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Authors: Manja Boehm, Ingrid Haenel, Benjamin Hoy, Lone Brøndsted, Todd G. Smith, Timothy Hoover, Silja Wessler and Nicole Tegtmeyer

Abstract

The serine protease HtrA of C. jejuni has been identified as a novel secreted virulence factor which opens cell-to-cell junctions by cleaving E-cadherin. Efficient C. jejuni transmigration across polarized human epithelial cells requires the intact flagellum and HtrA; however, the mechanism of HtrA secretion into the supernatant is unknown. Here we show that HtrA secretion is highly efficient and does not require its proteolytic activity because the protease-inactive S197A mutant is secreted like wild-type HtrA. In addition, the flagellar mutants ΔflaA/B, ΔfliI, ΔflgH, ΔflhA, ΔflhB, and ΔflgS were also able to secrete HtrA in high amounts, while they were strongly attenuated in secreting the well-known invasion antigen CiaB. We also tested several culture media and cell lines of different origin such as human, mouse, hamster, dog, and chicken for their ability to influence HtrA secretion. Interestingly, HtrA was effectively secreted in the presence of most but not all cell lines and media, albeit at different levels, but secretion was significantly higher when fetal calf serum (FCS) was added. These results demonstrate that HtrA secretion by Campylobacter proceeds independent of HtrA's protease activity, the flagellum and origin of cell lines, but can be strongly enhanced by molecular compound(s) present in FCS.

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Proteases constitute a significant part of cell wall-degrading enzymes (CWDEs) produced by fungal biocontrol agents and particularly crucial in mycoparasitism of fungal phytopathogens. Plate-based screening methods are routinely used for screening protease-producing microorganisms including fungi. Skim milk agar (SMA) is one of the most popular media for the detection of protease producing bacteria. However, SMA is not efficient to test fast growing fungi, because it does not give an estimation of the actual amount of secreted protease produced by fungal inocula. In the current study, the efficacy of two modified plate-screening methods, including split-SMA (SSMA) and minimal medium supplemented with skim milk (MSMW) was assessed for detection of protease production by three representative fungal strains including Trichoderma longibrachiatum strain N, Beauveria bassiana strain B and Purpureocillium lilacinum strain PL. Protease production was revealed on the three tested media by the three strains. However, the halo diameter of the fungal strains (a proxy for protease production) was the smallest on SMA. Furthermore, protease production could not be detected for T. longibrachiatum strain N on SMA due to its fast growth; while it showed the highest protease activity on both modified media compared with the other strains. According to the result of this study, the SSMA medium is an easy and more accurate method compared with the two other different methods as it displays the actual amount of protease produced by fungal strains and therefore this method is recommended for quantitative and qualitative detection of protease production by slow and fast growing fungi.

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The hypersensitive response (HR), a type of programmed cell death (PCD) during biotic stress is mediated by salicylic acid (SA). The aim of this work was to reveal the role of proteolysis and cysteine proteases in the execution of PCD in response of SA. Tomato plants were treated with sublethal (0.1 mM) and lethal (1 mM) SA concentrations through the root system. Treatment with 1 mM SA increased the electrolyte leakage and proteolytic activity and reduced the total protein content of roots after 6 h, while the proteolytic activity did not change in the leaves and in plants exposed to 0.1 mM SA. The expression of the papain-type cysteine protease SlCYP1, the vacuolar processing enzyme SlVPE1 and the tomato metacaspase SlMCA1 was induced within the first three hours in the leaves and after 0.5 h in the roots in the presence of 1 mM SA but the transcript levels did not increase significantly at sublethal SA. The Bax inhibitor-1 (SlBI-1), an antiapoptotic gene was over-expressed in the roots after SA treatments and it proved to be transient in the presence of sublethal SA. Protease inhibitors, SlPI2 and SlLTC were upregulated in the roots by sublethal SA but their expression remained low at 1 mM SA concentration. It is concluded that in contrast to leaves the SA-induced PCD is associated with increased proteolytic activity in the root tissues resulting from a fast up-regulation of specific cysteine proteases and down-regulation of protease inhibitors.

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The toxic effects of cylindrospermopsin (cyanobacterial toxin) on animals have been examined extensively, but little research has focused on their effects on plants. In this study cylindrospermopsin (CYN) caused alterations of growth, soluble protein content and protease enzyme activity were studied on two aquatic plants Lemna minor and Wolffia arrhiza in short-term (5 days) experiments. For the treatments we used CYN containing crude extracts of Aphanizomenon ovalisporum (BGSD-423) and purified CYN as well. The maximal inhibitory effects on fresh weight of L. minor and W. arrhiza caused by crude extract were 60% and 54%, respectively, while the maximum inhibitory effects were 30% and 43% in the case of purified CYN at 20 μg ml−1 CYN content of culture medium. In CYN-treated plants the concentration of soluble protein showed mild increases, especially in W. arrhiza. Protease isoenzyme activity gels showed significant alterations of enzyme activities under the influence of CYN. Several isoenzymes were far more active and new ones appeared in CYN-treated plants. Treatments with cyanobacterial crude extract caused stronger effects than the purified cyanobacterial toxins used in equivalent CYN concentrations.

