The Host Immune System Facilitates Disseminated Staphylococcus aureus Disease Due to Phagocytic Attraction to Candida albicans during Coinfection: a Case of Bait and Switch.

Invasive Staphylococcus aureus infections account for 15 to 50% of fatal bloodstream infections annually. These disseminated infections often arise without a defined portal of entry into the host but cause high rates of mortality. The fungus Candida albicans and the Gram-positive bacterium S. aureus can form polymicrobial biofilms on epithelial tissue, facilitated by the C. albicans adhesin encoded by ALS3 While a bacterium-fungus interaction is required for systemic infection, the mechanism by which bacteria disseminate from the epithelium to internal organs is unclear. In this study, we show that highly immunogenic C. albicans hyphae attract phagocytic cells, which rapidly engulf adherent S. aureus and subsequently migrate to cervical lymph nodes. Following S. aureus-loaded phagocyte translocation from the mucosal surface, S. aureus produces systemic disease with accompanying morbidity and mortality. Our results suggest a novel role for the host in facilitating a bacterium-fungus infectious synergy, leading to disseminated staphylococcal disease.

Staphylococcus-Candida Interaction Models: Antibiotic Resistance Testing and Host Interactions.

The fungus Candida albicans and bacterium Staphylococcus aureus can coexist in polymicrobial biofilms. S. aureus attaches strongly to hyphae, but not to the yeast form, of C. albicans with important consequences for virulence. Hyphae-associated S. aureus is less susceptible to antibiotic treatment. Furthermore, co-inoculation of C. albicans and S. aureus causes more severe and widespread infection than either microorganism alone. In this chapter, a basic in vitro model for studying the interaction between C. albicans hyphae and S. aureus is presented, which makes use of a fluorescently labeled S. aureus strain. Furthermore, two protocols are described that allow investigation of the effect of C. albicans and S. aureus interaction on antibiotic susceptibility or on interactions with the host. The latter focuses on phagocytosis of C. albicans-adhered S. aureus by macrophages. The protocols presented here may serve as a starting point to study the interaction of C. albicans with various other bacterial species.

LuxS signaling in Porphyromonas gingivalis-host interactions.

Dental plaque is a multispecies biofilm in the oral cavity that significantly influences oral health. The presence of the oral anaerobic pathogen Porphyromonas gingivalis is an important determinant in the development of periodontitis. Direct and indirect interactions between P. gingivalis and the host play a major role in disease development. Transcriptome analysis recently revealed that P. gingivalis gene-expression is regulated by LuxS in both an AI-2-dependent and an AI-2 independent manner. However, little is known about the role of LuxS-signaling in P. gingivalis-host interactions. Here, we investigated the effect of a luxS mutation on the ability of P. gingivalis to induce an inflammatory response in human oral cells in vitro. Primary periodontal ligament (PDL) fibroblasts were challenged with P. gingivalis ΔluxS or the wild-type parental strain and gene-expression of pro-inflammatory mediators IL-1ß, IL-6 and MCP-1 was determined by real-time PCR. The ability of P. gingivalis ΔluxS to induce an inflammatory response was severely impaired in PDL-fibroblasts. This phenotype could be restored by providing of LuxS in trans, but not by addition of the AI-2 precursor DPD. A similar phenomenon was observed in a previous transcriptome study showing that expression of PGN_0482 was reduced in the luxS mutant independently of AI-2. We therefore also analyzed the effect of a mutation in PGN_0482, which encodes an immuno-reactive, putative outer-membrane protein. Similar to P. gingivalis ΔluxS, the P. gingivalis Δ0482 mutant had an impaired ability to induce an inflammatory response in PDL fibroblasts. LuxS thus appears to influence the pro-inflammatory responses of host cells to P. gingivalis, likely through regulation of PGN_0482.

A challenge with Porphyromonas gingivalis differentially affects the osteoclastogenesis potential of periodontal ligament fibroblasts from periodontitis patients and non-periodontitis donors.

