Differential in vitro and in vivo effect of barley cysteine and serine protease inhibitors on phytopathogenic microorganisms

Protease inhibitors from plants have been involved in defence mechanisms against pests and pathogens. Phytocystatins and trypsin/α-amylase inhibitors are two of the best characterized protease inhibitor families in plants. In barley, thirteen cystatins (HvCPI-1 to 13) and the BTI-CMe trypsin inhibitor have been previously studied. Their capacity to inhibit pest digestive proteases, and the negative in vivo effect caused by plants expressing these inhibitors on pests support the defence function of these proteins. Barley cystatins are also able to inhibit in vitro fungal growth. However, the antifungal effect of these inhibitors in vivo had not been previously tested. Moreover, their in vitro and in vivo effect on plant pathogenous bacteria is still unknown. In order to obtain new insights on this feature, in vitro assays were made against different bacterial and fungal pathogens of plants using the trypsin inhibitor BTI-CMe and the thirteen barley cystatins. Most barley cystatins and the BTI-CMe inhibitor were able to inhibit mycelial growth but no bacterial growth. Transgenic Arabidopsis plants independently expressing the BTI-CMe inhibitor and the cystatin HvCPI-6 were tested against the same bacterial and fungal pathogens. Neither the HvCPI-6 expressing transgenic plants nor the BTI-CMe ones were more resistant to plant pathogen fungi and bacteria than control Arabidopsis plants. The differences observed between the in vitro and in planta assays against phytopathogenic fungi are discusse

Degradation of human kininogens with the release of kinin peptides by extracellular proteinases of Candida spp.

The secretion of proteolytic enzymes by pathogenic microorganisms is one of the most successful strategies used by pathogens to colonize and infect the host organism. The extracellular microbial proteinases can seriously deregulate the homeostatic proteolytic cascades of the host, including the kinin-forming system, repeatedly reported to he activated during bacterial infection. The current study assigns a kinin-releasing activity to secreted proteinases of Candida spp. yeasts, the major fungal pathogens of humans. Of several Candida species studied, C. parapsilosis and C. albicans in their invasive filamentous forms are shown to produce proteinases which most effectively degrade proteinaceous kinin precursors, the kininogens. These enzymes, classified as aspartyl proteinases, have the highest kininogen-degrading activity at low pH (approx. 3.5), but the associated production of bradykinin-related peptides from a small fraction of kininogen molecules is optimal at neutral pH (6.5). The peptides effectively interact with cellular B2-type kinin receptors. Moreover, kinin-related peptides capable of interacting with inflammation-induced B1-type receptors are also formed, but with a reversed pH dependence. The presented variability of the potential extracellular kinin production by secreted aspartyl proteinases of Candida spp. is consistent with the known adaptability of these opportunistic pathogens to different niches in the host organism

Characterization of serine proteinase expression in agaricus bisporus and coprinopsis cinerea by using green fluorescent protein and the A. bisporus SPR1 Promoter

The Agaricus bisporus serine proteinase 1 (SPR1) appears to be significant in both mycelial nutrition and senescence of the fruiting body. We report on the construction of an SPR promoter::green fluorescent protein (GFP) fusion cassette, pGreen_hph1_SPR_GFP, for the investigation of temporal and developmental expression of SPR1 in homobasidiomycetes and to determine how expression is linked to physiological and environmental stimuli. Monitoring of A. bisporus pGreen_hph1_SPR_GFP transformants on media rich in ammonia or containing different nitrogen sources demonstrated that SPR1 is produced in response to available nitrogen. In A. bisporus fruiting bodies, GFP activity was localized to the stipe of postharvest senescing sporophores. pGreen_hph1_SPR_GFP was also transformed into the model basidiomycete Coprinopsis cinerea. Endogenous C. cinerea proteinase activity was profiled during liquid culture and fruiting body development. Maximum activity was observed in the mature cap, while activity dropped during autolysis. Analysis of the C. cinerea genome revealed seven genes showing significant homology to the A. bisporus SPR1 and SPR2 genes. These genes contain the aspartic acid, histidine, and serine residues common to serine proteinases. Analysis of the promoter regions revealed at least one CreA and several AreA regulatory motifs in all sequences. Fruiting was induced in C. cinerea dikaryons, and fluorescence was determined in different developmental stages. GFP expression was observed throughout the life cycle, demonstrating that serine proteinase can be active in all stages of C. cinerea fruiting body development. Serine proteinase expression (GFP fluorescence) was most concentrated during development of young tissue, which may be indicative of high protein turnover during cell differentiatio

