Janina P. Lewis

A Porphyromonas gingivalis tyrosine phosphatase is a multifunctional regulator of virulence attributes

Low Molecular Weight Tyrosine Phosphatases (LMWTP) are widespread in prokaryotes; however, understanding of the signalling cascades controlled by these enzymes is still emerging. Porphyromonas gingivalis, an opportunistic oral pathogen, expresses a LMWTP, Ltp1, that is differentially regulated in biofilm communities. Here we characterize the enzymatic activity of Ltp1 and, through the use of mutants that lack Ltp1 or expresses catalytically defective Ltp1, show that tyrosine phosphatase activity constrains both monospecies biofilm development and community development with the antecedent oral biofilm constituent Streptococcus gordonii. Exopolysaccharide production is downregulated by Ltp1 through transcriptional regulation of multiple genes involved in biosynthesis and transport. Furthermore, Ltp1 regulates transcriptional activity of luxS and thus impacts AI‐2‐dependent signalling in biofilm communities. In the absence of Ltp1 transcription across the hmu haemin uptake locus is reduced, and consequently uptake of haemin is impaired in the Ltp1 mutant. The gingipain proteinases Kgp and RgpA/B remain phosphorylated in the Ltp1 mutant. Phosphorylated Rgps are poorly secreted, whereas cell surface activity of phosphorylated Kgp is enhanced. By controlling the activity of several virulence‐associated properties, Ltp1 may restrain the pathogenic potential of P. gingivalis and maintain a commensal interaction with the host.

The Capsule of Porphyromonas gingivalis Leads to a Reduction in the Host Inflammatory Response, Evasion of Phagocytosis, and Increase in Virulence

ABSTRACT Periodontal disease is a chronic oral inflammatory disease that is triggered by bacteria such as Porphyromonas gingivalis. P. gingivalis strains exhibit great heterogeneity, with some strains being encapsulated while others are nonencapsulated. Although the encapsulated strains have been shown to be more virulent in a mouse abscess model, so far the role of the capsule in P. gingivalis interactions with host cells is not well understood and its role in virulence has not been defined. Here, we investigated the contribution of the capsule to triggering a host response following microbial infection, as well as its protective role following bacterial internalization by host phagocytic cells with subsequent killing, using the encapsulated P. gingivalis strain W50 and its isogenic nonencapsulated mutant, PgC. Our study shows significant time-dependent upregulation of the expression of various groups of genes in macrophages challenged with both the encapsulated and nonencapsulated P. gingivalis strains. However, cells infected with the nonencapsulated strain showed significantly higher upregulation of 9 and 29 genes at 1 h and 8 h postinfection, respectively, than cells infected with the encapsulated strain. Among the genes highly upregulated by the nonencapsulated PgC strain were ones coding for cytokines and chemokines. Maturation markers were induced at a 2-fold higher rate in dendritic cells challenged with the nonencapsulated strain for 4 h than in dendritic cells challenged with the encapsulated strain. The rates of phagocytosis of the nonencapsulated P. gingivalis strain by both macrophages and dendritic cells were 4.5-fold and 7-fold higher, respectively, than the rates of phagocytosis of the encapsulated strain. On the contrary, the survival of the nonencapsulated P. gingivalis strain was drastically reduced compared to the survival of the encapsulated strain. Finally, the encapsulated strain exhibited greater virulence in a mouse abscess model. Our results indicate that the P. gingivalis capsule plays an important role in aiding evasion of host immune system activation, promoting survival of the bacterium within host cells, and increasing virulence. As such, it is a major virulence determinant of P. gingivalis.

Metal uptake in host-pathogen interactions: role of iron in Porphyromonas gingivalis interactions with host organisms.

