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Biomedical subjects

Jan Potempa

Publications and source records attributed to Jan Potempa.

At least 19 recordsLinked to original sources

Does the importance of the C-terminal residues in the maturation of RgpB from Porphyromonas gingivalis reveal a novel mechanism for protein export in a subgroup of Gram-Negative bacteria?

The mature 507-residue RgpB protein belongs to an important class of extracellular outer membrane-associated proteases, the gingipains, from the oral pathogen Porphyromonas gingivalis that has been shown to play a central role in the virulence of the organism. The C termini of these gingipains along with other outer membrane proteins from the organism share homologous sequences and have been suggested to function in attachment of these proteins to the outer membrane. In this report, we have created a series of truncated and site-directed mutants of the C terminus from a representative member of this class, the RgpB protease, to investigate its role in the maturation of these proteins. Truncation of the last two residues (valyl-lysine) from the C terminus is sufficient to create an inactive version of the protein that lacks the posttranslational glycosylation seen in the wild type, and the protein remains trapped behind the outer membrane. Alanine scanning of the last five residues revealed the importance of the C-terminal motif in mediating correct posttranslational modification of the protein. This result may have a wider implication in a novel secretory pathway in distinct members of the Cytophaga-Flavobacterium-Bacteroidetes phylum.

Adhesins, Bacterial↗

Thrombin-activable fibrinolysis inhibitor (TAFI) zymogen is an active carboxypeptidase.

Thrombin-activable fibrinolysis inhibitor (TAFI) is a carboxypeptidase found in human plasma, presumably as an inactive zymogen. The current dogma is that proteolytic activation by thrombin/thrombomodulin generates the active enzyme (TAFIa), which down-regulates fibrinolysis by removing C-terminal lysine residues from partially degraded fibrin. In this study, we have shown that the zymogen exhibits continuous and stable carboxypeptidase activity against large peptide substrates, and we suggest that the activity down-regulates fibrinolysis in vivo.

Amino Acid Sequence↗

Exploring the Sn binding pockets in gingipains by newly developed inhibitors: structure-based design, chemistry, and activity.

Arg-gingipains (Rgps) and Lys-gingipain (Kgp) are cysteine proteinases secreted by Porphyromonas gingivalis, the major pathogen implicated in periodontal disease. Gingipains are essential to the bacterium for its virulence and survival, and development of inhibitors targeting these proteins provides an approach to treat periodontal diseases. Here, we present the first example of structure-based design of gingipains inhibitors, with the use of the crystal structure of RgpB and the homology model of Kgp. Chloromethyl ketones were selected as suitable compounds to explore the specificity of the Sn binding region of both enzymes. Three series of inhibitors bearing Arg or Lys at P1 and different substituents at P2 and P3 were designed, synthesized, and tested. High potency (k(obs)/[I] approximately 10(7) M(-1) s(-1)) was achieved for small ligands, such as the dipeptide analogues. The detailed analysis of Sn binding pockets revealed the molecular basis of inhibitory affinity and provided insight into the structure-activity relationship.

Adhesins, Bacterial↗

Functional and structural characterization of Spl proteases from Staphylococcus aureus.

Staphylococcus aureus is the major cause of nosocomial infections world-wide, with increasing prevalence of community-acquired diseases. The recent dramatic increase in multi-antibiotic resistance, including resistance to the last-resort drug, vancomycin, together with the lack of an effective vaccine highlight the need for better understanding of S.aureus pathogenicity. Comparative analysis of available bacterial genomes allows for the identification of previously uncharacterized S.aureus genes with potential roles in pathogenicity. A good example is a cluster of six serine protease-like (spl) genes encompassed in one operon, which encode for putative proteases with similarity to staphylococcal glutamylendopeptidase (V8 protease). Here, we describe an efficient expression system for the production of recombinant SplB and SplC proteases in Escherichia coli, together with structural and functional characterization of the purified enzymes. A unique mechanism of cytoplasm protection against activity of misdirected SplB was uncovered. Apparently, the co-translated signal peptide maintains protease latency until it is cleaved by the signal peptidase during protein secretion. Furthermore, the crystal structure of the SplC protease revealed a fold resembling that of the V8 protease and epidermolytic toxins. Arrangement of the active site cleft and substrate-binding pocket of SplC explains the mechanism of enzyme latency and suggests that some Spl proteases possess restricted substrate specificity similar to that of the V8 protease and epidermolytic toxins.