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low protease activity were performed for high protease activity flour HPAWF in baking studies. In addition, effects of these applications were repeated for low protease activity flour (LPAWF). 1 Materials and methods 1.1 Materials A sound (undamaged

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. , Gu , Z. & Wen , H. ( 2011 ). A preliminary study of the protease activities in germinating brown rice (Oryza sativa L.) . J . Sci. Food Agr. , 91 , 915 – 920

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Authors: L. Kredics, Zsuzsanna Antal, A. Szekeres, L. Hatvani, L. Manczinger, Cs. Vágvölgyi and Erzsébet Nagy

Eur J Biochem 173 179 184 Hagspiel, K., Haab, D., Kubicek, C. P.: Protease activity and proteolytic

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The germination and water uptake of Vicia faba seeds were suppressed in response to the treatments with the different concentrations of sea water (5%, 25% and 50%). The following parameters were increased: the osmotic potential, Na+, Cl, proline and protease activity. While K+, K+/Na+ ratio, Ca2+, amylase activity and total soluble sugars were decreased. Gibberellic acid treatments to the seeds counteracted the harmful effect which were induced by sea water treatments. In turn the germination percentage, water uptake, K+, K+/Na+ ratio, Ca2+, total soluble sugars, a-amylase and protease activities were increased, while Na+, Cl, and proline were decreased. The changes in protein banding pattern in Vicia faba germinated seeds in sea water were investigated. Salinization induced de novo synthesis of some salt responsive proteins. The salt responsive proteins might be osmotin (M wt 23.39 and 26.86 KDa), dehydrin (36.79 and 40.63 KDa) and ubiquitin (8.81 KDa) which were apparent in Vicia faba seeds. The seeds priming soaking in GA and germinated in sea water induced de novo synthesis of some responsive proteins. This indicated that gibberellic acid had a synergistic effect on the induction of the salt gene which is responsible for the synthesis of the mentioned proteins.

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The ability of Kocuria varians to grow and produce protease when utilizing various local wastes was studied. Impact of cultivation pH on growth and enzyme production was also evaluated. Cassava waste combined with bambara nut waste (1:1) gave the best protease yield. Maximum enzyme production was attained when production medium was adjusted to pH 9. Highest protease concentration in the culture fluid was recorded at 20 h during the exponential phase of growth. The enzyme was optimally active and stable at 80 °C. Optimum pH for protease activity was at 11 with optimal stability at the alkaline range (pH 7–11) after incubation for 1 h. The enzyme was inhibited by EDTA, Hg2+, and Zn2+, but not by Pb2+, and was slightly stimulated by Cu2+. The properties of this protease make it a promising candidate for further studies and possible applications in processes involving extreme conditions of pH and temperature.

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Authors: L. Kredics, Zsuzsanna Antal, A. Szekeres, L. Manczinger, Ilona Dóczi, F. Kevei and Elisabeth Nagy

Species belonging to the filamentous fungal genus Trichoderma are well known as potential candidates for the biological control of plant pathogenic fungi and as cellulase producers of biotechnological importance. Several data were published in the last decade also about the clinical importance of this genus, indicating that Trichoderma strains may be potential opportunistic pathogens in immunocompromised patients. However, there is a lack of information about the potential virulence factors of clinical Trichoderma strains. This study was designed to examine the extracellular proteolytic enzymes of six clinical T. longibrachiatum isolates. Supernatants from induced liquid cultures of the examined strains were screened for proteolytic enzyme activities with 11 different chromogenic p-nitroaniline substrates. The production of trypsin-like, chymotrypsin-like and chymoelastase-like protease activities cleaving N-Benzoyl-L-Phe-L-Val-L-Arg-p-nitroanilide, N-Succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide,and N-Succinyl-L- Ala-L-Ala-L-Pro-L-Leu-p-nitroanilide, respectively, was common among the strains examined. Separation of trypsin- and chymotrypsin-like activities by column chromatography revealed, that both systems are complex consisting of several isoenzymes. The pH-dependence of these two protease systems was also studied. Based on the results, the different isoenzymes seem to have different optimal pH values. Extracellular proteolytic enzymes may be involved in the pathogenecity of Trichoderma strains as facultative human pathogens.

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