Iron is a nutrient that is indispensable for growth of almost all living organisms. The virulence of pathogenic organisms in the mammalian host is related to the availability of iron, therefore, microbial iron acquisition mechanisms are an important determinant of infection potential. Indeed, iron levels regulate 10–20% of all genes present on microbial genomes, including iron acquisition mechanism and virulence factor encoding genes. The association between iron and microbial virulence is well established (121,158). Many infectious diseases, such as gonorrhea, malaria, tuberculosis, and diarrheal infections, depend on the expression of microbial acquisition mechanisms capable of competing with the host’s iron scavenging mechanisms. The availability of iron in the mammalian host is extremely low, therefore, successful pathogens evolved strategies to overcome this limitation. Some pathogens produce high affinity metal chelators capable of competing with the host’s scavenging mechanisms, while others degrade and release mammalian proteins such as transferrin or hemoglobin that bind iron or hemin. Iron overload in the host from genetic predispositions, therapeutic intervention, or nutritional status can also increase the risk of infection by many pathogens, such as Plasmodium falciparum and Mycobacterium tuberculosis (121). Treating the host by administering iron chelators that capture free iron and compete with the acquisition system can limit microbial iron acquisition. However, this treatment also limits the availability of iron for the host, which can also have a damaging effect. Another way to alter iron availability is to interfere with the microbial iron acquisition mechanisms required by the pathogen to survive at specific stages of infection. Therefore, understanding the role of the iron acquisition mechanisms at various stages of bacterial infection may lead to the development of additional interventional strategies. The oral cavity harbors at least 700 different species of bacteria (1,19,104,105,139). The contribution of many of those species to onset and progression of oral diseases is still to be established. However, several bacterial species are strongly implicated as aetiological agents of periodontal diseases. Among them are members of the Bacteroidetes phylum: Porphyromonas gingivalis, Prevotella intermedia, and Tannerella forsythia ((18,40,105). Of these, P. gingivalis is the most intensively investigated oral bacterium, and significant progress has been made in recent years regarding its iron uptake mechanisms. Because of this, we chose P. gingivalis as a model to describe the role of iron in host-pathogen interactions in the oral cavity. P. gingivalis is a black pigmented, anaerobic, gram-negative bacterium that is implicated as a prime aetiological agent of initiation and progression of periodontal disease (40,46). As periodontal disease is also associated with increased risk of systemic conditions, such as coronary heart disease and preterm delivery of low birthweight infants (43,50,93,107,119,120,148), the burden of infection caused by P. gingivalis may be higher than previously estimated. The periodontal status of patients was significantly improved following elimination of the bacterium (reviewed in (40,113), however, current therapies still allow for recurrent infections (3,17,125). Currently, the standard therapeutic approach includes mechanical removal of the organism followed by antibiotic treatment. But rapidly increasing resistance to antibiotics and the potential for recurrent infection suggests more targeted and longer-lasting approaches are needed to reduce the levels of P. gingivalis in the oral cavity. To achieve this goal, a thorough knowledge of the biology of P. gingivalis is needed. Periodontal diseases are triggered by microorganisms and manifest themselves in inflammation, bleeding, and destruction of soft and tooth tissues. P. gingivalis is found in healthy sites such as the buccal mucosa, tongue, tonsils, palate and supragingival dental plaque on the tooth enamel surface (86), but its numbers are significantly elevated in the subgingival environment and especially in diseased areas (92), suggesting the bacterium is well adapted and benefits from the hostile inflammatory state of the host. To survive in the diseased periodontal pocket the pathogen must be able to escape host defenses and acquire sufficient nutrients that will sustain its growth. One such nutrient required for growth of P. gingivalis is iron, and its presence has a profound effect on the bacterium’s virulence. Using a mouse abscess model, the Ebersole group demonstrated that bacteria grown under hemin-limitation were less virulent than their counterparts grown in hemin-excess (64,83,87). In this review, we discuss the mechanisms of iron acquisition by P. gingivalis. We also describe regulatory mechanisms, because excess iron reacts with peroxide to generate hydroxyl radicals that are detrimental to the bacterial cell. P. gingivalis is distantly related to bacteria in which iron acquisition and homeostasis mechanisms are well known, so this review provides novel supportive information with regard to iron acquisition mechanisms in bacteria.

Adaptation of Porphyromonas gingivalis to microaerophilic conditions involves increased consumption of formate and reduced utilization of lactate

Porphyromonas gingivalis, previously classified as a strict anaerobe, can grow in the presence of low concentrations of oxygen. Microarray analysis revealed alteration in gene expression in the presence of 6 % oxygen. During the exponential growth phase, 96 genes were upregulated and 79 genes were downregulated 1.4-fold. Genes encoding proteins that play a role in oxidative stress protection were upregulated, including alkyl hydroperoxide reductase (ahpCF), superoxide dismutase (sod) and thiol peroxidase (tpx). Significant changes in gene expression of proteins that mediate oxidative metabolism, such as cytochrome d ubiquinol oxidase-encoding genes, cydA and cydB, were detected. The expression of genes encoding formate uptake transporter (PG0209) and formate tetrahydrofolate ligase (fhs) was drastically elevated, which indicates that formate metabolism plays a major role under aerobic conditions. The concomitant reduction of expression of a gene encoding the lactate transporter PG1340 suggests decreased utilization of this nutrient. The concentrations of both formate and lactate were assessed in culture supernatants and cells, and they were in agreement with the results obtained at the transcriptional level. Also, genes encoding gingipain protease secretion/maturation regulator (porR) and protease transporter (porT) had reduced expression in the presence of oxygen, which also correlated with reduced protease activities under aerobic conditions. In addition, metal transport was affected, and while iron-uptake genes such as the genes encoding the haemin uptake locus (hmu) were downregulated, expression of manganese transporter genes, such as feoB2, was elevated in the presence of oxygen. Finally, genes encoding putative regulatory proteins such as extracellular function (ECF) sigma factors as well as small proteins had elevated expression levels in the presence of oxygen. As P. gingivalis is distantly related to the well-studied model organism Escherichia coli, results from our work may provide further understanding of oxygen metabolism and protection in other related bacteria belonging to the phylum Bacteroidetes.

