Samir Jawhara

Colonization of Mice by Candida albicans Is Promoted by Chemically Induced Colitis and Augments Inflammatory Responses through Galectin-3

BACKGROUND Little is known about the relationship between colonic inflammation and Candida albicans colonization. Galectin-3 (Gal-3) is an intestinal lectin that binds to specific C. albicans glycans and is involved in inflammation. METHODS Colitis was experimentally induced in wild-type and Gal3(-/-) mice using dextran sulfate sodium (DSS) before oral administration of C. albicans. Yeast recovered from stools was quantified. The presence of yeast and inflammation were evaluated in sections of colon by histologic examination, quantification of myeloperoxidase (MPO) activity, and by gene expression for cytokines and innate immune receptors. Serum from mice was collected for determination of anti-yeast mannan antibodies, including anti-Saccharomyces cerevisiae antibodies (ASCA), which are biomarkers of an inflammatory bowel disease. RESULTS Inflammation strongly promoted C. albicans colonization. Conversely, C. albicans augmented inflammation induced by DSS, as assessed by histologic scores, MPO activity, and tumor necrosis factor (TNF)-alpha and Toll-like receptor (TLR)-2 expression. C. albicans colonization generated ASCA. The absence of Gal-3 reduced DSS inflammation and abolished the response of TLR-2 and TNF-alpha to C. albicans colonization. CONCLUSIONS DSS-induced colitis provides a model for establishing C. albicans colonization in mice. This model reveals that C. albicans augments inflammation and confirms the role of Gal-3 in both inflammation and the control of host responses to C. albicans.

Saccharomyces boulardii decreases inflammation and intestinal colonization by Candida albicans in a mouse model of chemically-induced colitis

The present study was designed to investigate the effects of Saccharomyces boulardii on inflammation and intestinal colonization by Candida albicans in a BALB/c mouse model of colitis that had been induced by dextran-sulfate-sodium (DSS). Colonization with C. albicans was established by oral gavage with a 200 microL suspension of 10(7) yeast cells. A 1.5% solution of DSS was administered in drinking water 1 h after C. albicans oral challenge, while 10(7) cells of S. boulardii was inoculated daily by oral gavage for 1 week. Faeces were collected daily for 2 weeks. Seven groups of mice consisting of those that were administered either C. albicans or S. boulardii or both were sacrificed after 14 days and samples of the colon were taken for histological scoring and real-time PCR (RT-PCR) analysis of inflammatory cytokines and toll-like receptors (TLRs). Compared to control animals that did not receive DSS, the number of C. albicans colonies recovered from faeces was significantly greater in mice receiving DSS. In contrast, the colony forming units (CFUs) of C. albicans were greatly reduced in mice receiving S. boulardii. The administration of this yeast decreased the severity of DSS-induced clinical scores and histological inflammation. At the mRNA expression level, an increase in TLR2 and TLR4 resulting from the presence of S. boulardii was associated with a reduction in the inflammatory cytokines TNFalpha and INFgamma. In mice receiving DSS and C. albicans, TLR4 was over-expressed by stimulation with both yeasts, but TLR2 and TNFalpha, which were increased by the administration of C. albicans alone, were decreased in the presence of S. boulardii. These results indicate that S. boulardii decreased inflammation and C. albicans colonization in this BALB/c mouse model of colitis.

Modulation of Intestinal Inflammation by Yeasts and Cell Wall Extracts: Strain Dependence and Unexpected Anti-Inflammatory Role of Glucan Fractions

Yeasts and their glycan components can have a beneficial or adverse effect on intestinal inflammation. Previous research has shown that the presence of Saccharomyces cerevisiae var. boulardii (Sb) reduces intestinal inflammation and colonization by Candida albicans. The aim of this study was to identify dietary yeasts, which have comparable effects to the anti-C. albicans and anti-inflammatory properties of Sb and to assess the capabilities of yeast cell wall components to modulate intestinal inflammation. Mice received a single oral challenge of C. albicans and were then given 1.5% dextran-sulphate-sodium (DSS) for 2 weeks followed by a 3-day restitution period. S. cerevisiae strains (Sb, Sc1 to Sc4), as well as mannoprotein (MP) and β-glucan crude fractions prepared from Sc2 and highly purified β-glucans prepared from C. albicans were used in this curative model, starting 3 days after C. albicans challenge. Mice were assessed for the clinical, histological and inflammatory responses related to DSS administration. Strain Sc1-1 gave the same level of protection against C. albicans as Sb when assessed by mortality, clinical scores, colonization levels, reduction of TNFα and increase in IL-10 transcription. When Sc1-1 was compared with the other S. cerevisiae strains, the preparation process had a strong influence on biological activity. Interestingly, some S. cerevisiae strains dramatically increased mortality and clinical scores. Strain Sc4 and MP fraction favoured C. albicans colonization and inflammation, whereas β-glucan fraction was protective against both. Surprisingly, purified β-glucans from C. albicans had the same protective effect. Thus, some yeasts appear to be strong modulators of intestinal inflammation. These effects are dependent on the strain, species, preparation process and cell wall fraction. It was striking that β-glucan fractions or pure β-glucans from C. albicans displayed the most potent anti-inflammatory effect in the DSS model.