Amino Acid Sequence↗

Selective inhibition of Porphyromonas gingivalis growth by a factor Xa inhibitor, DX-9065a.

BACKGROUND: Porphyromonas gingivalis is a causative bacterium of adult periodontitis. However, there is no drug specific for P. gingivalis and for its virulence factor. OBJECTIVES: The objective of this study was to examine the effects of a new selective inhibitor of activated factor X, DX-9065a, on growth of Porphyromonas gingivalis and other periodontopathic bacteria. METHODS: We incubated P. gingivalis and other periodontopathic bacteria in the presence or absence of DX-9065a and examined the effect of DX-9065a on bacterial growth and trypsin-like activity in its cultures. We also examined the effects of DX9065a on amidolytic activity of purified trypsin-like proteinases (gingipains RgpA and RgpB), from P. gingivalis and on trypsin-like activity in gingival crevicular fluids from patients with adult periodontitis. RESULTS: DX-9065a selectively inhibited the growth of P. gingivalis and Prevotella intermedia, and its effect on P. gingivalis was bactericidal. Trypsin-like proteinase activity was detected in P. gingivalis, and the activity was strongly inhibited by DX-9065a. DX-9065a even inhibited amidolytic activity of RgpA and RgpB from P. gingivalis. Furthermore, trypsin-like proteinase activity in gingival crevicular fluids was strongly inhibited by DX-9065a. CONCLUSIONS: DX-9065a inhibits P. gingivalis growth in part through to its ability to inhibit the trypsin-like proteinase activity in P. gingivalis and may be useful for a new drug for treatment of adult periodontitis.

Actinomyces↗

Gingipains from Porphyromonas gingivalis W83 synergistically disrupt endothelial cell adhesion and can induce caspase-independent apoptosis.

We have shown previously that gingipains from Porphyromonas gingivalis W83 can induce cell detachment, cell adhesion molecule (CAM) cleavage, and apoptosis in endothelial cells; however, the specific roles of the individual gingipains are unclear. Using purified gingipains, we determined that each of the gingipains can cleave CAMs to varying degrees with differing kinetics. Kgp and HRgpA work together to quickly detach endothelial cells. Interestingly, in the absence of active caspases, both gingipain-active W83 extracts and purified HRgpA and RgpB induce apoptotic morphology, suggesting that the gingipains can induce both caspase-dependent and caspase-independent apoptosis. Using z-VAD-FMK to inhibit Kgp activity and leupeptin to inhibit Rgp activity in gingipain-active W83 extracts, we investigated the relative significance of the synergistic role of the gingipains. z-VAD-FMK or leupeptin delayed, but did not inhibit, cell detachment induced by gingipain-active W83 extracts or purified gingipains. There was partial cleavage of N-cadherin and cleavage of VE-cadherin was not inhibited. Degradation of integrin beta1 was inhibited only in the presence of z-VAD-FMK. These results further clarify the role P. gingivalis plays in tissue destruction occurring in the periodontal pocket.

Adhesins, Bacterial↗

Investigations into sigmaB-modulated regulatory pathways governing extracellular virulence determinant production in Staphylococcus aureus.

