Amino acid sequence of a basic Agkistrodon halys blomhoffii phospholipase A2. Possible role of NH2-terminal lysines in action on phospholipids of Escherichia coli

A basic (pI = 10.2) phospholipase A2 of the venom of the snake Agkistrodon halys blomhoffii is one of a few phospholipases A2 capable of hydrolyzing the phospholipids of Escherichia coli killed by a bactericidal protein purified from human or rabbit neutrophil granules. We have shown that modification of as many as 4 mol of lysine per mole of the phospholipase A2, either by carbamylation or by reductive methylation [Forst, S., Weiss, J., & Elsbach, P. (1982) J. Biol. Chem. 257, 14055-14057], had no effect on catalytic activity toward extracted E. coli phospholipids or the phospholipids of autoclaved E. coli. In contrast, modification of 1 mol of lysine per mole of enzyme substantially reduced activity toward the phospholipids of E. coli killed by the neutrophil protein. To explore further the role of lysines in the function of this phospholipase A2, we determined the amino acid sequence of the enzyme and the incorporation of [14C]cyanate into individual lysines when, on average, 1 lysine per molecule of enzyme had been carbamylated. After incorporation of approximately 1 mol of [14C]cyanate per mole of protein, the phospholipase A2 was reduced, alkylated, and exhaustively carbamylated with unlabeled cyanate. The amino acid sequence was determined of the NH2-terminal 33 amino acids of the holoprotein and of peptides isolated after digestion with trypsin and Staphylococcus aureus V-8 protease. The protein contains 122 amino acid residues, 17 of which are lysines. The NH2-terminal region is unique among more than 30 phospholipases A2 previously sequenced because of its high content of basic residues (His-1, Arg-6, and Lys-7, -10, -11, and -15).(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of the histidine kinase, EnvZ, in the production of outer membrane proteins in the symbiotic-pathogenic bacterium Xenorhabdus nematophilus.

We show that inactivation of envZ, the gene encoding the histidine kinase sensor protein, EnvZ, of Xenorhabdus nematophilus, affected the production of several outer membrane proteins (Opns). X. nematophilus produced five major Opns during exponential growth. Insertional inactivation of envZ led to a decrease in the production of OpnP, the OmpF-like pore-forming protein which constitutes approximately 50% of the total outer membrane protein in X. nematophilus. OpnA production was also reduced, while the remaining Opns were produced normally. During the transition to stationary phase, three new outer membrane proteins, OpnB, OpnS, and OpnX, were induced in the wild-type strain. The envZ-minus strain, ANT1, did not produce OpnB and OpnX, while OpnS was induced at markedly reduced levels. These results suggest that EnvZ was required for the high-level production of OpnP during exponential growth and may be involved in the production of OpnB, OpnS, and OpnX during stationary-phase growth. We also show that ANT1 was more pathogenic than the wild-type strain when as few as five cells were injected into the hemolymph of the larval stage of the tobacco hornworm (Manduca sexta). The larvae died before significant numbers of bacteria were detectable in the hemolymph. These results are discussed in relation to the role of EnvZ in the life cycle of X. nematophilus.     

Characterization of two heparan sulphate-binding sites in the mycobacterial adhesin Hlp

Background  

The histone-like Hlp protein is emerging as a key component in mycobacterial pathogenesis, being involved in the initial events of host colonization by interacting with laminin and glycosaminoglycans (GAGs). In the present study, nuclear magnetic resonance (NMR) was used to map the binding site(s) of Hlp to heparan sulfate and identify the nature of the amino acid residues directly involved in this interaction.     

Xenorhabdus antibiotics: a comparative analysis and potential utility for controlling mastitis caused by bacteria

Aims: The role of antibiotics produced by bacterial symbionts of entomopathogenic nematodes is to suppress growth of microbes in the soil environment. These antibiotics are active against Gram‐positive and Gram‐negative bacteria, and were tested against mastitis isolates from dairy cows. Methods and Results: Two bioassays were adapted for Xenorhabdus antibiotics; an overlay method on agar plates, and serially diluted, cell‐free, Xenorhabdus cultures. The antimicrobial activities of the liquid cultures of 13 strains from five Xenorhabdus species were further evaluated. Antimicrobial activities of the type strains of X. nematophila, X. budapestensis and X. szentirmaii were tested on mastitis isolates of Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae with both bioassays. A previously reported antibiotic from X. nematophila, nematophin, was synthesized in three steps from tryptamine and 4‐methyl‐2‐oxovaleric acid sodium salt. Conclusions: The antibiotics of all three Xenorhabdus strains were powerful in either bioassay, but the sensitivity of the isolates differed from each other. While Kl. pneumoniae was the least susceptible, Staph. aureus had the highest sensitivity to each Xenorhabdus strain. Xenorhabdus szentirmaii and X. budapestensis were more potent antibiotic producers than X. nematophila, and raceme nematophin was ineffective against all mastitis isolates. Significance and Impact of the Study: These results indicate that Xenorhabdus antibiotics are effective against mastitis isolates and should be further evaluated for their potential in mastitis control or prevention.     

