Levels Of Extracellular Protease Research Articles

Role of sodium pyruvate in maintaining the survival and cytotoxicity of Staphylococcus aureus under high glucose conditions.

Glucose is a crucial carbon source for the growth of Staphylococcus aureus, but an excess of glucose is detrimental and even leads to cell death. Pyruvate, the central metabolite of glycolysis, has been shown to have anti-inflammatory and antioxidant properties. This study aimed to investigate the protective effect of pyruvate on S. aureus under high glucose conditions. Sodium pyruvate greatly increased the cytotoxicity of S. aureus strain BAA-1717 to human erythrocytes and neutrophils in vitro. However, the cytotoxicity and survival of S. aureus were significantly reduced by high glucose, which was restored to normal levels by the addition of sodium pyruvate. The expression of hlg and lukS in S. aureus was higher in the LB-GP cultures than that in LB-G cultures, but there was no significant difference in cytotoxicity between LB-GP and LB-G cultures. Furthermore, the hemolytic activity of S. aureus supernatants could be inhibited by the cell-free culture medium (CFCM) of LB-G cultures, suggesting that high levels of extracellular proteases were presence in the CFCM of LB-G cultures, resulting in degradation of the hemolytic factors. The expression of sarA, which negatively regulates extracellular protease secretion, was higher in LB-GP cultures than that in LB-G cultures. Additionally, sodium pyruvate increased acetate production in S. aureus, which helps maintain cell viability under acidic environment. In conclusion, pyruvate plays an important role in the survival and cytotoxicity of S. aureus under high glucose conditions. This finding may aid in the development of effective treatments for diabetic foot infections.

<p><strong>Statistical optimization of alkaline protease from <em>Bacillus amyloliquefaciens </em>SP1</strong></p>

Alkaline protease has tremendous applications in different industries, and to fulfil industrial and commercial enzyme requirements, overproduction and optimization of production medium are prerequisites. Protease production by Bacillus amyloliquefaciens SP1, isolated from apple tree rhizosphere was studied. Plackett Burmann design and response surface methodology was used to optimize medium for alkaline protease production. Higher levels of protease activity were observed in presence of disaccharides owing to diauxic growth and inorganic nitrogen sources proved to be less favourable. After screening through one-factor-at-a time experiments and Plackett Burmann design, casein, yeast extract, maltose and KH2PO4 were identified as the most significant variables. Statistical design based three-dimensional (3-D) and contour plots were generated to understand the relationship between enzyme activity and medium variables. The protease production was found to increase from 1730 to 3630 µg/ml/min in optimized medium i.e. approx. 2.1 fold increased compared with original medium. The study assumes significance in the ability of bacterium to survive in wide variety of salts and agricultural wastes and yield optimum levels of extracellular protease.

The rpf/DSF signalling system of Stenotrophomonas maltophilia positively regulates biofilm formation, production of virulence-associated factors and β-lactamase induction.

Stenotrophomonas maltophilia is a multidrug-resistant opportunistic pathogen. S. maltophilia quorum-sensing system is mediated by the diffusible signal factor (DSF), which synthesis depends on rpfF. It has been reported that rpfF disruption in S. maltophilia K279a leads to a loss of DSF synthesis, reduced levels of extracellular protease, swarming motility and virulence in the Galleria mellonella model. The aim of this work was to attain a deeper knowledge of the role of the rpf/DSF signalling system in S. maltophilia biofilm formation, phenotypic traits associated with biofilm development and virulence and antimicrobial susceptibility. To this end, comparative studies were conducted on S. maltophilia K279a and K279arpfF. The results presented here put in evidence the positive role of DSF in bacterial growth, biofilm formation, swimming and twitching motilities, DNAse, lipases and siderophores production as well as resistance to oxidative stress. Interestingly, DSF seems to be essential for the development of the spatially organised structure seen in mature biofilms. Therefore, DSF from S. maltophlia K279a positively regulates biofilm formation and virulence. Furthermore, DSF is necessary for the induction of L1 and L2 β-lactamase production in K279a. This is the first evidence of the role of the rpf/DSF signalling system in S. maltophilia β-lactam resistance.

