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.

Filifactor alocis enhances survival of Porphyromonas gingivalis W83 in response to H2 O2 -induced stress.

A dysbiotic microbial community whose members have specific/synergistic functions that are modulated by environmental conditions, can disturb homeostasis in the subgingival space leading to destructive inflammation, plays a role in the progression of periodontitis. Filifactor alocis, a gram-positive, anaerobic bacterium, is a newly recognized microbe that shows a strong correlation with periodontal disease. Our previous observations suggested F. alocis to be more resistant to oxidative stress compared to Porphyromonas gingivalis. The objective of this study is to further determine if F. alocis, because of its increased resistance to oxidative stress, can affect the survival of other 'established' periodontal pathogens under environmental stress conditions typical of the periodontal pocket. Here, we have shown that via their interaction, F. alocis protects P. gingivalis W83 under H2 O2 -induced oxidative stress conditions. Transcriptional profiling of the interaction of F. alocis and P. gingivalis in the presence of H2 O2 -induced stress revealed the modulation of several genes, including those with ABC transporter and other cellular functions. The ABC transporter operon (PG0682-PG0685) of P. gingivalis was not significant to its enhanced survival when cocultured with F. alocis under H2 O2 -induced oxidative stress. In F. alocis, one of the most highly up-regulated operons (FA0894-FA0897) is predicted to encode a putative manganese ABC transporter, which in other bacteria can play an essential role in oxidative stress protection. Collectively, the results may indicate that F. alocis could likely stabilize the microbial community in the inflammatory microenvironment of the periodontal pocket by reducing the oxidative environment. This strategy could be vital to the survival of other pathogens, such as P. gingivalis, and its ability to adapt and persist in the periodontal pocket.

Determining Susceptibility and Potential Mediators of Resistance for the Novel Polymyxin Derivative, SPR206, in Acinetobacter baumannii.

With the increase in carbapenem-resistant A. baumannii (CRAB) infections, there has been a resurgence in the use of polymyxins, specifically colistin (COL). Since the reintroduction of COL-based regimens in treating CRAB infections, several COL-resistant A. baumannii isolates have been identified, with the mechanism of resistance heavily linked with the loss of the lipopolysaccharide (LPS) layer of the bacterial outer membrane through mutations in lpxACD genes or the pmrCAB operon. SPR206, a novel polymyxin derivative, has exhibited robust activity against multidrug-resistant (MDR) A. baumannii. However, there is a dearth of knowledge regarding its efficacy in comparison with other A. baumannii-active therapeutics and whether traditional polymyxin (COL) mediators of A. baumannii resistance also translate to reduced SPR206 activity. Here, we conducted susceptibility testing using broth microdilution on 30 A. baumannii isolates (17 COL-resistant and 27 CRAB), selected 14 COL-resistant isolates for genomic sequencing analysis, and performed time-kill analyses on four COL-resistant isolates. In susceptibility testing, SPR206 demonstrated a lower range of minimum inhibitory concentrations (MICs) compared with COL, with a four-fold difference observed in MIC50 values. Mutations in lpxACD and/or pmrA and pmrB genes were detected in each of the 14 COL-resistant isolates; however, SPR206 maintained MICs ≤ 2 mg/L for 9/14 (64%) of the isolates. Finally, SPR206-based combination regimens exhibited increased synergistic and bactericidal activity compared with COL-based combination regimens irrespective of the multiple resistance genes detected. The results of this study highlight the potential utility of SPR206 in the treatment of COL-resistant A. baumannii infections.

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.

Local and Systemic Effects of Porphyromonas gingivalis Infection.

Porphyromonas gingivalis, a gram-negative anaerobe, is a leading etiological agent in periodontitis. This infectious pathogen can induce a dysbiotic, proinflammatory state within the oral cavity by disrupting commensal interactions between the host and oral microbiota. It is advantageous for P. gingivalis to avoid complete host immunosuppression, as inflammation-induced tissue damage provides essential nutrients necessary for robust bacterial proliferation. In this context, P. gingivalis can gain access to the systemic circulation, where it can promote a prothrombotic state. P. gingivalis expresses a number of virulence factors, which aid this pathogen toward infection of a variety of host cells, evasion of detection by the host immune system, subversion of the host immune responses, and activation of several humoral and cellular hemostatic factors.

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.

The Oxidative Stress-Induced Hypothetical Protein PG_0686 in Porphyromonas gingivalis W83 Is a Novel Diguanylate Cyclase.

