The Role of Ergosterol and Sphingolipids in the Localization and Activity of Candida albicans' Multidrug Transporter Cdr1p and Plasma Membrane ATPase Pma1p.

Opportunistic pathogen Candida albicans causes systemic infections named candidiasis. Due to the increasing number of multi-drug resistant clinical isolates of Candida sp., currently employed antifungals (e.g., azoles) are insufficient for combating fungal infection. One of the resistance mechanisms toward azoles is increased expression of plasma membrane (PM) transporters (e.g., Cdr1p), and such an effect was observed in C. albicans clinical isolates. At the same time, it has been proven that a decrease in PMs sphingolipids (SLs) content correlates with altered sensitivity to azoles and diminished Cdr1p levels. This indicates an important role for SL in maintaining the properties of PM and gaining resistance to antifungal agents. Here, we prove using a novel spot variation fluorescence correlation spectroscopy (svFCS) technique that CaCdr1p localizes in detergent resistant microdomains (DRMs). Immunoblot analysis confirmed the localization of CaCdr1p in DRMs fraction in both the C. albicans WT and erg11Δ/Δ strains after 14 and 24 h of culture. We also show that the C. albicanserg11Δ/Δ strain is more sensitive to the inhibitor of SLs synthesis; aureobasidin A (AbA). AbA treatment leads to a diminished amount of SLs in C. albicans WT and erg11Δ/Δ PM, while, for C. albicanserg11Δ/Δ, the general levels of mannose-inositol-P-ceramide and inositol-P-ceramide are significantly lower than for the C. albicans WT strain. Simultaneously, the level of ergosterol in the C. albicans WT strain after adding of AbA remains unchanged, compared to the control conditions. Analysis of PM permeabilization revealed that treatment with AbA correlates with the disruption of PM integrity in C. albicanserg11Δ/Δ but not in the C. albicans WT strain. Additionally, in the C. albicans WT strain, we observed lower activity of H+-ATPase, correlated with the delocalization of both CaCdr1p and CaPma1p.

Degradation of plastics and plastic-degrading bacteria in cold marine habitats.

Synthetic plastics present in everyday materials constitute the main anthropogenic debris entering the Earth's oceans. The oceans provide important and valuable resources such as food, energy, and water. They are also the main way of international trade and the main stabilizer of the climate. Hence, changes in the marine ecosystem caused by anthropogenic influences such as plastic pollution can have a dramatic impact on a global scale. Although the problem of plastics still remains unsolved, different ways are being considered to reduce their impact on the environment. One of them is to use microorganisms capable of degradation of plastic. A particularly interesting area is the application of microorganisms isolated from cold regions in view of their unique characteristics. Nevertheless, the interactions between plastic and microorganisms are still poorly known. Here, we present a review of current knowledge on plastic degradation and plastic-microorganism interactions in cold marine habitats. Moreover, we highlight the advantages of microorganisms isolated from this environment for eliminating plastic waste from ecosystems.

Zophobas morio versus Tenebrio molitor: Diversity in gut microbiota of larvae fed with polymers.

Plastics are common synthetic materials that have been abundantly present as pollutants in natural ecosystems for the past few decades. Thus scientists have investigated the capability of plastic digestion by insects. Here we compare the effectiveness of biodegradation of the specific polymers: expanded polystyrene (EPS), polyvinyl chloride (PVC), low-density polyethylene (LDPE) and polypropylene (PP) altogether with above variants of plastics with microelements and vitamins by the mealworm - the larval form of the beetle Tenebrio molitor - and larvae of the beetle Zophobas morio, known as superworms. Z. morio beetles on all diets were able to complete their life cycle from larvae through pupae and imago, gaining 19 % and 22 % in mass on LDPE and EPS; 8 % and 7 % on PVC and PP. Mealworms (T. molitor) reared on polymers had minimal weight gain, gaining 2 % on LDPE and EPS, and a slight reduction in mass was observed when reared on PP and PVC. Not all specimens of T. molitor were able to pupate and transform to the adult stage. The results suggest that larvae of Z. morio can eat and degrade some types of plastic compounds more effectively than T. molitor. The changes in microbial gut communities were compared between these two species. The highest mass gain for Z. morio is associated with higher diversity in gut microbia and it was more diverse than that of T. molitor. Citrobacter freundii, a bacterium recognized for its ability to degrade long-chain polymers, linear hydrocarbons and cyclic hydrocarbons, was found in the microflora of Z. morio. The results confirm that superworms can survive on polymer feed. Moreover, this diet supplemented with microelements and vitamins increases the number of bacterial species and the diversity in the microbial gut.

