Novel Naja atra cardiotoxin 1 (CTX-1) derived antimicrobial peptides with broad spectrum activity.

Naja atra subsp. atra cardiotoxin 1 (CTX-1), produced by Chinese cobra snakes, belonging to Elapidae family, is included in the three-finger toxin family and exerts high cytotoxicity and antimicrobial activity too. Using as template mainly the tip and the subsequent ß-strand of the first "finger" of this toxin, different sequences of 20 amino acids linear peptides have been designed in order to avoid toxic effects but to maintain or even strengthen the partial antimicrobial activity already seen for the complete toxin. As a result, the sequence NCP-0 (Naja Cardiotoxin Peptide-0) was designed as ancestor and subsequently 4 other variant sequences of NCP-0 were developed. These synthesized variant sequences have shown microbicidal activity towards a panel of reference and field strains of Gram-positive and Gram-negative bacteria. The sequence named NCP-3, and its variants NCP-3a and NCP-3b, have shown the best antimicrobial activity, together with low cytotoxicity against eukaryotic cells and low hemolytic activity. Bactericidal activity has been demonstrated by minimum bactericidal concentration (MBC) assay at values below 10 µg/ml for most of the tested bacterial strains. This potent antimicrobial activity was confirmed even for unicellular fungi Candida albicans, Candida glabrata and Malassezia pachydermatis (MBC 50-6.3 µg/ml), and against the fast-growing mycobacteria Mycobacterium smegmatis and Mycobacterium fortuitum. Moreover, NCP-3 has shown virucidal activity on Bovine Herpesvirus 1 (BoHV1) belonging to Herpesviridae family. The bactericidal activity is maintained even in a high salt concentration medium (125 and 250 mM NaCl) and phosphate buffer with 20% Mueller Hinton (MH) medium against E. coli, methicillin resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa reference strains. Considering these in vitro obtained data, the search for active sequences within proteins presenting an intrinsic microbicidal activity could provide a new way for discovering a large number of novel and promising antimicrobial peptides families.

Activity of AMP2041 against human and animal multidrug resistant Pseudomonas aeruginosa clinical isolates.

BACKGROUND: Antimicrobial resistance is a growing threat to public health. Pseudomonas aeruginosa is a relevant pathogen causing human and animal infections, frequently displaying high levels of resistance to commonly used antimicrobials. The increasing difficulty to develop new effective antibiotics have discouraged investment in this area and only a few new antibiotics are currently under development. An approach to overcome antibiotic resistance could be based on antimicrobial peptides since they offer advantages over currently used microbicides. METHODS: The antimicrobial activity of the synthetic peptide AMP2041 was evaluated against 49 P. aeruginosa clinical strains with high levels of antimicrobial resistance, isolated from humans (n = 19) and animals (n = 30). In vitro activity was evaluated by a microdilution assay for lethal dose 90% (LD90), while the activity over time was performed by time-kill assay with 12.5 µg/ml of AMP2014. Evidences for a direct membrane damage were investigated on P. aeruginosa ATCC 27853 reference strain, on animal isolate PA-VET 38 and on human isolate PA-H 24 by propidium iodide and on P. aeruginosa ATCC 27853 by scanning electron microscopy. RESULTS: AMP2041 showed a dose-dependent activity, with a mean (SEM) LD90 of 1.69 and 3.3 µg/ml for animal and human strains, respectively. AMP2041 showed microbicidal activity on P. aeruginosa isolates from a patient with cystic fibrosis (CF) and resistance increased from first infection isolate (LD90 = 0.3 µg/ml) to the mucoid phenotype (LD90 = 10.4 µg/ml). The time-kill assay showed a time-dependent bactericidal effect of AMP2041 and LD90 was reached within 20 min for all the strains. The stain-dead assay showed an increasing of membrane permeabilization and SEM analysis revealed holes, dents and bursts throughout bacterial cell wall after 30 min of incubation with AMP2041. CONCLUSIONS: The obtained results assessed for the first time the good antimicrobial activity of AMP2041 on P. aeruginosa strains of human origin, including those deriving from a CF patient. We confirmed the excellent antimicrobial activity of AMP2041 on P. aeruginosa strains derived from dog otitis. We also assessed that AMP2041 antimicrobial activity is linked to changes of the P. aeruginosa cell wall morphology and to the increasing of membrane permeability.

Establishment of a bovine herpesvirus 4 based vector expressing a secreted form of the bovine viral diarrhoea virus structural glycoprotein E2 for immunization purposes.