The survival/adaptation of Porphyromonas gingivalis to the inflammatory environment of the periodontal pocket requires an ability to overcome oxidative stress. Several functional classes of genes, depending on the severity and duration of the exposure, were induced in P. gingivalis under H2O2-induced oxidative stress. The PG_0686 gene was highly upregulated under prolonged oxidative stress. PG_0686, annotated as a hypothetical protein of unknown function, is a 60 kDa protein that carries several domains including hemerythrin, PAS10, and domain of unknown function (DUF)-1858. Although PG_0686 showed some relatedness to several diguanylate cyclases (DGCs), it is missing the classical conserved, active site sequence motif (GGD[/E]EF), commonly observed in other bacteria. PG_0686-related proteins are observed in other anaerobic bacterial species. The isogenic mutant P. gingivalis FLL361 (ΔPG_0686::ermF) showed increased sensitivity to H2O2, and decreased gingipain activity compared to the parental strain. Transcriptome analysis of P. gingivalis FLL361 showed the dysregulation of several gene clusters/operons, known oxidative stress resistance genes, and transcriptional regulators, including PG_2212, CdhR and PG_1181 that were upregulated under normal anaerobic conditions. The intracellular level of c-di-GMP in P. gingivalis FLL361 was significantly decreased compared to the parental strain. The purified recombinant PG_0686 (rPG_0686) protein catalyzed the formation of c-di-GMP from GTP. Collectively, our data suggest a global regulatory property for PG_0686 that may be part of an unconventional second messenger signaling system in P. gingivalis. Moreover, it may coordinately regulate a pathway(s) vital for protection against environmental stress, and is significant in the pathogenicity of P. gingivalis and other anaerobes. IMPORTANCE Porphyromonas gingivalis is an important etiological agent in periodontitis and other systemic diseases. There is still a gap in our understanding of the mechanisms that P. gingivalis uses to survive the inflammatory microenvironment of the periodontal pocket. The hypothetical PG_0686 gene was highly upregulated under prolonged oxidative stress. Although the tertiary structure of PG_0686 showed little relatedness to previously characterized diguanylate cyclases (DGCs), and does not contain the conserved GGD(/E)EF catalytic domain motif sequence, an ability to catalyze the formation of c-di-GMP from GTP is demonstrated. The second messenger pathway for c-di-GMP was previously predicted to be absent in P. gingivalis. PG_0686 paralogs are identified in other anaerobic bacteria. Thus, PG_0686 may represent a novel class of DGCs, which is yet to be characterized. In conclusion, we have shown, for the first time, evidence for the presence of c-di-GMP signaling with environmental stress protective function in P. gingivalis.

Nitric oxide stress resistance in Porphyromonas gingivalis is mediated by a putative hydroxylamine reductase.

Porphyromonas gingivalis, the causative agent of adult periodontitis, must maintain nitric oxide (NO) homeostasis and surmount nitric oxide stress from host immune responses or other oral bacteria to survive in the periodontal pocket. To determine the involvement of a putative hydroxylamine reductase (PG0893) and a putative nitrite reductase-related protein (PG2213) in P. gingivalis W83 NO stress resistance, genes encoding those proteins were inactivated by allelic exchange mutagenesis. The isogenic mutants P. gingivalis FLL455 (PG0893ermF) and FLL456 (PG2213ermF) were black pigmented and showed growth rates and gingipain and hemolytic activities similar to those of the wild-type strain. P. gingivalis FLL455 was more sensitive to NO than the wild type. Complementation of P. gingivalis FLL455 with the wild-type gene restored the level of NO sensitivity to a level similar to that of the parent strain. P. gingivalis FLL455 and FLL456 showed sensitivity to oxidative stress similar to that of the wild-type strain. DNA microarray analysis showed that PG0893 and PG2213 were upregulated 1.4- and 2-fold, respectively, in cells exposed to NO. In addition, 178 genes were upregulated and 201 genes downregulated more than 2-fold. The majority of these modulated genes were hypothetical or of unknown function. PG1181, predicted to encode a transcriptional regulator, was upregulated 76-fold. Transcriptome in silico analysis of the microarray data showed major metabolomic variations in key pathways. Collectively, these findings indicate that PG0893 and several other genes may play an important role in P. gingivalis NO stress resistance.

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.

Differential response of Porphyromonas gingivalis to varying levels and duration of hydrogen peroxide-induced oxidative stress.

