Increased Protease Production Research Articles

The major role of sarA in limiting Staphylococcus aureus extracellular protease production in vitro is correlated with decreased virulence in diverse clinical isolates in osteomyelitis

ABSTRACT We previously demonstrated that MgrA, SarA, SarR, SarS, SarZ, and Rot bind at least three of the four promoters associated with genes encoding primary extracellular proteases in Staphylococcus aureus (Aur, ScpA, SspA/SspB, SplA-F). We also showed that mutation of sarA results in a greater increase in protease production, and decrease in biofilm formation, than mutation of the loci encoding any of these other proteins. However, these conclusions were based on in vitro studies. Thus, the goal of the experiments reported here was to determine the relative impact of the regulatory loci encoding these proteins in vivo. To this end, we compared the virulence of mgrA, sarA, sarR, sarS, sarZ, and rot mutants in a murine osteomyelitis model. Mutants were generated in the methicillin-resistant USA300 strain LAC and the methicillin-sensitive USA200 strain UAMS-1, which was isolated directly from the bone of an osteomyelitis patient during surgical debridement. Mutation of mgrA and rot limited virulence to a statistically significant extent in UAMS-1, but not in LAC, while the sarA mutant exhibited reduced virulence in both strains. The reduced virulence of the sarA mutant was correlated with reduced cytotoxicity for osteoblasts and osteoclasts, reduced biofilm formation, and reduced sensitivity to the antimicrobial peptide indolicidin, all of which were directly attributable to increased protease production in both LAC and UAMS-1. These results illustrate the importance of considering diverse clinical isolates when evaluating the impact of regulatory mutations on virulence and demonstrate the significance of SarA in limiting protease production in vivo in S. aureus.

Agro-industrial by-products as substrates for the production of bacterial protease and antioxidant hydrolysates

Feather meal (FM), sweet whey (LW), lyophilized whey (WL), and cassava bagasse (CB) were investigated as substrates to obtain extracellular proteases and bioactive hydrolysates during cultivations with Bacillus sp. CL33A. Initial evaluations indicated that FM (10 g/L), as single substrate, resulted in higher protease production, and higher soluble protein contents and antioxidant activities in culture supernatants, in comparison to LW, WL (5, 10, 15 g/L) or CB (5, 10, 15 g/L plus 1 g/L (NH4)2SO4)). Increasing FM to 30 g/L enhanced protease production 6-fold, which peaked after 4 days of cultivation. Protease production was paralleled by increases in soluble protein content in supernatants, which demonstrated radical-scavenging, Fe3+-reducing, and Fe2+-chelating activities. Supplementation of 30 g/L FM with 5 g/L WL increased protease yields (1.8-fold) after 4 days; enzyme production peaked after 6 days of cultivation, corresponding to a 3-fold increase in comparison to FM-only medium at day 4. Adding CB (5 and 10 g/L) to 30 g/L FM increased protease production by 110–130% after 4 days of cultivation. LW-supplemented FM medium showed decreased protease production, postponing soluble protein release and increases on antioxidant activities. No marked influences of supplemental WL or CB were observed on the antioxidant activities of obtained protein hydrolysates. FM is a low-cost substrate to simultaneously obtain bioproducts. The use of WL or CB as co-substrates might be interesting to manage and add value to these by-products. The obtained protease might find applications in the enzymatic hydrolysis of food proteins, and antioxidant hydrolysates deserve investigations as animal feed.

