Replacement of histidine 340 with alanine inactivates the group A Streptococcus extracellular cysteine protease virulence factor.

Streptococcus pyogenes expresses a highly conserved extracellular cysteine protease that is a virulence factor for invasive disease, including soft tissue infection. Site-directed mutagenesis was used to generate a His340Ala recombinant mutant protein that was made as a stable 40-kDa zymogen by Escherichia coli. Purified His340Ala protein was proteolytically inactive when bovine casein and human fibronectin were used as substrates. Wild-type 28-kDa streptococcal protease purified from S. pyogenes processed the 40-kDa mutant zymogen to a 28-kDa mature form, a result suggesting that the derivative protein retained structural integrity. The data are consistent with the hypothesis that His340 is an enzyme active site residue, an idea confirmed by recent solution of the zymogen crystal structure (T. F. Kagawa, J. C. Cooney, H. M. Baker, S. McSweeney, M. Liu, S. Gubba, J. M. Musser, and E. N. Baker, Proc. Natl. Acad. Sci. USA 97:2235-2240, 2000). The data provide additional insight into structure-function relationships in this S. pyogenes virulence factor.

Crystal structure of the zymogen form of the group A Streptococcus virulence factor SpeB: an integrin-binding cysteine protease.

Pathogenic bacteria secrete protein toxins that weaken or disable their host, and thereby act as virulence factors. We have determined the crystal structure of streptococcal pyrogenic exotoxin B (SpeB), a cysteine protease that is a major virulence factor of the human pathogen Streptococcus pyogenes and participates in invasive disease episodes, including necrotizing fasciitis. The structure, determined for the 40-kDa precursor form of SpeB at 1.6-A resolution, reveals that the protein is a distant homologue of the papain superfamily that includes the mammalian cathepsins B, K, L, and S. Despite negligible sequence identity, the protease portion has the canonical papain fold, albeit with major loop insertions and deletions. The catalytic site differs from most other cysteine proteases in that it lacks the Asn residue of the Cys-His-Asn triad. The prosegment has a unique fold and inactivation mechanism that involves displacement of the catalytically essential His residue by a loop inserted into the active site. The structure also reveals the surface location of an integrin-binding Arg-Gly-Asp (RGD) motif that is a feature unique to SpeB among cysteine proteases and is linked to the pathogenesis of the most invasive strains of S. pyogenes.

Fibrinogen cleavage by the Streptococcus pyogenes extracellular cysteine protease and generation of antibodies that inhibit enzyme proteolytic activity.

The extracellular cysteine protease from Streptococcus pyogenes is a virulence factor that plays a significant role in host-pathogen interaction. Streptococcal protease is expressed as an inactive 40-kDa precursor that is autocatalytically converted into a 28-kDa mature (active) enzyme. Replacement of the single cysteine residue involved in formation of the enzyme active site with serine (C192S mutation) abolished detectable proteolytic activity and eliminated autocatalytic processing of zymogen to the mature form. In the present study, we investigated activity of the wild-type (wt) streptococcal protease toward human fibrinogen and bovine casein. The former is involved in blood coagulation, wound healing, and other aspects of hemostasis. Treatment with streptococcal protease resulted in degradation of the COOH-terminal region of fibrinogen alpha chain, indicating that fibrinogen may serve as an important substrate for this enzyme during the course of human infection. Polyclonal antibodies generated against recombinant 40- and 28-kDa (r40- and r28-kDa) forms of the C192S streptococcal protease mutant exhibited high enzyme-linked immunosorbent assay titers but demonstrated different inhibition activities toward proteolytic action of the wt enzyme. Activity of the wt protease was readily inhibited when the reaction was carried out in the presence of antibodies generated against r28-kDa C192S mutant. Antibodies produced against r40-kDa C192S mutant had no significant effect on proteolysis. These data suggest that the presence of the NH(2)-terminal prosegment prevents generation of functionally active antibodies and indicate that inhibition activity of antibodies most likely depends on their ability to bind the active-site region epitope(s) of the protein.

Overexpression of macrophage colony-stimulating factor (CSF-1) and its receptor, c-fms, in normal ovarian granulosa cells leads to cell proliferation and tumorigenesis.