The alternating 50 Hz magnetic field depending on the hydrophobicity of the strain affects the viability, filamentation and sensitivity to drugs of Candida albicans.

In recent decades, Candida albicans have been the main etiological agent of life-threatening invasive infections, characterized by various mechanisms of resistance to commonly used antifungals. One of the strategies to fight Candida infections may be the use of an electromagnetic field. In this study, we examined the influence of the alternating magnetic field of 50 Hz on the cells of C. albicans. We checked the impact of the alternating magnetic field of 50 Hz on the viability, filamentation and sensitivity to fluconazole and amphotericin B of two, differing in hydrophobicity, strains of C. albicans, CAF2-1 and CAF 4-2. Our results indicate that using the alternating magnetic field of 50 Hz reduces the growth of C. albicans. Interestingly, it presents a stronger effect on the hydrophobic strain CAF4-2 than on the hydrophilic CAF2-1. The applied electromagnetic field also affects the permeabilization of the cell membrane. However, it does not inhibit the transformation from yeast to hyphal forms. AMF is more effective in combination with fluconazole rather than amphotericin B. Our findings confirm the hypothesis that the application of the alternating magnetic field of 50 Hz in antifungal therapy may arise as a new option to support the treatment of Candida infections.

Production and characterization of lipopeptide biosurfactant from a new strain of Pseudomonas antarctica 28E using crude glycerol as a carbon source.

Pseudomonas is a cosmopolitan genus of bacteria found in soil, water, organic matter, plants and animals and known for the production of glycolipid and lipopeptide biosurfactants. In this study bacteria (laboratory collection number 28E) isolated from soil collected in Spitsbergen were used for biosurfactant production. 16S rRNA sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) revealed that this isolate belongs to the species Pseudomonas antarctica. In the present study, crude glycerol, a raw material obtained from several industrial processes, was evaluated as a potential low-cost carbon source to reduce the costs of lipopeptide production. Among several tested glycerols, a waste product of stearin production, rich in nitrogen, iron and calcium, ensured optimal conditions for bacterial growth. Biosurfactant production was evidenced by a reduction of surface tension (ST) and an increase in the emulsification index (E24%). According to Fourier-transform infrared spectroscopy (FTIR) and electrospray ionization mass spectrometry (ESI-MS), the biosurfactant was identified as viscosin. The critical micelle concentration (CMC) of lipopeptide was determined to be 20 mg L-1. Interestingly, viscosin production has been reported previously for Pseudomonas viscosa, Pseudomonas fluorescens and Pseudomonas libanensis. To the best of our knowledge, this is the first report on viscosin production by a P. antarctica 28E. The results indicated the potential of crude glycerol as a low-cost substrate to produce a lipopeptide biosurfactant with promising tensioactive and emulsifying properties.

The Role of ERG11 Point Mutations in the Resistance of Candida albicans to Fluconazole in the Presence of Lactate.

Candidiasis refers to both superficial and deep-tissue fungal infections often caused by Candida albicans. The treatment of choice for these infections is the use of azoles, such as fluconazole (FLC). However, the increased use of antifungal agents has led to the emergence of azole-resistant isolates of C. albicans. Thus, the development of alternative drugs that are more efficient and with a better toxicological profile is necessary. This study aimed to determine the susceptibility profile of C. albicans CAF2-1 strain to FLC in the presence of glucose or lactate. The research was also focused on single nucleotide polymorphism (SNP) and the determination of the effect of the identified point mutations on the amino acid sequence of the Erg11 protein. The results show the growth of C. albicans CAF2-1 in the presence of FLC was significantly lower in the presence of lactate than in glucose. As a result, among recorded 45 amino acid mutations, the following mutations may be associated with the reduced susceptibility of C. albicans to FLC: G10D, G10V, I11M, I11R, Y13N, F31V, L35F, A249D, Q250H, E266G, R267G, N273K, D275C, D275G, D275R. Moreover, a twice higher number of hot-spot mutations was found in the presence of glucose as a sole carbon source compared to cells grown on lactate.

