Antimicrobial Peptides (AMP) in the Cell-Free Culture Media of Xenorhabdus budapestensis and X. szentirmaii Exert Anti-Protist Activity against Eukaryotic Vertebrate Pathogens including Histomonas meleagridis and Leishmania donovani Species.

Anti-microbial peptides provide a powerful toolkit for combating multidrug resistance. Combating eukaryotic pathogens is complicated because the intracellular drug targets in the eukaryotic pathogen are frequently homologs of cellular structures of vital importance in the host organism. The entomopathogenic bacteria (EPB), symbionts of entomopathogenic-nematode species, release a series of non-ribosomal templated anti-microbial peptides. Some may be potential drug candidates. The ability of an entomopathogenic-nematode/entomopathogenic bacterium symbiotic complex to survive in a given polyxenic milieu is a coevolutionary product. This explains that those gene complexes that are responsible for the biosynthesis of different non-ribosomal templated anti-microbial protective peptides (including those that are potently capable of inactivating the protist mammalian pathogen Leishmania donovanii and the gallinaceous bird pathogen Histomonas meleagridis) are co-regulated. Our approach is based on comparative anti-microbial bioassays of the culture media of the wild-type and regulatory mutant strains. We concluded that Xenorhabdus budapestensis and X. szentirmaii are excellent sources of non-ribosomal templated anti-microbial peptides that are efficient antagonists of the mentioned pathogens. Data on selective cytotoxicity of different cell-free culture media encourage us to forecast that the recently discovered "easy-PACId" research strategy is suitable for constructing entomopathogenic-bacterium (EPB) strains producing and releasing single, harmless, non-ribosomal templated anti-microbial peptides with considerable drug, (probiotic)-candidate potential.

Metabolites of Xenorhabdus bacteria are potent candidates for mitigating amphibian chytridiomycosis.

Chytridiomycosis, caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd), has caused extreme losses in amphibian biodiversity. Finding bacteria that produce metabolites with antifungal properties may turn out to be invaluable in the fight against this devastating disease. The entomopathogenic bacteria, Xenorhabdus szentirmaii and X. budapestensis produce secondary metabolites that are effective against a wide range of fungal plant pathogens. To assess whether they may also be effective against Bd, we extracted cell-free culture media (CFCM) from liquid cultures of X. szentirmaii and X. budapestensis and tested their ability to inhibit Bd growth in vitro. As a second step, using juvenile common toads (Bufo bufo) experimentally infected with Bd we also tested the in vivo antifungal efficacy of X. szentirmaii CFCM diluted to 2 and 10% (v/v), while also assessing possible malign side effects on amphibians. Results of the in vitro experiment documented highly effective growth inhibition by CFCMs of both Xenorhabdus species. The in vivo experiment showed that treatment with CFCM of X. szentirmaii applied at a dilution of 10% resulted in infection intensities reduced by ca. 73% compared to controls and to juvenile toads treated with CFCM applied at a dilution of 2%. At the same time, we detected no negative side effects of treatment with CFCM on toad survival and development. Our results clearly support the idea that metabolites of X. szentirmaii, and perhaps of several other Xenorhabdus species as well, may prove highly useful for the treatment of Bd infected amphibians.

XENOFOOD-An Autoclaved Feed Supplement Containing Autoclavable Antimicrobial Peptides-Exerts Anticoccidial GI Activity, and Causes Bursa Enlargement, but Has No Detectable Harmful Effects in Broiler Cockerels despite In Vitro Detectable Cytotoxicity on LHM Cells.

Entomopathogenic bacteria are obligate symbionts of entomopathogenic nematode (EPN) species. These bacteria biosynthesize and release non-ribosomal-templated hybrid peptides (NR-AMPs), with strong, and large-spectral antimicrobial potential, capable of inactivating pathogens belonging to different prokaryote, and eukaryote taxa. The cell-free conditioned culture media (CFCM) of Xenorhabdus budapestensis and X. szentirmaii efficiently inactivate poultry pathogens like Clostridium, Histomonas, and Eimeria. To learn whether a bio-preparation containing antimicrobial peptides of Xenorhabdus origin with accompanying (in vitro detectable) cytotoxic effects could be considered a safely applicable preventive feed supplement, we conducted a 42-day feeding experiment on freshly hatched broiler cockerels. XENOFOOD (containing autoclaved X. budapestensis, and X. szentirmaii cultures developed on chicken food) were consumed by the birds. The XENOFOOD exerted detectable gastrointestinal (GI) activity (reducing the numbers of the colony-forming Clostridium perfringens units in the lower jejunum. No animal was lost in the experiment. Neither the body weight, growth rate, feed-conversion ratio, nor organ-weight data differed between the control (C) and treated (T) groups, indicating that the XENOFOOD diet did not result in any detectable adverse effects. We suppose that the parameters indicating a moderate enlargement of bursas of Fabricius (average weight, size, and individual bursa/spleen weight-ratios) in the XENOFOOD-fed group must be an indirect indication that the bursa-controlled humoral immune system neutralized the cytotoxic ingredients of the XENOFOOD in the blood, not allowing to reach their critical cytotoxic concentration in the sensitive tissues.

