Porphyromonas gingivalis infection increases osteoclastic bone resorption and osteoblastic bone formation in a periodontitis mouse model.

BACKGROUND: Porphyromonas gingivalis has been shown to invade osteoblasts and inhibit their differentiation and mineralization in vitro. However, it is unclear if P. gingivalis can invade osteoblasts in vivo and how this would affect alveolar osteoblast/osteoclast dynamics. This study aims to answer these questions using a periodontitis mouse model under repetitive P. gingivalis inoculations. METHODS: For 3-month-old BALB/cByJ female mice, 10(9) CFU of P. gingivalis were inoculated onto the gingival margin of maxillary molars 4 times at 2-day intervals. After 2 weeks, another 4 inoculations at 2-day intervals were applied. Calcein was injected 7 and 2 days before sacrificing animals to label the newly formed bone. Four weeks after final inoculation, mice were sacrificed and maxilla collected. Immunohistochemistry, micro-CT, and bone histomorphometry were performed on the specimens. Sham infection with only vehicle was the control. RESULTS: P. gingivalis was found to invade gingival epithelia, periodontal ligament fibroblasts, and alveolar osteoblasts. Micro-CT showed alveolar bone resorption and significant reduction of bone mineral density and content in the infected mice compared to the controls. Bone histomorphometry showed a decrease in osteoblasts, an increase in osteoclasts and bone resorption, and a surprisingly increased osteoblastic bone formation in the infected mice compared to the controls. CONCLUSIONS: P. gingivalis invades alveolar osteoblasts in the periodontitis mouse model and cause alveolar bone loss. Although P. gingivalis appears to suppress osteoblast pool and enhance osteoclastic bone resorption, the bone formation capacity is temporarily elevated in the infected mice, possibly via some anti-microbial compensational mechanisms.

Genetic exchange of fimbrial alleles exemplifies the adaptive virulence strategy of Porphyromonas gingivalis.

Porphyromonas gingivalis is a gram-negative anaerobic bacterium, a member of the human oral microbiome, and a proposed "keystone" pathogen in the development of chronic periodontitis, an inflammatory disease of the gingiva. P. gingivalis is a genetically diverse species, and is able to exchange chromosomal DNA between strains by natural competence and conjugation. In this study, we investigate the role of horizontal DNA transfer as an adaptive process to modify behavior, using the major fimbriae as our model system, due to their critical role in mediating interactions with the host environment. We show that P. gingivalis is able to exchange fimbrial allele types I and IV into four distinct strain backgrounds via natural competence. In all recombinants, we detected a complete exchange of the entire fimA allele, and the rate of exchange varies between the different strain backgrounds. In addition, gene exchange within other regions of the fimbrial genetic locus was identified. To measure the biological implications of these allele swaps we compared three genotypes of fimA in an isogenic background, strain ATCC 33277. We demonstrate that exchange of fimbrial allele type results in profound phenotypic changes, including the quantity of fimbriae elaborated, membrane blebbing, auto-aggregation and other virulence-associated phenotypes. Replacement of the type I allele with either the type III or IV allele resulted in increased invasion of gingival fibroblast cells relative to the isogenic parent strain. While genetic variability is known to impact host-microbiome interactions, this is the first study to quantitatively assess the adaptive effect of exchanging genes within the pan genome cloud. This is significant as it presents a potential mechanism by which opportunistic pathogens may acquire the traits necessary to modify host-microbial interactions.

Integrin α5ß1-fimbriae binding and actin rearrangement are essential for Porphyromonas gingivalis invasion of osteoblasts and subsequent activation of the JNK pathway.

BACKGROUND: Chronic periodontitis is an infectious disease of the periodontium, which includes the gingival epithelium, periodontal ligament and alveolar bone. The signature clinical feature of periodontitis is resorption of alveolar bone and subsequent tooth loss. The Gram-negative oral anaerobe, Porphyromonas gingivalis, is strongly associated with periodontitis, and it has been shown previously that P. gingivalis is capable of invading osteoblasts in a dose- and time-dependent manner resulting in inhibition of osteoblast differentiation and mineralization in vitro. It is not yet clear which receptors and cytoskeletal components mediate the invasive process, nor how the signaling pathways and viability of osteoblasts are affected by bacterial internalization. This study aimed to investigate these issues using an in vitro model system involving the inoculation of P. gingivalis ATCC 33277 into primary osteoblast cultures. RESULTS: It was found that binding between P. gingivalis fimbriae and integrin α5ß1 on osteoblasts, and subsequent peripheral condensation of actin, are essential for entry of P. gingivalis into osteoblasts. The JNK pathway was activated in invaded osteoblasts, and apoptosis was induced by repeated infections. CONCLUSIONS: These observations indicate that P. gingivalis manipulates osteoblast function to promote its initial intracellular persistence by prolonging the host cell life span prior to its intercellular dissemination via host cell lysis. The identification of molecules critical to the interaction between P. gingivalis and osteoblasts will facilitate the development of new therapeutic strategies for the prevention of periodontal bone loss.

Natural competence is a major mechanism for horizontal DNA transfer in the oral pathogen Porphyromonas gingivalis.

