Inhibition of ß-lactamase function by de novo designed peptide.

Antimicrobial resistance is a great public health concern that is now described as a "silent pandemic". The global burden of antimicrobial resistance requires new antibacterial treatments, especially for the most challenging multidrug-resistant bacteria. There are various mechanisms by which bacteria develop antimicrobial resistance including expression of ß-lactamase enzymes, overexpression of efflux pumps, reduced cell permeability through downregulation of porins required for ß-lactam entry, or modifications in penicillin-binding proteins. Inactivation of the ß-lactam antibiotics by ß-lactamase enzymes is the most common mechanism of bacterial resistance to these agents. Although several effective small-molecule inhibitors of ß-lactamases such as clavulanic acid and avibactam are clinically available, they act only on selected class A, C, and some class D enzymes. Currently, none of the clinically approved inhibitors can effectively inhibit Class B metallo-ß-lactamases. Additionally, there is increased resistance to these inhibitors reported in several bacteria. The objective of this study is to use the Resonant Recognition Model (RRM), as a novel strategy to inhibit/modulate specific antimicrobial resistance targets. The RRM is a bio-physical approach that analyzes the distribution of energies of free electrons and posits that there is a significant correlation between the spectra of this energy distribution and related protein biological activity. In this study, we have used the RRM concept to evaluate the structure-function properties of a group of 22 ß-lactamase proteins and designed 30-mer peptides with the desired RRM spectral periodicities (frequencies) to function as ß-lactamase inhibitors. In contrast to the controls, our results indicate 100% inhibition of the class A ß-lactamases from Escherichia coli and Enterobacter cloacae. Taken together, the RRM model can likely be utilized as a promising approach to design ß-lactamase inhibitors for any specific class. This may open a new direction to combat antimicrobial resistance.

The involvement of CdhR in Porphyromonas gingivalis during nitric oxide stress.

Porphyromonas gingivalis, the causative agent of adult periodontitis, must gain resistance to frequent oxidative and nitric oxide (NO) stress attacks from immune cells in the periodontal pocket to survive. Previously, we found that, in the wild-type and under NO stress, the expression of PG1237 (CdhR), the gene encoding for a putative LuxR transcriptional regulator previously called community development and hemin regulator (CdhR), was upregulated 7.7-fold, and its adjacent gene PG1236 11.9-fold. Isogenic mutants P. gingivalis FLL457 (ΔCdhR::ermF), FLL458 (ΔPG1236::ermF), and FLL459 (ΔPG1236-CdhR::ermF) were made by allelic exchange mutagenesis to determine the involvement of these genes in P. gingivalis W83 NO stress resistance. The mutants were black pigmented and ß hemolytic and their gingipain activities varied with strains. FLL457 and FLL459 mutants were more sensitive to NO compared to the wild type, and complementation restored NO sensitivity to that of the wild type. DNA microarray analysis of FLL457 showed that approximately 2% of the genes were upregulated and over 1% of the genes downregulated under NO stress conditions compared to the wild type. Transcriptome analysis of FLL458 and FLL459 under NO stress showed differences in their modulation patterns. Some similarities were also noticed between all mutants. The PG1236-CdhR gene cluster revealed increased expression under NO stress and may be part of the same transcriptional unit. Recombinant CdhR showed binding activity to the predicted promoter regions of PG1459 and PG0495. Taken together, the data indicate that CdhR may play a role in NO stress resistance and be involved in a regulatory network in P. gingivalis.

Characterization of FA1654: A putative DPS protein in Filifactor alocis.

The survival/adaptation of Filifactor alocis, a fastidious Gram-positive asaccharolytic anaerobe, to the inflammatory environment of the periodontal pocket requires an ability to overcome oxidative stress. Moreover, its pathogenic characteristics are highlighted by its capacity to survive in the oxidative-stress microenvironment of the periodontal pocket and a likely ability to modulate the microbial community dynamics. There is still a significant gap in our understanding of its mechanism of oxidative stress resistance and its impact on the virulence and pathogenicity of the microbial biofilm. Coinfection of epithelial cells with F. alocis and Porphyromonas gingivalis resulted in the upregulation of several genes, including HMPREF0389_01654 (FA1654). Bioinformatics analysis indicates that FA1654 has a "di-iron binding domain" and could function as a DNA starvation and stationary phase protection (DPS) protein. We have further characterized the FA1654 protein to determine its role in oxidative stress resistance in F. alocis. In the presence of hydrogen peroxide-induced oxidative stress, there was an ∼1.3 fold upregulation of the FA1654 gene in F. alocis. Incubation of the purified FA1654 protein with DNA in the presence of hydrogen peroxide and iron resulted in the protection of the DNA from Fenton-mediated degradation. Circular dichroism and differential scanning fluorimetry studies have documented the intrinsic ability of rFA1654 protein to bind iron; however, the rFA1654 protein is missing the intrinsic ability to reduce hydrogen peroxide. Collectively, the data may suggest that FA1654 in F. alocis is involved in oxidative stress resistance via an ability to protect against Fenton-mediated oxidative stress-induced damage.