Label-Free Proteomic Approach to Characterize Protease-Dependent and -Independent Effects of sarA Inactivation on the Staphylococcus aureus Exoproteome.

The staphylococcal accessory regulator A (sarA) impacts the extracellular accumulation of Staphylococcus aureus virulence factors at the level of intracellular production and extracellular protease-mediated degradation. We previously used a proteomics approach that measures protein abundance of all proteoforms to demonstrate that mutation of sarA results in increased levels of extracellular proteases and assesses the impact of this on the accumulation of S. aureus exoproteins. Our previous approach was limited as it did not take into account that large, stable proteolytic products from a given protein could result in false negatives when quantified by total proteoforms. Here, our goal was to use an expanded proteomics approach utilizing a dual quantitative method for measuring abundance at both the total proteoform and full-length exoprotein levels to alleviate these false negatives and thereby provide for characterization of protease-dependent and -independent effects of sarA mutation on the S. aureus exoproteome. Proteins present in conditioned medium from overnight, stationary phase cultures of the USA300 strain LAC, an isogenic sarA mutant, and a sarA mutant unable to produce any of the known extracellular proteases (sarA/protease) were resolved using one-dimensional gel electrophoresis. Quantitative proteomic comparisons of sarA versus sarA/protease mutants identified proteins that were cleaved in a protease-dependent manner owing to mutation of sarA, and comparisons of sarA/protease mutant versus the LAC parent strain identified proteins in which abundance was altered in a sarA mutant in a protease-independent manner. Furthermore, the proteins uniquely identified by the full-length data analysis approach eliminated false negatives observed in the total proteoform analysis. This expanded approach provided for a more comprehensive analysis of the impact of mutating sarA on the S. aureus exoproteome.

Cell Surface Human Airway Trypsin-Like Protease Is Lost During Squamous Cell Carcinogenesis.

Cancer progression is accompanied by increased levels of extracellular proteases that are capable of remodeling the extracellular matrix, as well as cleaving and activating growth factors and receptors that are involved in pro‐cancerous signaling pathways. Several members of the type II transmembrane serine protease (TTSP) family have been shown to play critical roles in cancer progression, however, the expression or function of the TTSP Human Airway Trypsin‐like protease (HAT) in carcinogenesis has not been examined. In the present study we aimed to determine the expression of HAT during squamous cell carcinogenesis. HAT transcript is present in several tissues containing stratified squamous epithelium and decreased expression is observed in carcinomas. We determined that HAT protein is consistently expressed on the cell surface in suprabasal/apical layers of squamous cells in healthy cervical and esophageal epithelia. To assess whether HAT protein is differentially expressed in normal tissue versus tissue in different stages of carcinogenesis, we performed a comprehensive immunohistochemical analysis of HAT protein expression levels and localization in arrays of paraffin embedded human cervical and esophageal carcinomas compared to the corresponding normal tissue. We found that HAT protein is expressed in the non‐proliferating, differentiated cellular strata and is lost during the dedifferentiation of epithelial cells, a hallmark of squamous cell carcinogenesis. Thus, HAT expression may potentially be useful as a marker for clinical grading and assessment of patient prognosis in squamous cell carcinomas. J. Cell. Physiol. 231: 1476–1483, 2016. © 2015 Wiley Periodicals, Inc.

Comparative impact of diverse regulatory loci on Staphylococcus aureus biofilm formation.