Pilot study with technosphere/PTH(1-34)--a new approach for effective pulmonary delivery of parathyroid hormone (1-34)

Sequential subcutaneous PTH injection therapy (repeated 14 days of PTH administration and a subsequent treatment pause for a few weeks) is known to increase bone mineral density in patients with osteopenic disorders. Alternative methods of drug delivery may be beneficial in increasing compliance. A pilot study was performed in 10 healthy volunteers (4 female/6-male, age: 25.6 +/- 3.5 years, BMI: 22.3 +/- 2.4 kg/m 2, mean +/- SD) to assess the pharmacokinetic profiles of 1600 IU of PTH(1 - 34) using the pulmonary Technosphere drug delivery system in comparison to a subcutaneous injection of 400 IU. The treatments were administered in the morning after an overnight fast and blood samples for measurement of PTH(1 - 34), PTH(1 - 84), and calcium and calcitonin were taken over a period of 6 hours. Both injection and pulmonary application of PTH(1 - 34) were well tolerated. After pulmonary administration of Technosphere/PTH(1 - 34), PTH(1 - 34) appeared in the serum with a faster concentration increase (T max: pulmonary 10 +/- 5 min vs. subcutaneous 28 +/- 8 min, p < 0.001) and with higher maximal concentrations (C max : pulmonary 309 +/- 215 pmol/l vs. subcutaneous 102 +/- 45 pmol/l, p < 0.05) as compared to the subcutaneous injection. The relative bioavailability of pulmonary Technosphere/PTH(1 - 34) was calculated to be 48 %. No differences were seen between pulmonary and subcutaneous application with regard to the PTH(1 - 84), calcitonin and calcium concentrations. In conclusion, pulmonary application of Technosphere/PTH(1 - 34) appears to be an effective and thus attractive candidate for PTH substitution therapy in osteoporosis and other conditions leading to a decrease in bone mineral density.     

Role of Secondary Metabolites in Establishment of the Mutualistic Partnership between Xenorhabdus nematophila and the Entomopathogenic Nematode Steinernema carpocapsae

Xenorhabdus nematophila engages in a mutualistic partnership with the nematode Steinernema carpocapsae, which invades insects, migrates through the gut, and penetrates into the hemocoel (body cavity). We showed previously that during invasion of Manduca sexta, the gut microbe Staphylococcus saprophyticus appeared transiently in the hemocoel, while Enterococcus faecalis proliferated as X. nematophila became dominant. X. nematophila produces diverse secondary metabolites, including the major water-soluble antimicrobial xenocoumacin. Here, we study the role of X. nematophila antimicrobials in interspecies competition under biologically relevant conditions using strains lacking either xenocoumacin (ΔxcnKL strain), xenocoumacin and the newly discovered antibiotic F (ΔxcnKL:F strain), or all ngrA-derived secondary metabolites (ngrA strain). Competition experiments were performed in Grace''s insect medium, which is based on lepidopteran hemolymph. S. saprophyticus was eliminated when inoculated into growing cultures of either the ΔxcnKL strain or ΔxcnKL:F strain but grew in the presence of the ngrA strain, indicating that ngrA-derived antimicrobials, excluding xenocoumacin or antibiotic F, were required to eliminate the competitor. In contrast, S. saprophyticus was eliminated when coinjected into M. sexta with either the ΔxcnKL or ngrA strain, indicating that ngrA-derived antimicrobials were not required to eliminate the competitor in vivo. E. faecalis growth was facilitated when coinjected with either of the mutant strains. Furthermore, nematode reproduction in M. sexta naturally infected with infective juveniles colonized with the ngrA strain was markedly reduced relative to the level of reproduction when infective juveniles were colonized with the wild-type strain. These findings provide new insights into interspecies competition in a host environment and suggest that ngrA-derived compounds serve as signals for in vivo nematode reproduction.     

New Insights into the Colonization and Release Processes of Xenorhabdus nematophila and the Morphology and Ultrastructure of the Bacterial Receptacle of Its Nematode Host, Steinernema carpocapsae

We present results from epifluorescence, differential interference contrast, and transmission electron microscopy showing that Xenorhabdus nematophila colonizes a receptacle in the anterior intestine of the infective juvenile (IJ) stage of Steinernema carpocapsae. This region is connected to the esophagus at the esophagointestinal junction. The process by which X. nematophila leaves this bacterial receptacle had not been analyzed previously. In this study we monitored the movement of green fluorescent protein-labeled bacteria during the release process. Our observations revealed that Xenorhabdus colonizes the distal region of the receptacle and that exposure to insect hemolymph stimulated forward movement of the bacteria to the esophagointestinal junction. Continued exposure to hemolymph caused a narrow passage in the distal receptacle to widen, allowing movement of Xenorhabdus down the intestine and out the anus. Efficient release of both the wild type and a nonmotile strain was evident in most of the IJs incubated in hemolymph, whereas only a few IJs incubated in nutrient-rich broth released bacterial cells. Incubation of IJs in hemolymph treated with agents that induce nematode paralysis dramatically inhibited the release process. These results suggest that bacterial motility is not required for movement out of the distal region of the receptacle and that hemolymph-induced esophageal pumping provides a force for the release of X. nematophila out of the receptacle and into the intestinal lumen.