Identification of novel extracellular putative chitinase and hydrolase from Geomyces sp. B10I with the biodegradation activity towards polyesters.

Cold-adapted filamentous fungal strain Geomyces sp. B10I has been reported to decompose polyesters such as poly(e-caprolactone) (PCL), poly(butylene succinate) (PBS) and poly(butylene succinate-co-butylene adipate) (PBSA). Here, we identified the enzymes of Geomyces sp. B10I, which appear to be responsible for its biodegradation activity. We compared their amino acid sequences with sequences of well-studied fungal enzymes. Partial purification of an extracellular mixture of the two enzymes, named hydrGB10I and chitGB10I, using ammonium sulfate precipitation and ionic exchange chromatography gave 14.16-fold purity. The amino acid sequence of the proteins obtained from the MALDI-TOF analysis determined the molecular mass of 77.2 kDa and 46.5 kDa, respectively. Conserved domain homology analysis revealed that both proteins belong to the class of hydrolases; hydrGB10I belongs to the glycosyl hydrolase 81 superfamily, while chitGB10I contains the domain of the glycosyl hydrolase 18 superfamily. Phylogenetic analysis suggests a distinct nature of the hydrGB10I and chitGB10I of Geomyces sp. B10I when compared with other fungal polyester-degrading enzymes described to date.

Current Knowledge on Polyethylene Terephthalate Degradation by Genetically Modified Microorganisms.

The global production of polyethylene terephthalate (PET) is estimated to reach 87.16 million metric tons by 2022. After a single use, a remarkable part of PET is accumulated in the natural environment as plastic waste. Due to high hydrophobicity and high molecular weight, PET is hardly biodegraded by wild-type microorganisms. To solve the global problem of uncontrolled pollution by PET, the degradation of plastic by genetically modified microorganisms has become a promising alternative for the plastic circular economy. In recent years many studies have been conducted to improve the microbial capacity for PET degradation. In this review, we summarize the current knowledge about metabolic engineering of microorganisms and protein engineering for increased biodegradation of PET. The focus is on mutations introduced to the enzymes of the hydrolase class-PETase, MHETase and cutinase-which in the last few years have attracted growing interest for the PET degradation processes. The modifications described in this work summarize the results obtained so far on the hydrolysis of polyethylene terephthalate based on the released degradation products of this polymer.

The potential of cold-adapted microorganisms for biodegradation of bioplastics.

Due to the extensive use of plastics, their quantity in the environment is constantly increasing, which creates a global problem. In the present study, we sought to isolate, test and identify Antarctic microorganisms which possess the ability to biodegrade bioplastics such as poly(ε-caprolactone) (PCL), poly(butylene succinate) (PBS) and poly(butylene succinate-co-butylene adipate) (PBSA) at low temperatures. 161 bacterial and 38 fungal isolates were isolated from 22 Antarctic soil samples. Among them, 92.16% of bacterial and 77.27% of fungal isolates formed a clear zone on emulsified PBSA, 98.04% and 81.82% on PBS and 100% and 77.27% on PCL as an additive to minimal medium at 20 °C. Based on the 16S and 18S rRNA sequences, bacterial strains were identified as species belonging to Pseudomonas and Bacillus and fungal strains as species belonging to Geomyces, Sclerotinia, Fusarium and Mortierella, while the yeast strain was identified as Hansenula anomala. In the biodegradation process conducted under laboratory conditions at 14, 20 and 28 °C, Sclerotinia sp. B11IV and Fusarium sp. B3'M strains showed the highest biodegradation activity at 20 °C (49.68% for PBSA and 33.7% for PCL, 45.99% for PBSA and 49.65% for PCL, respectively). The highest biodegradation rate for Geomyces sp. B10I was noted at 14 °C (25.67% for PBSA and 5.71% for PCL), which suggested a preference for lower temperatures (at 20 °C the biodegradation rate was only 11.34% for PBSA, and 4.46% for PCL). These data showed that microorganisms isolated from Antarctic regions are good candidates for effective plastic degradation at low temperatures.