Type Strains of Entomopathogenic Nematode-Symbiotic Bacterium Species, Xenorhabdus szentirmaii (EMC) and X. budapestensis (EMA), Are Exceptional Sources of Non-Ribosomal Templated, Large-Target-Spectral, Thermotolerant-Antimicrobial Peptides (by Both), and Iodinin (by EMC).

Antimicrobial multidrug resistance (MDR) is a global challenge, not only for public health, but also for sustainable agriculture. Antibiotics used in humans should be ruled out for use in veterinary or agricultural settings. Applying antimicrobial peptide (AMP) molecules, produced by soil-born organisms for protecting (soil-born) plants, seems a preferable alternative. The natural role of peptide-antimicrobials, produced by the prokaryotic partner of entomopathogenic-nematode/bacterium (EPN/EPB) symbiotic associations, is to sustain monoxenic conditions for the EPB in the gut of the semi-anabiotic infective dauer juvenile (IJ) EPN. They keep pathobiome conditions balanced for the EPN/EPB complex in polyxenic (soil, vanquished insect cadaver) niches. Xenorhabdus szentirmaii DSM16338(T) (EMC), and X. budapestensis DSM16342(T) (EMA), are the respective natural symbionts of EPN species Steinernema rarum and S. bicornutum. We identified and characterized both of these 15 years ago. The functional annotation of the draft genome of EMC revealed 71 genes encoding non-ribosomal peptide synthases, and polyketide synthases. The large spatial Xenorhabdus AMP (fabclavine), was discovered in EMA, and its biosynthetic pathway in EMC. The AMPs produced by EMA and EMC are promising candidates for controlling MDR prokaryotic and eukaryotic pathogens (bacteria, oomycetes, fungi, protozoa). EMC releases large quantity of iodinin (1,6-dihydroxyphenazine 5,10-dioxide) in a water-soluble form into the media, where it condenses to form spectacular water-insoluble, macroscopic crystals. This review evaluates the scientific impact of international research on EMA and EMC.

Multidrug Resistance (MDR) and Collateral Sensitivity in Bacteria, with Special Attention to Genetic and Evolutionary Aspects and to the Perspectives of Antimicrobial Peptides-A Review.

Antibiotic poly-resistance (multidrug-, extreme-, and pan-drug resistance) is controlled by adaptive evolution. Darwinian and Lamarckian interpretations of resistance evolution are discussed. Arguments for, and against, pessimistic forecasts on a fatal "post-antibiotic era" are evaluated. In commensal niches, the appearance of a new antibiotic resistance often reduces fitness, but compensatory mutations may counteract this tendency. The appearance of new antibiotic resistance is frequently accompanied by a collateral sensitivity to other resistances. Organisms with an expanding open pan-genome, such as Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae, can withstand an increased number of resistances by exploiting their evolutionary plasticity and disseminating clonally or poly-clonally. Multidrug-resistant pathogen clones can become predominant under antibiotic stress conditions but, under the influence of negative frequency-dependent selection, are prevented from rising to dominance in a population in a commensal niche. Antimicrobial peptides have a great potential to combat multidrug resistance, since antibiotic-resistant bacteria have shown a high frequency of collateral sensitivity to antimicrobial peptides. In addition, the mobility patterns of antibiotic resistance, and antimicrobial peptide resistance, genes are completely different. The integron trade in commensal niches is fortunately limited by the species-specificity of resistance genes. Hence, we theorize that the suggested post-antibiotic era has not yet come, and indeed might never come.

[Antimicrobial effect on some zoonotic bacteria, of the cell-free fermentation fluid and purified peptide fraction of the entomopathogenic bacterium, Xenorhabdus budapestensis]. / A Xenorhabdus budapestensis entomopatogén baktérium sejtmentes fermentlevének és tisztítottfehérje-frakciójának antimikrobiális hatása néhány zoonoticus baktériumra.