The role of Moraxella catarrhalis outer membrane proteins in pathogenesis

Moraxella catarrhalis is an important respiratory pathogen. It is a common cause of otitis media in children, a usual infections agent in sinusitis and makes up to 10% of all exacerbations of chronic obstructive pulmonary disease (COPD). It is known to cause more frequent disease in the colder season, and, generally, its importance is recognised mainly in countries exposed to moderate climate. M. catarrhalis widely colonises infants and children but it is very uncommon for the bacterium to colonise the mucosa in adults. The present thesis is concentrated on discussing two properties of M. catarrhalis, - binding and inactivation of alpha1-anticnymotrypsin (ACT) and complement component 3 (C3) for which M. catarrhalis outer membrane proteins “ubiquitous surface proteins” (Usp) A1/A2 are responsible. No other pathogenic bacterium is known to bind ACT. The ACT is a serine protease inhibitor playing important part in antiprotease lung protection and anti-inflammatory signalling. We established that M. catarrhalis is able to bind and inactivate ACT through the two active domains on both UspA1 and A2 proteins. The phenomenon is proposed to be important for a) protease-antiprotease balance in the lung, for b) exacerbating inflammation and for c) inhibition of phagocytic clearance of M. catarrhalis. The localization of ACT binding inside the UspA1 and A2 proteins provided us with means to develop a simple and quick method for purification of ACT from plasma or serum based on ACT affinity to a fragment of UspA2 protein of M. catarrhalis. The purification can be carried out practically in one step and the biological properties of ACT are retained. In the last part of the thesis the C3 binding to M. catarrhalis Usp A1 and A2 was studied. The binding has been attributed to a single fragment in the both UspA1 and A2. Importantly, C3d fragment of C3 has been found responsible for the binding to UspA1/A2. The UspA2 has been demonstrated to inhibit both the classical and the alternative pathways of the complement. The C3 binding by M. catarrhalis strongly correlated with serum resistance of the bacterium, as well as with the expression of UspA1/A2

Expressed sequence tags from the oomycete fish pathogen Saprolegnia parasitica reveal putative virulence factors

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The cell envelope subtilisin-like proteinase is a virulence determinant for Streptococcus suis

<p>Abstract</p> <p>Background</p> <p><it>Streptococcus suis </it>is a major swine pathogen and zoonotic agent that mainly causes septicemia, meningitis, and endocarditis. It has recently been suggested that proteinases produced by <it>S. suis </it>(serotype 2) are potential virulence determinants. In the present study, we screened a <it>S. suis </it>mutant library created by the insertion of Tn<it>917 </it>transposon in order to isolate a mutant deficient in a cell surface proteinase. We characterized the gene and assessed the proteinase for its potential as a virulence factor.</p> <p>Results</p> <p>Two mutants (G6G and M3G) possessing a single Tn<it>917 </it>insertion were isolated. The affected gene coded for a protein (SSU0757) that shared a high degree of identity with <it>Streptococccus thermophilus </it>PrtS (95.9%) and, to a lesser extent, with <it>Streptococcus agalactiae </it>CspA (49.5%), which are cell surface serine proteinases. The SSU0757 protein had a calculated molecular mass of 169.6 kDa and contained the catalytic triad characteristic of subtilisin family proteinases: motif I (Asp₂₀₀), motif II (His₂₃₉), and motif III (Ser₅₆₈). SSU0757 also had the Gram-positive cell wall anchoring motif (Leu-Pro-X-Thr-Gly) at the carboxy-terminus, which was followed by a hydrophobic domain. All the <it>S. suis </it>isolates tested, which belonged to different serotypes, possessed the gene encoding the SSU0757 protein. The two mutants devoid of subtilisin-like proteinase activity had longer generation times and were more susceptible to killing by whole blood than the wild-type parent strain P1/7. The virulence of the G6G and M3G mutants was compared to the wild-type strain in the CD1 mouse model. Significant differences in mortality rates were noted between the P1/7 group and the M3G and G6G groups (<it>p </it>< 0.001).</p> <p>Conclusion</p> <p>In summary, we identified a gene coding for a cell surface subtilisin-like serine proteinase that is widely distributed in <it>S. suis</it>. Evidences were brought for the involvement of this proteinase in <it>S. suis </it>virulence.</p

Alimentary tract proteinases of the Southern corn rootworm (Diabrotica undecimpunctata howardi) and the potential of potato Kunitz proteinase inhibitors for larval control.

Proteolytic digestion by larval Diabrotica undecimpunctata howardi (Barber) (D.undecimpunctata) has been investigated with the aim of producing transgenic plants possessing enhanced resistance to this economically important crop pest. Biochemical characterisation in vitro by pH dependent hydrolysis and inhibition assays incorporating E-64, pepstatin A and soybean Kunitz trypsin inhibitor showed the majority of hydrolytic activity occurs at pH 5.5 and is performed by cysteine and aspartic endopeptidases. Cysteine and aspartic proteinase encoding clones were isolated from a larval alimentary tract cDNA library. Four cathepsin L-like cysteine proteinases and two cathepsin D-like aspartic proteinase cDNA clones were identified by codmg homology to known proteinase sequences. Analysis of primary and secondary sequence features revealed D. undecimpunctata aspartic proteinase 1 exhibits features associated with cathepsins E and is proposed to be a D. undecimpunctata cathepsm E-like aspartic proteinase.Cathepsin D-like aspartic proteinase inhibitors of the potato Kunitz protemase inhibitor (PKPI) family have been isolated by PCR and expressed employing the pET expression system (Novagen). In vitro assays demonstrated the inhibitory activity of PKPI-A and PKPl-B inhibitors against larval D. undecimpunctata alimentary tract proteolytic enzymes. To the authors knowledge this work represents the first reporting of the expression and purification of biologically active PKPI proteins. In vitro assays incorporating oryzacystatin I and PKPI proteins resulted in increased inhibition of proteolytic activity compared to single inhibitor and uninhibited control reactions. Inhibition assays provide evidence for the potential of a dual protemase inhibitor strategy to arrest protein hydrolysis by larval D. undecimpunctata, preventing essential amino acid absorption. Further research is necessary to characterise the properties of the digestive enzymes isolated in this work and the inhibitory spectrum of PKPI proteins. Transgenic crops expressing a combination of oryzacystatin and PKPI proteins would be predicted to show enhanced resistance to insect herbivores by virtue of digestive proteolysis inhibition