Small molecule substrates for the rapid quantification of acyl transfer activity of nylon hydrolase NylCA.

The widespread use of polyamides such as nylons has led to the accumulation of nylon waste, which is particularly resistant to decomposition due to the intrinsic stability of the amide bond. New methods are required for the true recycling of these waste materials by depolymerization. Enzymes that are capable of hydrolyzing polyamides have been proposed as biocatalysts that may be suitable for this application. NylC is an enzyme that can mediate the hydrolysis of aminohexanoic acid oligomers, and to some extent, bulk polymers. However, current assays to characterize the activity of this enzyme require long reaction times and/or rely on secondary reactions to quantify hydrolysis. Herein, we have designed structurally-optimized small molecule chromogenic esters that serve as substrate analogues for monitoring NylC acyltransferase activity in a continuous manner. This assay can be performed in minutes at room temperature, and the substrate N-acetyl-GABA-pNP ester (kcat = 0.37 s-1, KM = 256 µM) shows selectivity for NylC in complex biological media. We also demonstrate that activity towards this substrate analogue correlates with amide hydrolysis, which is the primary activity of this enzyme. Furthermore, our screening of substrate analogues provides insight into the substrate specificity of NylC, which is relevant to biocatalytic applications.

Role of Acetyltransferase PG1842 in Gingipain Biogenesis in Porphyromonas gingivalis.

Porphyromonas gingivalis, the major etiologic agent in adult periodontitis, produces large amounts of proteases that are important for its survival and pathogenesis. The activation/maturation of gingipains, the major proteases, in P. gingivalis involves a complex network of processes which are not yet fully understood. VimA, a putative acetyltransferase and virulence-modulating protein in P. gingivalis, is known to be involved in gingipain biogenesis. P. gingivalis FLL92, a vimA-defective isogenic mutant (vimA::ermF-ermAM) showed late-onset gingipain activity at stationary phase, indicating the likelihood of a complementary functional VimA homolog in that growth phase. This study aimed to identify a functional homolog(s) that may activate the gingipains in the absence of VimA at stationary phase. A bioinformatics analysis showed five putative GCN5-related N-acetyltransferases (GNAT) encoded in the P. gingivalis genome that are structurally related to VimA. Allelic exchange mutagenesis was used to make deletion mutants for these acetyltransferases in the P. gingivalisvimA-defective mutant FLL102 (ΔvimA::ermF) genetic background. One of the mutants, designated P. gingivalis FLL126 (ΔvimA-ΔPG1842), did not show any late-onset gingipain activity at stationary phase compared to that of the parent strain P. gingivalis FLL102. A Western blot analysis of stationary-phase extracellular fractions with antigingipain antibodies showed immunoreactive bands that were similar in size to those for the progingipain species present only in the ΔvimA-ΔPG1842 isogenic mutant. Both recombinant VimA and PG1842 proteins acetylated Y230, K247, and K248 residues in the pro-RgpB substrate. Collectively, these findings indicate that PG1842 may play a significant role in the activation/maturation of gingipains in P. gingivalisIMPORTANCE Gingipain proteases are key virulence factors secreted by Porphyromonas gingivalis that cause periodontal tissue damage and the degradation of the host immune system proteins. Gingipains are translated as an inactive zymogen to restrict intracellular proteolytic activity before secretion. Posttranslational processing converts the inactive proenzyme to a catalytically active protease. Gingipain biogenesis, including its secretion and activation, is a complex process which is still not fully understood. One recent study identified acetylated lysine residues in the three gingipains RgpA, RgpB, and Kgp, thus indicating a role for acetylation in gingipain biogenesis. Here, we show that the acetyltransferases VimA and PG1842 can acetylate the pro-RgpB gingipain species. These findings further indicate that acetylation is a potential mechanism in the gingipain activation/maturation pathway in P. gingivalis.

Involvement of PG2212 zinc finger protein in the regulation of oxidative stress resistance in Porphyromonas gingivalis W83.

