Abstract: 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 peptide's 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.
Abstract: AIM: The aims of this prospective study were: 1) to assess the frequency, type and severity of comorbidities (COMs) and complications (COMPLs) in acute stroke patients, according to the weighted comorbidity index (w-CI) of Liu et al. and 2 new indices, respectively COM severity index (COM-SI) and COMPL severity index (COMPL-SI); 2) to separately analyse the interference of COMs and COMPLs with functional status and recovery during stroke rehabilitation treatment; 3) to compare the ability of COM-SI and COMPL-SI to predict functional independence at discharge with that of w-CI. METHODS: Eighty-five stroke rehabilitation inpatients participated in the study. The type, incidence and severity of COM at admission and of COMPL during the whole hospital stay were studied prospectively. The Functional Independence Measure (FIM) scale was administered at both admission and discharge. RESULTS: About 1/3 suffered from some significant COM and another 1/3 developed COMPLs needing specific medical treatment and/or clinical monitoring. The most frequent COMs and COMPLs were cardiovascular and psychiatric/psychological diseases. The odds of having a high efficiency in the daily functional gain (FIM score) were greater for patients without any COM (3.5) and/or COMPL (4.6). Similarly, the odds of having a high FIM score at discharge were greater (3.5) for patients without COM or COMPL. The COM-SI demonstrated a higher predictive capacity of the FIM score at discharge (5%) than w-CI (4%), and COMPL-SI (1%). CONCLUSIONS: COM-SI resulted as the most interesting predictive index of functional outcome at discharge, after accounting for the functional status at admission.
Abstract: OBJECTIVES: To investigate the in vitro antifungal activity of the structurally different cathelicidin peptides SMAP-29, BMAP-27, BMAP-28, protegrin-1 (PG-1) and indolicidin. METHODS: The in vitro antifungal and fungicidal activities of these antimicrobial peptides were respectively assessed via MIC determinations and killing kinetics assays. The effects of the peptides on membrane permeabilization and morphology were evaluated by flow cytometry, intracellular ATP release measurements and scanning electron microscopy. RESULTS: All five peptides showed a potent but differential antifungal activity against more than 70 clinical isolates belonging to over 20 different species of pathogenic fungi; some of which are resistant to amphotericin B and azoles. The MIC values of the peptides ranged between 0.5 and 32 microM, with PG-1 being the most effective and having the widest spectrum of activity. Filamentous fungi were instead found to be scarcely susceptible to the action of these cathelicidin peptides. All these cathelicidins rapidly killed Candida albicans and Cryptococcus neoformans cells in a dose- and time-dependent manner. The rapid uptake of propidium iodide into treated cells and morphological alterations apparent on their cellular surfaces suggest a killing mechanism based on membrane permeabilization and damage. CONCLUSIONS: This study indicates that these five structurally varied host defence peptides are all endowed with the capacity to inactivate a number of fungal pathogens, irrespectively of their resistance to antifungal drugs, and suggests they might be potentially useful leads for the development of novel fungicidal agents.
Abstract: Proline-rich peptides are a unique group of antimicrobial peptides that exert their activity selectively against Gram-negative bacteria through an apparently non-membranolytic mode of action that is not yet well understood. We have investigated the mechanism underlying the antibacterial activity of the proline-rich cathelicidin Bac7 against Salmonella enterica and Escherichia coli. The killing and membrane permeabilization kinetics as well as the cellular localization were assessed for the fully active N-terminal fragment Bac7(1-35), its all-D enantiomer and for differentially active shortened fragments. At sub-micromolar concentrations, Bac7(1-35) rapidly killed bacteria by a non-lytic, energy-dependent mechanism, whereas its D-enantiomer was inactive. Furthermore, while the L-enantiomer was rapidly internalized into bacterial cells, the D-enantiomer was virtually excluded. At higher concentrations (>or=64 microM), both L- and D-Bac7(1-35) were instead able to kill bacteria also via a lytic mechanism. Overall, these results suggest that Bac7 may inactivate bacteria via two different modes of action depending on its concentration: (i) at near-MIC concentrations via a mechanism based on a stereospecificity-dependent uptake that is likely followed by its binding to an intracellular target, and (ii) at concentrations several times the MIC value, via a non-stereoselective, membranolytic mechanism.