Limiting protease production plays a key role in the pathogenesis of the divergent clinical isolates of Staphylococcus aureus LAC and UAMS-1

ABSTRACT Using the USA300, methicillin-resistant Staphylococcus aureus strain LAC, we previously examined the impact of regulatory mutations implicated in biofilm formation on protease production and virulence in a murine sepsis model. Here we examined the impact of these mutations in the USA200, methicillin-sensitive strain UAMS-1. Mutation of agr, mgrA, rot, sarA and sigB attenuated the virulence of UAMS-1. A common characteristic of codY, rot, sigB, and sarA mutants was increased protease production, with mutation of rot having the least impact followed by mutation of codY, sigB and sarA, respectively. Protein A was undetectable in conditioned medium from all four mutants, while extracellular nuclease was only present in the proteolytically cleaved NucA form. The abundance of high molecular weight proteins was reduced in all four mutants. Biofilm formation was reduced in codY, sarA and sigB mutants, but not in the rot mutant. Eliminating protease production partially reversed these phenotypes and enhanced biofilm formation. This was also true in LAC codY, rot, sarA and sigB mutants. Eliminating protease production enhanced the virulence of LAC and UAMS-1 sarA, sigB and rot mutants in a murine sepsis model but did not significantly impact the virulence of the codY mutant in either strain. Nevertheless, these results demonstrate that repressing protease production plays an important role in defining critical phenotypes in diverse clinical isolates of S. aureus and that Rot, SigB and SarA play critical roles in this regard.

Rhamnolipid Enhances Burkholderia Pseudomallei Biofilm Susceptibility, Disassembly and Production of Virulence Factors

Aim: This study evaluated the effect of the biosurfactant rhamnolipid on the antimicrobial susceptibility, biofilm growth dynamics and production of virulence factors by Burkholderia pseudomallei. Materials & methods: The effects of rhamnolipid on planktonic and biofilm growth and its interaction with antibacterial drugs were evaluated. Then, its effects on growing and mature biofilms and on protease and siderophore production were assessed. Results: Rhamnolipid did not inhibit B. pseudomallei growth, but significantly enhanced the activity of meropenem and amoxicillin-clavulanate against mature biofilms. Rhamnolipid significantly reduced the biomass of mature biofilms, significantly increased protease production by growing and mature biofilms and siderophore release by growing biofilms. Conclusion: Rhamnolipid enhances the antimicrobial activity against B. pseudomallei, assists biofilm disassembly and alters protease and siderophore production by bacterial biofilms.

IL-15 and IL15RA in Osteoarthritis: Association With Symptoms and Protease Production, but Not Structural Severity.

Objective: Interleukin-15 (IL-15) is a pro-inflammatory cytokine that is increased in joint fluids of early-stage osteoarthritis (OA) patients, and has been associated with expression of proteases that can damage cartilage, and the development of neuropathic pain-like symptoms (NP) after nerve injury. The objective of this study was to further explore the role of IL-15 in the pathogenesis of OA cartilage degeneration and test genetic variation in the IL-15 receptor α gene (IL15RA) for an association with OA with radiographic severity and symptoms.Methods: Cartilage samples from donors (n = 10) were analyzed for expression of the IL15 receptor α-chain using immunohistochemistry, and for responses to IL-15 in vitro using explant cultures. Data from two independent Nottinghamshire-based studies (n = 795 and n = 613) were used to test genetic variants in the IL15RA gene (rs2228059 and rs7097780) for an association with radiographic severity, symptomatic vs. asymptomatic OA and NP.Results: IL-15Rα was expressed in chondrocytes from cartilage obtained from normal and degenerative knees. IL-15 significantly increased the release of matrix metalloproteinase-1 and -3 (MMP-1 and -3), but did not affect loss of proteoglycan from the articular matrix. Genetic variants in the IL15RA gene are associated with risk of symptomatic vs. asymptomatic OA (rs7097780 OR = 1.48 95% 1.10–1.98 p < 0.01) and with the risk of NP post-total joint replacement (rs2228059 OR = 0.76 95% 0.63–0.92 p < 0.01) but not with radiographic severity.Conclusions: In two different cohorts of patients, we show an association between genetic variation at the IL15 receptor and pain. Although ex vivo cartilage explants could respond to IL-15 with increased protease production, we found no effect of IL-15 on cartilage matrix loss and no association between IL15RA variants and radiographic severity. Together, these results suggest that IL-15 signaling may be a target for pain, but may not impact structural progression, in OA.

