The Extracellular Matrix of Candida albicans Biofilms Impairs Formation of Neutrophil Extracellular Traps.

Neutrophils release extracellular traps (NETs) in response to planktonic C. albicans. These complexes composed of DNA, histones, and proteins inhibit Candida growth and dissemination. Considering the resilience of Candida biofilms to host defenses, we examined the neutrophil response to C. albicans during biofilm growth. In contrast to planktonic C. albicans, biofilms triggered negligible release of NETs. Time lapse imaging confirmed the impairment in NET release and revealed neutrophils adhering to hyphae and migrating on the biofilm. NET inhibition depended on an intact extracellular biofilm matrix as physical or genetic disruption of this component resulted in NET release. Biofilm inhibition of NETosis could not be overcome by protein kinase C activation via phorbol myristate acetate (PMA) and was associated with suppression of neutrophil reactive oxygen species (ROS) production. The degree of impaired NET release correlated with resistance to neutrophil attack. The clinical relevance of the role for extracellular matrix in diminishing NET production was corroborated in vivo using a rat catheter model. The C. albicans pmr1Δ/Δ, defective in production of matrix mannan, appeared to elicit a greater abundance of NETs by scanning electron microscopy imaging, which correlated with a decreased fungal burden. Together, these findings show that C. albicans biofilms impair neutrophil response through an inhibitory pathway induced by the extracellular matrix.

Targeting Fibronectin To Disrupt In Vivo Candida albicans Biofilms.

New drug targets are of great interest for the treatment of fungal biofilms, which are routinely resistant to antifungal therapies. We theorized that the interaction of Candida albicans with matricellular host proteins would provide a novel target. Here, we show that an inhibitory protein (FUD) targeting Candida-fibronectin interactions disrupts biofilm formation in vitro and in vivo in a rat venous catheter model. The peptide appears to act by blocking the surface adhesion of Candida, halting biofilm formation.

Host contributions to construction of three device-associated Candida albicans biofilms.

Among the most fascinating virulence attributes of Candida is the ability to transition to a biofilm lifestyle. As a biofilm, Candida cells adhere to a surface, such as a vascular catheter, and become encased in an extracellular matrix. During this mode of growth, Candida resists the normal immune response, often causing devastating disease. Based on scanning electron microscopy images, we hypothesized that host cells and proteins become incorporated into clinical biofilms. As a means to gain an understanding of these host-biofilm interactions, we explored biofilm-associated host components by using microscopy and liquid chromatography-mass spectrometry. Here we characterize the host proteins associated with several in vivo rat Candida albicans biofilms, including those from vascular catheter, denture, and urinary catheter models as well as uninfected devices. A conserved group of 14 host proteins were found to be more abundant during infection at each of the niches. The host proteins were leukocyte and erythrocyte associated and included proteins involved in inflammation, such as C-reactive protein, myeloperoxidase, and alarmin S100-A9. A group of 59 proteins were associated with both infected and uninfected devices, and these included matricellular and inflammatory proteins. In addition, site-specific proteins were identified, such as amylase in association with the denture device. Cellular analysis revealed neutrophils as the predominant leukocytes associating with biofilms. These experiments demonstrate that host cells and proteins are key components of in vivo Candida biofilms, likely with one subset associating with the device and another being recruited by the proliferating biofilm.

Rat indwelling urinary catheter model of Candida albicans biofilm infection.

Indwelling urinary catheters are commonly used in the management of hospitalized patients. Candida can adhere to the device surface and propagate as a biofilm. These Candida biofilm communities differ from free-floating Candida, exhibiting high tolerance to antifungal therapy. The significance of catheter-associated candiduria is often unclear, and treatment may be problematic considering the biofilm drug-resistant phenotype. Here we describe a rodent model for the study of urinary catheter-associated Candida albicans biofilm infection that mimics this common process in patients. In the setting of a functioning, indwelling urinary catheter in a rat, Candida proliferated as a biofilm on the device surface. Characteristic biofilm architecture was observed, including adherent, filamentous cells embedded in an extracellular matrix. Similar to what occurs in human patients, animals with this infection developed candiduria and pyuria. Infection progressed to cystitis, and a biofilmlike covering was observed over the bladder surface. Furthermore, large numbers of C. albicans cells were dispersed into the urine from either the catheter or bladder wall biofilm over the infection period. We successfully utilized the model to test the efficacy of antifungals, analyze transcriptional patterns, and examine the phenotype of a genetic mutant. The model should be useful for future investigations involving the pathogenesis, diagnosis, therapy, prevention, and drug resistance of Candida biofilms in the urinary tract.