In Vivo Imaging of Bioluminescent Escherichia coli in a Cutaneous Wound Infection Model for Evaluation of an Antibiotic Therapy

ABSTRACT A rapid, continuous method for noninvasively monitoring the effectiveness of several antibacterial agents in real time by using a model of wound infection was developed. This study was divided into three steps: (i) construction of a plasmid to transform Escherichia coli into a bioluminescent variant, (ii) study of the bioluminescent E. coli in vitro as a function of temperature and pH, and (iii) determination of the MIC and the minimal bactericidal concentration of sulfamethoxazole-trimethoprim (SMX-TMP). Finally, the efficacy of SMX-TMP was monitored in vivo in a cutaneous wound model (hairless rat) infected with this bioluminescent bacterium by using a bioluminescence imaging system. E. coli was transformed by electroporation with a shuttle vector (pRB474) containing the firefly (Photinus pyralis) luciferase gene, resulting in a bioluminescent phenotype. It was found that pH 5.0 was optimal for incorporation of the susbstrate d-luciferin for the luciferase reaction. In vitro, when the agar dilution method, standard turbidity assays, and the bioluminescence imaging system were used, E. coli(pRB474) proved to be susceptible to SMX-TMP. In vivo, at 4 h, SMX-TMP treatment was already efficient compared to no treatment (P = 0.034). At 48 h, no bioluminescence was detected in the wound, demonstrating the susceptibility of E. coli to SMX-TMP. In conclusion, this study points out the advantage of using bioluminescence imaging to evaluate the effects of antibiotics for the treatment of acute infections in vivo in a nondestructive and noninvasive manner.

Short term Candida albicans colonization reduces Pseudomonas aeruginosa-related lung injury and bacterial burden in a murine model.

IntroductionPseudomonas aeruginosa is a frequent cause of ventilator-acquired pneumonia (VAP). Candida tracheobronchial colonization is associated with higher rates of VAP related to P. aeruginosa. This study was designed to investigate whether prior short term Candida albicans airway colonization modulates the pathogenicity of P. aeruginosa in a murine model of pneumonia and to evaluate the effect of fungicidal drug caspofungin.MethodsBALB/c mice received a single or a combined intratracheal administration of C. albicans (1 × 105 CFU/mouse) and P. aeruginosa (1 × 107 CFU/mouse) at time 0 (T0) upon C. albicans colonization, and Day 2. To evaluate the effect of antifungal therapy, mice received caspofungin intraperitoneally daily, either from T0 or from Day 1 post-colonization. After sacrifice at Day 4, lungs were analyzed for histological scoring, measurement of endothelial injury, and quantification of live P. aeruginosa and C. albicans. Blood samples were cultured for dissemination.ResultsA significant decrease in lung endothelial permeability, the amount of P. aeruginosa, and bronchiole inflammation was observed in case of prior C. albicans colonization. Mortality rate and bacterial dissemination were unchanged by prior C. albicans colonization. Caspofungin treatment from T0 (not from Day 1) increased their levels of endothelial permeability and lung P. aeruginosa load similarly to mice receiving P. aeruginosa alone.ConclusionsP. aeruginosa-induced lung injury is reduced when preceded by short term C. albicans airway colonization. Antifungal drug caspofungin reverses that effect when used from T0 and not from Day 1.

Murine Model of Dextran Sulfate Sodium-induced Colitis Reveals Candida glabrata Virulence and Contribution of β-Mannosyltransferases

Background: β-1,2-Linked mannosides (β-Mans) are adhesins present in the cell wall of the pathogenic yeast Candida glabrata. Results: β-Mans are associated with intensive gut colonization by C. glabrata, which exacerbates intestinal inflammation and induces mouse mortality. Conclusion: β-Mans affect the virulence of C. glabrata in the host. Significance: This study confirms and extends our knowledge about β-Mans as therapeutic targets for combating life-threatening Candida infections. Candida glabrata, like Candida albicans, is an opportunistic yeast pathogen that has adapted to colonize all segments of the human gastrointestinal tract and vagina. The C. albicans cell wall expresses β-1,2-linked mannosides (β-Mans), promoting its adherence to host cells and tissues. Because β-Mans are also present in C. glabrata, their role in C. glabrata colonization and virulence was investigated in a murine model of dextran sulfate sodium (DSS)-induced colitis. Five clustered genes of C. glabrata encoding β-mannosyltransferases, BMT2–BMT6, were deleted simultaneously. β-Man expression was studied by Western blotting, flow cytometry, and NMR analysis. Mortality, clinical, histologic, and colonization scores were determined in mice receiving DSS and different C. glabrata strains. The results show that C. glabrata bmt2–6 strains had a significant reduction in β-1,2-Man expression and a disappearance of β-1,2-mannobiose in the acid-stable domain. A single gavage of C. glabrata wild-type strain in mice with DSS-induced colitis caused a loss of body weight, colonic inflammation, and mortality. Mice receiving C. glabrata bmt2–6 mutant strains had normal body weight and reduced colonic inflammation. Lower numbers of colonies of C. glabrata bmt2–6 were recovered from stools and different parts of the gastrointestinal tract. Histopathologic examination revealed that the wild-type strain had a greater ability to colonize tissue and cause tissue damage. These results showed that C. glabrata has a high pathogenic potential in DSS-induced colitis, where β-Mans contribute to colonization and virulence.

