Candida albicans and Streptococcus salivarius modulate IL-6, IL-8, and TNF-alpha expression and secretion by engineered human oral mucosa cells

We investigated the involvement of oral epithelial cells via two cytokines (IL-6 and TNF-alpha) and one chemokine (IL-8) in local defences against live yeast (Candida albicans) and bacteria (Streptococcus salivarius) using an engineered human oral mucosa model. We report that the yeast changed from the blastospore to the hyphal form and induced significant tissue disorganization at later contact periods (24 and 48 h) compared to the bacteria. However, this effect did not reduce the viability or total number of epithelial cells. Gene activation analyses revealed that IL-6, IL-8 and TNF-alpha mRNA levels rose in tissues in contact with live C. albicans or S. salivarius. Gene activation was followed by an upregulation of protein secretion. IL-6 levels were higher after contact with C. albicans than with S. salivarius. IL-8 levels after contact with S. salivarius were higher than with C. albicans. Our study suggests that S. salivarius is more efficient at inducing proinflammatory mediator release than C. albicans. These results provide additional evidence for the contribution of oral epithelial cells to the inflammatory response against fungi and bacteria.     

Production and characterization of an in vitro engineered human oral mucosa.

The role of epithelial cells in oral pathologies is poorly understood. Until now, most studies have used normal or transformed epithelial cell monolayers, a system that largely bypasses oral mucosal complexity. To overcome these limitations, an engineered human oral mucosa (EHOM) model has been produced and characterized. Following histological and immunohistochemical analyses, EHOM showed well-organized and stratified tissues in which epithelial cells expressed proliferating keratins such as Ki-67, K14, and K19 and also differentiating keratin (K10). In this model, epithelial cells interacted with fibroblasts in the lamina propria by secreting basement membrane proteins (laminins) and by expressing integrins (beta1 and alpha2beta1). Cytokine analyses using cultured supernatants showed that cells in EHOM were able to secrete interleukins (IL) including IL-1beta and IL-8 and tumor necrosis factor alpha (TNF-alpha). Finally, cells in this engineered model were able to secrete different metalloproteinases such as gelatinase-A and gelatinase-B. In conclusion, using tissue engineering technology, we produced well-organized EHOM tissues. It is anticipated that this model will be useful for examining mechanisms involved in oral diseases under controlled conditions by modeling the interactions between mucosa and microorganisms in the oral cavity.     

Protection of mice from lethal endogenous Candida albicans infection by immunization with Candida membrane antigen

The protective effects of immunization with Candida membrane antigen (CMA) on a systemic infection originating from intestinally colonized Candida albicans were examined. The colonization of orally inoculated C. albicans in the intestinal tract was established in BALB/c mice that had been concomitantly treated with oral doses of antibacterial drugs. In these animals, a systemic dissemination of C. albicans with fatal outcome was induced by a repeated dosing of prednisolone. In this endogenous infection model, the effects of immunization by CMA on the infection were examined. CMA-immunized mice showed a longer lifespan than unimmunized mice. The protective effect of CMA immunization in immunosuppressed mice was also measured by a decrease in body weight loss after treatment with prednisolone and in the number of viable Candida cells in the target organs, the kidneys and livers. However, the CFU of C. albicans in the intestinal tract was not significantly lowered. These results suggest that CMA immunization inhibited the dissemination of systemic Candida infection from the intestinal tract induced by treatment with prednisolone.     

Cellular interactions of Candida albicans with human oral epithelial cells and enterocytes

The human pathogenic fungus Candida albicans can cause systemic infections by invading epithelial barriers to gain access to the bloodstream. One of the main reservoirs of C. albicans is the gastrointestinal tract and systemic infections predominantly originate from this niche. In this study, we used scanning electron and fluorescence microscopy, adhesion, invasion and damage assays, fungal mutants and a set of fungal and host cell inhibitors to investigate the interactions of C. albicans with oral epithelial cells and enterocytes. Our data demonstrate that adhesion, invasion and damage by C. albicans depend not only on fungal morphology and activity, but also on the epithelial cell type and the differentiation stage of the epithelial cells, indicating that epithelial cells differ in their susceptibility to the fungus. C. albicans can invade epithelial cells by induced endocytosis and/or active penetration. However, depending on the host cell faced by the fungus, these routes are exploited to a different extent. While invasion into oral cells occurs via both routes, invasion into intestinal cells occurs only via active penetration.     