Role of Porphyromonas gingivalis FeoB2 in metal uptake and oxidative stress protection.

ABSTRACT Porphyromonas gingivalis, a gram-negative anaerobic bacterium, is a recognized periodontopathogen. It exhibits a high degree of aerotolerance and is able to survive in host cells, indicating that efficient oxidative stress protection mechanisms must be present in this organism. Manganese homeostasis plays a major role in oxidative stress protection in a variety of organisms; however, the transport and role of this metal in P. gingivalis is not well understood. Analysis of the genome of P. gingivalis W83 revealed the presence of two genes encoding homologs of a ferrous iron transport protein, FeoB1 and FeoB2. FeoB2 has been implicated in manganese accumulation in P. gingivalis. We sought to determine the role of the FeoB2 protein in metal transport as well as its contribution to resistance to oxygen radicals. Quantitative reverse transcriptase PCR analyses demonstrated that expression of feoB2 is induced in the presence of oxygen. The role of FeoB2 was investigated using an isogenic mutant strain deficient in the putative transporter. We characterized the FeoB2-mediated metal transport using 55Fe2+ and 54Mn2+. The FeoB2-deficient mutant had dramatically reduced rates of manganese uptake (0.028 pmol/min/107 bacteria) compared with the parental strain (0.33 pmol/min/107 bacteria) (after 20 min of uptake using 50 nM of 54Mn2+). The iron uptake rates, however, were higher in the mutant strain (0.75 pmol/min/107 bacteria) than in the wild type (0.39 pmol/min/107 bacteria). Interestingly, reduced survival rates were also noted for the mutant strain after exposure to H2O2 and to atmospheric oxygen compared to the parental strain cultured under the same conditions. In addition, in vitro infection of host cells with the wild type, the FeoB2-deficient mutant, and the same-site revertant revealed that the mutant had a significantly decreased capability for intracellular survival in the host cells compared to the wild-type strain. Our results demonstrate that feoB2 encodes a major manganese transporter required for protection of the bacterium from oxidative stress generated by atmospheric oxygen and H2O2. Furthermore, we show that FeoB2 and acquisition of manganese are required for intracellular survival of P. gingivalis in host cells.

Role of the Porphyromonas gingivalis extracytoplasmic function sigma factor, SigH

Little is known about the regulatory mechanisms that allow Porphyromonas gingivalis to survive in the oral cavity. Here we characterize the sigma (σ) factor SigH, one of six extracytoplasmic function (ECF) σ factors encoded in the P. gingivalis genome. Our results indicate that sigH expression is upregulated by exposure to molecular oxygen, suggesting that sigH plays a role in adaptation of P. gingivalis to oxygen. Furthermore, several genes involved in oxidative stress protection, such as sod, trx, tpx, ftn, feoB2 and the hemin uptake hmu locus, are downregulated in a mutant deficient in SigH designated as V2948. ECF σ consensus sequences were identified upstream of the transcriptional start sites of these genes, consistent with the SigH-dependent regulation of these genes. Growth of V2948 was inhibited in the presence of 6% oxygen when compared with the wild-type W83 strain, whereas in anaerobic conditions both strains were able to grow. In addition, reduced growth of V2948 was observed in the presence of peroxide and the thiol-oxidizing reagent diamide when compared with the W83 strain. The SigH-deficient strain V2948 also exhibited reduced hemin uptake, consistent with the observed reduced expression of genes involved in hemin uptake. Finally, survival of V2948 was reduced in the presence of host cells compared with the wild-type W83 strain. Collectively, our studies demonstrate that SigH is a positive regulator of gene expression required for survival of the bacterium in the presence of oxygen and oxidative stress, hemin uptake and virulence.

HcpR of Porphyromonas gingivalis Is Required for Growth under Nitrosative Stress and Survival within Host Cells