INTRODUCTION: Many multi-resistant patogens appear continuously resulting in a permanent need for the development of novel antibiotics. A large number of antibiotics introduced in clinical and veterinary practices are not effective. Antibacterial peptides with unusual mode of action may represent a promising option against multi-resistant pathogens. The entomopathogenic Xenorhabdus budapestensis bacteria produce several different antimicrobial peptides compounds such as bicornutin-A and fabclavin. AIM: The aim of the authors was to evaluate the in vitro antibacterial effect of Xenorhabdus budapestensis using zoonotic patogen bacteria. METHOD: Cell-free conditioned media and purified peptide fractions of Xenorhabdus budapestensis were tested on Gram-positive (Rhodococcus equi, Erysipelothrix rhusiopathia, Staphylococcus aureus, Streptococcus equi, Corynebacterium pseudotuberculosis, Listeria monocytagenes) and Gram-negative bacteria (Salmonella gallinarum, Salmonella derbi, Bordatella bronchoseptica, Escherichia coli, Pasteurella multocida, Aeromonas hydrophila) using agar diffusion test on blood agar plates. RESULTS: It was found that Xenorhabdus budapestensis bacteria produced compounds with strong and dose-dependent effects on the tested organisms. Purified peptid fraction exerted a more marked effect than cell free conditioned media. Gram-positive bacteria were more sensitive to this antibacterial effect than Gram-negative bacteria. CONCLUSIONS: Antibacterial peptide compound from Xenorhabdus budapestensis exert marked antibacterial effect on zoonotic patogen bacteria and they should be further evaluated in future for their potential use in the control or prevention of zoonoses.

Proteolytic enzyme production by strains of the insect pathogen xenorhabdus and characterization of an early-log-phase-secreted protease as a potential virulence factor.

As a comparison to a similar study on Photorhabdus strains, 15 Xenorhabdus bacterial strains and secondary phenotypic variants of two strains were screened for proteolytic activity by five detection methods. Although the number and intensity of proteolytic activities were different, every strain was positive for proteolytic activity by several tests. Zymography following native PAGE detected two groups of activities with different substrate affinities and a higher and lower electrophoretic mobility that were distinguished as activity 1 and 2, respectively. Zymography following SDS-PAGE resolved three activities, which were provisionally named proteases A, B, and C. Only protease B, an ∼55-kDa enzyme, was produced by every strain. This enzyme exhibited higher affinity to the gelatin substrate than to the casein substrate. Of the chromogenic substrates used, three were hydrolyzed: furylacryloyl-Ala-Leu-Val-Tyr (Fua-ALVY), Fua-LGPA (LGPA is Leu-Gly-Pro-Ala) (a substrate for collagen peptidases), and succinyl-Ala-Ala-Pro-Phe-thiobenzyl (Succ-AAPF-SBzl). All but the Fua-LGPA-ase activity seemed to be from secreted enzymes. According to their substrate preference profiles and inhibitor sensitivities, at least six such proteolytic enzymes could be distinguished in the culture medium of Xenorhabdus strains. The proteolytic enzyme that was secreted the earliest, protease B and the Succ-AAPF-SBzl-hydrolyzing enzyme, appeared from the early logarithmic phase of growth. Protease B could also be detected in the hemolymph of Xenorhabdus-infected Galleria mellonella larvae from 15 h postinfection. The purified protease B hydrolyzed in vitro seven proteins in the hemolymph of Manduca sexta that were also cleaved by PrtA peptidase from Photorhabdus. The N-terminal sequence of protease B showed similarity to a 55-kDa serralysin type metalloprotease in Xenorhabdus nematophila, which had been identified as an orthologue of Photorhabdus PrtA peptidase.

A robust phylogenetic framework for the bacterial genus Photorhabdus and its use in studying the evolution and maintenance of bioluminescence: a case for 16S, gyrB, and glnA.