The relative impact of 23 mutations on biofilm formation was evaluated in the USA300, methicillin-resistant strain LAC. Mutation of sarA, atl, codY, rsbU, and sigB limited biofilm formation in comparison to the parent strain, but the limitation imposed by mutation of sarA was greater than that imposed by mutation of any of these other genes. The reduced biofilm formation of all mutants other than the atl mutant was correlated with increased levels of extracellular proteases. Mutation of fur- and mgrA-enhanced biofilm formation but in LAC had no impact on protease activity, nuclease activity, or accumulation of the polysaccharide intercellular adhesin (PIA). The increased capacity of these mutants to form a biofilm was reversed by mutation of sarA, and this was correlated with increased protease production. Mutation of sarA, mgrA, and sigB had the same phenotypic effect in the methicillin-sensitive strain UAMS-1, but mutation of codY increased rather than decreased biofilm formation. As with the UAMS-1 mgrA mutant, this was correlated with increased production of PIA. Examination of four additional clinical isolates suggests that the differential impact of codY on biofilm formation may be a conserved characteristic of methicillin-resistant versus methicillin-sensitive strains.

New Promoters to Improve Heterologous Protein Production in Aspergillus vadensis

Aspergillus is a widely used host organism for the industrial production of homologous and heterologous proteins. Although Aspergillus niger is most commonly used, a close relative of this species, Aspergillus vadensis, has been suggested as a suitable and more favourable alternative due in part, to the low levels of extracellular proteases which it produces. Despite much progress being made in the hyper production of homologous proteins, the yields obtained for heterologous proteins have still not reached a comparable level. Genetic strategies, including the development of strong constitutional promoters have been shown to lead to an increase in the levels of recombinant protein production. In this study, six novel constitutional promoters from A. niger (pef1α, ptktA, pef1β, ptal1, pcetA and ppgkA) and a further five from A. vadensis (pef1α, prps31, pgpdA, pubi1 and poliC) were tested in A. vadensis using a gene encoding a secreted arabinofuranosidase from Fusarium oxysporum as a reporter for heterologous protein production. Remarkably, 9 of the 11 promoter constructs tested resulted in higher ABF activity than the commonly used gpdA promoter. While this could partly be assigned to the number of copies of the expression cassette in the transformants, clear differences in productivity of the promoters could be observed.

Abstract 420: Critical role of MMP-2 and GATA-1 nuclear interaction in the regulation of ionizing radiation (IR)-induced IL-10/Stat3-mediated invasion and migration in glioblastoma.

Abstract Glioblastoma multiforme (GBM) is the most lethal human brain tumor with high recurrence rates despite the existence of multimodal treatments including chemotherapy and radiation after surgical tumor resection. Augmented tumor metastasis after irradiation (IR) has been attributed to enhanced extracellular protease levels in tumors. Secreted or cleaved matrix metalloproteinase-2 (MMP-2) levels were reported to be elevated in irradiated tumors. Interestingly, previous in silico prediction analyses identified the presence of a putative nuclear localization signal (NLS) in MMP-2 and reported the cleavage of PARP by nuclear-MMP-2. However, MMP-2 nuclear localization in possible correlation with tumor progression has not been identified to date. In addition to membrane and cytoplasmic expression of MMP-2, we observed prominent MMP-2 nuclear localization, which is further elevated in IR (8 Gy)-treated human glioma xenograft cells 4910 and 5310. Interestingly, The protein-transcription factor (TF) interaction arrays using 4910 nuclear extracts revealed the potential binding of nuclear-MMP-2 with several transcription factors including CEF-2, c-Rel, FOXO4, GAS, GATA-1, MUSF-1, p300, PARP and RREB1. Co-IP experiments showed the direct binding between MMP-2/GATA-1 in glioma. Further, we also observed the presence of MMP-2 in the GATA-1 DNA-binding complex and its recruitment onto the promoter of IL-10, a GATA-1 target gene. ChIP-cloning with anti-MMP-2 antibody confirmed the specific binding of MMP-2 with GATA-1 consensus elements. We also observed that the MMP-2/GATA-1 nuclear co-localization was elevated in correlation with increasing GBM pathological grades. Downregulation of MMP-2 inhibited the GATA-1 expression and DNA-binding activity and abrogated IR-induced migration and invasion in these cells. Conversely, treatment with recombinant MMP-2 protein elevated the GATA-1 DNA-binding activity and increased IL-10 and phospho-Stat3 expression in correlation with increased invasion and migration. In vivo orthotopic experiments with MMP-2.siRNA decreased tumor growth in nude mice and inhibited nuclear MMP-2 and GATA-1 expression levels in tumors. To our knowledge, this is the first study demonstrating significant IR-induced nuclear translocation of MMP-2 and shows the functional association between MMP-2/GATA-1 in the regulation of IL-10/Stat3-mediated migration and invasion. Our results suggest the potential therapeutic efficacy of targeting MMP-2/GATA-1 liaison in glioma treatment. Citation Format: Divya Kesanakurti, Dilip Maddirela, Meena Gujrati, Jasti S. Rao. Critical role of MMP-2 and GATA-1 nuclear interaction in the regulation of ionizing radiation (IR)-induced IL-10/Stat3-mediated invasion and migration in glioblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 420. doi:10.1158/1538-7445.AM2013-420