Porphyromonas gingivalis, an anaerobic oral pathogen implicated in adult periodontitis, can exist in an environment of oxidative stress. To evaluate its adaptation to this environment, we have assessed the response of P. gingivalis W83 to varying levels and durations of hydrogen peroxide (H(2)O(2))-induced stress. When P. gingivalis was initially exposed to a subinhibitory concentration of H(2)O(2) (0.1 mM), an adaptive response to higher concentrations could be induced. Transcriptome analysis demonstrated that oxidative stress can modulate several functional classes of genes depending on the severity and duration of the exposure. A 10 min exposure to H(2)O(2) revealed increased expression of genes involved in DNA damage and repair, while after 15 min, genes involved in protein fate, protein folding and stabilization were upregulated. Approximately 9 and 2.8% of the P. gingivalis genome displayed altered expression in response to H(2)O(2) exposure at 10 and 15 min, respectively. Substantially more genes were upregulated (109 at 10 min; 47 at 15 min) than downregulated (76 at 10 min; 11 at 15 min) by twofold or higher in response to H(2)O(2) exposure. The majority of these modulated genes were hypothetical or of unknown function. One of those genes (pg1372) with DNA-binding properties that was upregulated during prolonged oxidative stress was inactivated by allelic exchange mutagenesis. The isogenic mutant P. gingivalis FLL363 (pg1372 : : ermF) showed increased sensitivity to H(2)O(2) compared with the parent strain. Collectively, our data indicate the adaptive ability of P. gingivalis to oxidative stress and further underscore the complex nature of its resistance strategy under those conditions.

Nitrate reductase activity of bacteria in saliva of term and preterm infants.

The salivary glands of adults concentrate nitrate from plasma into saliva where it is converted to nitrite by bacterial nitrate reductases. Nitrite can play a beneficial role in adult gastrointestinal and cardiovascular physiology. When nitrite is swallowed, some of it is converted to nitric oxide (NO) in the stomach and may then exert protective effects in the gastrointestinal tract and throughout the body. It has yet to be determined either when newborn infants acquire oral nitrate reducing bacteria or what the effects of antimicrobial therapy or premature birth may be on the bacterial processing of nitrate to nitrite. We measured nitrate and nitrite levels in the saliva of adults and both preterm and term human infants in the early weeks of life. We also measured oral bacterial reductase activity in the saliva of both infants and adults, and characterized the species of nitrate reducing bacteria present. Oral bacterial conversion of nitrate to nitrite in infants was either undetectable or markedly lower than the conversion rates of adults. No measurable reductase activity was found in infants within the first two weeks of life, despite the presence of oral nitrate reducing bacteria such as Actinomyces odontolyticus, Veillonella atypica, and Rothia mucilaginosa. We conclude that relatively little nitrite reaches the infant gastrointestinal tract due to the lack of oral bacterial nitrate reductase activity. Given the importance of the nitrate-nitrite-NO axis in adults, the lack of oral nitrate-reducing bacteria in infants may be relevant to the vulnerability of newborns to hypoxic stress and gastrointestinal tract pathologies.

Can periodontal pockets and caries lesions act as reservoirs for coronavirus?

The periodontal pocket and likely caries lesions may act as a reservoir and source of dissemination and development of systemic infections. While periodontal pockets have been found to harbor several viral species, there is no information on its ability to serve as a reservoir for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We have used a real-time polymerase chain reaction (RT-PCR) approach to evaluate SARS-CoV-2 in periodontal pockets and cavitated caries lesions in a cross-sectional study of 72 participants who were divided into six groups: symptomatic positive COVID-19 cases with periodontal pockets, symptomatic positive with cavitated caries lesions, asymptomatic positive with periodontal pockets, asymptomatic positive with cavitated caries lesions, positive control, and negative control. A total of 180 samples were interrogated by RT-PCR to amplify the SARS-CoV-2 E and S genes. SARS-CoV-2 was present in 41.7% of symptomatic positive COVID-19 cases with periodontal pockets and 16.7% of symptomatic positive with cavitated caries lesions. The mean Ct value of E and S genes in periodontal pockets patients were 36.06±0.46 and 30.06±6.73, respectively, and the mean Ct value for both genes in caries lesions patients were 35.73±4.14, and 34.78±1.93, respectively. The sensitivity, specificity, and accuracy to detect SARS-CoV-2 among periodontal pockets were 20.8% (95% CI 7.13-42.15), 100% (95% CI 73.54-100.0), and 47.2% (95% CI 30.22-64.51), respectively. Among cavitated caries lesions patients, they were 8.3% (95% CI 1.03-27.0), 100% (95% CI 73.54-100.0), and 38.9% (95% CI 23.14-56.54), respectively. SARS-CoV-2 can be detected in periodontal pockets and caries lesions, and these sites may act as reservoirs for the virus. However, the sensitivity of SARS-CoV-2 detection is low compared with other methods. To our knowledge, this report is the first to investigate the relationship between SARS-CoV-2 and periodontal pockets and caries.