The adaptation of Porphyromonas gingivalis to H2O2-induced stress while inducible is modulated by an unknown OxyR-independent mechanism. Previously, we reported that the PG_2212 gene was highly upregulated in P. gingivalis under conditions of prolonged oxidative stress. Because this gene may have regulatory properties, its function in response to H2O2 was further characterized. PG2212, annotated as a hypothetical protein of unknown function, is a 10.3-kDa protein with a cysteine 2-histidine 2 (Cys2His2) zinc finger domain. The isogenic mutant P. gingivalis FLL366 (ΔPG_2212) showed increased sensitivity to H2O2 and decreased gingipain activity compared to the parent strain. Transcriptome analysis of P. gingivalis FLL366 revealed that approximately 11% of the genome displayed altered expression (130 downregulated genes and 120 upregulated genes) in response to prolonged H2O2-induced stress. The majority of the modulated genes were hypothetical or of unknown function, although some are known to participate in oxidative stress resistance. The promoter region of several of the most highly modulated genes contained conserved motifs. In electrophoretic mobility shift assays, the purified rPG2212 protein did not bind its own promoter region but bound a similar region in several of the genes modulated in the PG_2212-deficient mutant. A metabolome analysis revealed that PG2212 can regulate a number of genes coding for proteins involved in metabolic pathways critical for its survival under the conditions of oxidative stress. Collectively, our data suggest that PG2212 is a transcriptional regulator that plays an important role in oxidative stress resistance and virulence regulation in P. gingivalis.

Stroke rehabilitation in urban and rural settings in the Philippines: Protocol for an interview and visual elicitation study.

INTRODUCTION: There is a lack of community-based rehabilitation for stroke in the Philippines, and research on this topic is limited. Different challenges may be encountered in urban and rural settings. The aim of the Tulong, Ugnayan ng Lingap At gabaY (TULAY) project is to develop a context-appropriate, community-based stroke support programme, consisting of self-management and training resources to augment the rehabilitation and recovery process. An important stage in the development of this programme is to qualitatively explore the experiences and needs of all stakeholders. MATERIALS AND METHODS: Using co-designed and evidence-based topic guides, in-depth semi-structured interviews will be conducted with people living with stroke, household carers and care providers. We will aim to gain a representation of different regions (within Luzon, Visayas, and Northern Mindanao), socioeconomic levels, and urban and rural locations. For people with stroke and household carers, interviews will be supplemented by auto-photography and visual elicitation to widen access for those that prefer to share their experiences visually or have communication problems. An interpretivist paradigm will be applied across all interview data and the consolidated criteria for reporting qualitative research (COREQ) will be followed. Thematic analysis will be undertaken using guidance by Braun and Clarke. DISCUSSION: To our knowledge, this study is the first of its kind in the Philippines. It has several methodological strengths, including the capture of perspectives from multiple stakeholders in diverse settings, the inclusion of people with communication difficulties, use of visual methods, and analysis in the native language. The findings will have various applications, including the potential to influence policy, practice and guidelines, and to inform the development of the TULAY stroke support programme.

Proteome variation among Filifactor alocis strains.

Filifactor alocis, a Gram-positive anaerobic rod, is now considered one of the marker organisms associated with periodontal disease. Although there was heterogeneity in its virulence potential, this bacterium was shown to have virulence properties that may enhance its ability to survive and persist in the periodontal pocket. To gain further insight into a possible mechanism(s) of pathogenesis, the proteome of F. alocis strains was evaluated. Proteins including several proteases, neutrophil-activating protein A and calcium-binding acid repeat protein, were identified in F. alocis. During the invasion of HeLa cells, there was increased expression of several of the genes encoding these proteins in the potentially more virulent F. alocis D-62D compared to F. alocis ATCC 35896, the type strain. A comparative protein in silico analysis of the proteome revealed more cell wall anchoring proteins in the F. alocis D-62D compared to F. alocis ATCC 35896. Their expression was enhanced by coinfection with Porphyromonas gingivalis. Taken together, the variation in the pathogenic potential of the F. alocis strains may be related to the differential expression of several putative virulence factors.

Filifactor alocis has virulence attributes that can enhance its persistence under oxidative stress conditions and mediate invasion of epithelial cells by porphyromonas gingivalis.

Filifactor alocis, a Gram-positive anaerobic rod, is one of the most abundant bacteria identified in the periodontal pockets of periodontitis patients. There is a gap in our understanding of its pathogenicity and ability to interact with other periodontal pathogens. To evaluate the virulence potential of F. alocis and its ability to interact with Porphyromonas gingivalis W83, several clinical isolates of F. alocis were characterized. F. alocis showed nongingipain protease and sialidase activities. In silico analysis revealed the molecular relatedness of several virulence factors from F. alocis and P. gingivalis. In contrast to P. gingivalis, F. alocis was relatively resistant to oxidative stress and its growth was stimulated under those conditions. Biofilm formation was significantly increased in coculture. There was an increase in adherence and invasion of epithelial cells in coculture compared with P. gingivalis or F. alocis monocultures. In those epithelial cells, endocytic vesicle-mediated internalization was observed only during coculture. The F. alocis clinical isolate had an increased invasive capacity in coculture with P. gingivalis compared to the ATCC 35896 strain. In addition, there was variation in the proteomes of the clinical isolates compared to the ATCC 35896 strain. Hypothetical proteins and those known to be important virulence factors in other bacteria were identified. These results indicate that F. alocis has virulence properties that may enhance its ability to survive and persist in the periodontal pocket and may play an important role in infection-induced periodontal disease.

