Oxidative stress resistance in Porphyromonas gingivalis

Porphyromonas gingivalis, a black-pigmented, Gram-negative anaerobe, is an important etiologic agent of periodontal disease. The harsh inflammatory condition of the periodontal pocket implies that this organism has properties that will facilitate its ability to respond and adapt to oxidative stress. Because the stress response in the pathogen is a major determinant of its virulence, a comprehensive understanding of its oxidative stress resistance strategy is vital. We discuss multiple mechanisms and systems that clearly work in synergy to defend and protect P. gingivalis against oxidative damage caused by reactive oxygen species. The involvement of multiple hypothetical proteins and/or proteins of unknown function in this process may imply other unique mechanisms and potential therapeutic targets.     

Exit of intracellular Porphyromonas gingivalis from gingival epithelial cells is mediated by endocytic recycling pathway

Gingival epithelial cells function as an innate host defence system to prevent intrusion by periodontal bacteria. Nevertheless, Porphyromonas gingivalis, the most well‐known periodontal pathogen, can enter gingival epithelial cells and pass through the epithelial barrier into deeper tissues. However, it is poorly understood how this pathogen exits from infected cells for further transcellular spreading. The present study was performed to elucidate the cellular machinery exploited by P. gingivalis to exit from immortalized human gingival epithelial cells. P. gingivalis was shown to be internalized with early endosomes positive for the FYVE domain of EEA1 and transferrin receptor, and about half of the intracellular bacteria were then sorted to lytic compartments, including autolysosomes and late endosomes/lysosomes, while a considerable number of the remaining organisms were sorted to Rab11‐ and RalA‐positive recycling endosomes. Inhibition experiments revealed that bacterial exit was dependent on actin polymerization, lipid rafts and microtubule assembly. Dominant negative forms and RNAi knockdown of Rab11, RalA and exocyst complex subunits (Sec5, Sec6 and Exo84) significantly disturbed the exit of P. gingivalis. These results strongly suggest that the recycling pathway is exploited by intracellular P. gingivalis to exit from infected cells to neighbouring cells as a mechanism of cell‐to‐cell spreading.     

Proteomics-based analysis of a counter-oxidative stress system in Porphyromonas gingivalis

Porphyromonas gingivalis is a Gram-negative anaerobic pathogen associated with chronic periodontitis. Although anaerobic, P. gingivalis exhibits a high degree of aerotolerance, which enables it to survive within periodontal pockets. The aim of the present study was to examine the effect of oxidative stress on protein expression in P. gingivalis to obtain a better understanding of the mechanism underlying its aerotolerance. To accomplish this, P. gingivalis cells were grown under conditions of hemin limitation (0.01 microg/mL) to avoid the oxygen protective effect of hemin on oxidative stress. The proteins were then extracted from cultures either left untreated or subjected to oxidative stress and separated by 2-DE. The resultant protein expression profiles were examined by image scanning, and those found to differ depending on the presence or absence of aeration were subjected to MALDI-MS and then analyzed using the ORF database of P. gingivalis W83 from The Institute of Genomic Research. Oxidative stress was found to affect the expression of numerous proteins in P. gingivalis cells. In particular, the levels of HtpG, GroEL, DnaK, AhpC, TPR domain protein, and trigger factor were substantially increased.     

Coaggregation of Porphyromonas gingivalis and Fusobacterium nucleatum PK 1594 is mediated by capsular polysaccharide and lipopolysaccharide

Previous reports have shown that coaggregation between Porphyromonas gingivalis and Fusobacterium nucleatum, two important periodontopathogens, is mediated by a galactoside on the surface of P. gingivalis and a lectin on F. nucleatum. In the present study, purified capsular polysaccharide (CPS) and lipopolysaccharide (LPS) of P. gingivalis PK 1924 (serotype K5) were found to be able to bind to F. nucleatum cells and to inhibit binding of F. nucleatum to P. gingivalis serotype K5. Sugar binding studies showed that the requirements for binding of P. gingivalis serotype K5 CPS and LPS to the F. nucleatum lectin are: the presence of a metal divalent ion, an axial free hydroxyl group at position 4 and free equatorial hydroxyl groups at position 3 and 6 of d-galactose. These data suggest that P. gingivalis serotype K5- CPS and LPS act as receptors mediating coaggregation between P. gingivalis and fusobacteria.     