Photorhabdus spp., the only known bioluminescent terrestrial bacteria are well known for their symbiotic association with heterorhabditid nematodes. This association, along with their ability to kill insects, has aroused interest in the evolutionary relationships within this bacterial group. Currently, three species are recognized within the genus Photorhabdus; P. temperata and P. luminescens, which are endosymbionts of Heterorhabditis spp., and P. asymbiotica, which has been isolated from human wounds and has recently been shown to also have a heterorhabditid nematode vector. To examine phylogenetic relationships among these taxa, we utilize total evidence Bayesian, likelihood, and parsimony based analyses of three genetic loci (16S rRNA gene, gyrB, and glnA) to construct a robust evolutionary hypothesis for the genus Photorhabdus. Here we use this phylogeny to evaluate existing specific and sub-specific taxonomic statements within the genus, identify previously undescribed Photorhabdus strains, test the utility of 16S rRNA gene, gyrB, and glnA in resolving various levels of relationships within the genus, and, finally, to investigate the evolution of bioluminescence. The genes examined produced the most robust phylogenetic hypothesis to date for the genus Photorhabdus, as indicated by strong bootstrap and posterior probability values at previously unresolved or poorly resolved nodes. We show that glnA is particularly useful in resolving specific and intra-specific relationships poorly resolved in other studies. We conclude that P. asymbiotica is the sister group to P. luminescens and that the new strains HIT and JUN should be given a new group designation within P. asymbiotica. Furthermore, we reveal a pattern of decline in bioluminescent intensity through the evolution of Photorhabdus, suggesting that this may be a trait acquired and maintained under previous ecological (aquatic) selection pressures that is now gradually being lost in its terrestrial environment.

Cleavage site analysis of a serralysin-like protease, PrtA, from an insect pathogen Photorhabdus luminescens and development of a highly sensitive and specific substrate.

The aim of this study was the development of a sensitive and specific substrate for protease A (PrtA), a serralysin-like metzincin from the entomopathogenic microorganism, Photorhabdus. First, cleavage of three biological peptides, the A and B chains of insulin and beta-lipotropin, and of 15 synthetic peptides, was investigated. In the biological peptides, a preference for the hydrophobic residues Ala, Leu and Val was observed at three substrate positions, P2, P1' and P2'. At these positions in the synthetic peptides the preferred residues were Val, Ala and Val, respectively. They contributed to the efficiency of hydrolysis in the order P1' > P2 > P2'. Six amino acids of the synthetic peptides were sufficient to reach the maximum rate of hydrolysis, in accordance with the ability of PrtA to cleave three amino acids from both the N- and the C-terminus of some fragments of biological peptides. Using the best synthetic peptide, a fluorescence-quenched substrate, N-(4-[4'(dimethylamino)phenylazo]benzoyl-EVYAVES-5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid, was prepared. The approximately 4 x 10(6) M(-1) x s(-1) specificity constant of PrtA (at K(m) approximately 5 x 10(-5) M and k(cat) approximately 2 x 10(2) s(-1)) on this substrate was the highest activity for a serralysin-type enzyme, allowing precise measurement of the effects of several inhibitors and pH on PrtA activity. These showed the characteristics of a metalloenzyme and a wide range of optimum pH, similar to other serralysins. PrtA activity could be measured in biological samples (Photorhabdus-infected insect larvae) without interference from other enzymes, which indicates that substrate selectivity is high towards PrtA. The substrate sensitivity allowed early (14 h post infection) detection of PrtA, which might indicate PrtA's participation in the establishment of infection and not only, as it has been supposed, in bioconversion.

Xenofuranones A and B: phenylpyruvate dimers from Xenorhabdus szentirmaii.

Xenofuranones A (1) and B (2) have been isolated from cultures of the insect-pathogenic bacterium Xenorhabdus szentirmaii, and their structures were elucidated by NMR and mass spectroscopy. Both compounds show similarities to fungal furanones, and their biosynthesis was studied using a reversed approach by feeding putative 12C precursors to an overall 13C background in small-scale experiments followed by gas chromatographic analysis coupled to mass spectrometry.

Description of four novel species of Xenorhabdus, family Enterobacteriaceae: Xenorhabdus budapestensis sp. nov., Xenorhabdus ehlersii sp. nov., Xenorhabdus innexi sp. nov., and Xenorhabdus szentirmaii sp. nov.

The taxonomic affiliation was determined for four Xenorhabdus strains isolated from four Steinernema hosts from different countries. As compared to the five validly described Xenorhabdus species, i.e., X. nematophila, X. japonica, X. beddingii, X. bovienii and X. poinarii, these isolates represented novel species on the basis of 16S rRNA gene sequences and riboprint patterns, as well as by physiological and metabolic properties. They were named Xenorhabdus budapestensis sp. nov., type strain DSM 16342T, isolated from Steinernema bicornutam; Xenorhabdus ehlersii sp. nov., type strain DSM 16337T, isolated from Steinernema serratum; Xenorhabdus innexi sp. nov., type strain DSM 16336T isolated from Steinernema scapterisci; and Xenorhabdus szentirmaii sp. nov., type strain DSM 16338T, isolated from Steinernema rarum.