Sialidase and sialoglycoproteases can modulate virulence in Porphyromonas gingivalis.

The Porphyromonas gingivalis recombinant VimA can interact with the gingipains and several other proteins, including a sialidase. Sialylation can be involved in protein maturation; however, its role in virulence regulation in P. gingivalis is unknown. The three sialidase-related proteins in P. gingivalis showed the characteristic sialidase Asp signature motif (SXDXGXTW) and other unique domains. To evaluate the roles of the associated genes, randomly chosen P. gingivalis isogenic mutants created by allelic exchange and designated FLL401 (PG0778::ermF), FLL402 (PG1724::ermF), and FLL403 (PG0352::ermF-ermAM) were characterized. Similar to the wild-type strain, FLL402 and FLL403 displayed a black-pigmented phenotype in contrast to FLL401, which was not black pigmented. Sialidase activity in P. gingivalis FLL401 was reduced by approximately 70% in comparison to those in FLL402 and FLL403, which were reduced by approximately 42% and 5%, respectively. Although there were no changes in the expression of the gingipain genes, their activities were reduced by 60 to 90% in all the isogenic mutants compared to that for the wild type. Immunoreactive bands representing the catalytic domains for RgpA, RgpB, and Kgp were present in FLL402 and FLL403 but were missing in FLL401. While adhesion was decreased, the capacity for invasion of epithelial cells by the isogenic mutants was increased by 11 to 16% over that of the wild-type strain. Isogenic mutants defective in PG0778 and PG0352 were more sensitive to hydrogen peroxide than the wild type. Taken together, these results suggest that the P. gingivalis sialidase activity may be involved in regulating gingipain activity and other virulence factors and may be important in the pathogenesis of this organism.

Role of vimA in cell surface biogenesis in Porphyromonas gingivalis.

The Porphyromonas gingivalis vimA gene has been previously shown to play a significant role in the biogenesis of gingipains. Further, in P. gingivalis FLL92, a vimA-defective mutant, there was increased auto-aggregation, suggesting alteration in membrane surface proteins. In order to determine the role of the VimA protein in cell surface biogenesis, the surface morphology of P. gingivalis FLL92 was further characterized. Transmission electron microscopy demonstrated abundant fimbrial appendages and a less well defined and irregular capsule in FLL92 compared with the wild-type. In addition, atomic force microscopy showed that the wild-type had a smoother surface compared with FLL92. Western blot analysis using anti-FimA antibodies showed a 41 kDa immunoreactive protein band in P. gingivalis FLL92 which was missing in the wild-type P. gingivalis W83 strain. There was increased sensitivity to globomycin and vancomycin in FLL92 compared with the wild-type. Outer membrane fractions from FLL92 had a modified lectin-binding profile. Furthermore, in contrast with the wild-type strain, nine proteins were missing from the outer membrane fraction of FLL92, while 20 proteins present in that fraction from FLL92 were missing in the wild-type strain. Taken together, these results suggest that the VimA protein affects capsular synthesis and fimbrial phenotypic expression, and plays a role in the glycosylation and anchorage of several surface proteins.

Correction: In Porphyromonas gingivalis VimF Is Involved in Gingipain Maturation through the Transfer of Galactose.

[This corrects the article DOI: 10.1371/journal.pone.0063367.].

A single dose, three-arm, placebo-controlled, phase I study of the bradykinin B2 receptor antagonist Anatibant (LF16-0687Ms) in patients with severe traumatic brain injury.

