Marco Scocchi

Role of the Escherichia coli SbmA in the antimicrobial activity of proline‐rich peptides

In contrast to many antimicrobial peptides, members of the proline‐rich group of antimicrobial peptides inactivate Gram‐negative bacteria by a non‐lytic mechanism. Several lines of evidence indicate that they are internalized into bacteria and their activity mediated by interaction with unknown cellular components. With the aim of identifying such interactors, we selected mutagenized Escherichia coli clones resistant to the proline‐rich Bac7(1–35) peptide and analysed genes responsible for conferring resistance, whose products may thus be involved in the peptides mode of action. We isolated a number of genomic regions bearing such genes, and one in particular coding for SbmA, an inner membrane protein predicted to be part of an ABC transporter. An E. coli strain carrying a point mutation in sbmA, as well as other sbmA‐null mutants, in fact showed resistance to several proline‐rich peptides but not to representative membranolytic peptides. Use of fluorescently labelled Bac7(1–35) confirmed that resistance correlated with a decreased ability to internalize the peptide, suggesting that a bacterial protein, SbmA, is necessary for the transport of, and for susceptibility to, proline‐rich antimicrobial peptides of eukaryotic origin.

Structure and Biology of Cathelicidins

Cathelicidins are a recently discovered family of effector molecules in innate immunity. In the past few years, a great deal of investigations have elucidated several aspects of their biology, such as the gene structure and activation mechanism. Despite substantial progress in the field, several issues remain to be clarified, including the biological role of the conserved proregion and the molecular mechanisms responsible for diversification of the peptide domain. The cathelicidin-derived peptides have been deeply investigated with respect to structure, spectrum of activity and mechanism of action. In general, they show a potent in vitro activity against antibiotic-resistant microorganisms. The widespread diffusion of multi-resistant strains has highlighted their potential as lead compounds for the development of novel antiinfective agents. Indeed, some of these peptides, or analogs, are already under advanced clinical trials for the treatment of topical infections. Finally, several reports suggesting that cathelicidin peptides may play additional roles in host defense, such as wound healing and chemotactic activity, have opened new fields of investigations. Further studies however are required to clearly establish the physiological relevance of the observed effects.

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

Structural organization of the bovine cathelicidin gene family and identification of a novel member1

Cathelicidins are a group of myeloid antimicrobial peptide precursors found in a variety of mammalian species. Transcripts of this family show a highly conserved 5′ region corresponding to the 5′ untranslated region, signal peptide and propiece, and diverse 3′ regions encoding structurally varied C‐terminal sequences that correspond to mature antimicrobial peptides after proteolytic release from the precursors. To establish the size of the bovine gene family encoding these proteins, λ genomic clones were isolated by screening a bovine library with a probe based on the conserved cDNA region of bovine members. Restriction mapping, hybridization studies and sequence analysis identified 11 distinct genes that based on the intergenic distances of contiguous genes appear to be in close physical proximity. Among these, a novel gene encoding the precursor of a putative α‐helical antimicrobial peptide was recognized and sequenced. The novel gene appears to be expressed in bovine bone marrow myeloid cells, spleen and testis.

Chemical synthesis and biological activity of a novel antibacterial peptide deduced from a pig myeloid cDNA

Several myeloid precursors of antibacterial peptides have recently been shown to share homologous pre‐ and pro‐regions. Taking advantage of this homology, a novel cDNA was cloned from pig bone marrow RNA. This encodes a 166‐residue polypeptide with highly conserved pre‐ (29 residues) and pro‐ (101 residues) sequences, followed by a unique, 36‐residue C‐terminal sequence. Structure analyses of this C‐terminal region have identified a highly cationic sequence predicted to adopt an amphipathic α‐helical conformation. A peptide corresponding to this sequence was chemically synthesized and shown to arrest the growth of both Gram‐positive and Gram‐negative bacteria. At least for Escherichia coli, the activity of this peptide appears to be mediated by its ability to permeabilize the bacterial membranes.

Identification and characterization of a primary antibacterial domain in CAP18, a lipopolysaccharide binding protein from rabbit leukocytes

Secondary structure prediction studies on CAP18, a lipopolysaccharide binding protein from rabbit granulocytes, identified a highly cationic, 21‐residue sequence with the tendency to adopt an amphipathic α‐helical conformation, as observed in many antimicrobial peptides. The corresponding peptide was chemically synthesized and shown to exert a potent bacterieidal activity against both Gram‐negative and Gram‐positive bacteria, and a rapid permeabilization of the inner membrane of Escherichia coli. Five analogues were synthesized to elucidate structure/activity relationships. It was found that helix disruption virtually eliminates antibacterial activity, while the degree of amphipathicity and the presence of an aromatic residue greatly affect the kinetics of bacterial inner membrane permeabilization.