Comparison of proteolytic activities produced by entomopathogenic Photorhabdus bacteria: strain- and phase-dependent heterogeneity in composition and activity of four enzymes.

Twenty strains (including eight phase variant pairs) of nematode-symbiotic and insect-pathogenic Photorhabdus bacteria were examined for the production of proteolytic enzymes by using a combination of several methods, including gelatin liquefaction, zymography coupled to native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and activity measurement with two chromogen substrate types. Four protease activities (approximately 74, approximately 55, approximately 54, and approximately 37 kDa) could be separated. The N-terminal sequences of three of the proteases were determined, and a comparison with sequences in databases allowed identification of these proteases as HEXXH metallopeptidases. Thus, the 74-kDa protease (described formerly as Php-B [J. Marokhazi, G. Koczan, F. Hudecz, L. Graf, A. Fodor, and I. Venekei, Biochem. J. 379:633-640, 2004) is an ortholog of OpdA, a member the thimet oligopeptidase family, and the 55-kDa protease is an ortholog of PrtA, a HEXXH+H peptidase in clan MB (metzincins), while the 37-kDa protease (Php-C) belongs to the HEXXH+E peptidases in clan MA. The 54-kDa protease (Php-D) is a nonmetalloenzyme. PrtA and Php-C were zymographically detected, and they occurred in several smaller forms as well. OpdA could not be detected by zymography. PrtA, Php-C, and Php-D were secreted proteases; OpdA, in contrast, was an intracellular enzyme. OpdA activity was found in every strain tested, while Php-D was detected only in the Brecon/1 strain. There was significant strain variation in the secretion of PrtA and Php-C activities, but reduced activity or a lack of activity was not specific to secondary-phase variants. The presence of PrtA, OpdA, and Php-C activities could be detected in the hemolymph of Galleria melonella larvae 20 to 40 h postinfection. These proteases appear not to be directly involved in the pathogenicity of Photorhabdus, since strains or phase variants lacking any of these proteases do not show reduced virulence when they are injected into G. melonella larvae.

Enzymic characterization with progress curve analysis of a collagen peptidase from an enthomopathogenic bacterium, Photorhabdus luminescens.

A proteolytic enzyme, Php-B ( Photorhabdus protease B), was purified from the entomopathogenic bacterium, Photorhabdus luminescens. The enzyme is intracellular, and its molecular mass is 74 kDa. Tested on various peptide and oligopeptide substrates, Php-B hydrolysed only oligopeptides, with significant activity against bradykinin and a 2-furylacryloyl-blocked peptide, Fua-LGPA (2-furylacryloyl-Leu-Gly-Pro-Ala; kcat=3.6x10(2) s(-1), K(m)=5.8x10(-5) M(-1), pH optimum approx. 7.0). The p K(a1) and the p K(a2) values of the enzyme activity (6.1 and 7.9 respectively), as well as experiments with enzyme inhibitors and bivalent metal ions, suggest that the activity of Php-B is dependent on histidine and cysteine residues, but not on serine residues, and that it is a metalloprotease, which most probably uses Zn2+ as a catalytic ion. The enzyme's ability to cleave oligopeptides that contain a sequence similar to collagen repeat (-Pro-Xaa-Gly-), bradykinin and Fua-LGPA (a synthetic substrate for bacterial collagenases and collagen peptidases), but not native collagens (types I and IV) or denatured collagen (gelatin), indicates that Php-B is probably a collagen peptidase, the first enzyme of this type to be identified in an insect pathogen, that might have a role in the nutrition of P. luminescens by degrading small collagen fragments. For the determination of enzyme kinetic constants, we fitted a numerically integrated Michaelis-Menten model to the experimental progress curves. Since this approach has not been used before in the characterization of proteases that are specific for the P1'-P4' substrate sites (e.g. collagenolytic enzymes), we present a comparison of this method with more conventional ones. The results confirm the reliability of the numerical integration method in the kinetic analysis of collagen-peptide-hydrolysing enzymes.

Using a DNA microarray to investigate the distribution of insect virulence factors in strains of photorhabdus bacteria.

Photorhabdus is an insect-pathogenic bacterium in which oral toxicity to insects is found in two distinct taxonomic groups. Using a DNA microarray and comparative genomics, we show that oral toxicity is associated with toxin complex genes tcaABC and that this locus can be mobilized or deleted within different strains.