Biological phenotypes are important characteristics of microorganisms, and often reflect their genotype and genotype changes. Traditionally, Trichophyton rubrum (T. rubrum) phenotypes were detected using carbon source assimilation tests, during which the types of tested substances are limited. In addition, the operation is complicated, and only one substance can be tested at once. To observe the changes of the metabolic phenotype of T. rubrum after laser irradiation, a high-throughput phenotype microarray system was used to analyze the metabolism of different carbon, nitrogen, phosphorus and sulfur source substrates in a Biolog metabolic phenotyping system.
The strain of T. rubrum used in this study can effectively utilize 33 carbon, 20 nitrogen, 16 phosphorus, and 13 sulfur source substrates prior to laser irradiation. After laser irradiation, the strain was able to utilize 10 carbon, 12 nitrogen, 12 phosphorus, and 8 sulfur source substrates. The degree of utilization was significantly decreased compared with the control. Both groups efficiently utilized saccharides and organic acids as carbon sources as well as some amino acids as nitrogen sources for growth. The number of substrates utilized by T. rubrum after laser irradiation were significantly reduced, especially carbon substrates. Some substrates utilization degree in the laser treated group was higher than control, such as D-glucosamine, L-glutamine, D-2-Phospho-Glyceric Acid, D-glucosamine-6-phosphate, and D-methionine.
Laser irradiation of T. rubrum may lead to changes in the metabolic substrate and metabolic pathway, thus weakening the activity of the strain.
Trichophyton rubrum (T. rubrum) is a dermatophyte responsible for causing the majority of superficial fungal infections worldwide . It can cause infection on the skin, especially the scalp, the inguinal region, the feet, and the nails . These infections can be either chronic or acute with mild to moderate dermatological symptoms. Traditional treatment of T. rubrum infection includes both external antifungal creams, such as miconazole nitrate, and oral administration of, such as ketoconazole. Both treatments could cause side effects. Common side effects for oral administration include headaches, taste disturbance, dermatitis, anorexia, vomiting, epigastric pain and diarrhea. Generally, the side effects of topical antifungal agents include periungual erythema and burning at the application site .
Laser treatments for fungal infections have attracted attention from both clinicians and scientists in recent years, and the fungicidal effect and mechanism of laser therapy have become hot research topics. Vural and coauthors  investigated the mechanism of fungal inhibition by laser treatment and reported that, in addition to nonspecific thermal damage, pigments may be another factor influencing the fungicidal effects due to the cell walls of onychomycosis-causing Trichophyton species containing considerable melanin [4, 5]. In a similar study, Zhuo et al.  found that high dose of laser irradiation to T. rubrum caused an increase of reactive oxygen species (ROS), inhibiting the growth of T. rubrum. Subsequently, another study  confirmed that photoirradiation therapy stimulated massive ROS production in cells, causing oxidative damage, one of the main mechanisms inhibiting fungal growth.
Previous studies on fungal phenotypes are mostly limited to mycelial morphology, growth rate, drug sensitivity, and biofilm-forming ability but rarely focus on metabolic phenotypes. Phenotypic microarrays (PMs) are a new technology that complement genomics and proteomics, which have long been used to identify microbial strains and determine microbial cell metabolic phenotypes [8, 9]. Zhang et al.  analyzed about 2,000 phenotypes of Streptococcus mutans mutants using the Biolog PM technology and found that the mutants were more tolerant to various inhibitors of target protein synthesis, DNA synthesis, and cell wall biosynthesis. Additionally, Chen and coauthors  explored the differences in biological characteristics between wild-type and mutant strains of Vibrio cholerae using this technology. Furthermore, the PM technique was applied with genomics and proteomics to study the phenotypic and functional changes of conidia after gene alterations .
In the present study, we used PMs to further analyze the phenotypic changes in T. rubrum after laser irradiation. The utilization of four types of nutrition sources, including carbon (the Biolog FF microplate), nitrogen (the Biolog PM3 microplate), phosphorus and surfur (the Biolog PM4 microplate) were analyzed with and without laser irradiation. Carbon, nitrogen, phosphorus and surfur are the most important nutrients required for fungal growth. Our goals are to better understand the effects and mechanism of laser irradiation on T. rubrum physiology, and to provide new insights on treatment of T. rubrum infections.
The source of T. rubrum
A strain of T. rubrum was isolated from a patient with onychomycosis who has a good response to laser treatment at Beijing Friendship Hospital (Beijing, China) in December 2017. The strain was identified as T. rubrum by sequencing the ITS region from its ribosomal DNA using the primers ITS1 and ITS4 from White et al. . The sequence was deposited in the NCBI Nucleotide database with the access number of OP811248. Clinical trial revealed that the strain is sensitive to laser treatment. The strain is maintained at the Beijing Friendship Hospital and available upon request.