A universal stress protein of Porphyromonas gingivalis is involved in stress responses and biofilm formation

Porphyromonas gingivalis is recognized as one of the major periodontal pathogens in subgingival plaque, which is implicated in the progression of chronic periodontal disease. We analyzed the role of upsA in P. gingivalis 381 and its uspA-deficient mutant CW301 under various stress conditions. In general, the uspA mutant was less tolerant to a variety of environmental stresses relative to the parental strain. In addition, gene expression of uspA is upregulated during biofilm formation. Biofilm formation of the uspA mutant was also less than that of strain 381. In conclusion, the uspA gene affecting the stress responses of P. gingivalis is required for optimal biofilm formation.     

Nitric oxide generation from heme/copper assembly mediated nitrite reductase activity

Nitric oxide (NO) as a cellular signaling molecule and vasodilator regulates a range of physiological and pathological processes. Nitrite (NO₂ ^?) is recycled in vivo to generate nitric oxide, particularly in physiologic hypoxia and ischemia. The cytochrome c oxidase binuclear heme a ₃/Cu_B active site is one entity known to be responsible for conversion of cellular nitrite to nitric oxide. We recently reported that a partially reduced heme/copper assembly reduces nitrite ion, producing nitric oxide; the heme serves as the reductant and the cupric ion provides a Lewis acid interaction with nitrite, facilitating nitrite (N–O) bond cleavage (Hematian et al., J. Am. Chem. Soc. 134:18912–18915, 2012). To further investigate this nitrite reductase chemistry, copper(II)–nitrito complexes with tridentate and tetradentate ligands were used in this study, where either O,O′-bidentate or O-unidentate modes of nitrite binding to the cupric center are present. To study the role of the reducing ability of the ferrous heme center, two different tetraarylporphyrinate–iron(II) complexes, one with electron-donating para-methoxy peripheral substituents and the other with electron-withdrawing 2,6-difluorophenyl substituents, were used. The results show that differing modes of nitrite coordination to the copper(II) ion lead to differing kinetic behavior. Here, also, the ferrous heme is in all cases the source of the reducing equivalent required to convert nitrite to nitric oxide, but the reduction ability of the heme center does not play a key role in the observed overall reaction rate. On the basis of our observations, reaction mechanisms are proposed and discussed in terms of heme/copper heterobinuclear structures.     

The introduction of colonic-Bacteriodes shuttle plasmids into Porphyromonas gingivalis: Identification of a putative P. gingivalis insertion-sequence element

Two Escherichia coli-Bacteroides plasmid-shuttle vectors pNJR5 and pNJR12 were introduced for the first time into Porphyromonas gingivalis W83 by conjugal transfer from E. coli. The transfer frequencies were comparable to those obtained when using colonic Bacteroides as recipients. Both plasmids were maintained in P. gingivalis W83 and could be isolated and introduced back into E. coli. Plasmid DNA extracted from one P. gingivalis W83 pNJR12 transconjugant had an additional 1.5 kb of inserted DNA. Southern-blot analysis of P. gingivalis W83 chromosomal DNA using this inserted DNA as a probe revealed the presence of multiple copies of this sequence on the chromosome. We propose that this DNA represents a P. gingivalis insertion sequence (IS) element and should be referred to as IS1126. This is the first IS element to be isolated from a Gram-negative oral anaerobic bacterium.     

Nitric oxide production by bone cells is fluid shear stress rate dependent

Shear stress due to mechanical loading-induced flow of interstitial fluid through the lacuno-canalicular network is a likely signal for bone cell adaptive responses. Moreover, the rate (determined by frequency and magnitude) of mechanical loading determines the amount of bone formation. Whether the bone cells' response to fluid shear stress is rate dependent is unknown. Here we investigated whether bone cell activation by fluid shear stress is rate dependent. MC3T3-E1 osteoblastic cells were subjected for 15 min to fluid shear stress of varying frequencies and amplitudes, resulting in peak fluid shear stress rates ranging from 0 to 39.6 Pa-Hz. Nitric oxide production, a parameter for bone cell activation, was found to be linearly dependent on the fluid shear stress rate; the slope was steepest at 5 min (0.11 Pa-Hz(-1)) and decreased to 0.03 Pa-Hz(-1) at 15 min. We conclude that the fluid shear stress rate is an important parameter for bone cell activation.     

