Hypha essential genes in Candida albicans pathogenesis of oral lichen planus: an in-vitro study

Background Hypha essential genes (HEGs) of Candida Albicans have been emerging into scholar’s attention, little known about their functions in oral lichen planus (OLP) with an uncovered etiology. This research aimed to observe necessary genes in biphasic C. albicans from OLP and study their relevance in pathogenesis, so as to evaluate possible roles of morphologic switching in etiology of OLP. Methods Samples were collected from OLP lesions of patients, mycelia were cultured and total RNA was extracted then subjected to reverse transcription-PCR and real-time PCR. Results HWP1 and HGC1 were significantly expressed in hyphae phase and weakly detected in yeast phase, while there was no significant difference of EFG1, ALS3, and ECE1 between in yeast and mycelia. Conclusion HGC1 and HWP1 were confirmed to be hypha essential genes, with HGC1 for hypha morphogenesis and HWP1 for adhesion invasion in pathogenesis of C. albicans in OLP. ALS3, ECE1 and EFG1 played minor roles in hyphae maintenance and adhesion for hyphae. These might be deemed as hints for the etiology of OLP and indicate HGC1 and HWP1 to be a priority of potential drug target.


Background
The sense of Candida albicans (C. albicans) in oral lichen planus (OLP) has acquired extensive attentions [1][2][3]. The course of pathogenesis of C. albicans usually includes three stages: adhesion, invasion and tissue lesion, in which hypha growth is the vital stage, for that yeast cells and germ tube could be swallowed by neutrophils, while the hyphae and long germ tube wouldn't [4], with a consistence to the common knowledge that hyphae cells might inhibit chemotaxis, absorption and consuming of neutrophils, and avoid being disrupted by phagocytes. What is its role in OLP?
Probable genotypic mutations of C. albicans in the occurrence and development of OLP ( Fig. 1) were illustrated in previous researches [5,6]. Further studies about phase transformation of C. albicans in OLP progression have been conducted. Change of the gene expression was uncovered to be directly related to phase switching of C. albicans. Specific expressions of HGC1, HWP1, ECE1, ALS3, and EFG1 in yeast and hypha phase of C. albicans from OLP were detected after RNA isolation, function and significance of these hypha essential genes (HEGs) and signal molecule EFG1 were analyzed in this study.

Materials and methods
Enrolled subjects included 59 patients with non-erosive OLP and 41 patients with erosive OLP visited dental school of Zhejiang University during 2011.8.1-2015.6.1. The patients were clinically diagnosed and histopathologically confirmed as OLP, with no history of smoking and alcohol abuse; moreover, they hadn't got any visible oral lesions except OLP; besides, they were off systemic or topical anti-inflammatory or immunosuppression/ immunomodulatory drugs; furthermore, they received no treatments for OLP within 3 months prior to the specimen collection, and without any relevance to systematic infections, allergies, cardiovascular diseases, immunodeficient diseases and autoimmune diseases.
The non-erosive group of 59 patients included an average age of 53.24 years old, 23 males averaged 53.84 years old and 36 females averaged 52.62 years old. The erosion group of 41 patients included an average age of 52.98 years old, 14 males averaged 48.70 years old and 27 females averaged 55.99 years old. There were no significant differences of gender or age between the two groups.
The study was carried out in accordance with the Helsinki Declaration of 1975, as revised in 2000, and was reviewed and approved by the local ethic institutional review boards (IRB) raising the ethic codes as No. 2010 (137) from the second hospital and No. 20150012 from the stomatology hospital both affiliated to the School of Medicine Zhejiang University. All the patients delivered their written consent for participation in the study.

Methods
Samples were collected by rubbing with sterile cotton swab in lichen planus lesions, and then transferred within 30 min to clinical microbiology laboratories of Affiliated 2 nd Hospital, School of Medicine, Zhejiang University.