Traumatic brain injury (TBI) mortality and morbidity remains a public health challenge. Because experimental studies support an important role of bradykinin (BK) in the neurological deterioration that follows TBI, a double-blind, randomized, placebo-controlled study of Anatibant (LF16- 0687Ms), a selective and potent antagonist of the BK B(2) receptor, was conducted in severe (Glasgow Coma Scale [GCS] < 8) TBI patients (n = 25) at six sites in the United States. At 8-12 h after injury (9.9 +/- 2.8 h), patients received a single subcutaneous injection of Anatibant (3.75 mg or 22.5 mg, n = 10 each) or placebo (n = 5). The primary objective was to investigate the pharmacokinetics of Anatibant; general safety, local tolerability, levels of the bradykinin metabolite BK1-5 in plasma and cerebrospinal fluid (CSF), intracranial pressure (ICP), and cerebral perfusion pressure were also assessed. We observed a dose-proportionality of the pharmacokinetics, Cmax, and AUC of Anatibant. V(d)/F, Cl/F, and t(1/2) were independent on the dose and protein binding was >97.7%. Anatibant, administered as single subcutaneous injections of 3.75 g and 22.5 mg, was well tolerated in severe TBI patients with no unexpected clinical adverse events or biological abnormalities observed. Interestingly, plasma and CSF levels of BK1-5 were significantly and markedly increased after trauma (e.g., 34,700 +/- 35,300 fmol/mL in plasma vs. 34.9 +/- 5.6 fmol/mL previously reported for normal volunteers), supporting the use of Anatibant as a treatment of secondary brain damage. To address this issue, a dose-response trial that would investigate the effects of Anatibant on the incidence of raised ICP and on functional outcome in severe TBI patients is needed.

In Porphyromonas gingivalis VimF is involved in gingipain maturation through the transfer of galactose.

Previously, we have reported that gingipain activity in Porphyromonas gingivalis, the major causative agent in adult periodontitis, is post-translationally regulated by the unique Vim proteins including VimF, a putative glycosyltransferase. To further characterize VimF, an isogenic mutant defective in this gene in a different P. gingivalis genetic background was evaluated. In addition, the recombinant VimF protein was used to further confirm its glycosyltransferase function. The vimF-defective mutant (FLL476) in the P. gingivalis ATCC 33277 genetic background showed a phenotype similar to that of the vimF-defective mutant (FLL95) in the P. gingivalis W83 genetic background. While hemagglutination was not detected and autoaggregation was reduced, biofilm formation was increased in FLL476. HeLa cells incubated with P. gingivalis FLL95 and FLL476 showed a 45% decrease in their invasive capacity. Antibodies raised against the recombinant VimF protein in E. coli immunoreacted only with the deglycosylated native VimF protein from P. gingivalis. In vitro glycosyltransferase activity for rVimF was observed using UDP-galactose and N-acetylglucosamine as donor and acceptor substrates, respectively. In the presence of rVimF and UDP-galactose, a 60 kDa protein from the extracellular fraction of FLL95 which was identified by mass spectrometry as Rgp gingipain, immunoreacted with the glycan specific mAb 1B5 antibody. Taken together, these results suggest the VimF glycoprotein is a galactosyltransferase that may be specific for gingipain glycosylation. Moreover, galatose is vital for the growing glycan chain.

Inactivation of vimF, a putative glycosyltransferase gene downstream of vimE, alters glycosylation and activation of the gingipains in Porphyromonas gingivalis W83.

Regulation/activation of the Porphyromonas gingivalis gingipains is poorly understood. A 1.2-kb open reading frame, a putative glycosyltransferase, downstream of vimE, was cloned, insertionally inactivated using the ermF-ermAM antibiotic resistance cassette, and used to create a defective mutant by allelic exchange. In contrast to the wild-type W83 strain, this mutant, designated P. gingivalis FLL95, was nonpigmented and nonhemolytic when plated on Brucella blood agar. Arginine- and lysine-specific gingipain activities were reduced by approximately 97% and 96%, respectively, relative to that of the parent strain. These activities were unaffected by the growth phase, in contrast to the vimA-defective mutant P. gingivalis FLL92. Expression of the rgpA, rgpB, and kgp gingipain genes was unaffected in P. gingivalis FLL95 in comparison to the wild-type strain. In nonactive gingipain extracellular protein fractions, multiple high-molecular-weight proteins immunoreacted with gingipain-specific antibodies. The specific gingipain-associated sugar moiety recognized by monoclonal antibody 1B5 was absent in FLL95. Taken together, these results suggest that the vimE downstream gene, designated vimF (virulence modulating gene F), which is a putative glycosyltransferase group 1, is involved in the regulation of the major virulence factors of P. gingivalis.

Altered gingipain maturation in vimA- and vimE-defective isogenic mutants of Porphyromonas gingivalis.