Abstract: PMAP-36 is a cathelicidin-derived host defence peptide originally deduced by a transcript from pig bone marrow RNA. The expression of the propeptide in leukocytes, and the structure, antimicrobial activity, and mechanism of action of the mature peptide were investigated. The proform is stored as a dimeric precursor of 38 kDa formed by a dimerization site at its C-terminal cysteine residue; it is likely that the mature peptide is dimeric when released. Monomeric and dimeric forms of PMAP-36 were chemically synthesized and their activity compared. Both forms assumed an amphipathic alpha-helical conformation and exhibited a potent and rapid microbicidal activity against a wide spectrum of microorganisms, mediated by their ability to permeabilize the microbial membranes rapidly. A shortened fragment localized the helical region to the N terminus, but showed a significantly lower potency and slower permeabilization kinetics, indicating an important role of the nonhelical C-terminal hydrophobic portion of this molecule. Dimerization modulated the effectiveness of the peptide in terms of killing and permeabilization kinetics, and reduced medium dependence. It allows the molecule to achieve an impressive charge density (+28 in 70 residues), although the significance of this feature with respect to biological activity has yet to be determined.
Abstract: OBJECTIVE: To determine levels of plasma amino acid tyrosine and tryptophan, precursors of brain catecholamine and serotonin neurotransmitters, respectively, in rehabilitative patients with ischemic stroke. DESIGN: Controlled, pre-post analysis, consecutive sample. SETTING: Rehabilitation center. PARTICIPANTS: Twenty men with ischemic stroke (age, 68+/-11.3y) consecutively admitted into rehabilitation 15+/-10 days (range, 7-28d) after an acute cerebrovascular insult; 15 healthy sedentary subjects (controls 1); and 13 healthy hypoactive individuals who had recently had knee arthroplasty (controls 2). Both control groups were matched to stroke subjects for age, gender, and body weight. INTERVENTIONS: At 8:00 am, after overnight fasting, venous blood samples were drawn from patients to determine plasma tyrosine and tryptophan levels. A nutritional evaluation, including nitrogen balance, was made. The same procedures were repeated after 45 days of rehabilitation. Amino acid data were compared with those obtained from the controls. MAIN OUTCOME MEASURE: Plasma concentrations of amino acids. RESULTS: Patients with ischemic stroke, on admission, had lower plasma tyrosine concentration than did both controls 1 (P<.0005) and controls 2 (P<.001), but a similar tryptophan level. The plasma content of tyrosine was similar between the 2 control groups. After 45 days of rehabilitation, the stroke patients' tyrosine and tryptophan levels remained virtually unchanged, as did nutritional parameters. Nutritional intakes were adequate to meet body needs but insufficient to correct plasma tyrosine. CONCLUSIONS: Patients experiencing a recent stroke may have low plasma tyrosine levels and, therefore, reduced brain catecholamine formation. It is possible that an imbalance of brain neurotransmitters may occur.
Abstract: Most antimicrobial peptides (AMPs) impair the viability of target bacteria by permeabilizing bacterial membranes. However, the proline-rich AMPs have been shown to kill susceptible organisms without causing significant membrane perturbation and may act by inhibiting the activity of bacterial targets. To gain initial insight into the events that follow interaction of a proline-rich peptide with bacterial cells, we used DNA macroarray technology to monitor transcriptional alterations of Escherichia coli in response to challenge with a subinhibitory concentration of the proline-rich Bac7(1-35). Substantial changes in the expression levels of 70 bacterial genes from various functional categories were detected. Among these, 26 genes showed decreased expression, while 44 genes, including genes that are potentially involved in bacterial resistance to antimicrobials, showed increased expression. The generation of a transcriptional response under the experimental conditions used is consistent with the ability of Bac7(1-35) to interact with bacterial components and affect biological processes in this organism.
Abstract: 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.
Abstract: Epithelia- and leukocyte-associated antimicrobial peptides provide immediate protection against microbial infections by rapidly inactivating potential pathogens. Bac5 is a member of the cathelicidin family of antimicrobial peptides and is stored in the cytoplasmic granules of bovine neutrophils. We investigated the expression of this gene in airway and intestine, and although the gene was not found to be locally expressed in these tissues, a strong Bac5 induction signal was detected by in situ hybridization in neutrophils infiltrating infected lung, consistent with expression of this gene in activated neutrophils. The Bac5 gene was also induced in bovine peripheral neutrophils stimulated with Escherichia coli or purified lipopolysaccharide (LPS) but not in other blood cells and in resting neutrophils. The levels of Bac5 mRNA increased at 12-24 h post-stimulation, and a dose-dependent increase in Bac5 expression was determined in the presence of increasing amounts of LPS. A metabolically labeled product with a molecular weight compatible with that of proBac5 was immunoprecipitated from cell-free media of stimulated neutrophils, suggesting that the newly synthesized polypeptide is released extracellularly. Collectively, these results provide the first evidence that fully differentiated neutrophils are capable of de novo synthesis and secretion of a granule-associated antimicrobial peptide.