Protease production by the keratinolytic Bacillus sp. CL18 through feather bioprocessing

Bacillus sp. CL18 was investigated to propose a bioprocess for protease production using feathers as organic substrate. In feather broth (FB), containing feathers as sole organic substrate (1-100gl-1), maximal protease production was observed at 30gl-1 (FB30) after 6days of cultivation, whereas increased feather concentrations negatively affected protease production and feather degradation. Protease production peaks were always observed earlier during cultivations than maximal feather degradation. In FB30, 80% of initial feathers mass were degraded after 7days. Addition of glucose, sucrose, starch, yeast extract (2gl-1), CaCl2, or MgCl2 (10mmoll-1) to FB30 decreased protease production and feather degradation. FB30 supplementation with NH4Cl (1gl-1) resulted in less apparent negative effects on protease production, whereas peptone (2gl-1) increased protease yields earlier during cultivations (3days). Through a central composite design employed to investigate the effects of peptone and NH4Cl (0.5-4.5gl-1) on protease production and feather degradation, FB30 supplementation with peptone and NH4Cl (0.5-1.1gl-1) increased protease production within a shorter cultivation time (5days) and hastened complete feather degradation (6days). Feather bioconversion concurs with sustainable production of value-added products.

Medium Formulation and its optimization for increased protease production by Penicillium sp. LCJ228 and its potential in blood stain removal

The production of protease by Penicillium sp. LCJ228 was optimized under submerged fermentation. Nutritional and physical factors that influence protease production were optimized by one factor at a time (OFAT) method in order to achieve high yield of protease. Maximum protease production was obtained on the 4th day in a liquid medium containing glucose (15 g/L), yeast extract (15 g/L), black gram husk (10 g/L) with an initial pH of 10 and an inoculum size of 2 g/L of fungal mycelium. About 2.4 fold increase in protease production was observed in the optimized medium. The maximally yielded crude protease was then precipitated and characterized by SDS-PAGE and native-PAGE. The molecular weight of the ammonium sulphate precipitated protease was ~66 to 70 kDa and native-PAGE showed three isoenzymes. The crude and precipitated protease had the ability to completely remove blood stains on cotton fabric indicating its potential to be used as a stain remover in detergents.

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.

Relationships between the index of protein modification (Kolbach index) and hydrolytic enzyme production in a wheat malt

In this study, the relationships between protein degradation, enzyme activities and quality of EBC (European Brewery Convention) wort were investigated. The Kolbach index of wheat malt at 37.6–42.7% was found to be suitable for acquiring higher enzyme activities. A globulin increase was the main factor that promoted the activities of the degradation enzymes. Strong synergistic activity was observed among the polysaccharide degradation activities of enzymes [α-amylase, β-amylase, β-glucanase, β-d-xylosidase and β-(1,4)-endoxylanase]. Increased protease production improved the Kolbach index of a wheat malt, that is, promoted the degradation of gliadins into albumins and globulins. Increased albumins and globulins resulted in increased enzyme activity for polysaccharide degradation. Increased enzyme activities demonstrated synergistic actions that ultimately promoted the quality indices of an EBC wort, including extract yield, α-amino nitrogen, acidity and chromaticity. Copyright © 2014 The Institute of Brewing & Distilling

Impact of the functional status of saeRS on in vivo phenotypes of Staphylococcus aureus sarA mutants.