The strain T. rubrum was cultured in Sabouraud dextrose agar for 7 days at 25 °C. Fungal suspension was prepared with 1.0 McFarland turbidity. One µL of the culture was inoculated on two sides of a 2% Malt Extract Agar (MEA) plate (Biolog Inc., CA, USA). One side was used for the control group and the other side for the laser treatment. The plates were cultured in an incubator at 25℃, and colony growth was observed daily. When the colony grew to 6 mm in diameter (7 days), the laser side was irradiated using a long-pulsed Nd:YAG 1064 nm laser (Beijing Shiji Guangtong Biotechnology Co., Ltd.) with the following parameters: 3 mm spot size; 1 Hz frequency; 30 ms pulse width; 408 J/cm2 laser energy; and each colony was given 200 spots. At this condition, the growth of colony is inhibited, as indicated by destruction of membrane structure and apoptotic cell death .
Metabolic phenotype analysis
Spores of T. rubrum were collected by rolling a sterile cotton swab on the surface of the colony. They were then dispersed into the inoculum (0.25% Gellan Gum and 0.03% Tween-40) and the fungal suspension turbidity was determined. The transmittance of 75% for the FF microplate (carbon sources) and 62% for the PM microplates (nitrogen or phosphorus and sulfur source) was used following the instruction of the manufacturer (Biolog Inc). The prepared fungal suspensions were inoculated into FF (95 carbon sources), PM3 (95 nitrogen source), and PM4 (59 phosphorus source and 39 sulfur source) microplates following manufacturer’s instruction (Biolog Inc) and then cultured in an OmniLog phenotype analysis system (Biolog Inc). The degree of utilization was calculated based on the chromogenic reactions. The OmniLog system software measures the color intensity of the reaction wells and changes over time . Each plate contains a negative control to ensure the color intensity was read correctly. The color intensity above the negative control was considered as positive. Triplet experiments were performed from the 3 independently grown colony plates.
Data processing and statistical analysis
Three independent experiments were performed and data were presented as mean ± standard error. Data analysis for metabolic profiling of T. rubrum was conducted using Kinetic and Parametric software from Biolog Inc. D5E_OKA_data.exe was used to collect the color intensity data and OL_FM_1.2.exe was used for data analysis. Phenotypes were determined based on the area under the kinetic curve of dye formation, which is the sum of the color intensities from all time points .
A paired t-test was conducted to compare the control and the laser treated group, and P < 0.05 was considered as statistically significant. The substances metabolized in the control group and laser group were selected, and two independent sample t-tests were performed to compare the difference in the degree of metabolism between the two groups. P < 0.05 was considered statistically significant.
Laser irradiation causes reduction in carbon source utilization
In the FF microplate (carbon source), T. rubrum of the control group utilized 33 carbon source substrates, while only 10 for the laser treated group (Table 1). The substance utilization degree was higher in the control group than the laser group, except for D-Glucosamine (B11) (Figs. 1 and 2). These results suggest that laser irradiation causes reduction on carbon source substrate utilization.
Laser irradiation causes reduction in nitrogen substrate utilization
In the PM3 microplate (nitrogen sources), 20 substrates were utilized by T. rubrum of the control group, while only 12 in the laser treated group (Table 2). Among the substrates utilized by both groups, the degree of utilization of majority of the nitrogen substrates (83.3%, 10/12) was lower in the laser treated group. Only the utilization of L-Aspartic Acid (A10) and L-Glutamine (B1) was higher in the laser treated group. The utilization of Ala-Gly (H4), Ala-Thr (H7) and Gly-Met (H11) showed no statistical difference between the two groups (Figs. 3 and 4).
Laser irradiation has less effect in sulfur-phosphorus source substrate utilization
In the PM4 microplate (sulfur-phosphorus substrates), the control group utilized 29 substrates, while the laser treated group only utilized 20 (Table 3). Among the substrates utilized by both groups, almost half (8/20) showed no statistical difference between the two groups for their degree of sulfur-phosphorus substrate utilization. The utilization of D-2-Phospho-Glyceric Acid (B6), D-Glucosamine-6-Phosphate (C6), D-Methionine (G8), Glycyl-L-Methionine (G9) and N-Acety l-D, L-Methionine (G10) was higher in the laser treated group (Figs. 5 and 6).