Smoking increases oral mucosa susceptibility to Candida albicans infection via the Nrf2 pathway: In vitro and animal studies

Smoking and Candida albicans (C. albicans) infection are risk factors for many oral diseases. Several studies have reported a close relationship between smoking and the occurrence of C. albicans infection. However, the exact underlying mechanism of this relationship remains unclear. We established a rat infection model and a C. albicans-Leuk1 epithelial cell co-culture model with and without smoke exposure to investigate the mechanism by which smoking contributes to C. albicans infection. Oral mucosa samples from healthy individuals and patients with oral leucoplakia were also analysed according to their smoking status. Our results indicated that smoking induced oxidative stress and redox dysfunction in the oral mucosa. Smoking-induced Nrf2 negatively regulated the NLRP3 inflammasome, impaired the oral mucosal defence response and increased the oral mucosa susceptibility to C. albicans. The results suggest that the Nrf2 pathway could be involved in the pathogenesis of oral diseases by mediating an antioxidative response to cigarette smoke exposure and suppressing host immunity against C. albicans.     

A novel antimicrobial peptide derived from human BPIFA1 protein protects against Candida albicans infection

Bactericidal/permeability-increasing fold containing family A, member 1 (BPIFA1) is an innate immunity defense protein. Our previous studies proved its antibacterial and antiviral effects, but its role in fungi remains unknown. The study aimed to identify antifungal peptides (AFP) derived from BPIFA1, and three antimicrobial peptides (AMP1–3) were designed. The antifungal effects were proved by growth inhibition assay. AMP3 activity was confirmed by germ tube growth experiment and XTT assay. Its effects on cell wall and membrane of Candida albicans were assessed by tannic acid and Annexin V-FITC/PI double staining, respectively. Additionally, scanning electron microscope (SEM) and transmission electron microscopy (TEM) were used for morphological and ultrastructural observation. The expression of ALS1, EAP1, and SUN41 was tested by qPCR. Ultimately, three AMPs could fight against C. albicans in vitro, and AMP3 was highly effective. It functioned by destroying the integrity of cell wall and normal structure of cell membrane. It also inhibited biofilm formation of C. albicans. In addition, AMP3 down-regulated the expression of ALS1, EAP1, and SUN41, those are known to be involved in virulence of C. albicans. Altogether, the study reported successful development of a novel AFP, which could be used as a new strategy for antifungal therapy.     

Alterations of protein expression in macrophages in response to Candida albicans infection

Although macrophages are an important first line of cellular defense, they are unable to effectively kill phagocytosed C. albicans. To determine the physiological basis of this inability, we investigated the alterations of macrophage proteins caused by C. albicans infection. Since the formation of C. albicans hyphae caused cell death, proteins were prepared 3 h after infection and examined by two-dimensional gel electrophoresis (2-DE). The most prominent changes were in glycolytic enzymes, which could have caused energy depletion of the infected cells. Also changed were proteins involved in maintenance of cellular integrity and NO production. Treatment of the macrophages with either cytochalasin D or taxol did not alter their inability to kill C. albicans. Our results indicate that multiple factors contribute to cell death as the pathogenic form of C. albicans becomes fully active inside macrophage cells.     

Expression of human membrane type 1 matrix metalloproteinase in Pichia pastoris

A soluble, C-terminal truncated form of human membrane type 1 matrix metalloproteinase (MT1-MMP) containing the hemopexin-like domain was expressed in Pichia pastoris strain KM71. High levels of secreted protein were detected. Although the c-DNA for the proenzyme (Ala(21)-Glu(523) called DeltaTM-MT1-MMP) was cloned, almost only active MT1-MMP (Tyr(112)-Glu(523)) with identical N-terminus as described for the wild-type enzyme was isolated. This active enzyme was highly purified and characterized with respect to its biochemical properties. The recombinant protein showed high stability against autolysis and proteolysis by yeast proteases, although the calculated in vivo half-life is rather low. The biochemical properties of this new MT1-MMP species were compared with the well-characterized catalytic domain (Ile(114)-Ile(318)) of MT1-MMP. The novel form of MT1-MMP exhibited a higher stability against autolysis than the isolated catalytic domain (Ile(114)-Ile(318)).     