We investigated the in vivo relevance of the impact of sarA and saeRS on protease production using derivatives of the USA300 strain LAC. The results confirmed that mutation of saeRS or sarA reduces virulence in a bacteremia model to a comparable degree. However, while eliminating protease production restored virulence in the sarA mutant, it had little impact in the saeRS mutant. Additionally, constitutive activation of saeRS (saeRS(C)) enhanced the virulence of LAC and largely restored virulence in the isogenic sarA mutant. Based on these results, together with our analysis of the representative virulence factors alpha toxin, protein A (Spa), and extracellular nucleases, we propose a model in which the attenuation of saeRS mutants is defined primarily by decreased production of such factors, while constitutive activation of saeRS increases virulence, and reverses the attenuation of sarA mutants, because it results in both increased production and decreased protease-mediated degradation of these same factors. This regulatory balance was also apparent in a murine model of catheter-associated infection, with the results suggesting that the impact of saeRS on nuclease production plays an important role during the early stages of these infections that is partially offset by increased protease production in sarA mutants.

Impact of Vancomycin on sarA-Mediated Biofilm Formation: Role in Persistent Endovascular Infections Due to Methicillin-Resistant Staphylococcus aureus

Staphylococcus aureus is the most common cause of endovascular infections. The staphylococcal accessory regulator A locus (sarA) is a major virulence determinant that may potentially impact methicillin-resistant S. aureus (MRSA) persistence in such infections via its influence on biofilm formation. Two healthcare-associated MRSA isolates from patients with persistent bacteremia and 2 prototypical community-acquired MRSA strains, as well as their respective isogenic sarA mutants, were studied for in vitro biofilm formation, fibronectin-binding capacity, autolysis, and protease and nuclease activities. These assays were done in the presence or absence of sub-minimum inhibitory concentrations (MICs) of vancomycin. In addition, these strain pairs were compared for intrinsic virulence and responses to vancomycin therapy in experimental infective endocarditis, a prototypical biofilm model. All sarA mutants displayed significantly reduced biofilm formation and binding to fibronectin but increased protease production in vitro, compared with their respective parental strains. Interestingly, exposure to sub-MICs of vancomycin significantly promoted biofilm formation and fibronectin-binding in parental strains but not in sarA mutants. In addition, all sarA mutants became exquisitely susceptible to vancomycin therapy, compared with their respective parental strains, in the infective endocarditis model. These observations suggest that sarA activation is important in persistent MRSA endovascular infection, potentially in the setting of biofilm formation.

Abstract 405: Vascular Smooth Muscle Cells (VSMCs) from Abdominal Aortic Aneurysms (AAA) Avidly Take Up Oxidized Cholesterol (oxChol) with Unique Responses Compared with VSMCs from Nonaneurysmal Aorta (NAA)

Objective: We sought to determine if VSMCs from AAA respond to oxChol exposure in a manner which may promote aortic matrix damage. Methods: Early passage VSMCs (5x10 5 ) from human AAA, Carotid plaques (CP) and nAA were compared after exposure to media with oxChol for 72 hours. Cells were counted and stained with Oil Red O, and staining was graded on a 0-4 scale, by 5 blinded experts. Conditioned media (CM) was assayed by ELISA for MMP-2, and corrected for cell number (mean±SE). Results: The average grade of Oil Red O stain (Fig 1) of VSMCs from AAA was 2.89 +/- 0.225, CEA plaques was 3.14 +/- 0.151 and nAA was 1.76 +/- 0.258. Cholesterol uptake was significantly different for AAA vs. nAA (p=.015) and CEA vs. nAA (p=.0002), but not between the unstained controls. Cell number (x10 3 ) at harvest after oxChol exposure was significantly greater than media alone for CP (562±48 vs 338±79, P&lt;0.02) and nAA (343±49 vs 250±31), but there was no effect on AAA (339±49 vs 313±43). With exposure to OxChol, MMP-2 concentration in CM from AAA cells was similar to that from CP cells (224±51ng/ml vs. 352±46ng/ml), but production from nAA cells was significantly less than CP (178±61ng/ml, P&lt;0.05) but not compared to AAA. Conclusion: VSMCs from human AAA exposed to oxChol had significantly increased cholesterol uptake, with MMP-2 production comparable to CP cells. Exposure to oxChol resulted in a mitogenic response in the CEA and nAA cells not seen in the AAA cells. Hypercholesterolemia may promote AAA due to pathologic cellular responses of increased cholesterol uptake, decreased growth and increased protease production. Additional studies are needed to clarify these responses.