Short fungal fractions of β-1,3 glucans affect platelet activation

Platelets are capable of binding, aggregating, and internalizing microorganisms, which enhances the elimination of pathogens from the blood. The yeast Candida albicans is a pathobiont causing life-threatening invasive infections. Its cell wall contains β-1,3 glucans that are known to trigger a wide range of host cell activities and to circulate during infection. We studied the effect of β-1,3 glucan fractions (BGFs) consisting of diglucosides (Glc2), tetraglucosides (Glc4), and pentaglucosides (Glc5) on human platelets, their mechanisms of action, and their possible impact on host defenses. The effect of BGFs on the coagulation process was determined by measuring thrombin generation. Platelets pretreated with BGFs were analyzed in terms of activation, receptor expression, aggregation, and adhesion to neutrophils and to C. albicans The results show that BGFs affected the endogenous thrombin potential in a concentration-dependent manner. For platelet activation, BGFs at a low concentration (2 μmol/l) reduced ATP release and prevented the phosphorylation of protein kinase C. BGFs diminished the expression of P-selectin and the activation of αIIbβ3 BGFs decreased platelet aggregation and the interaction between thrombin-stimulated platelets and neutrophils, fibrinogen, and C. albicans GLc5 decreased ATP release and TGF-β1 production in response to TLR4 upregulation in thrombin-stimulated platelets, but TLR4 blockage abolished the effect of BGFs on platelets. This study provides evidence that fungal pentaglucosides modulate platelet activity mediated via TLR4 stimulation and reduce platelet-neutrophil interaction.

Remodeling of the Candida glabrata cell wall in the gastrointestinal tract affects the gut microbiota and the immune response

The gastrointestinal (GI) microbiota acts a natural barrier to the proliferation of opportunistic pathogens. Candida glabrata is an opportunistic yeast pathogen that has adapted to colonize all segments of the human GI tract. We observed an increase in Escherichia coli, Enterococcus faecalis, and Bacteroides vulgatus populations, and a decrease in Lactobacillus johnsonii, Bacteroides thetaiotaomicron, and Bifidobacterium animalis in mice with DSS-induced colitis. This reduction was more pronounced for L. johnsonii during C. glabrata overgrowth. In addition, C. glabrata overgrowth increased mouse mortality and inflammatory parameters, and modulated the expression of intestinal receptors and signaling pathways. The C. glabrata cell wall underwent various changes during the course of C. glabrata colonization, and showed a significant increase in chitin. C. glabrata deficient in chitin synthase-3 induced fewer inflammatory parameters than the parental strain during intestinal inflammation. Oral administration of chitin attenuated the impact of colitis, and reduced the number of aerobic bacteria and C. glabrata overgrowth, while chitinase-3-like protein-1 increased. This study provides evidence that inflammation of the gut alters the microbial balance and leads to C. glabrata cell wall remodeling through an increase in chitin, which is involved in promoting persistence of C. glabrata in the gut.

Polymorphisms in the Mannose-Binding Lectin Gene are Associated with Defective Mannose-Binding Lectin Functional Activity in Crohn's Disease Patients.

Mannose-binding lectin, together with mannose-associated serine proteases, activates the lectin pathway of the complement system and subsequent inflammatory mechanisms. An association between mannose-binding lectin deficiency and anti-Saccharomyces cerevisiae antibody levels is observed in Crohn’s disease and this deficiency is frequently associated with a severe Crohn’s disease phenotype. In the present study, we assessed the relationship between serum concentrations of mannose-binding lectin, mannose-binding lectin functional activity, MBL2 and NOD2 polymorphisms, anti-S. cerevisiae antibody levels and clinical Crohn’s disease phenotype in 69 Crohn’s disease patients and 30 age- and sex-matched healthy controls. The results show that the MBL2 variant rs5030737 at codon 52 was associated with a low level of mannose-binding lectin and impaired mannose-binding lectin–mannose-associated serine protease (MBL-MASP) functional activity in Crohn’s disease patients. This MBL2 variant was also associated with a higher level of anti-S. cerevisiae antibodies. In addition, the NOD2 variant rs2066844, which is associated with susceptibility to Crohn’s disease, was significantly correlated with an impairment in MBL-MASP functional activity. These results provide evidence that Crohn’s disease patients have an impairment in MBL-MASP functional activity and that this defect is associated with MBL2 and NOD2 variants.

Erratum: Corrigendum: Polymorphisms in the Mannose-Binding Lectin Gene are Associated with Defective Mannose-Binding Lectin Functional Activity in Crohn’s Disease Patients

Scientific Reports 6: Article number: 29636; published online: 12 July 2016; updated: 16 September 2016 .