Ion Beam Mutagenesis in Arthrobotrys oligospora Enhances Nematode-Trapping Ability

The nematode-trapping fungus Arthrobotrys oligospora is able to produce extracellular protease that degrades the body walls of parasitic nematode larvae found in livestock and immobilizes the nematodes. Our aim was to obtain a strain of A. oligospora with a strong ability to trap nematodes by production of high levels of extracellular protease. A wild type strain of A. oligospora was subjected to mutagenic treatments involving low-energy ion beam implantation to generate mutants. Among these mutants, A. oligospora N showed high efficiency in trapping nematodes and was also able to secrete more extracellular protease, helping it to penetrate and digest the body walls of larvae. This work represents the first application of low-energy ion beams to generate mutations in a nematode-trapping fungus, and provides a new method of obtaining a fungus with high potential application.

Strong expression association between matriptase and its substrate prostasin in breast cancer

Breast cancer tumorigenesis is accompanied by increased levels of extracellular proteases that are capable of remodeling the extracellular matrix as well as cleaving and activating growth factors and signaling receptors that are critically involved in neoplastic progression. Multiple studies implicate the membrane anchored serine protease matriptase (also known as MT-SP1 and epithin) in breast cancer. The pro-form of the GPI-anchored serine protease prostasin has recently been identified as a physiological substrate of matriptase and the two proteases are co-expressed in multiple healthy tissues. In this study, the inter-relationship between the two membrane-anchored serine proteases in breast cancer was investigated using breast cancer cell lines and breast cancer patient samples to delineate the association between matriptase and prostasin. We used Western blotting to determine the expression of matriptase and prostasin proteins in a panel of breast cancer cell lines and immunohistochemistry to assess the expression in serial sections from breast cancer tissue arrays. We demonstrate that the expression of matriptase and prostasin is closely correlated in breast cancer cell lines as well as in breast cancer tissue samples. Furthermore, matriptase and prostasin display a near identical spatial expression pattern in the epithelial compartment of breast cancer tissue. These data suggest that the matriptase-prostasin cascade might play a critical role in breast cancer.

Modification of genetic regulation of a heterologous chitosanase gene in Streptomyces lividans TK24 leads to chitosanase production in the absence of chitosan

BackgroundChitosanases are enzymes hydrolysing chitosan, a β-1,4 linked D-glucosamine bio-polymer. Chitosan oligosaccharides have numerous emerging applications and chitosanases can be used for industrial enzymatic hydrolysis of chitosan. These extracellular enzymes, produced by many organisms including fungi and bacteria, are well studied at the biochemical and enzymatic level but very few works were dedicated to the regulation of their gene expression. This is the first study on the genetic regulation of a heterologous chitosanase gene (csnN106) in Streptomyces lividans.ResultsTwo S. lividans strains were used for induction experiments: the wild type strain and its mutant (ΔcsnR), harbouring an in-frame deletion of the csnR gene, encoding a negative transcriptional regulator. Comparison of chitosanase levels in various media indicated that CsnR regulates negatively the expression of the heterologous chitosanase gene csnN106. Using the ΔcsnR host and a mutated csnN106 gene with a modified transcription operator, substantial levels of chitosanase could be produced in the absence of chitosan, using inexpensive medium components. Furthermore, chitosanase production was of higher quality as lower levels of extracellular protease and protein contaminants were observed.ConclusionsThis new chitosanase production system is of interest for biotechnology as only common media components are used and enzyme of high degree of purity is obtained directly in the culture supernatant.