The Machine-Learning-Mediated Interface of Microbiome and Genetic Risk Stratification in Neuroblastoma Reveals Molecular Pathways Related to Patient Survival.

Currently, most neuroblastoma patients are treated according to the Children's Oncology Group (COG) risk group assignment; however, neuroblastoma's heterogeneity renders only a few predictors for treatment response, resulting in excessive treatment. Here, we sought to couple COG risk classification with tumor intracellular microbiome, which is part of the molecular signature of a tumor. We determine that an intra-tumor microbial gene abundance score, namely M-score, separates the high COG-risk patients into two subpopulations (Mhigh and Mlow) with higher accuracy in risk stratification than the current COG risk assessment, thus sparing a subset of high COG-risk patients from being subjected to traditional high-risk therapies. Mechanistically, the classification power of M-scores implies the effect of CREB over-activation, which may influence the critical genes involved in cellular proliferation, anti-apoptosis, and angiogenesis, affecting tumor cell proliferation survival and metastasis. Thus, intracellular microbiota abundance in neuroblastoma regulates intracellular signals to affect patients' survival.

Platelet and neutrophil responses to Porphyromonas gingivalis in human whole blood.

Porphyromonas gingivalis is a causative agent for periodontal disease. Binding of platelets to this gram-negative anaerobe can regulate host hemostatic (thrombus forming) and immune (neutrophil interacting) responses during bacterial infection. Additionally, in response to bacterial pathogens neutrophils can release their DNA, forming highly prothrombotic neutrophil extracellular traps (NETs), which then further enhance platelet responses. This study evaluates the role of P. gingivalis on platelet expression of CD62P, platelet-neutrophil interactions, and labeled neutrophil-associated DNA. Human whole blood was preincubated with varying P. gingivalis concentrations, with or without subsequent addition of adenosine diphosphate (ADP). Flow cytometry was employed to measure platelet expression of CD62P using PerCP-anti-CD61 and PE-anti-CD62P, platelet-neutrophil interactions using PerCP-anti-CD61 and FITC-anti-CD16, and the release of neutrophil DNA using FITC-anti-CD16 and Sytox Blue labeling. Preincubation with a high (6.25 × 106  CFU/mL) level of P. gingivalis significantly increased platelet expression of CD62P in ADP treated and untreated whole blood. In addition, platelet-neutrophil interactions were significantly increased after ADP stimulation, following 5-22 min preincubation of blood with high P. gingivalis CFU. However, in the absence of added ADP, platelet-neutrophil interactions increased in a manner dependent on the preincubation time with P. gingivalis. Moreover, after ADP addition, 16 min preincubation of whole blood with P. gingivalis led to increased labeling of neutrophil-associated DNA. Taken together, the results suggest that the presence of P. gingivalis alters platelet and neutrophil responses to increase platelet activation, platelet interactions with neutrophils, and the level of neutrophil antimicrobial NETs.

Role of Superoxide Reductase FA796 in Oxidative Stress Resistance in Filifactor alocis.

Filifactor alocis, a Gram-positive anaerobic bacterium, is now a proposed diagnostic indicator of periodontal disease. Because the stress response of this bacterium to the oxidative environment of the periodontal pocket may impact its pathogenicity, an understanding of its oxidative stress resistance strategy is vital. Interrogation of the F. alocis genome identified the HMPREF0389_00796 gene that encodes for a putative superoxide reductase (SOR) enzyme. SORs are non-heme, iron-containing enzymes that can catalyze the reduction of superoxide radicals to hydrogen peroxide and are important in the protection against oxidative stress. In this study, we have functionally characterized the putative SOR (FA796) from F. alocis ATCC 35896. The recombinant FA796 protein, which is predicted to be a homotetramer of the 1Fe-SOR class, can reduce superoxide radicals. F. alocis FLL141 (∆FA796::ermF) was significantly more sensitive to oxygen/air exposure compared to the parent strain. Sensitivity correlated with the level of intracellular superoxide radicals. Additionally, the FA796-defective mutant had increased sensitivity to hydrogen peroxide-induced stress, was inhibited in its ability to form biofilm and had reduced survival in epithelial cells. Collectively, these results suggest that the F. alocis SOR protein is a key enzymatic scavenger of superoxide radicals and protects the bacterium from oxidative stress conditions.

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.

Role of the Porphyromonas gingivalis iron-binding protein PG1777 in oxidative stress resistance.