The commonly used Staphylococcus aureus laboratory strain 8325-4 bears a naturally occurring 11-bp deletion in the sigmaB-regulating phosphatase rsbU. We have previously published a report (M. J. Horsburgh, J. L. Aish, I. J. White, L. Shaw, J. K. Lithgow, and S. J. Foster, J. Bacteriol. 184:5457-5467, 2002) on restoring the rsbU deletion, producing a sigmaB-functional 8325-4 derivative, SH1000. SH1000 is pleiotropically altered in phenotype from 8325-4, displaying enhanced pigmentation, increased growth yields, and a marked decrease in secreted exoproteins. This reduction in exoprotein secretion appears to result from a sixfold reduction in agr expression. In this study we have undertaken transposon mutagenesis of SH1000 to identify components involved in the modulation of extracellular proteases and alpha-hemolysin compared to 8325-4. In total, 13 genes were identified displaying increased alpha-hemolysin transcription and extracellular proteolysis. Phenotypic analysis revealed that each mutant also had decreased pigmentation and a general increase in protein secretion. Interestingly this phenotype was not identical in each case but was variable from mutant to mutant. None of the genes identified encoded classic regulatory proteins but were predominantly metabolic enzymes involved in amino acid biosynthesis and transport. Further analysis revealed that all of these mutations were clustered in a 35-kb region of the chromosome. By complementation and genetic manipulation we were able to demonstrate the validity of these mutations. Interestingly transcriptional analysis revealed that rather than being regulated by sigmaB, these genes appeared to have a role in the regulation of sigmaB activity. Thus, we propose that the loss of individual genes in this chromosomal hot spot region results in a destabilization of cellular harmony and disruption of the sigmaB regulatory cascade.

Bacterial Proteins↗

Roles of the host oxidative immune response and bacterial antioxidant rubrerythrin during Porphyromonas gingivalis infection.

The efficient clearance of microbes by neutrophils requires the concerted action of reactive oxygen species and microbicidal components within leukocyte secretory granules. Rubrerythrin (Rbr) is a nonheme iron protein that protects many air-sensitive bacteria against oxidative stress. Using oxidative burst-knockout (NADPH oxidase-null) mice and an rbr gene knockout bacterial strain, we investigated the interplay between the phagocytic oxidative burst of the host and the oxidative stress response of the anaerobic periodontal pathogen Porphyromonas gingivalis. Rbr ensured the proliferation of P. gingivalis in mice that possessed a fully functional oxidative burst response, but not in NADPH oxidase-null mice. Furthermore, the in vivo protection afforded by Rbr was not associated with the oxidative burst responses of isolated neutrophils in vitro. Although the phagocyte-derived oxidative burst response was largely ineffective against P. gingivalis infection, the corresponding oxidative response to the Rbr-positive microbe contributed to host-induced pathology via potent mobilization and systemic activation of neutrophils. It appeared that Rbr also provided protection against reactive nitrogen species, thereby ensuring the survival of P. gingivalis in the infected host. The presence of the rbr gene in P. gingivalis also led to greater oral bone loss upon infection. Collectively, these results indicate that the host oxidative burst paradoxically enhances the survival of P. gingivalis by exacerbating local and systemic inflammation, thereby contributing to the morbidity and mortality associated with infection.

Animals↗

Interaction of a novel form of Pseudomonas aeruginosa alkaline protease (aeruginolysin) with interleukin-6 and interleukin-8.

Pseudomonas aeruginosa secretes several proteases considered as important virulence factors. In this report we present data indicating that two key proinflammatory cytokines, interleukin-6 (IL-6) and IL-8, are substrates for pseudolysin (elastase) and aeruginolysin (alkaline protease). While IL-6 was totally digested by both proteases, a long form of IL-8 (IL-8-77) was first rapidly processed into a 72-residue form with enhanced chemokine activity, then very slowly degraded. Interestingly, aeruginolysin bearing two additional residues at the N-terminus (Leu-Lys-aeruginolysin) in the absence of calcium degraded both IL-6 and IL-8-72 far more efficiently than the shorter form of the enzyme.

Bacterial Proteins↗

Isolation and properties of extracellular proteinases of Penicillium marneffei.

Penicillium marneffei is a dimorphic fungus native to Southeast Asia. Disease caused by this organism, until recently a very rare condition, has increased dramatically in parallel with the increase in the number of individuals in the region immunocompromised by AIDS and other conditions. While much research has been performed on the control of dimorphic switching in P. marneffei, there is a relative dearth of information regarding the proteinases secreted by this pathogen. Our laboratory has purified and characterized two proteinases produced by this organism in liquid culture and cloned the gene of a third. Both the recombinant enzyme expressed from the cloned gene and one of those purified from culture supernatants have been identified as members of the eqolisin family, a group of pepstatin-insensitive acid proteinases. The other enzyme purified from a culture supernatant is a serine proteinase with activity in the neutral pH range. These enzymes appear to be differentially expressed, depending on culture conditions.