Loss of the matriptase inhibitor HAI‐2 during prostate cancer progression

Prostate cancer progression is accompanied by increased levels of extracellular proteases that are capable of remodeling the extracellular matrix as well as cleaving and activating growth factors and their receptors that are critically involved in pro-cancerous signaling pathways. The membrane anchored serine protease matriptase (also known as MT-SP1, epithin, and TADG15) has been implicated in prostate cancer. Several studies have demonstrated that the expression of this protease, both on the RNA and protein level is significantly increased during prostate cancer progression. Hepatocyte activator growth factor inhibitor-2 (HAI-2) has recently been identified as a physiological inhibitor of matriptase. It has been proposed that the increase of matriptase with a concomitant loss of its inhibitors may play a critical role in cancer progression. In this study, we used immunohistochemistry to determine the expression of HAI-2 protein in 136 prostate cancer samples, 20 prostate benign prostatic hyperplasia (BPH) samples, and 31 normal or tumor-adjacent prostate samples. Specificity of detection was ensured by using two unrelated HAI-2 antibodies and corresponding non-immune IgG antibodies. We demonstrate that HAI-2 protein is significantly decreased in malignant lesions as compared to normal and BPH lesions, and that the most poorly differentiated tumors (Gleason score 8-10) have the lowest level of HAI-2 expression. These data suggest that the loss of HAI-2 may be actively involved in prostate cancer progression by causing a reduced inhibitory capacity of proteolysis possibly of the physiological target for HAI-2 matriptase.

Optimization of Protease Secretion in Bacillus subtilis and Bacillus licheniformis by Screening of Homologous and Heterologous Signal Peptides

Bacillus subtilis and Bacillus licheniformis are widely used for the large-scale industrial production of proteins. These strains can efficiently secrete proteins into the culture medium using the general secretion (Sec) pathway. A characteristic feature of all secreted proteins is their N-terminal signal peptides, which are recognized by the secretion machinery. Here, we have studied the production of an industrially important secreted protease, namely, subtilisin BPN' from Bacillus amyloliquefaciens. One hundred seventy-three signal peptides originating from B. subtilis and 220 signal peptides from the B. licheniformis type strain were fused to this secretion target and expressed in B. subtilis, and the resulting library was analyzed by high-throughput screening for extracellular proteolytic activity. We have identified a number of signal peptides originating from both organisms which produced significantly increased yield of the secreted protease. Interestingly, we observed that levels of extracellular protease were improved not only in B. subtilis, which was used as the screening host, but also in two different B. licheniformis strains. To date, it is impossible to predict which signal peptide will result in better secretion and thus an improved yield of a given extracellular target protein. Our data show that screening a library consisting of homologous and heterologous signal peptides fused to a target protein can identify more-effective signal peptides, resulting in improved protein export not only in the original screening host but also in different production strains.

Production of extracellular protease from halotolerant bacterium, Bacillus aquimaris strain VITP4 isolated from Kumta coast

A newly isolated halotolerant Bacillus sp. VITP4 was investigated for the production of extracellular protease. 16S rRNA gene analysis identified it as Bacillus aquimaris. Enzyme secretion corresponded with growth ( G t , 38 min) in the basal Zobell medium, reaching a maximum during stationary phase (630 U/ml, 48 h). Protease production was investigated in different salt concentrations (0–4 M). While growth was optimum in the basal medium, higher levels of protease activity were observed in 0.5 M salt medium (728 U/ml, 48 h) and 1 M salt medium (796 U/ml, 78 h) with 21% and 32% increase in production, respectively. Salt concentrations above 2.5 M did not support bacterial growth. The optimum pH and temperature for production were pH 7.5 and 37 °C, respectively. A combination of peptone and yeast extract yielded optimum protease secretion. Inorganic nitrogen sources proved to be less favourable. Production was reduced in the presence of readily available carbon sources owing to catabolic repression. Effect of various salts (1–6%) indicated favourable bacterial growth in these conditions for producing proteolytic molecules with increased activity. The study assumes significance in the ability of the halotolerant bacterium to survive in a wide range of salinity and yield optimum levels of extracellular protease.