Preliminary identification of the C. albicans strain
Twist cotton was coated on Saori Lloyd agar, cultured for 24-48 h at 37 °C until bacterial colonies appeared, and then transferred to French CHROMagar chromogenic medium. After 24 h, colonies showing emerald were initially identified as C. albicans. Colonies appeared dark green were suspected as C. parapsilosis. Suspected parapsilosis were rechecked after another 24-48 h culturation in SDA for the reason of similar color. If it showed emerald green, C. albicans was confirmed; if it remained dark green, C. parapsilosis was identified.

Further characterization, separation and purification
Typical colonies were picked and inoculated on Sabouraud Lloyd agar plates three times for separation and purification. After being cultured for 48 h in SDA, the colonies of the isolates were milky white cheese-like circular shape, formed germ tube after 4-6 h 37 °C in the rice Tween agar, grew well after being incubated for 48 h in SDA. Light microscopy showed Gram-positive structures with large cell volume, circular or oval layers by the modified Gram staining. API 20C AUX Candida identification system confirmed that the results were in accordance with what in databases provided by BioM6rieux. Finally, cinnamon peptone strains were stored in − 20 °C refrigerator and were regularly sub-cultivated.
Strains were marked as isolates a-h, among which a/c/g were collected from female non-erosive OLP patients, b/e/ f were from female erosive OLP, isolate d was from male-erosive group, and the last isolate h was a standard strain (ATCC16220) thankfully afforded by clinical microbiology laboratories of Affiliated 2nd Hospital, School of Medicine, Zhejiang University.

Yeast phase culture of C. albicans
Two mm diam. of yeast colonies were picked into cell culture flask with 5 ml YPD (121 °C, 30 min autoclaved) with an adjusted concentration of 1*10 6 /ml and were shaken in 37 °C, 200 rpm thermo shaker for 24 h. The yeast phase was cryo preserved in −20 °C.

Hyphal phase culture of C. albicans
Two mm diam. of hyphal colonies were picked to waterjacket thermostatic constant incubator with 4.5 ml Roswell Park Memorial Institute (RPMI) 1640 + 0.5 ml calf serum (56 °C, 30 min inactivation complement). The concentration was adjusted to 1*10 6 /ml for culture 7 days and were passaged 12 times (the 1st 2 days, passaged every 8 h; the medium 2 days, passaged every 12 h; the last 3 days, passaged every 24 h). Finally, the hyphal collection was up to 99%.
In addition, 5 ml RPMI 1640, 4.5 ml RPMI 1640 + 0.5 ml calf serum (no inactivation) and 4.5 ml RPMI 1640 + 0.5 ml calf serum (121 °C, 30 min inactivation) were set as controls, mycelium cell formation was observed. The number of cells forming hyphae among 100 cells was counted at 2 h, 3 h, 6 h, 8 h, 12 h, 24 h, and 7 days under high power microscope three times for an average hyphae formation rate.

RNA extraction
The total RNA of biphasic Candida albicans from eight strains of 16 samples (each 5 ml) was extracted by acidwashed glass bead method and modified hot acid phenol method. The yeast phase strains were labeled as isolates ay-hy and hyphal phase strains were labeled as isolates ah-hh. 260/280 and DNA concentrations and purities were averaged after three repeats of performance.

Reverse transcriptase-polymerase chain reaction (RT-PCR)
Total RNA (2 µl) was reverse-transcribed into complementary DNA (cDNA) by incubation with 1 µl of reverse transcriptase in 20 µl of reaction buffer containing 1 µl of random primers and 10 mM dNTPs at 42 °C for 1 h.  Table 1.

Real-time polymerase chain reaction (Real-time PCR)
Real-time PCR was performed using real-time PCR kit (Applied Biosystems, USA

Statistical analysis
A P value < 0.05 was set as the standard statistical significance. The statistical comparisons of the experimental data were performed by one-way ANOVA using SPSS statistical software (SPSS 25.0; SPSS Inc., Chicago, IL, USA).

