial adhesion to host cells requires Lf binding to bacteria and/or host cells. Lf hinders microbial internalization by binding to both glycosaminoglycans and bacterial proteins which can be degraded by Lf-mediated proteolysis. Moreover, Lf internalisation and localisation to the host cell nuclei could modulate bacterial entry into cells through gene regulation. Finally, the capability of Lf to exert antiviral activity, through its binding to host cells and/or viral particles, strengthens the idea that it is an important brick in the mucosal wall, effective against both microbial and viral attacks.     

Proline-rich antimicrobial peptides: converging to a non-lytic mechanism of action

Proline-rich antimicrobial peptides are a group of cationic host defense peptides of vertebrates and invertebrates characterized by a high content of proline residues, often associated with arginine residues in repeated motifs. Those isolated from some mammalian and insect species, although not evolutionarily related, use a similar mechanism to selectively kill Gram-negative bacteria, with a low toxicity to animals. Unlike other types of antimicrobial peptides, their mode of action does not involve the lysis of bacterial membranes but entails penetration into susceptible cells, where they then act intracellularly. Some aspects of the transport system and cytoplasmic targets have been elucidated. These features make them attractive both as anti-infective lead compounds and as a new class of potential cell-penetrating peptides capable of internalising membrane-impermeant drugs into both bacterial and eukaryotic cells     

Bacterial antibiotic efflux systems of medical importance

Multidrug efflux systems endow on bacterial cells the ability to limit the access of antimicrobial agents to their targets. By actively pumping out antibiotic molecules, these systems prevent the intracellular accumulation necessary for antibiotics to exert their lethal activity. Drug efflux appears to be one of the most widespread antibiotic resistance mechanisms among microorganisms, since it has been demonstrated to occur in many Gram-positive and Gram-negative bacteria including medically important species like staphylococci, streptococci, enterobacteria and opportunistic pathogens like Pseudomonas aeruginosa. Efflux pumps can be specific for only one substrate or accommodate a more or less wide range of noxious products. Export of structurally unrelated compounds confers a multidrug-resistance phenotype on bacterial cells. Therapeutically critical levels of resistance can be achieved by overexpression of efflux systems, especially in those species such as P. aeruginosa which possess a low outer membrane permeability. It is suspected that the dual physiological function of active efflux systems is both the secretion of intracellular metabolites and the protection against a variety of harmful substances that the microorganism may encounter in its natural environment.     

Timing is everything: the regulation of type III secretion

Type Three Secretion Systems (T3SSs) are essential virulence determinants of many Gram-negative bacteria. The T3SS is an injection device that can transfer bacterial virulence proteins directly into host cells. The apparatus is made up of a basal body that spans both bacterial membranes and an extracellular needle that possesses a channel that is thought to act as a conduit for protein secretion. Contact with a host-cell membrane triggers the insertion of a pore into the target membrane, and effectors are translocated through this pore into the host cell. To assemble a functional T3SS, specific substrates must be targeted to the apparatus in the correct order. Recently, there have been many developments in our structural and functional understanding of the proteins involved in the regulation of secretion. Here we review the current understanding of protein components of the system thought to be involved in switching between different stages of secretion.     

Antifungal proteins: targets,mechanisms and prospective applications

All organisms have evolved several defence systems in order to protect themselves against bacteria, fungi and viruses. Higher organisms have developed a complex network of humoral and cellular responses, called adaptive immunity. A second defence system, innate immunity, was discovered in the early 1980s, consisting of small cationic peptides with a broad antimicrobial spectrum. These proteins act immediately at sites of infection or inflammation. The production of proteins with antimicrobial activity was not limited to higher organisms but was also found in insects, plants and microorganisms. During the last 2decades a broad range of proteins with very different structural features have been isolated and characterised from differing organisms ranging from bacteria to human beings. Over 500cationic membrane-acting proteins with antimicrobial and antifungal activities have been identified to date. Apart from these proteins, a very large number of antifungal proteins active on the fungal cell wall, on enzymes of the cell wall synthesis machinery, the plasma membrane and on intracellular targets have been characterised.Received 17 June 2003; received after revision 4 August 2003; accepted 18 August 2003     

Ammonia and amino acid transport across symbiotic membranes in nitrogen-fixing legume nodules

Biological nitrogen fixation involves the reduction of atmospheric N₂ to ammonia by the bacterial enzyme nitrogenase. In legume-rhizobium symbioses, the nitrogenase-producing bacteria (bacteroids) are contained in the infected cells of root nodules within which they are enclosed by a plant membrane to form a structure known as the symbiosome. The plant provides reduced carbon to the bacteroids in exchange for fixed nitrogen, which is exported to the rest of the plant. This exchange is controlled by plant-synthesised transport proteins on the symbiosome membranes. This review summarises our current understanding of these transport processes, focusing on ammonia and amino acid transport. Received 5 June 2000; revised 13 July 2000; accepted 14 July 2000     

Stimulation of pyruvate carboxylation by gastric secretagogues

Summary Pyruvate carboxylation was stimulated by 2 gastric secretagogues, histamine and dibutyryl cyclic AMP, and by butyrate. Thiocyanate, an inhibitor of acid secretion, produced a slight decrease. Avidin significantly reduced acid secretion and this effect was overcome by biotin and oxalacetate. The results suggest that carboxylation of pyruvate is one of the reactions controlling oxidative metabolism and acid secretion in toad gastric mucosa.This investigation was supported by Consejo de Desarrollo Científico y Humanístico de la Universidad del Zulia, and by CONICIT Grant S1-0455.