Interconnections between Sigma B, agr , and Proteolytic Activity in Staphylococcus aureus Biofilm Maturation

Staphylococcus aureus is a proficient biofilm former on host tissues and medical implants. We mutagenized S. aureus strain SH1000 to identify loci essential for ica-independent mechanisms of biofilm maturation and identified multiple insertions in the rsbUVW-sigB operon. Following construction and characterization of a sigB deletion, we determined that the biofilm phenotype was due to a lack of sigma factor B (SigB) activity. The phenotype was conserved in a sigB mutant of USA300 strain LAC, a well-studied community-associated methicillin-resistant S. aureus isolate. We determined that agr RNAIII levels were elevated in the sigB mutants, and high levels of RNAIII expression are known to have antibiofilm effects. By introducing an agr mutation into the SH1000 or LAC sigB deletion strain, S. aureus regained biofilm capacity, indicating that the biofilm phenotype was agr dependent. Protease activity is linked to agr activity and ica-independent biofilm formation, and we observed that the protease inhibitors phenylmethylsulfonyl fluoride and alpha-macroglobulin could reverse the sigB biofilm defect. Similarly, inactivating genes encoding both the aureolysin and Spl extracellular proteases in the sigB mutant restored biofilm capacity. Due to the growing link between murein hydrolase activity and biofilm maturation, autolysin zymography was performed, which revealed an altered profile in the sigB mutant; again, the phenotype could be repaired through protease inactivation. These findings indicate that the lack of SigB activity results in increased RNAIII expression, thus elevating extracellular protease levels and altering the murein hydrolase activity profile. Altogether, our observations demonstrate that SigB is an essential regulator of S. aureus biofilm maturation.

The interaction of induction, repression and starvation in the regulation of extracellular proteases in Aspergillus nidulans: evidence for a role for CreA in the response to carbon starvation

In Aspergillus nidulans, production of extracellular proteases in response to carbon starvation and to a lesser extent nitrogen starvation is controlled by XprG, a putative transcriptional activator. In this study the role of genes involved in carbon catabolite repression and the role of protein as an inducer of extracellular protease gene expression were examined. The addition of exogenous protein to the growth medium did not increase extracellular protease activity whether or not additional carbon or nitrogen sources were present indicating that induction does not play a major role in the regulation of extracellular proteases. Northern blot analysis confirmed that protein is not an inducer of the major A. nidulans protease, PrtA. Mutations in the creA, creB and creC genes increased extracellular protease levels in medium lacking a carbon source suggesting that they may have a role in the response to carbon starvation as well as carbon catabolite repression. Analysis of glkA4 frA2 and creADelta4 mutants showed that the loss of glucose signalling or the DNA-binding protein which mediates carbon catabolite repression did not abolish glucose repression but did increase extracellular protease activity. This increase was XprG-dependent indicating that the effect of these genes may be through modulation of XprG activity.