Abstract 3277: Increased mitogen-activated protein kinase kinase 4 (MEK4) induces metastasis in an orthotopic murine model of prostate cancer

Abstract Mitogen-activated protein kinase kinase 4 (MEK4) is a dual-specificity kinase that has been implicated in prostate cancer (PCa) progression. Increased MEK4 expression is observed in invasive cancer lesions in human prostate tissue. We have demonstrated the clinical importance of this in vitro, as MEK4 increases human PCa invasion. This phenotype is driven by increased protease production, and not via changes in cell migration. However, it is not known whether MEK4 regulates human PCa metastasis. This is of high importance as MEK4 has shown differential effects in various cancer models in a cell type specific fashion, and may therefore represent an important human PCa specific target. To emulate the situation of sustained altered MEK4 expression in humans, we engineered MEK4 clonal variant cell lines, generating multiple clones each for vector control (VC), increased wild type (WT), and increased constitutively-active mutant MEK4 (CA). Using a murine orthotopic implantation model designed to characterize in vivo behavior, we implanted 75 mice with these clones and demonstrated that both WT and CA MEK4 increase human PCa metastasis to the lung, a clinically relevant site in humans. In the primary tumor, interestingly, WT tumors were not increased compared to VC, while CA tumors were. Additionally, increased MMP-2 and MMP-10 were observed in both WT and CA tumors. However, MMP-9 was increased in WT tumors only, and MMP-13 in CA tumors only. Currently, we are expanding our efforts at characterizing additional molecular changes in primary tumor between these groups. Outside of the primary tumor, we isolated circulating tumor cells in the blood and bone marrow. We are currently characterizing their relevant cellular and molecular parameters. Using a clinically relevant murine model we have shown that increased MEK4 increases human PCa metastasis to the lung and protease production within the primary tumor. Further, a constitutively active phenotype drives tumor growth and circulating tumor cell formation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3277. doi:1538-7445.AM2012-3277

Improvement of Desferrioxamine B Production of Streptomyces pilosus ATCC 19797 With Use of Protease Inhibitor and Minerals Related to Its Activity

The relationship between protease and Desferal production was assayed. Experiments were performed using a cultivation of Streptomyces pilosus ATCC 19797 in soybean broth medium containing 2% soybean flour and 2% mannitol. The metabolism of the trihydroxamic acid sidrophore desferrioxamine B and protease production by a S. pilosus in nine continues days after culture were investigated as well as the effect of protease inhibitors was examined. It is found that the Desferal formation decreased with increased protease production. Also the effect of protease inhibitors and minerals in determined day of protease production in the culture medium by S. pilosus has been investigated.

Production and optimization of thermophilic alkaline protease in solid-state fermentation by Streptomyces sp. CN902

The purpose of the present research is to study the production of thermophilic alkaline protease by a local isolate, Streptomyces sp. CN902, under solid state fermentation (SSF). Optimum SSF parameters for enzyme production have been determined. Various locally available agro-industrial residues have been screened individually or as mixtures for alkaline protease production in SSF. The combination of wheat bran (WB) with chopped date stones (CDS) (5:5) proved to be an efficient mixture for protease production as it gave the highest enzyme activity (90.50 U g(-1)) when compared to individual WB (74.50 U g(-1)) or CDS (69.50 U g(-1)) substrates. This mixed solid substrate was used for the production of protease from Streptomyces sp. CN902 under SSF. Maximal protease production (220.50 U g(-1)) was obtained with an initial moisture content of 60%, an inoculum level of 1 x 10(8) (spore g(-1) substrate) when incubated at 45 degrees C for 5 days. Supplementation of WB and CDS mixtures with yeast extract as a nitrogen source further increased protease production to 245.50 U g(-1) under SSF. Our data demonstrated the usefulness of solid-state fermentation in the production of alkaline protease using WB and CDS mixtures as substrate. Moreover, this approach offered significant benefits due to abundant agro-industrial substrate availability and cheaper cost.