Abstract: Cathelicidin-derived antimicrobial peptides are a component of the peptide-based host defense of neutrophils and epithelia, with a widespread distribution in mammals. We recently reported the cDNA sequences of three putative horse myeloid cathelicidins, named eCATH-1, -2, and -3. A Western analysis was performed to investigate their presence in neutrophils and processing to mature peptides. eCATH-2 and eCATH-3, but not eCATH-1, were found to be present in uncleaved forms in horse neutrophils. The corresponding mature peptides were detected in inflammatory sites, suggesting that processing of the propeptides takes place upon neutrophil activation. A functional characterization was then performed with synthetic eCATH peptides. Circular dichroism measurements indicated an amphipathic alpha-helical conformation of these peptides in an anisotropic environment, and in vitro assays revealed a potent activity and a broad spectrum of antimicrobial activity for eCATH-1 and a somewhat more restricted spectrum of activity for eCATH-2. Conversely, a strong dependence on salt concentration was observed when the activity of eCATH-3 was tested. This peptide efficiently killed bacteria and some fungal species, i.e., Cryptococcus neoformans and Rhodotorula rubra, in low-ionic-strength media, but the activity was inhibited in the presence of physiological salt medium. This behavior could be modified by modulating the amphipathicity of the molecule. In fact, the synthetic analogue LLK-eCATH-3, with a slightly modified sequence that increases the hydrophobic moment of the peptide, displayed a potent activity in physiological salt medium against the strains resistant to eCATH-3 under these conditions.
Abstract: 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.
Abstract: Bac4 is a bovine cathelicidin gene contiguous to another member of this family named Bac7. Although mutations in the sequence suggested that Bac4 gene might be non-functional, primers based on Bac4 specific sequences allowed amplification of a 900 bp cDNA. The transcript comprises the sequences of exons 1, 2 and 3 of Bac7, and of exons 2, 3 and 4 of Bac4 gene and may result from a weak termination control of the transcription of the upstream Bac7 gene.
Abstract: A putative antimicrobial peptide of 34 residues was recently deduced from a bovine cathelicidin gene sequence and named BMAP-34. A peptide based on the deduced sequence was chemically synthesized and used to study the localization, structure and biological activities of BMAP-34. A Western blot analysis using antibodies raised to the synthetic peptide showed that BMAP-34 is stored as proform in the cytoplasmic granules of bovine neutrophils. CD spectroscopy indicates that the peptide assumes an amphipathic alpha-helical conformation, as also predicted by secondary structure analysis. The peptide exerts a broad spectrum antimicrobial activity against both Gram-negative and Gram-positive organisms, and is not active against eukaryotic cells. When tested on Escherichia coli ML-35, the kinetics of bacterial killing and of inner membrane permeabilization are slower than those observed for other alpha-helical peptides derived from cathelicidins.
Abstract: 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, lambda 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 alpha-helical antimicrobial peptide was recognized and sequenced. The novel gene appears to be expressed in bovine bone marrow myeloid cells, spleen and testis.
Abstract: Six phage clones containing gene members of the family of antimicrobial peptides named cathelicidins, were mapped to bovine chromosome 22q24, by means of fluorescence in situ hybridization. The mapping data suggest the clustering of cathelicidins into a CATHL@ locus, in a similar manner as for beta-defensins, another family of antimicrobial peptides, defining the locus DEFB@ mapped to 27q13-->q14.
Abstract: A molecular biological approach, based on preproregion homology in the precursors of several diverse antibacterial peptides, was used to clone a pig bone marrow cDNA encoding a novel 167-residue polypeptide. The preproregion of this polypeptide is highly similar to corresponding regions in congeners from pig, cattle and rabbit. It is followed by a unique, cationic, 37-residue sequence, which was predicted to have a high propensity for an alpha-helical conformation. A peptide, termed PMAP-37, corresponding to this sequence, was chemically synthesized and shown to undergo a transition from a random coil to an ordered, mainly helical, conformation on addition of trifluoroethanol. This behaviour is typical of an amphipathic alpha helix, a structure common to several membrane-active, antimicrobial peptides. In vitro experiments showed that PMAP-37 strongly inhibits the growth of several strains of Gram-negative and Gram-positive bacteria, with minimal inhibitory concentrations ranging over 1-4 microM, and permeabilizes the inner membrane of Escherichia coli. Interestingly, the 15-32 stretch of PMAP-37 show a remarkable similarity to N-terminal stretches in cecropins B and A from Drosophila melanogaster and Cecropia hyalophora, respectively. This affords an uncommon example of sequence convergence.
