Lipoprotein Extraction from Microbial Membrane and Lipoprotein/Lipopeptide Transfection into Mammalian Cells.

Microbial lipoproteins/lipopeptides are important virulence factors for periodontal diseases. The membrane lipoproteins from Mycoplasma salivarium or Tannerella forsythia can be easily extracted by exploiting a characteristic feature of Triton X-114: its aqueous nature at low temperatures (0-4 °C), which is absent at room temperature (25-37 °C). Transfection of these lipopeptides into macrophages was performed using the protein transfection reagent, PULSin.

A Novel Prosthetic Joint Infection Pathogen, Mycoplasma salivarium, Identified by Metagenomic Shotgun Sequencing.

Defining the microbial etiology of culture-negative prosthetic joint infection (PJI) can be challenging. Metagenomic shotgun sequencing is a new tool to identify organisms undetected by conventional methods. We present a case where metagenomics was used to identify Mycoplasma salivarium as a novel PJI pathogen in a patient with hypogammaglobulinemia.

Mycoplasma salivarium as a dominant coloniser of Fanconi anaemia associated oral carcinoma.

Mycoplasma salivarium belongs to the class of the smallest self-replicating Tenericutes and is predominantly found in the oral cavity of humans. In general it is considered as a non-pathogenic commensal. However, some reports point to an association with human diseases. M. salivarium was found e.g. as causative agent of a submasseteric abscess, in necrotic dental pulp, in brain abscess and clogged biliary stent. Here we describe the detection of M. salivarium on the surface of a squamous cell carcinoma of the tongue of a patient with Fanconi anaemia (FA). FA is an inherited bone marrow failure syndrome based on defective DNA-repair that increases the risk of carcinomas especially oral squamous cell carcinoma. Employing high coverage, massive parallel Roche/454-next-generation-sequencing of 16S rRNA gene amplicons we analysed the oral microbiome of this FA patient in comparison to that of an FA patient with a benign leukoplakia and five healthy individuals. The microbiota of the FA patient with leukoplakia correlated well with that of the healthy controls. A dominance of Streptococcus, Veillonella and Neisseria species was typically observed. In contrast, the microbiome of the cancer bearing FA patient was dominated by Pseudomonas aeruginosa at the healthy sites, which changed to a predominance of 98% M. salivarium on the tumour surface. Quantification of the mycoplasma load in five healthy, two tumour- and two leukoplakia-FA patients by TaqMan-PCR confirmed the prevalence of M. salivarium at the tumour sites. These new findings suggest that this mycoplasma species with its reduced coding capacity found ideal breeding grounds at the tumour sites. Interestingly, the oral cavity of all FA patients and especially samples at the tumour sites were in addition positive for Candida albicans. It remains to be elucidated in further studies whether M. salivarium can be used as a predictive biomarker for tumour development in these patients.

Empyema caused by Mycoplasma salivarium.

Mycoplasma salivarium infections outside the oral cavity are rare. We describe a 49-year-old man with laryngeal cancer and right pleural space infection with M. salivarium. To our knowledge, this is the first report of empyema due to Mycoplasma salivarium.

Mycoplasma salivarium isolated from brain abscesses.

We report two cases of cerebral abscesses with polymicrobial aetiology including Mycoplasma salivarium. In both cases, Mycoplasma was found incidentally, suggesting that a broader aetiological spectrum could be found in brain abscesses by use of molecular techniques targeting fastidious pathogens.

Distribution of Mycoplasma pneumoniae and Mycoplasma salivarium in the synovial fluid of arthritis patients.

By use of a very sensitive nested PCR method targeting part of the strongly conserved mycoplasmal 16S RNA genes, Mycoplasma pneumoniae was found in the synovial fluid of 19/24 (79%) of rheumatoid arthritis patients, 6/6 (100%) of patients with nonrheumatoid inflammatory arthritis, and 8/10 (80%) of osteoarthritis patients attending the rheumatology clinic for drainage of joint effusions. It was not found in the synovial exudates of 13 people attending the orthopedic clinic with traumatic knee injuries or undergoing surgery for knee replacement. However, M. pneumoniae was detected in 2/4 synovial biopsy specimens from orthopedic patients with traumatic knee injuries. M. pneumoniae was associated with the increased synovial fluids found in arthritic flares but was not found in the synovial fluids of trauma patients. Mycoplasma salivarium occurred sporadically. Mycoplasma fermentans had previously been isolated from patients with inflammatory cellular infiltrates, such as rheumatoid arthritis, but it was not detected for osteoarthritic patients from either clinic. It is possible that these organisms may contribute to chronic inflammation within the joints.

Presence of human mycoplasma DNA in gastric tissue samples from Korean chronic gastritis patients.

We aimed to determine whether mycoplasmas are present in Korean chronic gastritis, and to understand their roles in gastric cancer tumorigenesis, because mycoplasmas resemble Helicobacter pylori in terms of ammonia production and induction of inflammatory cytokines in immune and non-immune cells. The presence and identity of mycoplasmas were assessed by semi-nested PCR and sequencing, and the results were compared with pathologic data. Fifty-six samples collected from Korean chronic gastritis patients were used for this study. Twenty-three (41.1%) were positive for mycoplasmas. Eighteen sequenced samples contained a single human mycoplasma or two mycoplasmas, which were identified as Mycoplasma faucium (13/18), M. fermentans (3/18), M. orale (1/18), M. salivarium (2/18), and M. spermatophilum (1/18). Mycoplasma-infected chronic gastritis samples showed significantly more severe neutrophil infiltration than non-infected samples (P = 0.0135). Mycoplasma profiles in the oral cavity (M. salivarium is major) and stomach were different, and the presence of significant proinflammatory responses in mycoplasma-positive patients suggests that the mycoplasmas are not simply contaminants. Further studies are required to understand whether mycoplasmas play a role in gastric tumorigenesis.

Relationship between structures and biological activities of mycoplasmal diacylated lipopeptides and their recognition by toll-like receptors 2 and 6.

The lipopeptide FSL-1 [S-(2,3-bispalmitoyloxypropyl)-Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe, Pam(2)CGDPKHPKSF] synthesized on the basis of the N-terminal structure of a Mycoplasma salivarium lipoprotein capable of activating normal human gingival fibroblasts to induce the cell surface expression of ICAM-1 revealed an activity to induce production of monocyte chemoattractant protein 1, interleukin-6 (IL-6), and IL-8. FSL-1 also activated macrophages to produce tumor necrosis factor alpha as the Mycoplasma fermentans-derived lipopeptide MALP-2 (Pam(2)CGNNDESNISFKEK), a potent macrophage-activating lipopeptide, did. The level of the activity of FSL-1 was higher than that of MALP-2. This result suggests that the difference in the amino acid sequence of the peptide portion affects the activity because the framework structure other than the amino acid sequence of the former is the same as that of the latter. To determine minimal structural requirements for the activity of FSL-1, the diacylglyceryl Cys and the peptide portions were examined for this activity. Both portions did not reveal the activity. A single amino acid substitution from Phe to Arg and a fatty acid substitution from palmitic acid to stearic acid drastically reduced the activity. Similar results were obtained in measuring the NF-kappaB reporter activity of FSL-1 to human embryonic kidney 293 cells transfected with Toll-like receptor 2 and 6, together with a NF-kappaB-dependent luciferase reporter plasmid. These results suggest that both the diacylglyceryl and the peptide portions of FSL-1 are indispensable for the expression of biological activities and for the recognition by Toll-like receptors 2 and 6 and that the recognition of FSL-1 by Toll-like receptors 2 and 6 appears to be hydrophobic.