All of the authors contributed substantially to the conception and design of the study. H.A. and M.R. contributed to the data acquisition, analysis, interpretation, and manuscript preparation, while W.C. and A.S. validated the data analysis and interpretation. The authors also ensured the manuscript was critically revised for important intellectual content. Each author approved the final version of the manuscript for submission.

This research was funded by Fonds Émile-Beaulieu, Fondation de l’Université Laval, Quebec, Canada; grant number FO117430 and the PhD student, Humidah Alanazi, was funded by King Saud University, Saudi Arabia for research training. The Réseau de recherché en santé buccodentaire et osseuse (RSBO) supported the Open Access publication fee. The funders had no role in the study design, data collection and analysis, preparation of the manuscript, or decision to publish.

The authors thank The Réseau de recherche en santé buccodentaire et osseuse (RSBO) for its financial support (open access fee) to publish this manuscript. The authors thank M. Amine Belmadani for his technical assistance with the qRT-PCR analyses.

Figure 1. Exposure protocol of Candida albicans to e-cigarette vapor or combustible cigarette smoke.

Figure 1. Exposure protocol of Candida albicans to e-cigarette vapor or combustible cigarette smoke.

Figure 2. The growth of C. albicans was promoted by e-cigarette vapor. Cells were exposed or not for 15 min twice a day for 2 (panel a) or 3 (panel b) days, with the growth determined by MTT assay. Results are means ± SD ( n = 5). A significant difference was observed when comparing the C. albicans cells exposed to CCS, NR e-vapor, or NF e-vapor and those of the control (non-exposed cells). We also compared NF to NR, NR to CCS, and NF to CCS. ** p < 0.01; *** p < 0.001. Free asterisks refer to the statistical difference when comparing exposed (e-vapors/CCS) with non-exposed (Ctrl) C. albicans cells. Bars with asterisks show the comparison of NF to NR, NR to CCS, and NF to CCS.

Figure 2. The growth of C. albicans was promoted by e-cigarette vapor. Cells were exposed or not for 15 min twice a day for 2 (panel a) or 3 (panel b) days, with the growth determined by MTT assay. Results are means ± SD ( n = 5). A significant difference was observed when comparing the C. albicans cells exposed to CCS, NR e-vapor, or NF e-vapor and those of the control (non-exposed cells). We also compared NF to NR, NR to CCS, and NF to CCS. ** p < 0.01; *** p < 0.001. Free asterisks refer to the statistical difference when comparing exposed (e-vapors/CCS) with non-exposed (Ctrl) C. albicans cells. Bars with asterisks show the comparison of NF to NR, NR to CCS, and NF to CCS.

Figure 3. E-cigarette vapor increased the level of chitin produced by C. albicans . Following exposure or not to CCS, NR e-vapor, or NF e-vapor, cell wall proteins were extracted and subjected to chitin level quantification (see Materials and Methods section). Chitin levels are presented. Statistical significance was obtained by comparing the cells exposed to CCS or to NR or NF e-vapor and those of the control (non-exposed cells). *** p < 0.001; Free asterisks refer to the statistical difference when comparing exposed (e-vapors/CCS) to non-exposed (Ctrl) C. albicans . Bars with asterisks show the comparison of NR to CCS, and NF to CCS.

Figure 3. E-cigarette vapor increased the level of chitin produced by C. albicans . Following exposure or not to CCS, NR e-vapor, or NF e-vapor, cell wall proteins were extracted and subjected to chitin level quantification (see Materials and Methods section). Chitin levels are presented. Statistical significance was obtained by comparing the cells exposed to CCS or to NR or NF e-vapor and those of the control (non-exposed cells). *** p < 0.001; Free asterisks refer to the statistical difference when comparing exposed (e-vapors/CCS) to non-exposed (Ctrl) C. albicans . Bars with asterisks show the comparison of NR to CCS, and NF to CCS.

Figure 4. E-cigarette vapor increased the hyphal length of C. albicans cultured under cell morphology transition conditions. C. albicans cells were exposed or not to CCS, NF e-vapor, or NR e-vapor, then cultured at 37 °C in the presence of 10% fetal calf serum. Hyphal tube length was measured after 3 and 6 h using NIH-ImageJ software ( n = 5). * p < 0.05; ** p < 0.01, *** p < 0.001 (r. u = relative unit). Free asterisks refer to the statistical difference when comparing exposed (e-vapors/CCS) to non-exposed (Ctrl) C . albicans . Bars with asterisks show the comparison of NF to NR, and NF to CCS.