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 the Filifactor alocis Hypothetical Protein FA519 in Oxidative Stress Resistance.

In the periodontal pocket, there is a direct correlation between environmental conditions, the dynamic oral microbial flora, and disease. The relative abundance of several newly recognized microbial species in the oral microenvironment has raised questions on their impact on disease development. One such organism, Filifactor alocis, is significant to the pathogenic biofilm structure. Moreover, its pathogenic characteristics are highlighted by its ability to survive in the oxidative-stress microenvironment of the periodontal pocket and alter the microbial community dynamics. There is a gap in our understanding of its mechanism(s) of oxidative stress resistance and impact on pathogenicity. Several proteins, including HMPRFF0389-00519 (FA519), were observed in high abundance in F. alocis during coinfection of epithelial cells with Porphyromonas gingivalis W83. Bioinformatics analysis shows that FA519 contains a "Cys-X-X-Cys zinc ribbon domain" which could be involved in DNA binding and oxidative stress resistance. We have characterized FA519 to elucidate its roles in the oxidative stress resistance and virulence of F. alocis. Compared to the wild-type strain, the F. alocis isogenic gene deletion mutant, FLL1013 (ΔFA519::ermF), showed significantly reduced sensitivity to hydrogen peroxide and nitric oxide-induced stress. The ability to form biofilm and adhere to and invade gingival epithelial cells was also reduced in the isogenic mutant. The recombinant FA519 protein was shown to protect DNA from Fenton-mediated damage with an intrinsic ability to reduce hydrogen peroxide and disulfide bonds. Collectively, these results suggest that FA519 is involved in oxidative stress resistance and can modulate important virulence attributes in F. alocis. IMPORTANCE Filifactor alocis is an emerging member of the periodontal community and is now proposed to be a diagnostic indicator of periodontal disease. However, due to the lack of genetic tools available to study this organism, not much is known about its virulence attributes. The mechanism(s) of oxidative stress resistance in F. alocis is unknown. Therefore, identifying the adaptive mechanisms utilized by F. alocis to survive in the oxidative stress environment of the periodontal pocket would lead to understanding its virulence regulation, which could help develop novel therapeutic treatments to combat the effects of periodontal disease. This study is focused on the characterization of FA519, a hypothetical protein in F. alocis, as a multifunctional protein that plays an important role in the reactive oxygen species-detoxification pathway. Collectively, our results suggest that FA519 is involved in oxidative stress resistance and can modulate important virulence attributes in F. alocis.

PG1659 functions as anti-sigma factor to extracytoplasmic function sigma factor RpoE in Porphyromonas gingivalis W83.

Anti-sigma factors play a critical role in regulating the expression of sigma factors in response to environmental stress signals. PG1659 is cotranscribed with an upstream gene PG1660 (rpoE), which encodes for a sigma factor that plays an important role in oxidative stress resistance and the virulence regulatory network of P. gingivalis. PG1659, which is annotated as a hypothetical gene, is evaluated in this study. PG1659, composed of 130 amino acids, is predicted to be transmembrane protein with a single calcium (Ca2+ ) binding site. In P. gingivalis FLL358 (ΔPG1659::ermF), the rpoE gene was highly upregulated compared to the wild-type W83 strain. RpoE-induced genes were also upregulated in the PG1659-defective isogenic mutant. Both protein-protein pull-down and bacterial two-hybrid assays revealed that the PG1659 protein could interact with/bind RpoE. The N-terminal domain of PG1659, representing the cytoplasmic fragment of the protein, is critical for interaction with RpoE. In the presence of PG1659, the initiation of transcription by the RpoE sigma factor was inhibited. Taken together, our data suggest that PG1659 is an anti-sigma factor which plays an important regulatory role in the modulation of the sigma factor RpoE in P. gingivalis.

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 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.