Partial complementation of Sinorhizobium meliloti bacA mutant phenotypes by the Mycobacterium tuberculosis BacA protein.

The Sinorhizobium meliloti BacA ABC transporter protein plays an important role in its nodulating symbiosis with the legume alfalfa (Medicago sativa). The Mycobacterium tuberculosis BacA homolog was found to be important for the maintenance of chronic murine infections, yet its in vivo function is unknown. In the legume plant as well as in the mammalian host, bacteria encounter host antimicrobial peptides (AMPs). We found that the M. tuberculosis BacA protein was able to partially complement the symbiotic defect of an S. meliloti BacA-deficient mutant on alfalfa plants and to protect this mutant in vitro from the antimicrobial activity of a synthetic legume peptide, NCR247, and a recombinant human ß-defensin 2 (HBD2). This finding was also confirmed using an M. tuberculosis insertion mutant. Furthermore, M. tuberculosis BacA-mediated protection of the legume symbiont S. meliloti against legume defensins as well as HBD2 is dependent on its attached ATPase domain. In addition, we show that M. tuberculosis BacA mediates peptide uptake of the truncated bovine AMP, Bac7(1-16). This process required a functional ATPase domain. We therefore suggest that M. tuberculosis BacA is important for the transport of peptides across the cytoplasmic membrane and is part of a complete ABC transporter. Hence, BacA-mediated protection against host AMPs might be important for the maintenance of latent infections.

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.

The influence of comorbidities and complications on discharge function in stroke rehabilitation inpatients.

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.

Structural and functional analysis of horse cathelicidin peptides.

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.

Novel cathelicidins in horse leukocytes(1).

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.

Biological characterization of a novel mammalian antimicrobial peptide.

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.

Cloning and analysis of a transcript derived from two contiguous genes of the cathelicidin family.

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.

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

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.

Six antimicrobial peptide genes of the cathelicidin family map to bovine chromosome 22q24 by fluorescence in situ hybridization.

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.

cDNA sequences of three sheep myeloid cathelicidins.

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.

PMAP-37, a novel antibacterial peptide from pig myeloid cells. cDNA cloning, chemical synthesis and activity.

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.

Molecular cloning of Bac7, a proline- and arginine-rich antimicrobial peptide from bovine neutrophils.

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.

Molecular cloning and chemical synthesis of a novel antibacterial peptide derived from pig myeloid cells.

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.

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 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.

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 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.

Antimicrobial activity of two bactenecins against spirochetes.

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.

Proteolytic cleavage by neutrophil elastase converts inactive storage proforms to antibacterial bactenecins.

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.

NMR structural characterization of the reaction product between d(GpG) and the octahedral antitumor complex trans-RuCl2(DMSO)4.

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.