Xenorhabdus nematophila, a bacterium pathogenic for insects associated with the nematode Steinernema carpocapsae, releases high quantities of proteases, which may participate in the virulence against insects. Zymogram assays and cross-reactions of antibodies suggested that two distinct proteases were present. The major one, protease II, was purified and shown to have a molecular mass of 60 kDa and an estimated isoelectric point of 8.5. Protease II digested the chromogenic substrate N-tosyl-Gly-Pro-Arg-paranitroanilide (pNA) with V max and K m values of 0.0551 M/min and 234 M, respectively, and the substrate DL-Val-Leu-Arg-pNA with V max and K m values of 0.3830 M/min and 429 M, respectively. Protease II activity was inhibited 93% by Pefabloc SC and 45% by chymostatin. The optimum pH for protease II was 7, and the optimum temperature was 23°C. Proteolytic activity was reduced by 90% at 60°C for 10 min. Sequence analysis was performed on four internal peptides that resulted from the digestion of protease II. Fragments 29 and 45 are 75 and 68% identical to alkaline metalloproteinase produced by Pseudomonas aeruginosa. Fragment 29 is 79% identical to a metalloprotease of Erwinia amylovora and 75% identical to the protease C precursor of Erwinia chrysanthemi. Protease II showed no toxicity to hemocytes but destroyed antibacterial activity on the hemolymph of inoculated insects' larvae and reduced 97% of the cecropin A bacteriolytic activity.

Bacterial pathogenicity is a result of a combination of factors, including resistance to environmental threats and to the host's defenses, growth capability, localization in the host, tissue specificity, resource obtaining mechanisms and the bacterium's own defenses to aggression. A variety of bacterial components, often specific to each strain, are involved in the microorganism's survival, adhesion and growth in the host. Many of them are harmful and, therefore, are called virulence factors. The effects caused by the virulence factors determine the degree of aggressivity of the strain. In many cases the virulence factors are secreted proteins or enzymes, sometimes performing very specific functions. The enzymatic activity is directed to specific proteins from cell membranes, synaptic vesicle fusion proteins, among other important targets. One of the most toxic bacterial proteins is secreted by Clostridium botulinum, targeted to synaptic vesicle fusion proteins, cleaving them with a zinc-metalloprotease activity, which results in severe neurotoxic effects with a lethal dose as low as eight nanograms per kilogram of body weight. The tetanus neurotoxin acts in a similar way but is less active and Bacillus anthracis also presents a potent metalloprotease activity. In this work we describe a selection of these specially interesting and important bacterial proteins and proteases, stressing their relevance in the pathological process and in medical studies.

Photorhabdus sp. strain Az29 is symbiotic with an Azorean nematode of the genus Heterorhabditis in a complex that is highly virulent to insects even at low temperatures. The virulence of the bacteria is mainly attributed to toxins and bacterial enzymes secreted during parasitism. The bacteria secrete proteases during growth, with a peak at the end of the exponential growth phase. Protease secretion was higher in cultures growing at lower temperatures. At 10°C the activity was highest and remained constant for over 7 days, whereas at 23 and 28°C it showed a steady decrease. Two proteases, PrtA and PrtS, that are produced in the growth medium were purified by liquid chromatography. PrtA was inhibited by 1,10-phenantroline and by EDTA and had a molecular mass of 56 kDa and an optimal activity at pH 9 and 50°C. Sequences of three peptides of PrtA showed strong homologies with alkaline metalloproteases from Photorhabdus temperata K122 and Photorhabdus luminescens W14. Peptide PrtA-36 contained the residues characteristic of metzincins, known to be involved in bacterial virulence. In vitro, PrtA inhibited antibacterial factors of inoculated Lepidoptera and of cecropins A and B. PrtS had a molecular mass of 38 kDa and was inhibited by 1,10-phenanthroline but not by EDTA. Its activity ranged between 10 and 80°C and was optimal at pH 7 and 50°C. PrtS also destroyed insect antibacterial factors. Three fragments of PrtS showed homology with a putative metalloprotease of P. luminescens TTO1. Polyclonal antibody raised against PrtA did not recognize PrtS, showing they are distinct molecules.Photorhabdus spp. are non-free-living Enterobacteriaceae (6, 19). The entomopathogen species of Photorhabdus have a symbiotic relation with nematodes of the genus Heterorhabditis for transport with their infective juveniles (IJs). The IJs actively seek for an insect host, penetrating through its natural openings and cuticle. Inside the insect hemocoel, the bacteria are released and actively multiply avoiding the host defenses and causing an acute disease condition that is followed by insect death within 48 h. The bacteria also create the nutritional conditions and protective environment for the development of its nematode symbiont (2, 21).Bacteria of the Photorhabdus genus produce toxins and other potentially virulent factors (16). The characterized toxins are organized in pathogenic islands (53) and include the Tc complex, which is responsible for oral toxicity (9, 52), and the Mcf toxin, which causes loss of insect body turgor, followed by death (14). Among other potential virulence factors, there is a complex set of extracellular enzymes, including proteases, lipases, lecithinases, chitinases, and phosphatases (5,12,20). Proteases represent an important part of these enzymes, although their role on the virulence process is yet unclear. Until now, all extracellular proteases purified and characterized from Photorhabdus were classified as metalloproteases, but in reports on proteases from different strains it has been suggested that ther...

Snakebites are still a critical public health problem in developing countries or isolated areas. In Brazil, the North Region has a high distribution coefficient worsened by the significant number of eventually unreported cases, due to difficulties in access to health services, to the natural geographic barriers and the vast territory. In the Rio Negro area, the species Bothrops atrox, Bothrops brazili, Lachesis muta muta and Bothriopsis taeniata are thought to be the major species responsible for snakebites. The aim of this study was to qualitatively and quantitatively determine the antigenic cross-reactivity and expression of toxins and the immunogenicity of Bothrops venom species of the Amazon and to evaluate the general efficacy of the therapeutic sera. The in vivo assays demonstrated that the defibrinating activity of B. taeniata venom was absent but that the lethal and hemorrhagic properties were more intense than in the B. atrox venom. The results evidence venom variability among the two B. atrox populations from two distinct Amazonian regions, which may reveal a subjacent speciation process. The results point to new aspects that may guide the improvement of anti-Bothropic therapeutic serum.

The potential for reusability of whey is of concern due to its substantial nutritional value and, second, in view of the need to reduce environmental impact. The use of goat whey powder in the production of a fermented dairy beverage using a Lactobacillus casei culture as adjunct shows a good prospect of a food product with hypotensive activity. This study investigated the microbial viability, proteolysis and angiotensin-converting enzyme (ACE) inhibitory activity of a fermented dairy beverage produced with goat whey powder and a probiotic culture of L. casei BGP93 co-cultured with Streptococcus thermophilus TA-40. The probiotic beverage exhibited no significant difference from the control (absence of L. casei) with regard to titratable acidity, S. thermophilus viability and proteolysis degree during 21 days of storage (P > 0.05). During this period, the beverage maintained L. casei at appropriate levels (> 7 log cfu ml), thereby qualifying as a potential probiotic product. Although both control and probiotic beverages exhibited ACE inhibitory activity, as a result of proteolysis of whey proteins during fermentation, significant increased ACE inhibitory activity was found for the beverage with added probiotic L. casei (P < 0.05). The probiotic beverage has potency as a functional food candidate to be included in a dietary strategy aiming at prevention and control of hypertension.