UNLABELLED: Porphyromonas gingivalis is a Gram-negative anaerobe that resides exclusively in the human oral cavity. Long-term colonization by P. gingivalis requires the bacteria to evade host immune responses while adapting to the changing host physiology and alterations in the composition of the oral microflora. The genetic diversity of P. gingivalis appears to reflect the variability of its habitat; however, little is known about the molecular mechanisms generating this diversity. Previously, our research group established that chromosomal DNA transfer occurs between P. gingivalis strains. In this study, we examine the role of putative DNA transfer genes in conjugation and transformation and demonstrate that natural competence mediated by comF is the dominant form of chromosomal DNA transfer, with transfer by a conjugation-like mechanism playing a minor role. Our results reveal that natural competence mechanisms are present in multiple strains of P. gingivalis, and DNA uptake is not sensitive to DNA source or modification status. Furthermore, extracellular DNA was observed for the first time in P. gingivalis biofilms and is predicted to be the major DNA source for horizontal transfer and allelic exchange between strains. We propose that exchange of DNA in plaque biofilms by a transformation-like process is of major ecological importance in the survival and persistence of P. gingivalis in the challenging oral environment. IMPORTANCE: P. gingivalis colonizes the oral cavities of humans worldwide. The long-term persistence of these bacteria can lead to the development of chronic periodontitis and host morbidity associated with tooth loss. P. gingivalis is a genetically diverse species, and this variability is believed to contribute to its successful colonization and survival in diverse human hosts, as well as evasion of host immune defenses and immunization strategies. We establish here that natural competence is the major driving force behind P. gingivalis DNA exchange and that conjugative DNA transfer plays a minor role. Furthermore, we reveal for the first time the presence of extracellular DNA in P. gingivalis biofilms, which is most likely the source of DNA exchanged between strains within dental plaque. These studies expand our understanding of the mechanisms used by this important member of the human oral flora to transition its relationship with the host from a commensal to a pathogenic relationship.

Fimbriae of Porphyromonas gingivalis are important for initial invasion of osteoblasts, but not for inhibition of their differentiation and mineralization.

BACKGROUND: Porphyromonas gingivalis is etiologically associated with chronic periodontitis. The major fimbriae of this periodontal pathogen mediate binding to host gingival epithelial cells and fibroblasts, a critical function in the initiation of periodontitis. However, the role of fimbriae in P. gingivalis-osteoblast interactions remains unknown. In the present study, the involvement of major fimbriae in the initial and long-term interactions between P. gingivalis and osteoblasts is investigated. METHODS: Primary mouse calvarial osteoblast cultures were established and inoculated with P. gingivalis ATCC 33277 or YPF1, a major fimbriae-deficient mutant of P. gingivalis. Confocal microscopy images were acquired to assess bacterial invasion. DNA content measurement, real-time polymerase chain reaction, and alizarin red S staining and calcium content analysis were used to study the impact of bacteria on the proliferation, differentiation, and mineralization of osteoblasts, respectively. RESULTS: Compared to the parent strain, YPF1 was significantly reduced in invasion of osteoblasts after 3 hours interaction. However, extended culture of infected osteoblasts did not reveal significant differences in persistence between the two strains. Proliferation of osteoblasts was not affected by either strain, and differentiation and mineralization of osteoblasts were inhibited by both strains to comparable levels. CONCLUSION: This study reveals that major fimbriae are involved in the initial invasion of osteoblasts by P. gingivalis, but are not essential for the subsequent inhibition of osteoblast differentiation and mineralization in long-term culture.

Genetic analysis of mobile tetQ elements in oral Prevotella species.

Prevotella species are members of the bacterial oral flora and are opportunistic pathogens in polymicrobial infections of soft tissues. Antibiotic resistance to tetracyclines is common in these bacteria, and the gene encoding this resistance has been previously identified as tetQ. The tetQ gene is also found on conjugative transposons in the intestinal Bacteroides species; whether these related bacteria have transmitted tetQ to Prevotella is unknown. In this study, we describe our genetic analysis of mobile tetQ elements in oral Prevotella species. Our results indicate that the mobile elements encoding tetQ in oral species are distinct from those found in the Bacteroides. The intestinal bacteria may act as a reservoir for the tetQ gene, but Prevotella has incorporated this gene into an IS21-family transposon. This transposon is present in Prevotella species from more than one geographical location, implying that the mechanism of tetQ spread between oral Prevotella species is highly conserved.

Porphyromonas gingivalis invades osteoblasts and inhibits bone formation.

Porphyromonas gingivalis is etiologically associated with adult periodontitis, but it is unclear how P. gingivalis long-term interactions with bone cells contribute to this disease. This study investigates P. gingivalis interactions with osteoblasts over an extended time course. A primary mouse calvarial osteoblast culture was established and inoculated with P. gingivalis 33277 repeatedly every other day for up to four weeks. Invasion of osteoblasts by P. gingivalis, and the resulting effects on the proliferation, differentiation, and mineralization of osteoblasts were evaluated. P. gingivalis was found to invade osteoblasts in a dose-dependent manner, and repetitive inoculation increased the percentage of osteoblasts with internalized P. gingivalis. P. gingivalis did not affect osteoblast proliferation, but inhibited their differentiation and mineralization, partially via an inhibition of the differentiation regulatory transcription factors Cbfa-1 and osterix. In conclusion, P. gingivalis invades osteoblasts and inhibits bone formation, which likely contributes to alveolar bone loss in chronic periodontitis.