Candida and C. albicans detection results from OLP
After the phenotypic characterization from Sand type medium cultivation, Secco ma jia chromogenic cultivation, and separation, there were 5 cases of C. albicans and 1 case of Candida tropicalis from 59 cases outcomes of non-erosive OLP, and 7 cases of C. albicans, 1 case of Candida krusei and 1 case of C. parapsilosis from 41 cases of OLP erosive type. The Candida and C. albicans detection results were reported in Table 2. Additionally, the test results of CD3 + CD4 + T h cells in patients were also recorded in Table 3.

The electrophoresis for RT-PCR products of HEGs and EFG1
Figure 2a-f was the electrophoresis of the expression of HEGs, in which EFG1, ALS3, ECE1 were expressed in both phases; HGC1 and HWP1 showed no expression in yeast phase.

The real-time PCR results of each HEGs and EFG1
The results of real-time PCR for HEGs (HWP1, ECE1, ALS3, HGC1, EFG1) were shown in Table 4, Fig. 3a-h and 4 which showed that EFG1, ECE1, ALS3 got a ratio as P > 0.05 in biphasic cells comparing with the internal reference genes with no significant difference, while HGC1 and HWP1 got P < 0.05, namely there were significant differences for HGC1 and HWP1 between biphasic cells.

Existence of C. albicans in OLP
Researches about C. albicans as an opportunistically infectious fungus started from 1940, and its relationships with OLP has also drawn attention of scholars since 1974 [1,7]. Non obvious correlation between C. albicans and OLP was collected through a phenotypic characterization and in reports of Lundstrom [8] later in 1984, Lipperheide [9] in1996, and Mehdipour [10] in 2010.
On the other hand, genotype identification of clinical isolates by Jainkittivon [11] in 2007, Zeng [12] in 2009, and phenotypic characterization by Hatchuel [13] in 1990, as well as the computer selection by Li [5] in 2011 and our studies etc. have come to show that Candida comorbid rate of OLP was obviously higher than that of ordinary normal crowd, and certain differences of Candida comorbid rates have been also discovered between erosive and non-erosive OLP. These results of clinical detection rates of C. albicans were illustrated in Table 2.
Here reported, the Candida infection rates for 59 cases of non-erosive OLP group and 41 cases of erosive OLP group were 10.17% and 21.95% respectively, and the C. albicans infection rates were 8.47% and 17.07%, being consistent with the results of previous research [12].

Phase switching of C. albicans in OLP
After colonizing in the surface of oral mucosa, C. albicans would adhere to the epithelium with the help of mycelium and germ tube. When host defense gets weaker, the fungi would invade the epithelium, escaping from host defense by mycelium morphology to cause opportunistic infection [14,15]. Correspondingly, the average age of patients with OLP in this experiment was 52.23 years old, and the women patients aged 45-52 were relative to be menopausal. Literatures showed that estrogen receptor [16] (ER) levels in male and middle-aged women patients with OLP were both lower than healthy individuals, and   their cortisol levels [17,18] were higher than normal control group. Additionally, in this research, we collected the levels of CD3 + / CD4 + T h cells in these patients with OLP and found that the Studies revealed [19,20] that hyphae growth and stability to maintain mycelial morphology were attributable to serum. Besides white plaque lesions, congestion, erosion, seepage and ulcer also exist among the lines of OLP lesions. Environmental conditions of these pathological states for Candida phase switching were all different. We preliminarily deem that this might be the reason why the Candida detection rates from erosive OLP were higher than non-erosive OLP, and such phenotypic variability of OLP might just be a competition between host's immune defense and stimulation from pathogen C. albicans.

Biphasic expression of HEGs in C. albicans from OLP Biphasic expression of HWP1 and HGC1
To date, HGC1 is acknowledged as the uniquely essential gene for hypha growth, which is an adjusting protein gene of cellular cycle G1 for mycelium morphology [21,22]. In this study, the electrophoresis results of the RT-PCR products showed a full expression of HWP1 and HGC1 mRNAs in mycelial phase cells (Fig. 2a-f ), and the real-time PCR measurement further presented trace expression of HWP1 and HGC1 mRNAs in yeast cells (Table 4 and Fig. 3a-h), indicating that HWP1 and HGC1 might be essential genes respectively for adhesion and morphologic function in pathogenicity of C. albicans in OLP. HGC1 and HWP1 confer respective function in hyphae morphogenesis and invasion into host epithelia cell to induce OLP.