Figure 4. E-cigarette vapor increased the hyphal length of C. albicans cultured under cell morphology transition conditions. C. albicans cells were exposed or not to CCS, NF e-vapor, or NR e-vapor, then cultured at 37 °C in the presence of 10% fetal calf serum. Hyphal tube length was measured after 3 and 6 h using NIH-ImageJ software ( n = 5). * p < 0.05; ** p < 0.01, *** p < 0.001 (r. u = relative unit). Free asterisks refer to the statistical difference when comparing exposed (e-vapors/CCS) to non-exposed (Ctrl) C . albicans . Bars with asterisks show the comparison of NF to NR, and NF to CCS.

Figure 5. E-cigarette vapor increased the expression of secreted aspartyl proteinases SAPs 2 , 3 , and 9 . C. albicans cells were exposed or not twice a day for 15 min to CCS, NF e-vapor, or NR e-vapor, then incubated for 16 h at 37 °C prior to the extraction of total RNA and analysis by qRT-PCR ( n = 5). The expression was normalized to the GAPDH (housekeeping gene). Statistical significance was obtained by comparing the cells exposed to CCS or to NR or NF e-vapor and those of the control (non-exposed cells). * p < 0.05; ** p < 0.01; *** p < 0.001. Free asterisks refer to the statistical difference when comparing exposed (e-vapors/CCS) to non-exposed (Ctrl) C. albicans . Bars with asterisks show the comparison of NF to NR, NR to CCS, and NF to CCS.

Figure 5. E-cigarette vapor increased the expression of secreted aspartyl proteinases SAPs 2 , 3 , and 9 . C. albicans cells were exposed or not twice a day for 15 min to CCS, NF e-vapor, or NR e-vapor, then incubated for 16 h at 37 °C prior to the extraction of total RNA and analysis by qRT-PCR ( n = 5). The expression was normalized to the GAPDH (housekeeping gene). Statistical significance was obtained by comparing the cells exposed to CCS or to NR or NF e-vapor and those of the control (non-exposed cells). * p < 0.05; ** p < 0.01; *** p < 0.001. Free asterisks refer to the statistical difference when comparing exposed (e-vapors/CCS) to non-exposed (Ctrl) C. albicans . Bars with asterisks show the comparison of NF to NR, NR to CCS, and NF to CCS.

Figure 6. C. albicans pre-exposed to e-cigarette vapor adhered better to gingival epithelial cells cultures. Gingival epithelial cells were seeded in 6-well plates and cultured for 24 h. The cell monolayers were then co-cultured with e-vapor pre-exposed C. albicans . Adhesion of C. albicans to the gingival cells was assessed after 6 and 24 h using the crystal violet staining assay. Representative images are from four independent experiments, with each experiment performed in duplicate. Scale bars = 50 μm.

Figure 6. C. albicans pre-exposed to e-cigarette vapor adhered better to gingival epithelial cells cultures. Gingival epithelial cells were seeded in 6-well plates and cultured for 24 h. The cell monolayers were then co-cultured with e-vapor pre-exposed C. albicans . Adhesion of C. albicans to the gingival cells was assessed after 6 and 24 h using the crystal violet staining assay. Representative images are from four independent experiments, with each experiment performed in duplicate. Scale bars = 50 μm.

Figure 7. Growth and transition of C. albicans pre-exposed to e-cigarette vapor then co-cultured with gingival epithelial cells. C. albicans cells were exposed twice a day for 15 min to CCS, NR e-vapor, or NF e-vapor, followed by co-culture with gingival epithelial cells in a trans-well culture system. After 24 h, the C. albicans cells in the upper chamber were collected and used to determine their growth (upper figure) and transition (lower figure) by optical microscope analysis ( n = 4). * p < 0.05; ** p < 0.01; *** p < 0.001. Free asterisks refer to the statistical difference when comparing exposed (e-vapors/CCS) to the non-exposed (Ctrl) C. albicans . Bars with asterisks showed the comparison of NF to NR, and NR to CCS.

Figure 7. Growth and transition of C. albicans pre-exposed to e-cigarette vapor then co-cultured with gingival epithelial cells. C. albicans cells were exposed twice a day for 15 min to CCS, NR e-vapor, or NF e-vapor, followed by co-culture with gingival epithelial cells in a trans-well culture system. After 24 h, the C. albicans cells in the upper chamber were collected and used to determine their growth (upper figure) and transition (lower figure) by optical microscope analysis ( n = 4). * p < 0.05; ** p < 0.01; *** p < 0.001. Free asterisks refer to the statistical difference when comparing exposed (e-vapors/CCS) to the non-exposed (Ctrl) C. albicans . Bars with asterisks showed the comparison of NF to NR, and NR to CCS.