Amino Acid Sequence↗

Arginine-specific gingipains from Porphyromonas gingivalis stimulate production of hepatocyte growth factor (scatter factor) through protease-activated receptors in human gingival fibroblasts in culture.

Cystein proteinases (gingipains) from Porphyromonas gingivalis cleave a broad range of in-host proteins and are considered to be key virulence factors in the onset and development of adult periodontitis and host defense evasion. In periodontitis, an inflammatory disease triggered by bacterial infection, the production of hepatocyte growth factor (HGF) is induced not only by various factors derived from the host, such as inflammatory cytokines, but also by bacterial components. In this study we examined the possible enhanced production of HGF produced by human gingival fibroblasts upon stimulation with gingipains. Arginine-specific gingipain (Rgp) caused a marked production of HGF into the supernatant, the induction of HGF expression on the cell surface, and the up-regulation of HGF mRNA expression in a dose-dependent and an enzymatic activity-dependent manner. Because it has been reported that Rgp activated protease-activated receptors (PARs), we examined whether the induction of HGF triggered by Rgps on human gingival fibroblasts occurred through PARs. An RNA interference assay targeted to PAR-1 and PAR-2 mRNA revealed that gingipains-induced secretion of HGF was significantly inhibited by RNA interference targeted to PAR-1 and PAR-2. In addition, the Rgps-mediated HGF induction was completely inhibited by the inhibition of phospholipase C and was clearly inhibited by RNA interference targeted to p65, which is an NF-kappaB component. These results suggest that Rgps activated human gingival fibroblasts to secrete HGF in the inflamed sites and the mechanism(s) involved may actively participate in both inflammatory and reparative processes in periodontal diseases.

Adhesins, Bacterial↗

Inhibition of tumor necrosis factor-alpha-induced RANTES secretion by alkaline protease in A549 cells.

Pseudomonas aeruginosa is a gram-negative bacterium that is an opportunistic pathogen in patients with cystic fibrosis and in immunocompromised hosts. This bacterium produces a variety of proteolytic enzymes, including alkaline protease (AP), which has multiple biological effects. This study investigated the effects of AP on the A549 pulmonary epithelial cell line. Results demonstrate that AP inhibited tumor necrosis factor (TNF)-alpha-induced RANTES gene expression and secretion in a concentration-dependent manner. The TNF-alpha-induced RANTES gene expression and secretion was attenuated with a neutralizing monoclonal antibody directed against the TNF receptor type 1 (TNFR1). Conversely, a neutralizing monoclonal antibody directed against TNF receptor type II had no effect, suggesting that these events were regulated through the TNFR1 receptor. In addition, we observed that soluble TNF receptor type 1 (sTNFR1) levels were significantly increased in culture supernatants of AP-treated cells in a concentration-dependent manner. Finally, membrane-associated TNFR1 was decreased after AP exposures. In these studies, the enzymatically inactive form of AP had no effect on TNF-alpha-induced RANTES secretion, shedding of sTNFR1, or membrane-associated TNFR1. These results demonstrate that AP stimulates shedding of cell-surface TNFR1, resulting in an increase in sTNFR1. Consequently, these events decrease the cells' ability to stimulate RANTES gene expression and secretion through TNFR1.

Antibodies, Blocking↗

Induction of vascular leakage through release of bradykinin and a novel kinin by cysteine proteinases from Staphylococcus aureus.