OBJECTIVE: To investigate the interdependent role of macrophage colony-stimulating factor (CSF-1) and its receptor (c-fms) on their induction and their role in granulosa cell tumorigenesis. METHODS: Normal ovarian granulosa cells were used to develop stable transfectants that overexpress CSF-1 or CSF-1/c-fms. CSF-1 was expressed under the control of tissue/cell specific alpha-inhibin promoter, and c-fms was expressed constitutively using a viral promoter. Stable transfectants were used to examine the effect of overexpression of these molecules on the proliferation, induction of autocrine loop, and tumorigenesis. RESULTS: Expression vectors were developed for CSF-1 and its receptor, c-fms, and used to generate stable transfects overexpressing these genes in granulosa cells. Data show that overexpression of CSF-1 leads to the induction of its receptor. Stable transfectants that overexpress CSF-1 show about a 2.5-fold increase in cell proliferation compared with normal granulosa cells, and these cells are also converted to anchorage-independent and tumorigenic phenotype. Using an antisense RNA approach, we also demonstrated that the increased cell proliferation is CSF-1 specific. Concomitant overexpression of CSF-1 and c-fms further results in increased cell proliferation (sixfold), rapid anchorage-independent growth, and aggressive tumor formation. CONCLUSION: CSF-1 is capable of inducing its own receptor, and, similarly, the CSF-1 receptor, c-fms, can also induce its growth factor ligand. These studies also demonstrate the interdependent role of these genes in transformation of normal ovarian granulosa cells to a tumorigenic phenotype and suggest the possibility of a similar role for these genes in progression of ovarian cancer.

A natural variant of the cysteine protease virulence factor of group A Streptococcus with an arginine-glycine-aspartic acid (RGD) motif preferentially binds human integrins alphavbeta3 and alphaIIbbeta3.

The human pathogenic bacterium group A Streptococcus produces an extracellular cysteine protease [streptococcal pyrogenic exotoxin B (SpeB)] that is a critical virulence factor for invasive disease episodes. Sequence analysis of the speB gene from 200 group A Streptococcus isolates collected worldwide identified three main mature SpeB (mSpeB) variants. One of these variants (mSpeB2) contains an Arg-Gly-Asp (RGD) sequence, a tripeptide motif that is commonly recognized by integrin receptors. mSpeB2 is made by all isolates of the unusually virulent serotype M1 and several other geographically widespread clones that frequently cause invasive infections. Only the mSpeB2 variant bound to transfected cells expressing integrin alphavbeta3 (also known as the vitronectin receptor) or alphaIIbbeta3 (platelet glycoprotein IIb-IIIa), and binding was blocked by a mAb that recognizes the streptococcal protease RGD motif region. In addition, mSpeB2 bound purified platelet integrin alphaIIbbeta3. Defined beta3 mutants that are altered for fibrinogen binding were defective for SpeB binding. Synthetic peptides with the mSpeB2 RGD motif, but not the RSD sequence present in other mSpeB variants, blocked binding of mSpeB2 to transfected cells expressing alphavbeta3 and caused detachment of cultured human umbilical vein endothelial cells. The results (i) identify a Gram-positive virulence factor that directly binds integrins, (ii) identify naturally occurring variants of a documented Gram-positive virulence factor with biomedically relevant differences in their interactions with host cells, and (iii) add to the theme that subtle natural variation in microbial virulence factor structure alters the character of host-pathogen interactions.

Expression and characterization of group A Streptococcus extracellular cysteine protease recombinant mutant proteins and documentation of seroconversion during human invasive disease episodes.

A recent study with isogenic strains constructed by recombinant DNA strategies unambiguously documented that a highly conserved extracellular cysteine protease expressed by Streptococcus pyogenes (group A Streptococcus [GAS]) is a critical virulence factor in a mouse model of invasive disease (S. Lukomski, S. Sreevatsan, C. Amberg, W. Reichardt, M. Woischnik, A. Podbielski, and J. M. Musser, J. Clin. Invest. 99:2574-2580, 1997). To facilitate further investigations of the streptococcal cysteine protease, recombinant proteins composed of a 40-kDa zymogen containing a C192S amino acid substitution that ablates enzymatic activity, a 28-kDa mature protein with the C192S replacement, and a 12-kDa propeptide were purified from Escherichia coli containing His tag expression vectors. The recombinant C192S zymogen retained apparently normal structural integrity, as assessed by the ability of purified wild-type streptococcal cysteine protease to process the 40-kDa molecule to the 28-kDa mature form. All three recombinant purified proteins retained immunologic reactivity with polyclonal and monoclonal antibodies. Humans with a diverse range of invasive disease episodes (erysipelas, cellulitis, pneumonia, bacteremia, septic arthritis, streptococcal toxic shock syndrome, and necrotizing fasciitis) caused by six distinct M types of GAS seroconverted to the streptococcal cysteine protease. These results demonstrate that this GAS protein is expressed in vivo during the course of human infections and thereby provide additional evidence that the cysteine protease participates in host-pathogen interactions in some patients.

Regrowth of the rat biliary tree after 70% partial hepatectomy is coupled to increased secretin-induced ductal secretion.