AIM: Porphyromonas gingivalis (Pg) may cause an immune-inflammatory response in host cells leading to bone degradation by osteoclasts. We investigated the osteoclast-inducing capacity of periodontal ligament fibroblasts from periodontitis patients and non-periodontitis donors after a challenge with viable Pg. MATERIALS AND METHODS: PDLFs from periodontitis patients (n = 8) and non-periodontitis donors (n = 7) were incubated for 6 h with or without viable Pg and subsequently co-cultured with osteoclast precursors from peripheral blood mononuclear cells (PBMCs). The number of multinucleated tartrate-resistant acid phosphatase-positive cells was determined at 21 days. Expression of osteoclastogenesis-associated genes was assessed after infection of PDLFs mono-cultures and in PDLFs-PBMCs co-cultures. Resorption activity was analysed on bone slices. RESULTS: Pg induced the expression of osteoclastogenesis-associated genes by PDLFs. After bacterial challenge the formation of osteoclast-like cell was decreased in co-cultures of PBMCs with non-periodontitis PDLFs, but not with PDLFs from periodontitis patients. CONCLUSION: PDLFs from sites free of periodontitis respond to an infection with Pg by tempering formation of osteoclast-like cells, probably promoting clearance of the infection. PDLFs from periodontitis sites are desensitized to a Pg challenge in terms of their osteoclast-inducing capacity.

Influence of titanium on in vitro fibroblast-Porphyromonas gingivalis interaction in peri-implantitis.

AIM: Titanium wear particles have been found in peri-implant tissues, but their role in the pathogenesis of peri-implantitis remains unclear. We aimed to determine the in vitro inflammatory responses of peri-implant granulation tissue fibroblasts (PIGFs) to titanium particles alone and in the presence of viable Porphyromonas gingivalis. MATERIALS & METHODS: Peri-implant granulation tissue fibroblasts were challenged either with TiO2 particles, P. gingivalis or a combination of TiO2 particles and P. gingivalis. Gene expression and protein production of pro-inflammatory mediators by PIGFs were measured with PCR and ELISA, respectively. RESULTS: Higher doses of TiO2 were toxic to PIGFs and in sub-toxic doses, TiO2 caused an increase in gene expression of tumour necrosis factor (TNF)-A and increased protein production of TNF-α, interleukin (IL)-6 and IL-8. A challenge with P. gingivalis alone induced gene expression of TNF-A, IL-1ß, IL-6 and IL-8. A combined challenge with TiO2 and P. gingivalis caused a stronger increase in gene expression of TNF-A and protein production of TNF-α and MCP-1 than P. gingivalis alone. CONCLUSIONS: TiO2 particles and P. gingivalis, individually, can induce pro-inflammatory responses in PIGFs. Furthermore, TiO2 particles and viable P. gingivalis further enhance gene expression and production of TNF-α by PIGFs. Therefore, Ti wear particles in the peri-implant tissues in combination with P. gingivalis infection may contribute to the pathogenesis of peri-implantitis by enhancing the inflammation in peri-implant tissues.

Diverse effects of Porphyromonas gingivalis on human osteoclast formation.

Porphyromonas gingivalis is associated with periodontitis, a chronic inflammatory disease of the tooth-supporting tissues. A major clinical symptom is alveolar bone loss due to excessive resorption by osteoclasts. P. gingivalis may influence osteoclast formation in diverse ways; by interacting directly with osteoclast precursors that likely originate from peripheral blood, or indirectly by activating gingival fibroblasts, cells that can support osteoclast formation. In the present study we investigated these possibilities. Conditioned medium from viable or dead P. gingivalis, or from gingival fibroblasts challenged with viable or dead P. gingivalis were added to human mononuclear osteoclast precursors. After 21 days of culture the number of multinucleated (≥3 nuclei) tartrate resistant acid phosphatase (TRACP)-positive cells was determined as a measure for osteoclast formation. Conditioned medium from viable P. gingivalis, and from fibroblasts with viable P. gingivalis stimulated osteoclast formation (1.6-fold increase p < 0.05). Conditioned medium from dead bacteria had no effect on osteoclast formation, whereas conditioned medium from fibroblasts with dead bacteria stimulated formation (1.4-fold increase, p < 0.05). Inhibition of P. gingivalis LPS activity by Polymyxin B reduced the stimulatory effect of conditioned medium. Interestingly, when RANKL and M-CSF were added to cultures, conditioned media inhibited osteoclast formation (0.6-0.7-fold decrease, p < 0.05). Our results indicate that P. gingivalis influences osteoclast formation in vitro in different ways. Directly, by bacterial factors, likely LPS, or indirectly, by cytokines produced by gingival fibroblasts in response to P. gingivalis. Depending on the presence of RANKL and M-CSF, the effect of P. gingivalis is either stimulatory or inhibitory.

