Sequence and characterization of shuttle vectors for molecular cloning in Porphyromonas,Bacteroides and related bacteria

There is a lack of shuttle vectors to be needed for investigations into the genetics of Porphyromonas gingivalis and related species. To better understand the prevalence of candidates for such tools, we have examined multiple strains of black‐pigmented anaerobes (clinical and laboratory isolates) for plasmids. As no plasmids were found in P. gingivalis strains, we have used the pYH420 plasmid, derived from P. asaccharolytica, as backbone to construct a shuttle vector in combination with pUC19 from Escherichia coli. Nucleotide sequence determination of the pYH420 plasmid revealed that it contained a gene with similarity to rep from plasmid pTS1 (isolated from Treponema denticola) as well as a homolog of mobA, a member of a gene family found on mobilizable genetic elements found in the genus Bacteroides. We constructed the pG106 and pG108 shuttle vectors using parts of the pUC19 and pYH420 vectors. This resulted in a vector with a multiple cloning site (MCS) in the lacZ gene enabling us to perform blue–white colony selection. The pG106 and pG108 shuttle vectors are electro‐transformable into E. coli, P. gingivalis and B. thetaiotaomicron, where they are stable. We demonstrated that these vectors were suitable in these species for applications of molecular cloning including complementation and gene expression studies. Using the pG108 vector, we complement the hcpR mutant strain of P. gingivalis and rescued its ‐sensitive phenotype. We also performed a gene expression study using the P‐glow BS2 fluorescent reporter gene and the ahpC promoter in B. thetaiotaomicron.     

Erbium,Chromium:Yttrium‐Scandium‐Gallium‐Garnet Laser Effectively Ablates Single‐Species Biofilms on Titanium Disks Without Detectable Surface Damage

Background: Increasing evidence implicates biofilms, consisting of species such as Porphyromonas gingivalis (Pg), in the etiology of peri‐implantitis. Multiple approaches to ablate biofilms on failing implants have been proposed and include use of lasers, most recently the erbium, chromium:yttrium‐scandium‐gallium‐garnet (Er,Cr:YSGG) laser. The purpose of this study is to establish an in vitro single‐species biofilm model on implant surfaces and determine power settings of the Er,Cr:YSGG laser that remove biofilm without causing physical damage to disks. Methods: Single‐species biofilms consisting of Pg strain 381 were grown on titanium disks, including: 1) sandblasted, large‐grit, acid‐etched (SLA); 2) calcium phosphate nano‐coated (CaP); 3) anodized; or 4) machined surfaces. Power settings from 0 to 1.5 W using an Er,Cr:YSGG laser equipped with radial firing tip were used. Biofilm formation/removal was quantitated using confocal and scanning electron microscopy. Surface changes in temperature, microroughness, and water contact angle were analyzed. Results: Results show confluent Pg biofilm coating all disk surfaces. The laser removed biofilms from all surfaces, with CaP and SLA surfaces requiring power setting of 1.0 to 1.5 W for ablation of bacteria coating the disks. Within this power range, and with water spray, there were no changes in surface temperature, surface roughness, or contact angle on any surfaces tested. Conclusion: The Er,Cr:YSGG laser with radial firing tip and water spray was able to effectively ablate ≥95% of biofilm on all types of tested titanium surfaces, using clinically relevant power settings, without causing measurable physical changes to surfaces.     

The GroEL protein of Porphyromonas gingivalis accelerates tumor growth by enhancing endothelial progenitor cell function and neovascularization

Porphyromonas gingivalis is a bacterial species that causes destruction of periodontal tissues. Additionally, previous evidence indicates that GroEL from P. gingivalis may possess biological activities involved in systemic inflammation, especially inflammation involved in the progression of periodontal diseases. The literature has established a relationship between periodontal disease and cancer. However, it is unclear whether P. gingivalis GroEL enhances tumor growth. Here, we investigated the effects of P. gingivalis GroEL on neovasculogenesis in C26 carcinoma cell‐carrying BALB/c mice and chick eggs in vivo as well as its effect on human endothelial progenitor cells (EPC) in vitro. We found that GroEL treatment accelerated tumor growth (tumor volume and weight) and increased the mortality rate in C26 cell‐carrying BALB/c mice. GroEL promoted neovasculogenesis in chicken embryonic allantois and increased the circulating EPC level in BALB/c mice. Furthermore, GroEL effectively stimulated EPC migration and tube formation and increased E‐selectin expression, which is mediated by eNOS production and p38 mitogen‐activated protein kinase activation. Additionally, GroEL may enhance resistance against paclitaxel‐induced cell cytotoxicity and senescence in EPC. In conclusion, P. gingivalis GroEL may act as a potent virulence factor, contributing to the neovasculogenesis of tumor cells and resulting in accelerated tumor growth.