A comprehensive assessment of microbiome diversity in Tenebrio molitor fed with polystyrene waste.

Recently it was demonstrated that mealworm (Tenebrio molitor) larvae consume and biodegrade polystyrene. Thus, in this study a breeding investigation with various types of polystyrene was performed to follow the changes in the gut microbiome diversity. Polystyrene used for packaging purposes (PSp) and expanded polystyrene (EPS) were perceived as more favorable and attacked more frequently by mealworms compared to raw polystyrene (PS) and material commercially available for parcels (PSp). Although our studies showed that larvae could bite and chew selected materials, they are not able to degrade and use them for consumption purposes. In a next-generation sequencing experiment, among all samples, seven classes, Gammaproteobacteria, Bacilli, Clostridia, Acidobacteria, Actinobacteria, Alphaproteobacteria and Flavobacteria, were indicated as the most abundant, whereas the predominant genera were Enterobacter, Lactococcus and Enterococcus. Additionally, we isolated three bacteria strains able to use diverse types of bioplastic as a sole carbon source. The strains with biodegradable activity against bioplastic were identified as species of the genera Klebsiella, Pseudomonas and Serratia. The presence of a bacterial strain able to degrade bioplastic may suggest a potential niche for further investigations.

Biochemical properties and biotechnological applications of microbial enzymes involved in the degradation of polyester-type plastics.

Application of polyester-degrading enzymes should be considered as an eco-friendly alternative to chemical recycling due to the huge plastic waste disposal nowadays. Many hydrolases from several fungi and bacteria have been discovered and successfully evaluated for their activity towards different aliphatic polyesters (PHA, PBS, PBSA, PCL, PLA), aromatic polyesters (PET, PBT, PMT) as well as their co-polyesters (PBST, PBAT, PBSTIL). This revision gives an up-to-date overview on the main biochemical features and biotechnological applications of those reported enzymes which are able to degrade polyester-based plastics, including different microbial polyester depolymerases, esterases, cutinase-like enzymes and lipases. Summarized information includes available protein sequences with the corresponding accession numbers deposited in NCBI server, 3D resolved structures, and data about optimal conditions for enzymatic activity and stability of many of these microbial enzymes that would be helpful for researchers in this topic. Although screening and identification of new native polyester hydrolases from microbial sources is undeniable according to literature, we briefly highlight the importance of the design of improved enzymes towards recalcitrant aromatic polyesters through different approaches that include site-directed mutagenesis and surface protein engineering.

Isolation and characterization of Arctic microorganisms decomposing bioplastics.

The increasing amount of plastic waste causes significant environmental pollution. In this study, screening of Arctic microorganisms which are able to degrade bioplastics was performed. In total, 313 microorganisms were isolated from 52 soil samples from the Arctic region (Spitsbergen). Among the isolated microorganisms, 121 (38.66%) showed biodegradation activity. The ability of clear zone formation on emulsified poly(butylene succinate-co-adipate) (PBSA) was observed for 116 microorganisms (95.87%), on poly(butylene succinate) (PBS) for 73 microorganisms (60.33%), and on poly(ɛ-caprolactone) (PCL) for 102 microorganisms (84.3%). Moreover, the growth of microorganisms on poly(lactic acid) (PLA) agar plates was observed for 56 microorganisms (46.28%). Based on the 16S rRNA sequence, 10 bacterial strains which showed the highest ability for biodegradation were identified as species belonging to Pseudomonas sp. and Rhodococcus sp. The isolated fungal strains were tested for polycaprolactone films and commercial corn and potato starch bags degradation under laboratory conditions. Strains 16G (based on the analysis of a partial 18S rRNA sequence, identified as Clonostachys rosea) and 16H (identified as Trichoderma sp.) showed the highest capability for biodegradation. A particularly high capability for biodegradation was observed for the strain Clonostachys rosea, which showed 100% degradation of starch films and 52.91% degradation of PCL films in a 30-day shake flask experiment. The main advantage of the microorganisms isolated from Arctic environment is the ability to grow at low temperature and efficient biodegradation under this condition. The data suggest that C. rosea can be used in natural and laboratory conditions for degradations of bioplastics.