Antimicrobial activity of Bac7 fragments against drug-resistant clinical isolates

Ten peptides from 13 to 35 residues in length and covering the whole sequence of the Pro-rich peptide Bac7 were synthesized to identify the domain responsible for its antimicrobial activity. At least 16 residues of the highly cationic N-terminal sequence were required to maintain the activity against Gram-negative bacteria. The fragments Bac7(1-35) and, to a lesser extent, Bac7(1-16) proved active against a panel of antibiotic-resistant clinical isolates of Gram-negative bacteria, with the notable exception of Burkholderia cepacia. In addition, when tested against fungi, the longer fragment was also active against collection strains and clinical isolates of Cryptococcus neoformans, but not towards clinical isolates of Candida albicans.

Essential Role for the BacA Protein in the Uptake of a Truncated Eukaryotic Peptide in Sinorhizobium meliloti

The inner membrane BacA protein is essential for the establishment of chronic intracellular infections by Sinorhizobium meliloti and Brucella abortus within plant and mammalian hosts, respectively. In their free-living state, S. meliloti and B. abortus mutants lacking BacA have reductions in their outer membrane lipid A very-long-chain fatty acid (VLCFA) contents and exhibit low-level resistance to the glycopeptide bleomycin in comparison to their respective parent strains. In this paper we investigate the hypothesis that BacA is involved in peptide uptake in S. meliloti. We determined that an S. meliloti DeltabacA mutant is completely resistant to a truncated form of the eukaryotic peptide Bac7, Bac7(1-16), and this phenotype appears to be independent of its lipid A alteration. Subsequently, we discovered that BacA and/or Escherichia coli SbmA is essential for fluorescently labeled Bac7(1-16) uptake in S. meliloti. Given that there are hundreds of root nodule-specific peptides within the legume host, our data suggest that BacA-mediated peptide uptake could play a central role in the chronic infection process of S. meliloti. However, since we determined that two symbiotically defective S. meliloti bacA site-directed mutants (with the Q193G and R389G mutations, respectively) with known reductions in their lipid A VLCFA contents are still capable of peptide uptake, these findings suggest that BacA-dependent peptide uptake cannot fully account for the essential role of BacA in the legume symbiosis. Further, they provide evidence that the BacA function that leads to the S. meliloti lipid A VLCFA modification plays a key role in the chronic infection of legumes.

Antibacterial and anti-biofilm effects of cathelicidin peptides against pathogens isolated from cystic fibrosis patients

Six different cathelicidin-derived peptides were compared to tobramycin for antibacterial and anti-biofilm effects against S. aureus, P. aeruginosa, and S. maltophilia strains isolated from cystic fibrosis patients. Overall, SMAP-29, BMAP-28, and BMAP-27 showed relevant antibacterial activity (MIC(50) 4-8μg/ml), and in some cases higher than tobramycin. In contrast, indolicidin, LL-37, and Bac7(1-35) showed no significant antimicrobial activity (MIC(50)>32μg/ml). Killing kinetics experiments showed that in contrast to tobramycin the active cathelicidin peptides exert a rapid bactericidal activity regardless of the species tested. All three peptides significantly reduced biofilm formation by S. maltophilia and P. aeruginosa strains at 1/2× MIC, although at a lower extent than tobramycin. In addition, BMAP-28, as well as tobramycin, was also active against S. aureus biofilm formation. Preformed biofilms were significantly affected by bactericidal SMAP-29, BMAP-27 and BMAP-28 concentrations, although at a lesser extent than tobramycin. Overall, our results indicate the potential of some cathelicidin-derived peptides for the development of novel therapeutic agents for cystic fibrosis lung disease.

Novel cathelicidins in horse leukocytes

Cathelicidins are precursors of defense peptides of the innate immunity and are widespread in mammals. Their structure comprises a conserved prepropiece and an antimicrobial domain that is structurally varied both intra‐ and inter‐species. We investigated the complexity of the cathelicidin family in horse by a reverse transcription‐PCR‐based cloning strategy of myeloid mRNA and by Southern and Western analyses. Three novel cathelicidin sequences were deduced from bone marrow mRNA and designated equine cathelicidins eCATH‐1, eCATH‐2 and eCATH‐3. Putative antimicrobial domains of 26, 27 and 40 residues with no significant sequence homology to other peptides were inferred at the C‐terminus of the sequences. Southern analysis of genomic DNA using a probe based on the cathelicidin‐conserved propiece revealed a polymorphic DNA region with several hybridization‐positive fragments and suggested the presence of additional genes. A null eCATH‐1 allele was also demonstrated with a frequency of 0.71 in the horse population analyzed and low amounts of eCATH‐1‐specific mRNA were found in myeloid cells of gene‐positive animals. A Western analysis using antibodies to synthetic eCATH peptides revealed the presence of eCATH‐2 and eCATH‐3 propeptides, but not of eCATH‐1‐related polypeptides, in horse neutrophil granules and in the secretions of phorbol myristate acetate‐stimulated neutrophils. These results thus suggest that eCATH‐2 and eCATH‐3 are functional genes, whereas eCATH‐1 is unable to encode a polypeptide.