Abstract: Several myeloid antimicrobial peptide precursors have been shown to consist of a N-terminal proregion similar to a protein named cathelin and a structurally varied C-terminal antimicrobial domain. Proteins with these features have been named cathelicidins. In this paper we report the cDNA sequences of three ovine cathelicidins of 155, 160 and 190 residues, respectively, with cationic C-terminal sequences corresponding to putative antimicrobial domains. These are structurally varied and include a Cys-rich sequence of 12 residues, which is similar to the bovine antimicrobial cyclic dodecapeptide, a novel 29 residue sequence named SMAP-29 with a possible alpha-helical conformation, and a 60 residue sequence named Bac7.5, which appears to be a new member of the Pro- and Arg-rich group of mammalian antimicrobial peptides.
Abstract: 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 alpha-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.
Abstract: 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 alpha-helical conformation, as observed in many antimicrobial peptides. The corresponding peptide was chemically synthesized and shown to exert a potent bactericidal 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.
Abstract: Bac7 is a 7 kDa proline- and arginine-rich antimicrobial peptide which was purified from bovine neutrophils. We have used PCR to clone the cDNA of Bac7 precursor, a polypeptide of 21,569 Da. This cDNA is highly conserved in the 5' region, with respect to the corresponding region in the precursors of several other structurally unrelated myeloid antimicrobial peptides. Furthermore, a 148 nt non-coding region at the 3' end is 75% homologous to a corresponding region of the cDNA of the precursor of PR-39, a porcine antibacterial peptide which is also proline- and arginine-rich.
Abstract: A group of myeloid precursors of defense peptides has recently been shown to have highly homologous N-terminal regions. Using a strategy based on this homology, a novel cDNA was cloned from pig bone marrow RNA and found to encode a 153-residue polypeptide. This comprises a highly conserved region encompassing a 29-residue signal peptide and a 101-residue prosequence, followed by a unique, 23-residue, cationic, C-terminal sequence. A peptide corresponding to this C-terminal sequence was chemically synthesized and shown to exert antimicrobial activity against both Gram positive and negative bacteria at concentrations of 2-16 microM. The activity of this potent and structurally novel antibacterial peptide appears to be mediated by its ability to damage bacterial membranes, as shown by the rapid permeabilization of the inner membrane of Escherichia coli.
Abstract: Bac5 and Bac7 are antimicrobial peptides of bovine neutrophils that act on enteric gram-negative bacteria. We report here that these two peptides immobilize and kill Leptospira interrogans and Leptospira biflexa with MBCs of 6 to 25 micrograms/ml. Conversely, although both peptides bind to Borrelia burgdorferi, the organism is resistant to their action.
Abstract: The reaction between the antitumor octahedral complex trans-RuCl2(DMSO)4 and d(GpG) leads to the formation of a stable compound characterized by a covalent bifunctional coordination of the bases to the metal center. The structure of the compound has been fully characterized by NMR and molecular modeling studies, showing the presence of two N7-coordinated guanine moieties in a head to head conformation, two dimethyl sulfoxide molecules, and one halogen atom in the coordination sphere of the ruthenium. The glycosidic chi angles are essentially in the anti range, the sugar puckering of the 5'G is 3'-endo (100% N), whereas that of the 3'G is more flexible but mainly in 2'-endo conformation (85% S), the two bases are strongly destacked. The compound shows structural features which are surprisingly similar to those exhibited by the corresponding cisplatin complex, indicating that such a way of interaction with DNA is not exclusive to Pt or to metals with square planar coordination geometries.
Abstract: Bac5 and Bac7, antibiotics of the bactenecin (proline/arginine-rich peptide) family, are stored as proforms in the large granules of bovine neutrophils [Zanetti, M., Litteri, L., Gennaro, R., Horstmann, H. and Romeo, D. (1990) J. Cell Biol. 111, 1363-1371]. These proforms have been purified to homogeneity from granule extracts by immunoaffinity and reverse-phase chromatography. While mature bactenecins efficiently kill Escherichia coli, Klebsiella pneumoniae and Salmonella typhimurium with minimal inhibitory concentrations of 6-12 micrograms/ml, proBac5 and proBac7 do not affect the growth of the same microorganisms, even at 500 micrograms/ml. Previous investigations have suggested that the conversion of probactenecins into mature antimicrobial peptides is catalyzed by a neutral serine protease stored in the azurophil granules. Purified proBac5 and proBac7 were thus treated with elastase, cathepsin G or proteinase 3, which constitute the pool of neutral serine proteases of the azurophils, and the reaction products were identified by Western blot analysis, mass spectrometry, and N-terminal sequence analysis. Of the three proteases, only elastase is able to catalyze the stepwise cleavage of probactenecins into the corresponding mature peptides, which have the same mass, N-terminal sequence and antibiotic activity of authentic Bac5 and Bac7. These results point to the importance of cooperation between azurophils and large granules in mounting a defense reaction.