Staphylococcus aureus is a major pathogen of gram-positive septic shock and frequently is associated with consumption of plasma kininogen. We examined the vascular leakage (VL) activity of two cysteine proteinases that are secreted by S. aureus. Proteolytically active staphopain A (ScpA) induced VL in a bradykinin (BK) B(2)-receptor-dependent manner in guinea pig skin. This effect was augmented by staphopain B (SspB), which, by itself, had no VL activity. ScpA also produced VL activity from human plasma, apparently by acting directly on kininogens to release BK, which again was augmented significantly by SspB. Intravenous injection of ScpA into a guinea pig caused BK B(2)-receptor-dependent hypotension. ScpA and SspB together induced the release of leucyl-methionyl-lysyl-BK, a novel kinin with VL and blood pressure-lowering activities that are equivalent to BK. Collectively, these data suggest that production of BK and leucyl-methionyl-lysyl-BK by staphopains is a new mechanism of S. aureus virulence and bacterial shock. Therefore, staphopain-specific inhibitors and kinin-receptor antagonists could be used to treat this disease.

Animals↗

Neuropeptide release from dental pulp cells by RgpB via proteinase-activated receptor-2 signaling.

Dental pulp inflammation often results from dissemination of periodontitis caused mostly by Porphyromonas gingivalis infection. Calcitonin gene-related peptide and substance P are proinflammatory neuropeptides that increase in inflamed pulp tissue. To study an involvement of the periodontitis pathogen and neuropeptides in pulp inflammation, we investigated human dental pulp cell neuropeptide release by arginine-specific cysteine protease (RgpB), a cysteine proteinase of P. gingivalis, and participating signaling pathways. RgpB induced neuropeptide release from cultured human pulp cells (HPCs) in a proteolytic activity-dependent manner at a range of 12.5-200 nM. HPCs expressed both mRNA and the products of calcitonin gene-related peptide, substance P, and proteinase-activated receptor-2 (PAR-2) that were also found in dental pulp fibroblast-like cells. The PAR-2 agonists, SLIGKV and trypsin, also induced neuropeptide release from HPCs, and HPC PAR-2 gene knockout by transfection of PAR-2 antisense oligonucleotides inhibited significantly the RgpB-elicited neuropeptide release. These results indicated that RgpB-induced neuropeptide release was dependent on PAR-2 activation. The kinase inhibitor profile on the RgpB-neuropeptide release from HPC revealed a new PAR-2 signaling pathway that was mediated by p38 MAPK and activated transcription factor-2 activation, in addition to the PAR-2-p44/42 p38MAPK and -AP-1 pathway. This new RgpB activity suggests a possible link between periodontitis and pulp inflammation, which may be modulated by neuropeptides released in the lesion.

Activating Transcription Factor 2↗

A comparison of staphostatin B with standard mechanism serine protease inhibitors.

Staphostatins are the endogenous, highly specific inhibitors of staphopains, the major secreted cysteine proteases from Staphylococcus aureus. We have previously shown that staphostatins A and B are competitive, active site-directed inhibitors that span the active site clefts of their target proteases in the same orientation as substrates. We now report the crystal structure of staphostatin B in complex with wild-type staphopain B at 1.9 A resolution. In the complex structure, the catalytic residues are found in exactly the positions that would be expected for uncomplexed papain-type proteases. There is robust, continuous density for the staphostatin B binding loop and no indication for cleavage of the peptide bond that comes closest to the active site cysteine of staphopain B. The carbonyl carbon atom C of this peptide bond is 4.1 A away from the active site cysteine sulfur Sgamma atom. The carbonyl oxygen atom O of this peptide bond points away from the putative oxyanion hole and lies almost on a line from the Sgamma atom to the C atom. The arrangement is strikingly similar to the "ionmolecule" arrangement for the complex of papain-type enzymes with their substrates but differs significantly from the arrangement conventionally assumed for the Michaelis complex of papain-type enzymes with their substrates and also from the arrangement that is crystallographically observed for complexes of standard mechanism inhibitors and their target serine proteases.

Bacterial Proteins↗

Fighting an enemy within: cytoplasmic inhibitors of bacterial cysteine proteases.