Nitric oxide uptake by erythrocytes is primarily limited by extracellular diffusion not membrane resistance

The process of NO transfer into erythrocytes (RBCs) is of critical biological importance because it regulates the bioavailability and diffusional distance of endothelial-derived NO. It has been reported that the rate of NO reaction with oxyhemoglobin (Hb) within RBCs is nearly three orders of magnitude slower than that by equal amounts of free oxyhemoglobin. Consistent with early studies on oxygen uptake by RBCs, the process of extracellular diffusion was reported to explain this much lower NO uptake by RBC encapsulated Hb (Liu, X., Miller, M. J., Joshi, M. S., Sadowska-Krowicka, H., Clark, D. A., and Lancaster, J. R., Jr. (1998) J. Biol. Chem. 273, 18709-18713). However, it was subsequently proposed that the RBC membrane provides the main resistance to NO uptake rather than the process of extracellular diffusion (Vaughn, M. W., Huang, K. T., Kuo, L., and Liao, J. C. (2000) J. Biol. Chem. 275, 2342-2348). This conclusion was based on competition experiments that were assumed to be able to determine the rate constant of NO uptake by RBCs without extracellular diffusion limitation. To test the validity of this hypothesis, we theoretically analyzed competition experiments. Here, we show that competition experiments do not eliminate the extracellular diffusion limitation. Simulation of the competition data indicates that the main resistance to NO uptake by RBCs is caused by extracellular diffusion in the unstirred layer surrounding each RBC but not by the RBC membrane. This extracellular diffusion resistance is responsible for preventing interference of NO signaling in the endothelium without the need for special NO uptake by intracellular hemoglobin or a unique membrane resistance mechanism.     

Enhanced biofilm formation and loss of capsule synthesis: deletion of a putative glycosyltransferase in Porphyromonas gingivalis

Periodontitis is a biofilm-mediated disease. Porphyromonas gingivalis is an obligate anaerobe consistently associated with severe manifestations of this disease. As an opportunistic pathogen, the ability to proliferate within and disseminate from subgingival biofilm (plaque) is central to its virulence. Here, we report the isolation of a P. gingivalis transposon insertion mutant altered in biofilm development and the reconstruction and characterization of this mutation in three different wild-type strains. The mutation responsible for the altered biofilm phenotype was in a gene with high sequence similarity ( approximately 61%) to a glycosyltransferase gene. The gene is located in a region of the chromosome that includes up to 16 genes predicted to be involved in the synthesis and transport of capsular polysaccharide. The phenotype of the reconstructed mutation in all three wild-type backgrounds is that of enhanced biofilm formation. In addition, in strain W83, a strain that is encapsulated, the glycosyltransferase mutation resulted in a loss of capsule. Further experiments showed that the W83 mutant strain was more hydrophobic and exhibited increased auto-aggregation. Our results indicate that we have identified a gene involved in capsular-polysaccharide synthesis in P. gingivalis and that the production of capsule prevented attachment and the initiation of in vitro biofilm formation on polystyrene microtiter plates.     

A 35-kDa co-aggregation factor is a hemin binding protein in Porphyromonas gingivalis

It has been known that Porphyromonas gingivalis has an obligate requirement for hemin or selected heme- or Fe-containing compounds for its growth. In addition, the influence of hemin on the expression of several putative virulence factors produced by this bacterium has also been recently documented; however, the mechanisms involved in hemin uptake are poorly defined. We succeeded in cloning the gene coding for the 35-kDa protein, which was specifically expressed in P. gingivalis and seemed to confer colonizing activities. Recently, we have constructed the P. gingivalis 381 mutant defective in the 35-kDa protein by insertion mutagenesis. The beige mutant exhibited little co-aggregation and the virulence was also decreased. Based on these results and homology search analysis, we focused on assessing the hemin bindings and found the heme regulatory motif (HRM) as a hemin direct binding site. The 35-kDa protein did possess the binding ability of selected protoporphyrins involving the hemin. These results demonstrated that 35-kDa protein is one of the hemin binding proteins in P. gingivalis and suggested that hemin binding ability of 35-kDa protein is important for the expression of virulence in P. gingivalis.     