Whole genome sequencing of the response of Porphyromonas gingivalis W83 to hydrogen peroxide revealed an upregulation of several uncharacterized, novel genes. Under conditions of prolonged oxidative stress in P. gingivalis, increased expression of a unique transcriptional unit carrying the grpE, dnaJ and three other hypothetical genes (PG1777, PG1778 and PG1779) was observed. The transcriptional start site of this operon appears to be located 91 bp upstream of the translational start, with a potential -10 region at -3 nt and a -35 region at -39 nt. Isogenic P. gingivalis mutants FLL273 (PG1777 : : ermF-ermAM) and FLL293 (PG1779 : : ermF-ermAM) showed increased sensitivity to and decreased survival after treatment with hydrogen peroxide. P. gingivalis FLL273 showed a fivefold increase in the formation of spontaneous mutants when compared with the parent strain after exposure to hydrogen peroxide. The recombinant PG1777 protein displayed iron-binding properties when incubated with FeSO4 and Fe(NH4)2(SO4).6H2O. The rPG1777 protein protected DNA from degradation when exposed to hydrogen peroxide in the presence of iron. Taken together, the data suggest that the grpE-dnaJ-PG1777-PG1778-PG1779 transcriptional unit may play an important role in oxidative stress resistance in P. gingivalis via its ability to protect against DNA damage.

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.

Proteome analysis of coinfection of epithelial cells with Filifactor alocis and Porphyromonas gingivalis shows modulation of pathogen and host regulatory pathways.

Changes in periodontal status are associated with shifts in the composition of the bacterial community in the periodontal pocket. The relative abundances of several newly recognized microbial species, including Filifactor alocis, as-yet-unculturable organisms, and other fastidious organisms have raised questions on their impact on disease development. We have previously reported that the virulence attributes of F. alocis are enhanced in coculture with Porphyromonas gingivalis. We have evaluated the proteome of host cells and F. alocis during a polymicrobial infection. Coinfection of epithelial cells with F. alocis and P. gingivalis strains showed approximately 20% to 30% more proteins than a monoinfection. Unlike F. alocis ATCC 35896, the D-62D strain expressed more proteins during coculture with P. gingivalis W83 than with P. gingivalis 33277. Proteins designated microbial surface component-recognizing adhesion matrix molecules (MSCRAMMs) and cell wall anchor proteins were highly upregulated during the polymicrobial infection. Ultrastructural analysis of the epithelial cells showed formation of membrane microdomains only during coinfection. The proteome profile of epithelial cells showed proteins related to cytoskeletal organization and gene expression and epigenetic modification to be in high abundance. Modulation of proteins involved in apoptotic and cell signaling pathways was noted during coinfection. The enhanced virulence potential of F. alocis may be related to the differential expression levels of several putative virulence factors and their effects on specific host cell pathways.

Protective role of the PG1036-PG1037-PG1038 operon in oxidative stress in Porphyromonas gingivalis W83.

As an anaerobe, Porphyromonas gingivalis is significantly affected by the harsh inflammatory environment of the periodontal pocket during initial colonization and active periodontal disease. We reported previously that the repair of oxidative stress-induced DNA damage involving 8-oxo-7,8-dihydroguanine (8-oxoG) may occur by an undescribed mechanism in P. gingivalis. DNA affinity fractionation identified PG1037, a conserved hypothetical protein, among other proteins, that were bound to the 8-oxoG lesion. PG1037 is part of the uvrA-PG1037-pcrA operon in P. gingivalis which is known to be upregulated under H2O2 induced stress. A PCR-based linear transformation method was used to inactivate the uvrA and pcrA genes by allelic exchange mutagenesis. Several attempts to inactivate PG1037 were unsuccessful. Similar to the wild-type when plated on Brucella blood agar, the uvrA and pcrA-defective mutants were black-pigmented and beta-hemolytic. These isogenic mutants also had reduced gingipain activities and were more sensitive to H2O2 and UV irradiation compared to the parent strain. Additionally, glycosylase assays revealed that 8-oxoG repair activities were similar in both wild-type and mutant P. gingivalis strains. Several proteins, some of which are known to have oxidoreducatse activity, were shown to interact with PG1037. The purified recombinant PG1037 protein could protect DNA from H2O2-induced damage. Collectively, these findings suggest that the uvrA-PG1037-pcrA operon may play an important role in hydrogen peroxide stress-induced resistance in P. gingivalis.

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.

The effect of intraoperative mupirocin irrigation on Staphylococcus aureus within the maxillary sinus.