We have previously shown that gingipain activity in Porphyromonas gingivalis is modulated by the unique vimA and vimE genes. To determine if these genes had a similar phenotypic effect on protease maturation and activation, isogenic mutants defective in those genes were further characterized. Western blot analyses with antigingipain antibodies showed RgpA-, RgpB-, and Kgp-immunoreactive bands in membrane fractions as well as the culture supernatant of both P. gingivalis W83 and FLL93, the vimE-defective mutant. In contrast, the membrane of P. gingivalis FLL92, the vimA-defective mutant, demonstrated immunoreactivity only with RgpB antibodies. With mass spectrometry or Western blots, full-length RgpA and RgpB were identified from extracellular fractions. In similar extracellular fractions from P. gingivalis FLL92 and FLL93, purified RgpB activated only arginine-specific activity. In addition, the lipopolysaccharide profiles of the vimA and vimE mutants were truncated in comparison to that of W83. While glycosylated proteins were detected in the membrane and extracellular fractions from the vimA- and vimE-defective mutants, a monoclonal antibody (1B5) that reacts with specific sugar moieties of the P. gingivalis cell surface polysaccharide and membrane-associated Rgp gingipain showed no immunoreactivity with these fractions. Taken together, these results indicate a possible defect in sugar biogenesis in both the vimA- and vimE-defective mutants. These modulating genes play a role in the secretion, processing, and/or anchorage of gingipains on the cell surface.

The vimE gene downstream of vimA is independently expressed and is involved in modulating proteolytic activity in Porphyromonas gingivalis W83.

Regulation/activation of the Porphyromonas gingivalis gingipains is poorly understood. A unique 1.3-kb open reading frame downstream of the bcp-recA-vimA transcriptional unit was cloned, insertionally inactivated with the ermF-ermAM antibiotic resistance cassette, and used to create a defective mutant by allelic exchange. In contrast to the wild-type W83 strain, the growth rate of the mutant strain (designated FLL93) was reduced, and when plated on Brucella blood agar it was nonpigmented and nonhemolytic. Arginine- and lysine-specific gingipain activities were reduced by approximately 90 and 85%, respectively, relative to activities of the parent strain. These activities were unaffected by the culture's growth phase, in contrast to the vimA-defective mutant P. gingivalis FLL92, which has increased proteolytic activity in stationary phase. Expression of the rgpA, rgpB, and kgp gingipain genes was unaltered in P. gingivalis FLL93 compared to that of the wild-type strain. Further, in extracellular protein fractions a 64-kDa band was identified that was immunoreactive with the RgpB-specific proenzyme antibodies. Active-site labeling with dansyl-glutamyl-glycyl-arginyl chloromethyl ketone or immunoblot analysis showed no detectable protein band representing the gingipain catalytic domain. In vitro protease activity could be slightly induced by a urea denaturation-renaturation cycle in an extracellular protein fraction, in contrast to the vimA-defective mutant P. gingivalis FLL92. Expression of flanking genes, including recA, vimA, and Pg0792, was unaltered by the mutation. Taken together, these results suggest that the vimA downstream gene, designated vimE (for virulence-modulating gene E), is involved in the regulation of protease activity in P. gingivalis.

The Aer protein of Escherichia coli forms a homodimer independent of the signaling domain and flavin adenine dinucleotide binding.

In vivo cross-linking between native cysteines in the Aer receptor of Escherichia coli showed dimer formation at the membrane anchor and in the putative HAMP domain. Dimers also formed in mutants that did not bind flavin adenine dinucleotide and in truncated peptides without a signaling domain and part of the HAMP domain.

Gingipain RgpB is excreted as a proenzyme in the vimA-defective mutant Porphyromonas gingivalis FLL92.

We have previously shown that the unique vimA (virulence-modulating) gene could modulate proteolytic activity in Porphyromomas gingivalis. Although a reduction in cysteine protease activity was observed in the vimA-defective mutant, P. gingivalis FLL92, compared to that of the wild-type strain, no changes were seen in the expression of the gingipain genes. This result might suggest posttranscriptional regulation of protease expression. To determine whether there was a defect in the translation, transport, or maturation of the gingipains, P. gingivalis FLL92 was further characterized. In contrast to the wild-type strain, a 90% reduction was seen in both Rgp and Kgp protease activities in strain FLL92 during the exponential growth phase. These activities, however, increased to approximately 60% of that of the wild-type strain during stationary phase. Throughout all the growth phases, Rgp and Kgp activities were mostly soluble, in contrast to those of the wild-type strain. Western blot analyses identified unique Rgp- and Kgp-immunoreactive bands in extracellular protein fractions from FLL92 grown to late exponential phase. Also, the RgpB proenzyme was identified in this fraction by mass spectrometry. In addition, in vitro protease activity could be induced by a urea denaturation-renaturation cycle in this fraction. These results indicate that protease activity in P. gingivalis may be growth phase regulated, possibly by multiple mechanisms. Furthermore, the gingipain RgpB is excreted in an inactive form in the vimA mutant. In addition, these results provide the first evidence of posttranslational regulation of protease activity in P. gingivalis and may suggest an important role for the vimA gene in protease activation in this organism.