An alternative insect pathogenic strategy in an Aspergillus flavus auxotroph

In order to study fungal pathogen evolution, we used a model system whereby the opportunistic fungus Aspergillus flavus was serially propagated through the insect ( Galleria mellonella) larvae, yielding a cysteine/methionine auxotroph of A. flavus with properties of an obligate insect pathogen. The auxotroph exhibited insect host restriction but did not show any difference in virulence when compared with the wild-type (Scully LR, Bidochka MJ, 2006. Microbiology 152, 223–232). Here, we report that on 1 % insect cuticle medium and synthetic Galleria medium, the auxotroph displayed increased extracellular protease production, a virulence factor necessary for insect pathogenesis. In the wild-type strain, protease production was deregulated during carbon (glucose), nitrogen (nitrate), or sulphate deprivation. If all three were present, protease production was vastly reduced. However, in the cysteine/methionine auxotroph, protease production was deregulated in complete medium. We suggest that the deficiency in sulphate assimilation in the auxotroph resulted in deregulation of protease production. The auxotroph exhibited delayed germination and slower hyphal growth when compared to the wild-type but there were no differences in virulence or cuticle penetration, suggesting a shift in pathogenic strategy that compensated decreased growth with increased virulence factor (extracellular protease) production. We concluded that the biosynthetic deficiency that mediated insect host restriction also increased protease production in the slow-growing auxotroph, resulting in an alternate, more host-specific pathogenic strategy. However, we argue that transmission is not necessarily correlated with virulence as competition bioassays in insect larvae showed that the wild-type generally out-competed the auxotroph by producing the majority of the conidia on the sporulating cadavers. This is one of the few examples that highlight the effect of genome decay on nutrition acquisition, virulence, and transmission in fungal pathogen evolution.

Effects of degU32(Hy), degQa and degR Pleiotropic Regulatory Genes on the Growth and Protease Fermentation of Bacillus Subtilis Ki-2-132

Effects of degU32 (Hy), degR genes from Bacillus subtilis 168 and deg Qa gene from Bacillus amyloliquefaciens on Bacillus subtilis Ki-2-132 cell growth, sporulation and protease fermentation were investigated by introducing these genes into B. subtilis Ki-2-132 chromosome and/or cytoplasm. Although the genes come from different species and strains, they showed pleiotropic effects in B. subtilis Ki-2-132. B. subtilis Ki-2-132 degU32 (Hy) showed increased protease production, and when cooperating with deg Qa either in plasmid or in chromosome, further altered cell growth, increased protease production and affected the spore formation in a glucose and dosage dependent manner. By contrast, degR did not significantly affect the protease productivity in degU32 (Hy) mutant, consisting with that DegR was used to stabilise DegU-phosphate, which in degU32 (Hy) strain no longer further amplify the DegU-phosphate effect.