The genes encoding secreted, broad-spectrum activity cysteine proteases of Staphylococcus spp. (staphopains) and Streptococcus pyogenes (streptopain, SpeB) are genetically linked to genes encoding cytoplasmic inhibitors. While staphopain inhibitors have lipocalin-like folds, streptopain is inhibited by a protein bearing the scaffold of the enzyme profragment. Bioinformatic analysis of other prokaryotic genomes has revealed that two more species may utilize this same genetic arrangement to control streptopain-like proteases with lipocalin-like inhibitors, while three other species may employ a C-terminally located domain that resembles the profragment. This apparently represents a novel system that bacteria use to control the intracellular activity of their proteases.

Amino Acid Sequence↗

Inactivation of membrane tumor necrosis factor alpha by gingipains from Porphyromonas gingivalis.

Gingipains are cysteine proteinases produced by Porphyromonas gingivalis, a major causative bacterium of adult periodontitis. They consist of arginine-specific (HRgpA and RgpB) and lysine-specific (Kgp) proteinases. Gingipains strongly affect the host defense system by degrading some cytokines, components of the complement system, and several immune cell receptors. In an in vitro model, gingipains were shown to degrade soluble tumor necrosis factor alpha (TNF-alpha). However, since membrane TNF-alpha shows strong biological activity, especially in local inflammatory lesions, it was worth investigating whether gingipains might also destroy membrane TNF-alpha and limit its biological activities. To avoid a possible influence of gingipains on ADAM17, the secretase of TNF-alpha, the majority of experiments were performed using ADAM17-/- fibroblasts stably transfected with cDNA of human pro-TNF-alpha (ADAM17-/- TNF+). Arginine-specific gingipains (Rgp's) strongly diminished the level of TNF-alpha on the cell surface as measured by flow cytometry, and this process was not accompanied by an increased concentration of soluble TNF-alpha in the culture medium. Degradation of membrane TNF-alpha by Rgp's correlated with a strong decrease in TNF-alpha-mediated biological activities of ADAM17-/- TNF+ cells. First, the activation state of transcription factor NF-kappaB was suppressed; second, the cells were no longer able to induce apoptosis in HL-60 cells. Kgp was also able to cleave membrane TNF-alpha, but its effect was much weaker than that of Rgp's. Gingipains also limited the binding of native TNF-alpha to the target cells. Thus, gingipains are able not only to cleave soluble TNF-alpha but also to destroy the membrane form of the cytokine, which may additionally dysregulate the cytokine network.

Adhesins, Bacterial↗

Gingipains from Porphyromonas gingivalis W83 induce cell adhesion molecule cleavage and apoptosis in endothelial cells.

The presence of Porphyromonas gingivalis in the periodontal pocket and the high levels of gingipain activity detected in gingival crevicular fluid could implicate a role for gingipains in the destruction of the highly vascular periodontal tissue. To explore the effects of these proteases on endothelial cells, we exposed bovine coronary artery endothelial cells and human microvascular endothelial cells to gingipain-active extracellular protein preparations and/or purified gingipains from P. gingivalis. Treated cells exhibited a rapid loss of cell adhesion properties that was followed by apoptotic cell death. Cleavage of N- and VE-cadherin and integrin beta1 was observed in immunoblots of cell lysates. There was a direct correlation between the kinetics of cleavage of N- and VE-cadherin and loss of cell adhesion properties. Loss of cell adhesion, as well as N- and VE-cadherin and integrin beta1 cleavage, could be inhibited or significantly delayed by preincubation of P. gingivalis W83 gingipain-active extracellular extracts with the cysteine protease inhibitor Nalpha-p-tosyl-l-lysine chloromethylketone. Furthermore, purified gingipains also induced endothelial cell detachment and apoptosis. Apoptosis-associated events, including annexin V positivity, caspase-3 activation, and cleavage of the caspase substrates poly(ADP-ribose) polymerase and topoisomerase I (Topo I), were observed in endothelial cells after detachment. All of the effects observed were correlated with the different levels of cysteine-dependent proteolytic activity of the extracts tested. Taken together, these results indicate that gingipains from P. gingivalis can alter cell adhesion molecules and induce endothelial cell death, which could have implications for the pathogenicity of this organism.

Adhesins, Bacterial↗