BACKGROUND: Antibiotic irrigations are occasionally used during endoscopic sinus surgery when gross mucosal infection is present. These irrigations are thought to flush out pathogenic bacteria and decrease the bacterial load within the mucosal surfaces. This treatment, however, has not been studied in vivo and it is unknown whether antibiotic rinses produce a quantitative reduction in pathologic bacteria within the sinus mucosa. The objective of this study was to determine the relative abundance of Staphylococcus aureus within the maxillary sinus and to evaluate the impact of intraoperative mupirocin irrigation on bacterial burden. METHODS: Sixteen patients with symmetric maxillary chronic rhinosinusitis were prospectively enrolled. After bilateral maxillary antrostomies, biopsies were taken of the maxillary sinus mucosa on both sides. In each patient, the right side was irrigated with 240 mL of normal saline (NS) and the left side was irrigated with 240 mL of NS mixed with 60 mg mupirocin. Repeat maxillary sinus mucosal biopsies were taken from each side 7 to 10 days postsurgery. Each biopsy was analyzed using quantitative polymerase chain reaction to determine the presence and relative amount of S. aureus. RESULTS: Mupirocin irrigations were found to significantly reduce the amount of S. aureus found within the maxillary sinus mucosa compared to NS alone. The average fold change between the pre- and posttreatment biopsies on the right and left was 9.05 and 97.42, respectively (p < 0.01). CONCLUSION: Intraoperative mupirocin irrigations significantly reduce the amount of S. aureus detected within the diseased sinus mucosa at up to 10 days postoperatively.

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.

Involvement of extracytoplasmic function sigma factors in virulence regulation in Porphyromonas gingivalis W83.

Extracytoplasmic function (ECF) sigma factors are known to play an important role in the bacterial response to various environmental stresses and can significantly modulate their pathogenic potential. In the genome of Porphyromonas gingivalis W83, six putative ECF sigma factors were identified. To further evaluate their role in this organism, a PCR-based linear transformation method was used to inactivate five ECF sigma factor genes (PG0162, PG0214, PG0985, PG1660, and PG1827) by allelic exchange mutagenesis. All five isogenic mutants formed black-pigmented colonies on blood agar. Mutants defective in PG0985, PG1660, and PG1827 genes were more sensitive to 0.25 mM of hydrogen peroxide compared with the wild-type strain. Isogenic mutants of PG0162 and PG1660 showed a 50% decrease in gingipain activity. Reverse transcription-PCR analysis showed that there was no alteration in the expression of rgpA, rgpB, and kgp gingipain genes in these mutants. Hemolytic and hemagglutination activities were decreased by more than 50% in the PG0162 mutant compared with the wild type. Taken together, these findings suggest that ECF sigma factors can modulate important virulence factors in P. gingivalis. ECF sigma factors encoded by the PG0162 and PG1660 genes might also be involved in the post-transcriptional regulation of the gingipains.

DNA repair of 8-oxo-7,8-dihydroguanine lesions in Porphyromonas gingivalis.

The persistence of Porphyromonas gingivalis in the inflammatory environment of the periodontal pocket requires an ability to overcome oxidative stress. DNA damage is a major consequence of oxidative stress. Unlike the case for other organisms, our previous report suggests a role for a non-base excision repair mechanism for the removal of 8-oxo-7,8-dihydroguanine (8-oxo-G) in P. gingivalis. Because the uvrB gene is known to be important in nucleotide excision repair, the role of this gene in the repair of oxidative stress-induced DNA damage was investigated. A 3.1-kb fragment containing the uvrB gene was PCR amplified from the chromosomal DNA of P. gingivalis W83. This gene was insertionally inactivated using the ermF-ermAM antibiotic cassette and used to create a uvrB-deficient mutant by allelic exchange. When plated on brucella blood agar, the mutant strain, designated P. gingivalis FLL144, was similar in black pigmentation and beta-hemolysis to the parent strain. In addition, P. gingivalis FLL144 demonstrated no significant difference in growth rate, proteolytic activity, or sensitivity to hydrogen peroxide from that of the parent strain. However, in contrast to the wild type, P. gingivalis FLL144 was significantly sensitive to UV irradiation. The enzymatic removal of 8-oxo-G from duplex DNA was unaffected by the inactivation of the uvrB gene. DNA affinity fractionation identified unique proteins that preferentially bound to the oligonucleotide fragment carrying the 8-oxo-G lesion. Collectively, these results suggest that the repair of oxidative stress-induced DNA damage involving 8-oxo-G may occur by a still undescribed mechanism in P. gingivalis.

Gingipain-dependent interactions with the host are important for survival of Porphyromonas gingivalis.

Porphyromonas gingivalis, a major periodontal pathogen, must acquire nutrients from host derived substrates, overcome oxidative stress and subvert the immune system. These activities can be coordinated via the gingipains which represent the most significant virulence factor produced by this organism. In the context of our contribution to this field, we will review the current understanding of gingipain biogenesis, glycosylation, and regulation, as well as discuss their role in oxidative stress resistance and apoptosis. We can postulate a model, in which gingipains may be part of the mechanism for P. gingivalis virulence.

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.

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.