Traditionally, T. rubrum phenotypes were mostly detected using carbon source assimilation tests, during which the types of tested substances are limited, the operation is complicated, and only one substance can be tested at once. PMs overcome this defect by detecting the color response during the respiratory metabolism of living cells, enabling the collection of large amounts of data on the microbial utilization of various nutrients. In the present study, we analyzed the metabolic differences between the laser treated and non-treated (control) T. rubrum groups. First, compared with laser-irradiated T. rubrum, the control group efficiently utilized saccharides (e.g., arabinose, D-fructose, sucrose) and organic acids (e.g., glucuronic acid, D-malic acid, and aminobutyric acid) as carbon sources as well as some amino acids (e.g., alanine, arginine, serine) as nitrogen sources for growth. Many sulfur and phosphorus source substrates were also utilized, but their utilization degree was lower than that of the carbon and nitrogen source substrates. Second, compared with control T. rubrum, the number of substrates utilized by T. rubrum after laser irradiation were significantly reduced, including saccharides, organic acids, and a variety of amino acids, indicating that the process of energy generation may be damaged by laser . Third, the substrate utilization degree in the laser treated group was higher than that in the control group for several substances, such as carbon source substrate D-glucosamine, nitrogen source substrates L-aspartic acid and L-glutamine, phosphorus source substrate D-glucosamine-6-phosphate, and sulfur source substrate D-methionine. This could be related to a self-protection mechanism or the fact that laser irradiation promotes T. rubrum apoptosis. Further studies could help us better understand whether this is self-protection or cell death, and the associated molecular pathway.
Results from the carbon source test showed that the types of metabolized substrates were similar between the two groups, but the number of metabolized substrates was different. The laser treated group only used 30% of the substrates used by the control group (Figs. 1 and 2). Saccharides are the most abundant carbon source and may play a key role in the survival of T. rubrum. The tricarboxylic acid cycle (TCA) is not only a common metabolic pathway in aerobic organisms but also the final metabolic pathway of three major nutrients (saccharides, lipids, and amino acids). After the laser irradiation of T. rubrum, a variety of metabolites involved in the TCA were inhibited, including alpha-ketoglutarate, D-malic acid, succinic acid, L-asparagine, L-proline, L-threonine, and putrescine. Therefore, we speculate that after laser irradiation, some substrates cannot be utilized by T. rubrum due to the damages of genes that modulate the TCA, reducing the metabolic capacity of T. rubrum. In addition, laser irradiation reduced the metabolic capacity of T. rubrum for some substrates involved in glycolysis, such as D-glucuronic acid and maltitol. On this basis, it is speculated that laser irradiation may destroy multiple key genes involved in glycolysis or the TCA of T. rubrum, thus affecting the metabolism of corresponding substrates. Although the TCA is the main glucose metabolism pathway, it is not the only one. When the TCA is inhibited, the pentose phosphate pathway can be used to metabolize carbohydrates . In this study, T. rubrum irradiated by laser could still utilize some carbon source substrates in the pentose phosphate pathway, including D-arabinose, L-arabinose, D-ribose, and D-xylose.
D-glucosamine exists widely in the chitin and glycoproteins of bacterial cell walls and the chitin of fungal cell walls in the form of N-acetylglucosamine . The transport of glucosamine to cells through glucose transporters  will directly increase the flux through the hexosamine biosynthesis pathway, enhancing O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation . The O-GlcNAc protein is an endogenous protective mechanism triggered by stress and is considered an “emergency receptor” . Increases in O-GlcNAc levels have been shown to contribute to higher heat resistance in cells . In our study, we showed that the utilization of D-glucosamine is increased after laser irradiation. We suspect that laser irradiation of T. rubrum triggers this self-protection mechanism, resulting in a higher degree of D-glucosamine utilization.
Microbial growth and product synthesis require nitrogen sources, which are mainly used for the biosynthesis of amino acids, proteins, nucleic acids, and nitrogen metabolites. Research on the sporulation of T. rubrum under different nitrogen sources has shown that T. rubrum can grow normally without a carbon source when nitrogen sources are available , suggesting that nitrogen substrates are vital to T. rubrum. In our PM3 microplate with nitrogen sources, the control group effectively utilized 20 nitrogen-containing substances, mainly amino acids involved in glucose and nucleic acid metabolism and a few dipeptides. However, only 12 nitrogen substrates were utilized by T. rubrum after laser irradiation, and the degree of metabolism of most substrates was significantly lower than that of the control group.
