Extracellular polysaccharides are key constituents of the biofilm matrix of many microorganisms. One critical carbohydrate component of Candida albicans biofilms, β-1,3 glucan, has been linked to biofilm protection from antifungal agents. In this study, we identify three glucan modification enzymes that function to deliver glucan from the cell to the extracellular matrix. These enzymes include two predicted glucan transferases and an exo-glucanase, encoded by BGL2, PHR1, and XOG1, respectively. We show that the enzymes are crucial for both delivery of β-1,3 glucan to the biofilm matrix and for accumulation of mature matrix biomass. The enzymes do not appear to impact cell wall glucan content of biofilm cells, nor are they necessary for filamentation or biofilm formation. We demonstrate that mutants lacking these genes exhibit enhanced susceptibility to the commonly used antifungal, fluconazole, during biofilm growth only. Transcriptional analysis and biofilm phenotypes of strains with multiple mutations suggest that these enzymes act in a complementary fashion to distribute matrix downstream of the primary β-1,3 glucan synthase encoded by FKS1. Furthermore, our observations suggest that this matrix delivery pathway works independently from the C. albicans ZAP1 matrix formation regulatory pathway. These glucan modification enzymes appear to play a biofilm-specific role in mediating the delivery and organization of mature biofilm matrix. We propose that the discovery of inhibitors for these enzymes would provide promising anti-biofilm therapeutics.

Medical devices provide an ecological niche for microbes to flourish as a biofilm community, protected from antimicrobials and host defenses. Biofilms formed by Candida albicans, the most common fungal pathogen, survive exposure to extraordinary high drug concentrations. Here we show β-glucan synthase Fks1p produces glucan which is deposited in the biofilm matrix. The extracellular glucan is required for biofilm resistance and acts by sequestering antifungals, rendering cells resistant to their action. These findings provide the genetic basis for how biofilm matrix production governs drug resistance by impeding drug diffusion and identify a useful biofilm drug target.

Candida spp. infect medical devices, such as venous and urinary catheters, by adhering to the surface and forming a community of drug-resistant cells surrounded by a matrix. The ability to measure drug activity during this biofilm mode of growth is of interest for the investigation of resistance mechanisms and novel antifungal therapies. The tetrazolium salt (XTT) reduction assay is the test most commonly used to estimate viable biofilm growth and to examine the impact of biofilm therapies. The primary goal of the current experiments was to identify assay variables that affect the XTT assay result in order to improve assay reproducibility, sensitivity, and throughput for the study of antifungal activity. The species used in the current studies included Candida albicans, C. parapsilosis, and C. glabrata. The assay variables that were studied included the impact of culture conditions, the duration of biofilm growth, the timing and frequency of drug administration, the XTT source and concentration, and the duration of XTT incubation. The conditions that impacted the assay readout and altered assay sensitivity included the duration of biofilm growth, the frequency of drug dosing, and the duration of XTT incubation. Several factors were found to reduce time and assay expense, including the elimination of washing steps, the shortening of incubation times, and the use of lower XTT concentrations. A description of assay pitfalls and troubleshooting is included. Recognition of these technical variables should allow investigators to better design reproducible biofilm therapeutic studies.The most clinically important phenotype of Candida biofilm cells is their remarkable resistance to antifungal drugs (1,4,17,29,39). Cells in this environment can survive up to 1,000-foldhigher concentrations of antifungals than nonbiofilm, planktonic cells. Because antifungal drugs typically are not effective against biofilm organisms, the recommended therapy for Candida biofilm infection of a medical device includes device removal, which is associated with increased procedural morbidity and health care expenditures (25). Novel drug targets and the development of new antifungal agents for the treatment of these recalcitrant infections are therefore of interest.The use of the tetrazolium salt 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT) reduction assay to study Candida biofilms has been pioneered by labs in the mycology community (7,10,13,33,38). It is the method most commonly utilized for quantitative measurement of Candida biofilm mass, growth, and response to drug therapy (1,9,12,18,20,26,28,37). Other techniques used to assay the biofilm cell burden include [ 3 H]leucine incorporation, fluorescein diacetate, crystal violet staining, viable counts, dry weight measurements, and imaging using confocal or electron microscopy (5,8,10,35,36). The XTT assay has become the preferred tool due to the rapidity of the assay, the ability to use a high-throughput format (e.g., a 96-well plate), and more im...