Regulation of olfactomedin 4 by Porphyromonas gingivalis in a community context

At mucosal barriers, the virulence of microbial communities reflects the outcome of both dysbiotic and eubiotic interactions with the host, with commensal species mitigating or potentiating the action of pathogens. We examined epithelial responses to the oral pathogen Porphyromonas gingivalis as a monoinfection and in association with a community partner, Streptococcus gordonii. RNA-Seq of oral epithelial cells showed that the Notch signaling pathway, including the downstream effector olfactomedin 4 (OLFM4), was differentially regulated by P. gingivalis alone; however, regulation was overridden by S. gordonii. OLFM4 was required for epithelial cell migratory, proliferative and inflammatory responses to P. gingivalis. Activation of Notch signaling was induced through increased expression of the Notch1 receptor and the Jagged1 (Jag1) agonist. In addition, Jag1 was released in response to P. gingivalis, leading to paracrine activation. Following Jag1-Notch1 engagement, the Notch1 extracellular domain was cleaved by P. gingivalis gingipain proteases. Antagonism by S. gordonii involved inhibition of gingipain activity by secreted hydrogen peroxide. The results establish a novel mechanism by which P. gingivalis modulates epithelial cell function which is dependent on community context. These interrelationships have relevance for innate inflammatory responses and epithelial cell fate decisions in oral health and disease.Subject terms: Microbial ecology, Microbial ecology     

Autoaggregation of Lactobacillus reuteri is mediated by a putative DEAD-box helicase

We have cloned and sequenced a gene from Lactobacillus reuteri that encodes a 56 kDa protein, which mediates autoaggregation of the bacteria. Using an antiserum raised against extracellular proteins from the pig intestinal isolate L. reuteri 1063, we screened a genomic lambda library derived from the same strain. Affinity purification of recombinant protein from the isolated lambda clones showed that one type of clone expressed a protein that efficiently aggregated the parental strain when added to the bacteria. Subcloning and introduction of the corresponding gene, here denoted aggHinto the L. reuteri type strain markedly enhanced aggregation. Furthermore, insertional inactivation of aggH in strain 1063 resulted in an autoaggregation-deficient phenotype. Finally, an affinity-purified and cleaved fusion of AggH protein and the maltose-binding protein, MBP, strongly promoted aggregation of L. reuteri 1063, whereas the uncleaved fusion protein was inactive. Sequencing of aggH revealed that the corresponding protein has extensive sequence homology to the large family of ATP-dependent DEAD-box helicases. These results are intriguing in view of earlier data on the promotion of genetic exchange in Lactobacillus by aggregation.     

Myoepithelial-specific CD44 shedding is mediated by a putative chymotrypsin-like sheddase

Our previous studies have demonstrated that myoepithelial cells, which surround incipient carcinomas in situ of the breast and other organs, exert antiinvasive and antiangiogenic effects in vitro through the elaboration of a number of different suppressor molecules among which include the shed membrane CD44. The present study addresses the mechanism of this myoepithelial CD44 shedding. This CD44 shedding is enhanced by PMA pretreatment, is specific for myoepithelial CD44, and inhibited by chymotrypsin-like inhibitors (chymostatin, alpha(1)-antichymotrypsin, TPCK, and SCCA-2) but not by trypsin-like inhibitors (TLCK), nor papain-like inhibitors (SCCA-1) nor hydroxamate-based or general metalloproteinase inhibitors (BB2516 (marimastat), 1,10-phenanthroline, and TIMP-1). The effect of PMA can be mimicked by exogenous chymotrypsin but not by other proteases. The CD44 shedding activity cannot be transferred by conditioned media, cell-cell contact, peripheral membrane, or integral membrane fractions. However, cell-free purified integral plasma membrane fractions obtained from myoepithelial cells pretreated with PMA also exhibit CD44 shedding which is inhibited by chymotrypsin-like inhibitors. These findings support the presence and activation of a putative chymotrypsin-like sheddase as the mechanism of CD44 shedding in myoepithelial cells.