Fusobacterium nucleatum is a gram-negative anaerobe that is prevalent in periodontal disease and infections of different parts of the body. The organism has remarkable adherence properties, binding to partners ranging from eukaryotic and prokaryotic cells to extracellular macromolecules. Understanding its adherence is important for understanding the pathogenesis of F. nucleatum. In this study, a novel adhesin, FadA (Fusobacterium adhesin A), was demonstrated to bind to the surface proteins of the oral mucosal KB cells. FadA is composed of 129 amino acid (aa) residues, including an 18-aa signal peptide, with calculated molecular masses of 13.6 kDa for the intact form and 12.6 kDa for the secreted form. It is highly conserved among F. nucleatum, Fusobacterium periodonticum, and Fusobacterium simiae, the three most closely related oral species, but is absent in the nonoral species, including Fusobacterium gonidiaformans, Fusobacterium mortiferum, Fusobacterium naviforme, Fusobacterium russii, and Fusobacterium ulcerans. In addition to FadA, F. nucleatum ATCC 25586 and ATCC 49256 also encode two paralogues, FN1529 and FNV2159, each sharing 31% identity with FadA. A double-crossover fadA deletion mutant, F. nucleatum 12230-US1, was constructed by utilizing a novel sonoporation procedure. The mutant had a slightly slower growth rate, yet its binding to KB and Chinese hamster ovarian cells was reduced by 70 to 80% compared to that of the wild type, indicating that FadA plays an important role in fusobacterial colonization in the host. Furthermore, due to its uniqueness to oral Fusobacterium species, fadA may be used as a marker to detect orally related fusobacteria. F. nucleatum isolated from other parts of the body may originate from the oral cavity.

BackgroundTumor-associated macrophages (TAMs) constitute a significant part of infiltrating inflammatory cells that are frequently correlated with progression and poor prognosis of a variety of cancers. Tumor cell-produced human β-defensin-3 (hBD-3) has been associated with TAM trafficking in oral cancer; however, its involvement in tumor-related inflammatory processes remains largely unknown.MethodologyThe relationship between hBD-3, monocyte chemoattractant protein-1 (MCP-1), TAMs, and CCR2 was examined using immunofluorescence microscopy in normal and oral carcinoma in situ biopsy specimens. The ability of hBD-3 to chemoattract host macrophages in vivo using a nude mouse model and analysis of hBD-3 on monocytic cell migration in vitro, applying a cross-desensitization strategy of CCR2 and its pharmacological inhibitor (RS102895), respectively, was also carried out.Conclusions/FindingsMCP-1, the most frequently expressed tumor cell-associated chemokine, was not produced by tumor cells nor correlated with the recruitment of macrophages in oral carcinoma in situ lesions. However, hBD-3 was associated with macrophage recruitment in these lesions and hBD-3-expressing tumorigenic cells induced massive tumor infiltration of host macrophages in nude mice. HBD-3 stimulated the expression of tumor-promoting cytokines, including interleukin-1α (IL-1α), IL-6, IL-8, CCL18, and tumor necrosis factor-α (TNF-α) in macrophages derived from human peripheral blood monocytes. Monocytic cell migration in response to hBD-3 was inhibited by cross-desensitization with MCP-1 and the specific CCR2 inhibitor, RS102895, suggesting that CCR2 mediates monocyte/macrophage migration in response to hBD-3. Collectively, these results indicate that hBD-3 utilizes CCR2 to regulate monocyte/macrophage trafficking and may act as a tumor cell-produced chemoattractant to recruit TAMs. This novel mechanism is the first evidence of an hBD molecule orchestrating an in vivo outcome and demonstrates the importance of the innate immune system in the development of tumors.

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We analyzed the transcriptional regulation of the putative virulence genes encoded on the plasmid pCP13 from Clostridium perfringens strain 13. The transcription of the beta2-toxin (cpb2) and possible collagen adhesin (cna) genes were regulated in both a positive and negative manner, respectively, by the two-component VirR/VirS system. The secondary regulator of the VirR/VirS system, VR-RNA, also affects the expression of both of these genes in the same fashion as the VirR/VirS system. This indicates that the global regulatory cascade of the VirR/VirS system controls the expression of virulence genes located on the plasmid, as well as those found chromosomally in C. perfringens strain 13. ß

Human β-defensin-3 (hBD3) is an epithelial cell-derived innate immune regulatory molecule overexpressed in oral dysplastic lesions and fosters a tumor-promoting microenvironment. Expression of hBD3 is induced by the epidermal growth factor receptor signaling pathway. Here we describe a novel pathway through which the high-risk human papillomavirus type-16 (HPV-16) oncoprotein E6 induces hBD3 expression in mucosal keratinocytes. Ablation of E6 by siRNA induces the tumor suppressor p53 and diminishes hBD3 in HPV-16 positive CaSki cervical cancer cells and UM-SCC-104 head and neck cancer cells. Malignant cells in HPV-16-associated oropharyngeal cancer overexpress hBD3. HPV-16 E6 induces hBD3 mRNA expression, peptide production and gene promoter activity in mucosal keratinocytes. Reduction of cellular levels of p53 stimulates hBD3 expression, while activation of p53 by doxorubicin inhibits its expression in primary oral keratinocytes and CaSki cells, suggesting that p53 represses hBD3 expression. A p53 binding site in the hBD3 gene promoter has been identified by using electrophoretic mobility shift assays and chromatin immunoprecipitation (ChIP). In addition, the p63 protein isoform ΔNp63α, but not TAp63, stimulated transactivation of the hBD3 gene and was co-expressed with hBD3 in head and neck cancer specimens. Therefore, high-risk HPV E6 oncoproteins may stimulate hBD3 expression in tumor cells to facilitate tumorigenesis of HPV-associated head and neck cancer.

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Completion of the whole genome sequence of Clostridium perfringens strain 13 revealed the presence of an extracellular nuclease gene, cadA. Transcriptional analysis showed that the cadA gene is negatively regulated by the two-component VirR/VirS system and its secondary regulator VR-RNA. The CadA protein possesses an N-terminal signal sequence and a Gram-positive cell wall anchoring motif consisting of a sorting signal (LPXTG motif), a hydrophobic domain, and positively charged residues at the end of C-terminus. By comparing the DNase production between the wild type and the cadA mutant, and DNase activity assay with the recombinant truncated CadA protein, we confirmed that the cadA gene product is one of the DNases produced by C. perfringens.

At our resident-run clinic in an underserved community, laboratory test costs in 2013 exceeded the government subsidy by $400 000. To optimize limited resources and improve patient care, an education program to reduce testing was implemented.Between November 2014 and January 2015, residents attended lectures on utilization of laboratory testing, focusing on standard practice guidelines, and analyses of unnecessary tests. Multivariate nonparametric statistical methods and subgroup analysis were used to evaluate cost reduction.There were 453 clinic visits during the intervention period and 471 visits during the control period. Lectures were independently associated with a significant laboratory cost reduction. Median laboratory cost per visit decreased from $106.00 to $74.00. Total cost in the study period decreased from $79 403 to $51 463. There were similar reductions of laboratory costs in two subgroups: age groups of <50 years and ≥50 years, new encounters, and follow-up visits . In the analysis of individual tests, the cost of TSH and Vitamin D tests had the greatest reduction ($8176 and $5088 respectively).An appropriate physician education program can reduce laboratory tests and costs. Screening tests with inadequate evidence support were reduced most, whereas those with proven benefits did not decrease significantly.