Candida albicans frequently infects medical devices by growing as a biofilm, i.e., a community of adherent organisms entrenched in an extracellular matrix. During biofilm growth, Candida spp. acquire the ability to resist high concentrations of antifungal drugs. One recently recognized biofilm resistance mechanism involves drug sequestration by matrix ␤-1,3 glucan. Using a candidate gene approach, we investigated potential C. albicans ␤-1,3-glucan regulators, based on their homology to Saccharomyces cerevisiae, including SMI1 and protein kinase C (PKC) pathway components. We identified a role for the SMI1 in biofilm matrix glucan production and development of the associated drug resistance phenotype. This pathway appears to act through transcription factor Rlmp and glucan synthase Fks1p. The phenotypes of these mutant biofilms mimicked those of the smi1⌬/smi1⌬ biofilm, and overexpression of FKS1 in the smi1⌬/smi1⌬ mutant restored the biofilm resistant phenotype. However, control of this pathway is distinct from that of the upstream PKC pathway because the pkc1⌬/pkc1⌬, bck1⌬/bck1⌬, mkk2⌬/mkk2⌬, and mkc1⌬/mkc1⌬ biofilms retained the resistant phenotype of the parent strain. In addition, resistance to cell-perturbing agents and gene expression data do not support a significant role for the cell wall integrity pathway during the biofilm formation. Here we show that Smi1p functions in conjunction with Rlm1p and Fks1p to produce drug-sequestering biofilm ␤-glucan. Our work provides new insight into how the C. albicans biofilm matrix production and drug resistance pathways intersect with the planktonic cell wall integrity pathway. This novel connection helps explain how pathogens in a multicellular biofilm community are protected from anti-infective therapy.

Background: Right ventricular failure is an underrecognized consequence of COVID-19 pneumonia. Those with severe disease are treated with extracorporeal membrane oxygenation (ECMO) but with poor outcomes. Concomitant right ventricular assist device (RVAD) may be beneficial. Methods: A retrospective analysis of intensive care unit patients admitted with COVID-19 ARDS (Acute Respiratory Distress Syndrome) was performed. Non-intubated patients, those with acute kidney injury, and age > 75 were excluded. Patients who underwent RVAD/ECMO support were compared with those managed via invasive mechanical ventilation (IMV) alone. The primary outcome was in-hospital mortality. Secondary outcomes included 30-day mortality, acute kidney injury, length of ICU stay, and duration of mechanical ventilation. Results: A total of 145 patients were admitted to the ICU with COVID-19. Thirty-nine patients met inclusion criteria. Of these, 21 received IMV, and 18 received RVAD/ECMO. In-hospital (52.4 vs 11.1%, p=0.008) and 30-day mortality (42.9 vs 5.6%, p=0.011) were significantly lower in patients treated with RVAD/ECMO. Acute kidney injury occurred in 15 (71.4%) patients in the IMV group and zero RVAD/ECMO patients (p<0.001). ICU (11.5 vs 21 days, p=0.067) and hospital (14 vs 25.5 days, p=0.054) length of stay were not significantly different. There were no RVAD/ECMO device complications. The duration of mechanical ventilation was not significantly different (10 vs 5 days, p=0.44). Conclusions: RVAD support at the time of ECMO initiation resulted in the no secondary end-organ damage and higher in-hospital and 30-day survival versus IMV in specially selected patients with severe COVID-19 ARDS. Management of severe COVID-19 ARDS should prioritize right ventricular support.