BACKGROUND & AIMS: After partial hepatectomy, liver regeneration occurs with the return of hepatocyte mass to normal, Limited data exist regarding the renewal of the biliary tree after partial hepatectomy. This study tested the hypothesis that, after partial hepatectomy, the biliary tree regenerates by proliferation of the remaining cholangiocytes, leading to an increase in secretin-induced ductal bile secretion. METHODS: After 70% partial hepatectomy, cholangiocyte proliferation was assessed in situ by morphometric analysis and In vitro by measurement of 3H-thymidine incorporation. Ductal secretion was estimated by measurement of secretin receptor gene expression and adenosine 3',5'-cyclic monophosphate (cAMP) levels in vitro and by the effect of secretin on ductal bile secretion in vivo. RESULTS: DNA synthesis was undetectable in control cholangiocytes, increased and peaked at day 3 after partial hepatectomy, and returned to normal by day 28. Morphometric analysis showed regrowth of the biliary tree beginning at day 1 with restoration by day 10. The expression of secretin receptor gene and secretin-induced cAMP levels and secretin-induced bicarbonate-rich choleresis increased during the period of bile duct renewal. CONCLUSIONS: After partial hepatectomy, the increase in secretin-induced ductal bile secretion observed during bile duct renewal results from proliferation of remaining cholangiocytes.

Overexpression of int-5/aromatase in mammary glands of transgenic mice results in the induction of hyperplasia and nuclear abnormalities.

Our recent studies have shown that the cellular gene at the mouse mammary tumor virus integration site in the int-5 locus is aromatase. To study the role of int-5/aromatase in normal mammary development and mammary neoplasia, we have generated transgenic mice that overexpress int-5/aromatase under the control of mouse mammary tumor virus enhancer/promoter. All the transgenic virgin (n = 10) and postlactational (n = 15) females that overexpress int-5/aromatase show various histological abnormalities. Overexpression of int-5/aromatase in mammary glands of virgin females leads to the enlargement of 40% of ducts, of which 65% had hyperplastic lesions, 20% had dysplastic lesions, and 15% had fibroadenoma lesions. Overexpression of int-5/aromatase in involuted mammary glands of transgenic females induces hyperplasia in 75-80% of ducts and glands that exhibit a range of morphological abnormalities, including formation of hyperplastic alveolar nodule (10%), ductal and glandular hyperplasia (70-80%), ductal and lobular dysplasia (15%), and nuclear abnormalities (2-5%) such as multinucleation and karyomegaly, which are all indicative of preneoplastic changes. Our results show that early exposure of mammary epithelium to in situ estrogen and continued exposure to in situ estrogen as a result of overexpression of int-5/aromatase appears to predispose mammary tissue to preneoplastic changes, which may, in turn, increase the risk of developing neoplasia and increase susceptibility to environmental carcinogens. These findings support clinical and experimental data that suggest that early estrogen exposure increases breast cancer risk.

Morphological, molecular, and functional heterogeneity of cholangiocytes from normal rat liver.

BACKGROUND & AIMS: While the lobular heterogeneity of hepatocytes is established, limited data suggest that bile duct epithelial cells, of cholangiocytes, are heterogeneous along the normal intrahepatic biliary tree. Thus, we tested the hypothesis that cholangiocytes are structurally and functionally heterogeneous in the biliary tract of normal rats. METHODS: A series of in situ and in vitro experiments was performed in normal rats using complementary morphometric, molecular, and functional approaches. RESULTS: In situ morphometry showed that (1) intrahepatic bile ducts are heterogeneous in external diameter (5-200 mum); (2) individual cholangiocytes lining bile ducts are heterogeneous in area (3-80 mum2); and (3) a significant relationship exists between bile duct diameter and cholangiocyte area. Using a novel approach developed by us, we isolated three subpopulations of small, medium, and large cholangiocytes and compared them at the molecular and functional level. The expression of two cholangiocyte-specific genes (gamma-glutamyl transpeptidase and cytokeratin 19) was similar among the three subpopulations. In contrast, secretion receptor, Cl-/HCO3- exchanger, and cystic fibrosis transmembrane conductance regulator messenger RNAs were differentially expressed, being present on medium and large but not small cholangiocytes. At the functional level, adenosine 3', 5'-cyclic monophosphate and intracellular pH responses and Cl-/HCO3- exchanger activity was enhanced by secretion in medium and large but not small cholangiocytes. CONCLUSIONS: These data indicate that cholangiocytes are morphologically and functionally heterogeneous along the normal intrahepatic biliary tree and suggest that secretion-regulated transport of water and electrolytes occurs principally in medium and large ducts.

Regulation of Agrobacterium tumefaciens virulence gene expression: isolation of a mutation that restores virGD52E function.

Expression of Agrobacterium tumefaciens virulence (vir) genes is controlled by virA, virG, and a plant inducer. Isolation of two constitutive mutants of the transcriptional activator virG, virGN54D, and virGI106L, that support vir gene expression in a virA independent manner has previously been reported. Characterization of virGN54D by several groups showed considerable variation in its ability to activate vir gene transcription. In this study we demonstrate that these differences can be accounted for by plasmid copy number. We report the isolation of a third constitutive mutation, virGI77V, that partially restores transcription activation function of a nonfunctional virG mutant, virGD52E. The second regulator, VirA, in its extreme C-terminus, contains a domain that is homologous to the N-terminal domain of VirG. Deletion of this domain of VirA leads to a fully constitutive phenotype.