Novel alkaline proteases from alkaliphilic bacteria grown on chicken feather

Two alkaline protease producing alkaliphilic bacterial strains, designated as AL-20 and AL-89, were isolated from a naturally occurring alkaline habitat. The two strains were identified as Nesternkonia sp. and Bacillus pseudofirmus, respectively. Both strains grew and produced alkaline protease using feather as the sole source of carbon and nitrogen. Addition of 0.5% glucose to the feather medium increased protease production by B. pseudofirmus AL-89 and suppressed enzyme production by Nesternkonia sp. AL-20. The enzymes from both organisms were purified to electrophoretic homogeneity following ammonium sulphate precipitation, ion exchange, hydrophobic interaction, and gel filtration chromatography. The molecular weight, determined using SDS–PAGE, was 23 kDa for protease AL-20 and 24 kDa for protease AL-89. Protease AL-20 was active in a broad pH range displaying over 90% of its maximum activity between pH 7.5 and 11.5 with a peak at pH 10. The enzyme is unique in that unlike all other microbial serine proteases known so far, it did not require Ca 2+ for activity and thermal stability. Its optimum temperature for activity was at 70 °C and was stable after 1 h incubation at 65 °C both in the presence and absence of Ca 2+. These properties make protease AL-20 an ideal candidate for detergent application. Protease AL-89 on the other hand require Ca 2+ for activity and stability at temperature values above 50 °C. Its optimum activity was at 60 and 70 °C in the absence and presence of Ca 2+, respectively. It displayed a pH optimum of 11 and retained about 70% or more of its original activity between pH 6.5 and 11. B. pseudofirmus AL-89, and the protease it produce offers an interesting potential for the enzymatic and/or microbiological hydrolysis of feather to be used as animal feed supplement.

Microenvironmental Regulation of the Initiated Cell

In the classical skin model of tumor initiation, keratinocytes treated once with carcinogen retain their normal appearance and growth behavior indefinitely unless promoted to growth into papillomas. Because many of the papillomas regress and may recur with further promotion, their cells can also be considered as initiated. The growth of initiated keratinocytes can be inhibited either in vitro or in vivo by close association with an excess of normal keratinocytes, but it is enhanced by dermal fibroblasts. Chick embryo fibroblasts (CEF) in culture produce transformed foci after infection with Rous sarcoma virus (RSV) on a background of normal CEF in a medium containing 10% or less calf serum (CS), but they retain normal appearance and growth regulation in 10% fetal bovine serum (FBS) or 20% CS. Transformation of a carcinogen-treated line of mouse embryo fibroblasts is prevented, and can be reversed, in high concentrations of FBS in the presence of an excess of normal cells. FBS has high, broad-spectrum antiprotease activity. Increased protease production occurs in a variety of transformed cells and is correlated with progression in tumors. Protease treatment stimulates DNA synthesis and mitosis in confluent, contact-inhibited normal cell cultures. Synthetic inhibitors of proteases suppress transformation in carcinogen-treated cultures and inhibit tumor formation in animals. Several different classes of protease may be overexpressed in the same transformed cells. It is proposed that excessive protease production accounts for major features of neoplastic transformation of initiated cells, but that transformation can be held in check by protease inhibitors present in serum and released from surrounding cells. It would be informative to determine whether high concentrations of FBS would inhibit the neoplastic development of initiated keratinocytes.

Variation in extracellular protease production among clinical isolates of Staphylococcus aureus due to different levels of expression of the protease repressor sarA.

Staphylococcus aureus produces four major extracellular proteases: staphylococcal serine protease (V8 protease; SspA), cysteine protease (SspB), metalloprotease (aureolysin; Aur), and staphopain (Scp). Several in vitro studies have suggested that these enzymes are important virulence factors. Here we analyzed the protease production of 92 S. aureus strains from infected human soft tissue. Twenty-one strains produced variable zones of proteolysis on casein agar plates, while the remaining 71 strains appeared to be protease negative. The major protease genes were present in all protease-positive (n = 5) and protease-negative (n = 12) strains analyzed. Northern blotting showed that transcription of the protease genes was suppressed due to increased sigma factor B (SigB)-dependent expression of the protease repressor SarA. Other SigB-dependent traits such as pigmentation and expression of asp 23 were also increased in protease-negative compared to protease-positive strains. Inactivation of sarA in three protease-negative strains resulted in increased transcription of all protease genes and increased protease production, while overexpression of sarA in a strain producing protease at high levels repressed protease production. Our results suggest that the protease genes are conserved among clinical S. aureus strains and that the level of SigB-dependent expression of the protease repressor sarA determines the level of protease production in each strain.