Abstract
Fungal infections caused by Candida albicans poses a great threat to human health. The ability of biofilm formation is believed to be associated with resistance-related Candida infections. Currently, knowledge on extracellular matrix (EM) of C. albicans biofilm is limited. In this study, we introduced ion exchange resin, i.e., cation exchange resin (CER) and anion exchange resin (AER), in EM extraction of C. albicans biofilm as well as several non-albicans Candida (NAC) biofilms under static and dynamic states in combination with vortexing and ultrasonication (VU). The metabolites extracted from the dynamic C. albicans biofilm matrix using the CER-VU and VU were identified with ultra-high-performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) via untargeted filtration. Compared with other physical and chemical extraction methods, CER-VU was demonstrated to be an ideal approach with high-yield acquisitions of EM constituents including proteins, triglycerides and carbohydrates and low-level damages on fungal cell viability and integrity. The untargeted MS analysis further showed the high efficacy of CER-VU, as a large quantity of metabolites (217 versus 198) was matched comprising a great number of lipids, carbohydrates, amino acids, nucleic acids and their derivatives together with a high involvement of signaling pathways compared with the VU alone. However, combining the results from both the CER-VU and VU methods could generate more metabolites. In summary, the EM analysis of the dynamic C. albicans biofilm expands our understanding upon a comprehensive depiction of matrix components and provides another effective approach for EM extraction.
Highlights
Candida albicans, a commonly isolated fungus normally colonizing the human intestinal tract, skin and vagina, can be transformed into an opportunistic pathogen which is able to cause anything from mild mucosal discomforts to deadly systematic infections when the host endures long-time antibiotic treatment or immunosuppressive difficulty (Gow et al, 2012)
This study aims to investigate the efficiencies of seven methods in C. albicans biofilm extracellular matrix (EM) extraction, namely vortexing plus ultrasonication (VU), formaldehyde plus vortexing and ultrasonication (Formaldehyde-VU), EDTA plus vortexing and ultrasonication (EDTA-VU), NaOH plus vortexing and ultrasonication (NaOH-VU), ethanol plus vortexing and ultrasonication (Ethanol-VU), ion exchange resin plus vortexing and ultrasonication (IER-VU)—including cation/anion-exchange resin plus vortexing and ultrasonication (CER/anion exchange resin (AER)-VU)—under the static and dynamic states
The study on the static biofilm extractions showed a desired extraction efficiency with the cation exchange resin (CER)/AER-VU compared with the other methods including the VU
Summary
A commonly isolated fungus normally colonizing the human intestinal tract, skin and vagina, can be transformed into an opportunistic pathogen which is able to cause anything from mild mucosal discomforts to deadly systematic infections when the host endures long-time antibiotic treatment or immunosuppressive difficulty (Gow et al, 2012). The C. albicans biofilm is a heterogeneous, complex and threedimensional architecture conferring protections to fungal cells encased by the secreted extracellular matrix (EM) from attacks of host immune system and antifungal agents (Tobudic et al, 2012). As for the first contact point with environmental cues, comprehensive information related to the delicate components of C. albicans biofilm EM is a primary task to search for potential antifungal targets (Bjarnsholt et al, 2013). (Villas-Boas et al, 2005; Pinu et al, 2017) These methods aim to lyse target cell wall/membrane, release intracellular metabolic substances, and avoid metabolite degradations. The biofilm EM is a congregate of extracellular metabolites, requiring an ideal extraction technique to maximize the secreted matrix (number and quantity) and minimize cell damage which is dependent on extraction procedures and operating conditions (Sun et al, 2012). As an advanced combination technique with high separation capacity and high efficiency in compound identification, ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) accelerates the elucidation of characteristic compound structures and accurate molecular weights compared to traditional HPLC-MS/MS (Zhao et al, 2014)
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