Fungicidal monoclonal antibody C7 interferes with iron acquisition in Candida albicans.

We have developed a monoclonal antibody (MAb), C7, that reacts with the Als3p and enolase present in the Candida albicans cell wall and exerts three anti-Candida activities: candidacidal activity and inhibition of both adhesion and filamentation. To investigate the mode of action of MAb C7 on fungal viability, we examined changes in the genome-wide gene expression profile of C. albicans grown in the presence of a subinhibitory concentration of MAb C7 (12.5 µg/ml) by using microarrays. A total of 49 genes were found to be differentially expressed upon treatment with MAb C7. Of these, 28 were found to be upregulated and 21 were found to be downregulated. The categories of upregulated genes with the largest number of variations were those involved in iron uptake or related to iron homeostasis (42.86%), while the energy-related group accounted for 38.10% of the downregulated genes (8/21). Results were validated by real-time PCR. Since these effects resembled those found under iron-limited conditions, the activity of MAb C7 on C. albicans mutants with deletions in key genes implicated in the three iron acquisition systems described in this yeast was also assessed. Only mutants lacking the TPK1 gene and, to a lesser extent, the TPK2 gene were less sensitive to the candidacidal effect of MAb C7. FeCl(3) or hemin at concentrations of ≥ 7.8 µM reversed the candidacidal effect of MAb C7 on C. albicans in a concentration-dependent manner. The results presented in this study provide evidence that the candidacidal effect of MAb C7 is related to the blockage of the reductive iron uptake pathway of C. albicans.

Bone microbial decontamination agents in osseous grafting: an in vitro study with fresh human explants.

BACKGROUND: Establishing a safe prophylactic antimicrobial protocol in bone grafting may enhance osseous volume outcomes. The purpose of this in vitro study is to assess human osteoblast response and safety after explant antimicrobial exposure. METHODS: Fresh human bone explants were exposed to three antimicrobials: povidone-iodine (PovI; 0.05%, 1%, and 5%), chlorhexidine (CHX; 0.2% and 1%), and sodium hypochlorite (NaOCl; 2.5%, 4.5%, and 5.25%) at different times (15, 30, 45, and 60 seconds) and concentrations to assess cellular toxicity. Explants were washed three times with saline after exposure. Controls, explants cultured in the absence of antimicrobials, were performed for all experimental situations tested. Trials were conducted in triplicate. Particle size influence on osteoblast growth was determined between bone fragments with a diameter <2 and ≥2 to 5 mm. Test and control groups were monitored by light microscopy to evaluate cellular growth. Osteoblast differentiation and morphology was assessed by alkaline phosphatase activity and scanning electron microscopy (SEM). RESULTS: Osteoblast growth was similar for particles <2 and ≥2 to 5 mm. Alkaline phosphatase control reference values were not significantly different from test groups (0.35 mU/mL ± 0.004 versus 0.34 mU/mL ± 0.009; P >0.05). Light microscopy showed on average 97% osteoblastic growth for bone particles exposed to PovI 5% and CHX 0.2% for all times and CHX 1% up to 30 seconds. The odds ratio of positive osteoblastic growth after a 30-second 2.5% NaOCl exposure was 2.4 times higher than after 5.25%. On average, one of two replicas yielded positive growth with 2.5% NaOCl and one of three with 5.25%. After 60-second explant exposure, positive osteoblastic growth was 7.7 times more likely to occur with 5% PovI or 0.2% CHX than with 5.25% NaOCl (P <0.05). SEM analysis confirmed light microscopy similar cellular adhesion and osteoblast phenotypic features between test and control groups. CONCLUSIONS: Best osteoblastic growth occurred after bone PovI exposure and CHX 0.2%. Cellular toxicity seems to be influenced by the type of antimicrobial, concentration, and exposure time. SEM analysis confirmed absence of osteoblast phenotypic alterations after exposure. Decontamination agents can safely be used in bone transplantation using up to 5% PovI and 0.2% CHX for 1 minute and CHX 1% for 30 seconds.