The role of salivary histatin and the human cathelicidin LL-37 in wound healing and innate immunity.

Antimicrobial peptides are multifunctional in innate immunity and wound repair of multicellular organisms. We were the first to discover that histatins, a family of salivary antimicrobial peptides, enhance epithelial cell migration, suggesting a role in oral wound healing. It is unknown whether histatins display innate-immunity activities, similar to other antimicrobial peptides such as LL-37. Therefore, we compared the effect of Histatin-2 and LL-37 on several activities within the context of wound healing and innate immunity. We found that Histatin-2 enhances fibroblast migration, but only weakly induces proliferation. LL-37 enhances both fibroblast migration and proliferation, but only at a narrow concentration optimum (approximately 1 microm). At higher concentrations LL-37 causes cell death, whereas Histatin-2 is not cytotoxic. Both peptides do not alter fibroblast-to-myofibroblast differentiation. Histatin-2 does not alter interleukin-8 (IL-8) expression and lipopolysaccharide (LPS)-elevated cytokine and chemokine expression. In contrast, LL-37 induces IL-8 expression, but dampens the LPS-induced immune response. Neither Histatin-2 nor LL-37 affects human-neutrophil migration. Histatins are, unlike other antimicrobial peptides, not cytotoxic or proinflammatory. It seems that they are important for the initial stage of wound healing in which fast wound coverage is important for healing without infection, inflammation, or fibrosis development. Interestingly, these characteristics are more typical for the mouth than for skin.

The capsule of Porphyromonas gingivalis reduces the immune response of human gingival fibroblasts.

BACKGROUND: Periodontitis is a bacterial infection of the periodontal tissues. The Gram-negative anaerobic bacterium Porphyromonas gingivalis is considered a major causative agent. One of the virulence factors of P. gingivalis is capsular polysaccharide (CPS). Non-encapsulated strains have been shown to be less virulent in mouse models than encapsulated strains. RESULTS: To examine the role of the CPS in host-pathogen interactions we constructed an insertional isogenic P. gingivalis knockout in the epimerase-coding gene epsC that is located at the end of the CPS biosynthesis locus. This mutant was subsequently shown to be non-encapsulated. K1 capsule biosynthesis could be restored by in trans expression of an intact epsC gene. We used the epsC mutant, the W83 wild type strain and the complemented mutant to challenge human gingival fibroblasts to examine the immune response by quantification of IL-1beta, IL-6 and IL-8 transcription levels. For each of the cytokines significantly higher expression levels were found when fibroblasts were challenged with the epsC mutant compared to those challenged with the W83 wild type, ranging from two times higher for IL-1beta to five times higher for IL-8. CONCLUSIONS: These experiments provide the first evidence that P. gingivalis CPS acts as an interface between the pathogen and the host that may reduce the host's pro-inflammatory immune response. The higher virulence of encapsulated strains may be caused by this phenomenon which enables the bacteria to evade the immune system.

Rac1-induced cell migration requires membrane recruitment of the nuclear oncogene SET.

The Rho GTPase Rac1 controls cell adhesion and motility. The effector loop of Rac1 mediates interactions with downstream effectors, whereas its C-terminus binds the exchange factor beta-Pix, which mediates Rac1 targeting and activation. Here, we report that Rac1, through its C-terminus, also binds the nuclear oncogene SET/I2PP2A, an inhibitor of the serine/threonine phosphatase PP2A. We found that SET translocates to the plasma membrane in cells that express active Rac1 as well as in migrating cells. Membrane targeting of SET stimulates cell migration in a Rac1-dependent manner. Conversely, reduction of SET expression inhibits Rac1-induced migration, indicating that efficient Rac1 signalling requires membrane recruitment of SET. The recruitment of the SET oncogene to the plasma membrane represents a new feature of Rac1 signalling. Our results suggest a model in which Rac1-stimulated cell motility requires both effector loop-based downstream signalling and recruitment of a signalling amplifier, that is, SET, through the hypervariable C-terminus.