Biphasic expression of ALS3, ECE1
ALS3 is the gene which encodes the surface protein of cell wall. Its expression level is relatively high in cells, deeming its important significance in maintaining hyphae. It was once even acknowledged as the target gene in cellular immunity and antibody induction in C. albicans. In fact, ALS3-deficient mutants could normally adhere to early biofilm at the beginning of hypha formation, but the adhesion time is short, which makes cells fall off easily [23].
ECE1 is proved to encode cell membrane protein. According to previous literature [24,25], it is a polypeptide sequence composed of 271 amino acid residues and 34 amino acids, with no obvious correlation with the formation of hyphae, but formation of hyphea is incomplete in ECE1-deficient cell, with reduction of adhesion ability, demonstrating that ECE1 might play an integral role in morphology maintenance and function improvement for hypha growth. However, later explorations tended to reveal that ECE1 correlated closely to the extension of hyphae, with an increasing expression in the process of mycelial grow [26]. Briefly, it has been inferred consensually that ECE1 does not participate in morphogenesis of hypha formation. Consistent to that ALS3, ECE1 are not deemed to be the essential genes of Candida growth and the morphogenesis of hyphae production, the results in this research showed that ALS3 and ECE1 both expressed no obvious difference in yeast and mycelium phases ( Fig. 2a-f ), and might not be the essential genes for hyphae of C. albicans in OLP, while their roles in hyphae maintenance and adhesion could be indicated or further cared possibly.

Biphasic expression of EFG1
In fact, the morphologic phase switching of C. albicans is regulated by many signal pathways to ensure the genes HEGs to express. Among those signal pathways, EFG1 is a star molecule currently. It is a feasible idea to control C. albicans through regulating EFG1 to alter phase morphology [27], which might be potentially positive clues or basis for researches on susceptive drugs according to resistant genes. EFG1 plays a crucial role in multiple signaling pathways [28,29] to regulate HEGs with other different signaling molecules. These pathways include endocytosis effect in the substrate: endocytosis effect in the substrate → Dck1 → Rac1 Efg1/Fol8 → Czf1 → HEGs; Temperature/serum pathways: t emp era ture → H sp90 → serum → Ras1 → Cyr1 → cAMP → Tpk1/ Tpk2 → Efg1 → Flo8 → HEGs; the classic pH signaling pathways: pH → Rim21 → Rim8 → Rim13/20 → Ef g1 → HEGs; N-acetyl glucosamine signaling pathways: N-acetyl glucosamine → Ngt1 → Efg1 → HEGs, and additionally the hormone levels, serum nitrogen concentration, methyl-methionine, and methionine etc.
In this research, EFG1 mRNA expressions in yeast and mycelial phase cells showed no significant differences by the real-time PCR quantitative detection (Table 4 and Fig. 3a-h) indicating it was not HEGs. However, the experiments presented biofilms and hyphae forms were incomplete in EFG1 expression inhibited cells, implicating that although EFG1 mRNA had normal expression, it couldn't illustrate the specific expression of EFG1. Further experiments about qualitative and quantitative detection of EFG1 are meaningful.

Conclusions
C. albicans and Candida prevalence in patients with OLP showed that isolation ratio from erosive group overweighed the non-erosive OLP patients within this data. HWP1 was essential for adhesion and HGC1 was essential for morphogenesis in pathogenicity of C. albicans in OLP. HGC1 might be a unique essential gene for hypha morphogenesis, and HWP1 was crucial in hyphae maintenance and adhesion. Besides, ALS3, ECE1, and EFG1 played assistant roles in hyphae maintenance and adhesion. This experiment didn't included other pathways such as Tup1, NRG1, Rfg1 [30] on the reverse biphasic state of expression. Follow-up researches could be launched to explore how to block the development of Candida pathogenesis in OLP.

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