BACKGROUND: The biological characteristics of BoHV-4 make it a good candidate as a gene delivery vector for vaccination purposes. These characteristics include little or no pathogenicity, unlikely oncogenicity, the capability to accommodate large amounts of foreign genetic material, the ability to infect several cell types from different animal species, and the ability to maintain transgene expression in both undifferentiated and differentiated cells. RESULTS: A recombinant bovine herpesvirus 4 (BoHV-4CMV-IgKE2-14 Delta TK) expressing an enhanced secreted form of the bovine viral diarrhea virus (BVDV) structural glycoprotein E2 (gE2-14), obtained by the removal of the putative transmembrane domain and addition of a 14 amino acids peptide at its carboxyl terminal and an immunoglobulin K signal peptide to the amino terminal, was successfully constructed using a Recombineering (recombination -mediated genetic engineering) approach on BoHV-4 cloned as bacterial artificial chromosome. The galactokinase - based recombineering system was modified by the introduction of a kanamycin expression cassette and a kanamycin selection step that allowed a significant reduction of the untargeted background clones. BoHV-4CMV-IgKE2-14 Delta TK infected cell lines highly expressed gE2-14, which maintained native antigenic properties in a serum neutralization inhibition test. When rabbits and sheep were immunized with BoHV-4CMV-IgKE2-14 Delta TK, high levels of serum neutralized antibodies against BVDV were generated. CONCLUSION: This work highlights the engineerization of BoHV-4 genome as a vector for vaccine purposes and may provide the basis for BVDV vaccination exploiting the BoHV-4- based vector that delivers an improved secreted version of the BVDV structural glycoprotein E2.

Bovine herpesvirus 4 infects differentiated neuronal cells in culture and establish persistent infection upon selection.

Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus with no clear disease association. Although BoHV-4 is not considered a neurotropic virus, it has been detected in peripheral and/or central nervous system tissues during persistent infection (Lopez et al, 1996, Microb Pathogen 21: 47-58; Yamamoto et al, 2000, Arch Virol 145: 2363-2370; Asano et al, 2003, J Vet Med Sci 65: 87-93). However, the direct interaction between BoHV-4 and neurons has not been studied so far. The authors investigated the interaction of BoHV-4 with N2a (neuroblastoma cell line) cells through the use of two recombinant viruses (BoHV-4/26A3 neo and BoHV-4EGFP?TK). Because of the unique biological characteristics of N2a cells, which differentiate in neuron-like cells producing dendrites, axon, and specific neuronal markers, the authors found that BoHV-4 infects differentiated N2a cells and a persistent infection can be established. BoHV-4 persistently infected N2a cells produce infectious viral particles, which do not interfere with cellular differentiation.

Activation of bovine herpesvirus 4 lytic replication in a non-permissive cell line by overexpression of BoHV-4 immediate early (IE) 2 gene.

Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus with no clear disease association, it establishes persistent infections in its natural host, the bovine, and in an experimental host, the rabbit. BoHV-4 immediate early 2 (IE2) RNA is the less abundant, spliced, 1.8 kb RNA. The predicted amino acid sequence, of the IE2 protein, reveals that it could encode a 61 kDa protein with amino acid sequence homology to the Epstein-Barr virus (EBV) transactivator R protein and its homologues including, herpesvirus saimiri (HVS), equine herpesvirus 2 (EHV-2), murine herpesvirus 68 and Kaposi's sarcoma-associated herpesvirus (KSHV). We examined recently the interaction of BoHV-4 with a human rhabdomyosarcoma cell line, RD-4, and found that although some infectious viruses can be produced, no cytopathic effect (CPE) was observed [J. Gen. Virol. 81 (2000) 1807]. Because IE2 could play a critical role in BoHV-4 productive infection and its overexpression in RD-4 cells could switch the non-permissive RD-4 status to a permissive one. RD-4 cells expressing stably BoHV-4 IE2 gene were generated. BoHV-4 IE2 induced an increased production of infectious viral particles sufficient to obtain an apparent cytopathic effect. It is concluded that BoHV-4 IE2 is a key factor in determining the outcome of BoHV-4 infection.

Alkylation of sulfhydryl groups on Galpha(s/olf) subunits by N-ethylmaleimide: regulation by guanine nucleotides.

In rat striatum A(2A) adenosine receptors activate adenylyl cyclase through coupling to G(s)-like proteins, mainly G(olf) that is expressed at high levels in this brain region. In this study we report that the sulfhydryl alkylating reagent, N-ethylmaleimide (NEM), causes a concentration- and time-dependent inhibition of [3H] 2-p-(2-carboxyethyl)phenylethylamino)-5'-N-ethylcarboxamido adenosine ([3H]CGS21680) binding to rat striatal membranes. Membrane treatment with [14C]N-ethylmaleimide ([14C]NEM) labels numerous proteins while addition of 5'-guanylylimidodiphosphate (Gpp(NH)p) reduces labeling of only three protein bands that migrate in SDS-polyacrylamide gel electrophoresis with apparent molecular masses of approximately 52, 45 and 39 kDa, respectively. The 52- and 45-kDa labeled bands show electrophoretic motilities as Galpha(s)-long and Galpha(s)-short/Galpha(olf) subunits. An anti-Galpha(s/olf) antiserum immunoprecipitates two 14C labeled bands of 44 and 39 kDa. The band density decreases by 21-26% when membranes are treated with NEM in the presence of Gpp(NH)p. An anti-A(2A) receptor antibody also immunoprecipitates two 14C labeled bands of 40 and 38 kDa, respectively. However, such protein bands do not show any decrease of their density upon membrane treatment with NEM plus Gpp(NH)p. These results indicate that in rat striatal membranes NEM alkylates sulfhydryl groups of both Galpha(s/olf) subunits and A(2A) adenosine receptors. In addition, cysteine residues of Galpha(s/olf) are easily accessible to modification when the subunit is in the GDP-bound form. The 39- and 38-kDa labeled proteins may represent proteolytic fragments of Galpha(s/olf) and A(2A) adenosine receptor, respectively.