ABSTRACT Although the Gram-negative, anaerobic periodontopathogen Porphyromonas gingivalis must withstand nitrosative stress, which is particularly high in the oral cavity, the mechanisms allowing for protection against such stress are not known in this organism. In this study, microarray analysis of P. gingivalis transcriptional response to nitrite and nitric oxide showed drastic upregulation of the PG0893 gene coding for hybrid cluster protein (Hcp), which is a putative hydroxylamine reductase. Although regulation of hcp has been shown to be OxyR dependent in Escherichia coli, here we show that in P. gingivalis its expression is dependent on the Fnr-like regulator designated HcpR. Growth of the isogenic mutant V2807, containing an ermF-ermAM insertion within the hcpR (PG1053) gene, was significantly reduced in the presence of nitrite (P < 0.002) and nitric oxide-generating nitrosoglutathione (GSNO) (P < 0.001), compared to that of the wild-type W83 strain. Furthermore, the upregulation of PG0893 (hcp) was abrogated in V2807 exposed to nitrosative stress. In addition, recombinant HcpR bound DNA containing the hcp promoter sequence, and the binding was hemin dependent. Finally, V2807 was not able to survive with host cells, demonstrating that HcpR plays an important role in P. gingivalis virulence. This work gives insight into the molecular mechanisms of protection against nitrosative stress in P. gingivalis and shows that the regulatory mechanisms differ from those in E. coli.

AdpC Is a Prevotella intermedia 17 Leucine-Rich Repeat Internalin-Like Protein

ABSTRACT The oral bacterium Prevotella intermedia attaches to and invades gingival epithelial cells, fibroblasts, and endothelial cells. Several genes encoding proteins that mediate both the adhesion and invasion processes are carried on the genome of this bacterium. Here, we characterized one such protein, AdpC, belonging to the leucine-rich repeat (LRR) protein family. Bioinformatics analysis revealed that this protein shares similarity with the Treponema pallidum LRR (LRRTP) family of proteins and contains six LRRs. Despite the absence of a signal peptide, this protein is localized on the bacterial outer membrane, indicating that it is transported through an atypical secretion mechanism. The recombinant form of this protein (rAdpC) was shown to bind fibrinogen. In addition, the heterologous host strain Escherichia coli BL21 expressing rAdpC (V2846) invaded fibroblast NIH 3T3 cells at a 40-fold-higher frequency than control E. coli BL21 cells expressing a sham P. intermedia 17 protein. Although similar results were obtained by using human umbilical vein endothelial cells (HUVECs), only a 3-fold-increased invasion of V2846 into oral epithelial HN4 cells was observed. Thus, AdpC-mediated invasion is cell specific. This work demonstrated that AdpC is an important invasin protein of P. intermedia 17.

Characterization of Porphyromonas gingivalis Insertion Sequence-Like Element ISPg5

ABSTRACT Porphyromonas gingivalis, a black-pigmented, gram-negative anaerobe, is found in periodontitis lesions, and its presence in subgingival plaque significantly increases the risk for periodontitis. In contrast to many bacterial pathogens, P. gingivalis strains display considerable variability, which is likely due to genetic exchange and intragenomic changes. To explore the latter possibility, we have studied the occurrence of insertion sequence (IS)-like elements in P. gingivalis W83 by utilizing a convenient and rapid method of capturing IS-like sequences and through analysis of the genome sequence of P. gingivalis strain W83. We adapted the method of Matsutani et al. (S. Matsutani, H. Ohtsubo, Y. Maeda, and E. Ohtsubo, J. Mol. Biol. 196:445–455, 1987) to isolate and clone rapidly annealing DNA sequences characteristic of repetitive regions within a genome. We show that in P. gingivalis strain W83, such sequences include (i) nucleotide sequence with homology to tRNA genes, (ii) a previously described IS element, and (iii) a novel IS-like element. Analysis of the P. gingivalis genome sequence for the distribution of the least used tetranucleotide, CTAG, identified regions in many of the initial 218 contigs which contained CTAG clusters. Examination of these CTAG clusters led to the discovery of 11 copies of the same novel IS-like element identified by the repeated sequence capture method of Matsutani et al. This new 1,512-bp IS-like element, designated ISPg5, has features of the IS3 family of IS elements. When a recombinant plasmid containing much of ISPg5 was used in Southern analysis of several P. gingivalis strains, including clinical isolates, diversity among strains was apparent. This suggests that ISPg5 and other IS elements may contribute to strain diversity and can be used for strain fingerprinting.

Porphyromonas gingivalis Ferrous Iron Transporter FeoB1 Influences Sensitivity to Oxidative Stress

ABSTRACT Porphyromonas gingivalis FeoB1 is a ferrous iron transporter. Analysis of parental and feoB1-deficient strains of the periodontal pathogen revealed that the feoB1-deficient mutant strain had an increased ability to survive oxidative stress. Specifically, survival of the mutant strain was increased 33% with exposure to peroxide and 5% with exposure to atmospheric oxygen compared to the parental strain. Interestingly, the ability to survive intracellularly also increased fivefold in the case of the feoB1-deficient mutant. Our data suggest that although the FeoB1 protein is required for ferrous iron acquisition in P. gingivalis, it also has an adverse effect on survival of the bacterium under oxidative stress conditions. Finally, we show that feoB1 expression is not iron dependent and is dramatically reduced in the presence of host cells, consistent with the observed deleterious role it plays in bacterial survival.