Amino acids are precursors for purine and pyrimidine biosynthesis. Amino acid metabolism participates in the TCA and is the central point of glucose, lipid, and amino acid metabolism. After laser irradiation, T. rubrum lost the ability to utilize alanine, arginine, asparaginate, isoleucine, proline and ornithine, and its degree of utilization of glutamate, glycine, and serine decreased, indicating that amino acid metabolism was inhibited. Moreover, laser irradiation increased the utilization of L-aspartic acid and L-glutamine by T. rubrum, which may be due to the key roles of glutamine and aspartic acid in cell growth and proliferation. Glutamine and aspartic acid, as intermediate metabolites of glucose metabolism, can participate in the TCA and provide energy for cells . They also provide nitrogen sources for nucleotides, proteins, and other biological macromolecules . The efficient utilization of glutamine and aspartic acid can also affect DNA repair and replication  to maintain the survival of bacteria.
Phosphorus absorption and utilization play important roles in biological processes, such as heredity, energy metabolism, cell membrane integrity, and intracellular signal transduction. Organisms have formed a complex phosphate system to regulate phosphorus metabolism . Our analysis showed that without laser irradiation, T. rubrum effectively utilized phosphate, trimetaphosphate, hypophosphate, adenosine-3’-monophosphate, adenosine-2’,5’-cyclic monophosphate, D-2-phosphoglyceric acid, D-glucose-6-phosphoric acid, 2-deoxy-D-glucose-6-phosphoric acid, 6-phosphoric acid-gluconic acid, creatine phosphate, and choline phosphate, which are involved in gluconeogenesis, phospholipid metabolism, nucleotide metabolism, energy transport, and signal transduction. After laser irradiation, the ability of T. rubrum to utilize these phosphorous substances was lost or significantly reduced.
Sulfur-containing amino acids include methionine, cysteine, and cystine. Methionine is one of the most easily oxidized amino acids in organisms and the activity center of proteins. Methionine in proteins can function normally only if the correct structure is maintained. However, under oxidative stress, methionine is very likely to be oxidized to methionine sulfoxide . The increase in methionine utilization capability in the laser treated group may be related to increased ROS levels after laser irradiation [6, 27]. ROS can oxidize methionine to methionine sulfoxide, which may affect a variety of biological functions . Methionine sulfoxide reductase is widely distributed in pathogens and can reduce methionine sulfoxide to methionine. Under normal conditions, methionine sulfoxide reductase can reverse the above-mentioned oxidation reaction, protecting against oxidative stress . However, after laser irradiation, the capability of T. rubrum to utilize methionine sulfoxide decreased, and this oxidation could not be prevented or reversed (Fig. 7).
Glutathione is a low-molecular-mass polypeptide composed of glycine, cysteine, and glutamic acid. Glutathione can remove superoxide ions and other free radicals to prevent damage to the cell . Studies have shown that the laser irradiation of T. rubrum leads to an increase in ROS [6, 31]. We expect T. rubrum in the laser treated group to utilize more glutathione to resist the damage caused by superoxidation. However, our results revealed that the degree of glutathione utilization in the laser group was lower than that in the control group, which may be because glutathione is affected by multiple metabolic enzymes. The laser irradiation of T. rubrum could lead to glutathione synthesis dysfunction, limiting the ability to maintain the reduction under oxidative stress and reducing the glutathione utilization capacity of the laser group.
In conclusion, this study demonstrated that the overall metabolic capacity of T. rubrum decreased after laser irradiation, but not all substances were inhibited. Therefore, we believe that the laser irradiation parameters used in our experiment have a fungistatic effect, rather than a fungicidal effect, consistent with the results of our previous work. Our study provides new insights on the fungistatic and fungicidal effects of laser treatment. The limitation of this study lies in that we could only measure the degree of metabolic capacity of T. rubrum irradiated by laser. More experimental studies are needed to explore the specific mechanisms underlying these changes to confirm the effect of laser treatment on the metabolic phenotypes of T. rubrum.
Availability of data and materials
The data during the current study are available in the national center for biotechnology information (NCBI) under accessions number OP811248 (https://www.ncbi.nlm.nih.gov/nuccore/OP811248). Other data generated or analyzed during this study are included in the published article and its supplementary.
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The study conception and design were performed by Linfeng Li, Junying Zhao. Material preparation, data collection and analysis were performed by Ruina Zhang, The first draft of the manuscript was written by Ruina Zhang. All authors read and approved the final manuscript.
This research was performed according to the ethical guidelines in the Declaration of Helsinki and was approved by the institutional review board of Beijing Friendship Hospital Ethics Committee (Approval #:2011-1003-03). The participants in this study signed the informed consent form before the initiation of this study. The methods were carried out in accordance with relevant guidelines and regulations.
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