Figure 8. C. albicans pre-exposed to e-cigarette vapor promoted gingival epithelial cell differentiation. Gingival epithelial cell monolayers were co-cultured with C. albicans pre-exposed to e-vapor twice a day for 15 min. Following co-culture for 24 h in a trans-well culture system, the epithelial cell monolayers in the lower culture chambers were observed under an inverted optical microscope and photographed. Representative images are from four independent experiments, with each experiment performed in duplicate. Arrows indicate the differentiated cells. Scale bars = 50 μm.

Figure 8. C. albicans pre-exposed to e-cigarette vapor promoted gingival epithelial cell differentiation. Gingival epithelial cell monolayers were co-cultured with C. albicans pre-exposed to e-vapor twice a day for 15 min. Following co-culture for 24 h in a trans-well culture system, the epithelial cell monolayers in the lower culture chambers were observed under an inverted optical microscope and photographed. Representative images are from four independent experiments, with each experiment performed in duplicate. Arrows indicate the differentiated cells. Scale bars = 50 μm.

Figure 9. E-vapor pre-exposed C. albicans decreased gingival epithelial cell viability. Gingival epithelial cells were co-cultured for 24 h in the presence of e-vapor pre-exposed C. albicans . Epithelial cells were then detached, and their viability was determined by trypan blue exclusion assay ( n = 4). Statistical significance was obtained by comparing the cells exposed to CCS, NR e-vapor, or NF e-vapor with those of the control (non-exposed cells). * p < 0.05; ** p < 0.01; *** p < 0.001; ns = non-significant as compared to the control. Free asterisks refer to the statistical difference when comparing exposed (e-vapors/CCS) to the non-exposed (Ctrl) C. albicans . Bars with asterisks show the comparison of NF to NR, NR to CCS.

Figure 9. E-vapor pre-exposed C. albicans decreased gingival epithelial cell viability. Gingival epithelial cells were co-cultured for 24 h in the presence of e-vapor pre-exposed C. albicans . Epithelial cells were then detached, and their viability was determined by trypan blue exclusion assay ( n = 4). Statistical significance was obtained by comparing the cells exposed to CCS, NR e-vapor, or NF e-vapor with those of the control (non-exposed cells). * p < 0.05; ** p < 0.01; *** p < 0.001; ns = non-significant as compared to the control. Free asterisks refer to the statistical difference when comparing exposed (e-vapors/CCS) to the non-exposed (Ctrl) C. albicans . Bars with asterisks show the comparison of NF to NR, NR to CCS.

Figure 10. Epithelial cells co-cultured with e-vapor pre-exposed C. albicans displayed high levels of lactate dehydrogenase (LDH) activity. Gingival epithelial cells were co-cultured for 24 h in the presence of e-vapor pre-exposed C. albicans . Culture supernatants were collected and used to measure LDH activity, as described in the Materials and Methods section ( n = 4). Statistical significance was obtained by comparing the cells exposed to CCS, NR e-vapor, or NF e-vapor with those of the control (non-exposed cells). *** p < 0.001; ns = non-significant. Free asterisks refer to the statistical difference when comparing exposed (e-vapors/CCS) to non-exposed (Ctrl) C. albicans . Bars with asterisks show the comparison of NF to NR, NR to CCS.

Figure 10. Epithelial cells co-cultured with e-vapor pre-exposed C. albicans displayed high levels of lactate dehydrogenase (LDH) activity. Gingival epithelial cells were co-cultured for 24 h in the presence of e-vapor pre-exposed C. albicans . Culture supernatants were collected and used to measure LDH activity, as described in the Materials and Methods section ( n = 4). Statistical significance was obtained by comparing the cells exposed to CCS, NR e-vapor, or NF e-vapor with those of the control (non-exposed cells). *** p < 0.001; ns = non-significant. Free asterisks refer to the statistical difference when comparing exposed (e-vapors/CCS) to non-exposed (Ctrl) C. albicans . Bars with asterisks show the comparison of NF to NR, NR to CCS.

Table 1. Primer sequences used for the qRT-PCR. Primers were optimized previously [ Primer sequences used for the qRT-PCR. Primers were optimized previously [ 10 15 ].