In vitro analyses of tissue structure and interleukin-1beta expression and production by human oral mucosa in response to Candida albicans infections

Clinical and experimental observations suggest that oral epithelial cells play a key role in host defenses against candidal infections through cytokines and chemokines. We thus attempted to determine whether oral epithelial cells convey IL-1beta as a pro-inflammatory cytokine in response to Candida albicans infections. We created engineered human oral mucosa (EHOM), put them in contact with live and heat-inactivated C. albicans (10(5) yeast/cm2), and measured the expression of IL-1beta mRNA and protein. Tissue structure and C. albicans morphology were also evaluated. Only live C. albicans modulated IL-1beta expression and secretion. IL-1beta mRNA expression significantly increased during the early stages of infection and decreased during the later stages. The modulatory effect of C. albicans on IL-1beta expression was confirmed by the fact that increased amounts of inactive IL-1beta (33 kDa) were detected early during the infection which then dropped dramatically. There was a significant and time-dependent increase in the amount of the active form of IL-1beta (17 kDa) secreted into the supernatant by epithelial cells infected with live C. albicans. Histological features revealed damage to infected tissues (separation of epithelial cells, edema, vacuolization, reduction in thickness) compared to uninfected ones. Morphological analyses showed that C. albicans changed from a blastospore to a hyphal form at later infection periods. This transformation was very pronounced at 8 and 24 h post-infection. These results provide additional evidence for the contribution of oral epithelial cells to local defenses against exogenous stimulations such as C. albicans infections.     

Characterization of a truncated recombinant form of human membrane type 3 matrix metalloproteinase.

Membrane type 3 matrix metalloproteinase (MT3-MMP), an activator for the zymogen of MMP-2 (proMMP-2, or progelatinase A), is known to be expressed in human placenta, brain, lung and rat vascular smooth muscle cells, but information about its biochemical properties is limited. In the present study, we expressed and purified a truncated form of MT3-MMP lacking the transmembrane and intracytoplasmic domain (DeltaMT3) and characterized the enzyme biochemically. DeltaMT3 digested type III collagen into characteristic 3/4- and 1/4-fragments by cleaving the Gly781-Ile782 and Gly784-Ile785 bonds of alpha1(III) chains. Although DeltaMT3 did not have such an activity against type I collagen, it attacked the Gly4-Ile5 bond of the triple helical portion of alpha2(I) chains, leading to removal of the crosslink containing N-terminal telopeptides. By quantitative analyses of the activities of DeltaMT3 and a similar deletion mutant of MT1-MMP (DeltaMT1), DeltaMT3 was approximately fivefold more efficient at cleaving type III collagen. DeltaMT3 also digested cartilage proteoglycan, gelatin, fibronectin, vitronectin, laminin-1, alpha1-proteinase inhibitor and alpha2-macroglobulin into almost identical fragments to those given by DeltaMT1, although carboxymethylated transferrin digestion by DeltaMT3 generated some extra fragments. The activity of DeltaMT3 was inhibited by tissue inhibitor of metalloproteinases-2 (TIMP-2) and TIMP-3 in a 1 : 1 stoichiometry, but not by TIMP-1. ProMMP-2 was partially activated by DeltaMT3 to give the intermediate form. These results indicate that, like MT1-MMP, MT3-MMP exhibits proteolytic activities against a wide range of extracellular matrix molecules. However, differences in the proMMP-2 activation and tissue distribution suggest that MT3-MMP and MT1-MMP play different roles in the pathophysiological digestion of extracellular matrix.     

Phenotypic aspects of oral strains of Candida albicans in children with Down's syndrome.

The aim of this article is to characterize the biological aspects of oral strains of C. albicans in children with Down's syndrome. These yeasts were analyzed as to their macromorphological and enzymatic aspects and were tested as to their in vitro susceptibility to antifungal drugs using broth microdilution to determine the minimum inhibitory concentration (MIC). The morphotyping revealed that all oral C. albicans isolates from children with Down's syndrome promoted the formation of fringes regardless of size, while the control group presented smaller fringes. All oral C. albicans strains produced proteinase, but those with phospholipolytic activity showed greater enzyme capacity in the test group. In vitro susceptibility showed that all oral C. albicans isolates were sensitive to the drugs used.     

Environmental pH modulates biofilm formation and matrix composition in Candida albicans and Candida glabrata

Abstract

Candida species are fungal opportunistic pathogens capable of colonizing and infecting various human anatomical sites, where they have to adapt to distinct niche-specific pH conditions. The aim of this study was to analyse the features of Candida albicans and Candida glabrata biofilms developed under neutral and vaginal acidic (pH 4) conditions. C. albicans produced thicker and more filamentous biofilms under neutral than under acidic conditions. On the other hand, the formation of biofilms by C. glabrata was potentiated by the acidic conditions suggesting the high adaptability of this species to the vaginal environment. In general, both species developed biofilms containing higher amounts of matrix components (protein and carbohydrate) under neutral than acidic conditions, although the opposite result was found for one C. glabrata strain. Overall, this study contributes to a better understanding of the modulation of C. albicans and C. glabrata virulence by specific pH conditions.