Characterization of regulatory non-catalytic hexokinases in Aspergillus nidulans

Hexokinases catalyse the first step in glucose metabolism and play a role in glucose sensing in mammals, plants and fungi. We describe a new class of hexokinases that appear to be solely regulatory in function. The Aspergillus nidulans hxkD gene (formerly named xprF) encodes a hexokinase-like protein. We constructed hxkDDelta gene disruption mutants which showed increased levels of extracellular protease in response to carbon starvation. The hxkDDelta mutations are not completely recessive, indicating that the level of the gene product is critical. Transcript levels of hxkD increase during carbon starvation and this response is not dependent on functional HxkD. A gene encoding a second atypical hexokinase (HxkC) was identified. The hxkCDelta gene disruption mutant exhibits a phenotype similar, but not identical, to hxkDDelta mutants. As with hxkD, mutations in hxkC are suppressed by loss-of-function mutations in xprG, which encodes a putative transcriptional activator involved in the response to nutrient limitation. We show that GFP-tagged HxkD was found only in nuclei suggesting a regulatory role for HxkD. GFP-tagged HxkC was associated with mitochondria. Homologs of hxkC and hxkD are conserved in multi-cellular fungi. Genes encoding atypical hexokinases are present in many genome sequence databases. Thus, non-catalytic hexokinases may be widespread.

RNA interference (RNAi) for the silencing of extracellular serine proteases genes in Acanthamoeba: Molecular analysis and effect on pathogenecity

Silencing of extracellular serine protease genes was undertaken by interference RNA (RNAi). Chemically synthesized, small interfering RNA (siRNA) were highly specific and efficient in silencing the catalytic domain of extracellular serine proteases of Acanthamoeba. In order to confirm the silencing phenomenon, the extracellular serine protease activities in RNAi-treated parasites were compared to non-treated parasites, using zymography profiles, Acanthamoeba-conditioned medium (ACM) protease activity, cytotoxicity assays and extracellular serine protease mRNA levels analysis. Zymography profiles showed a decrease in the extracellular protease levels in the moderate pathogenic and pathogenic strains, after treatment with siRNA. These results were supported after the ACM protease activity and CPE assays were performed in all studied isolates, showing a lower protease activity or cytotoxicity both in the pathogenic and moderate pathogenic strains treated with RNAi. These results support that extracellular serine proteases are directly involved in the pathogenesis and virulence of Acanthamoeba.

A new black Aspergillus species, A. vadensis, is a promising host for homologous and heterologous protein production.

A new species of the group of black aspergilli, Aspergillus vadensis, was analyzed for its potential as a host for homologous and heterologous protein production. Unlike the other black aspergilli, this strain does not acidify the culture medium when nitrate is the nitrogen source and only produces very low levels of extracellular proteases, mainly serine metalloproteases. The stability of A. tubingensis feruloyl esterase A (FaeA) was compared upon production in wild-type A. vadensis, A. tubingensis, and an A. niger strain in which the three main protease-encoding genes were disrupted. The production of FaeA in A. vadensis resulted in larger amounts of intact protein than production in A. tubingensis and was similar to production in an A. niger protease disruptant, confirming in vivo the low proteolytic activity of A. vadensis. The protoplast formation and transformation efficiencies of A. vadensis were much higher than those of A. niger. These characteristics make A. vadensis a very promising candidate for homologous, and possibly heterologous, protein production.

The type IV fimbrial subunit gene (fimA) of Dichelobacter nodosus is essential for virulence, protease secretion, and natural competence.

Dichelobacter nodosus is the essential causative agent of footrot in sheep. The major D. nodosus-encoded virulence factors that have been implicated in the disease are type IV fimbriae and extracellular proteases. To examine the role of the fimbriae in virulence, allelic exchange was used to insertionally inactivate the fimA gene, which encodes the fimbrial subunit protein, from the virulent type G D. nodosus strain VCS1703A. Detailed analysis of two independently derived fimA mutants revealed that they no longer produced the fimbrial subunit protein or intact fimbriae and did not exhibit twitching motility. In addition, these mutants were no longer capable of undergoing natural transformation and did not secrete wild-type levels of extracellular proteases. These effects were not due to polar effects on the downstream fimB gene because insertionally inactivated fimB mutants were not defective in any of these phenotypic tests. Virulence testing of the mutants in a sheep pen trial conducted under controlled environmental conditions showed that the fimA mutants were avirulent, providing evidence that the fimA gene is an essential D. nodosus virulence gene. These studies represent the first time that molecular genetics has been used to determine the role of virulence genes in this slow growing anaerobic bacterium.