Morphological and physiological changes induced by contact-dependent interaction between Candida albicans and Fusobacterium nucleatum

Abstract

Candida albicans and Fusobacterium nucleatum are well-studied oral commensal microbes with pathogenic potential that are involved in various oral polymicrobial infectious diseases. Recently, we demonstrated that F. nucleatum ATCC 23726 coaggregates with C. albicans SN152, a process mainly mediated by fusobacterial membrane protein RadD and Candida cell wall protein Flo9. The aim of this study was to investigate the potential biological impact of this inter-kingdom interaction. We found that F. nucleatum ATCC 23726 inhibits growth and hyphal morphogenesis of C. albicans SN152 in a contact-dependent manner. Further analysis revealed that the inhibition of Candida hyphal morphogenesis is mediated via RadD and Flo9 protein pair. Using a murine macrophage cell line, we showed that the F. nucleatum-induced inhibition of Candida hyphal morphogenesis promotes C. albicans survival and negatively impacts the macrophage-killing capability of C. albicans. Furthermore, the yeast form of C. albicans repressed F. nucleatum-induced MCP-1 and TNFα production in macrophages. Our study suggests that the interaction between C. albicans and F. nucleatum leads to a mutual attenuation of virulence, which may function to promote a long-term commensal lifestyle within the oral cavity. This finding has significant implications for our understanding of inter-kingdom interaction and may impact clinical treatment strategies.

Figure 1. Effects of F. nucleatum (Fn) on the growth and hyphal morphogenesis of C. albicans (Ca).

After 4-hour incubation under Ca hyphae-inducing conditions described in the Materials and Methods, samples from monoculture of Ca yeast cells (A1), or Ca yeast/Fn (A2) and Ca hyphae/Fn (A3) co-cultures were taken and visualized under the microscope. At least 10 images were taken for each sample and representative images are shown. The viability of Ca (B1) and Fn (B2) was monitored before and after their 4-hour incubation as mono- and duo-species. The viability of Ca was also determined when pre-developed Ca hyphae were co-cultured with Fn (B3). The lower-right inset in A1 shows Ca yeast cells before cultivation. Error bars = SD. A star indicates P < 0.05. The scale bar is 10 μm.

Figure 2. Contact-dependent hyphal morphogenesis inhibition of C. albicans.

C. albicans (Ca) wt (A–C) or Ca flo9 mutant (D) yeast cells were inoculated into the upper chamber, while mono-culture of F. nucleatum (Fn) wt (A), co-cultures of Ca wt yeast cells and Fn wt (B); Ca wt yeast cells and Fn radD mutants (C); or Ca flo9 yeast cells and Fn wt (D) were inoculated into the lower chamber. Microbes in the two chambers are physically separated by a 0.4-μm pore-size membrane, but share the same culture medium. Chambers were incubated as described in Materials and Methods. Samples in both chambers were taken 4 hours after cultivation and visualized under the microscope. All assays were performed in duplicate and repeated three times on different days. At least 10 images were taken for each sample and representative images are presented. The scale bar is 10 μm.

Figure 3. Contact-dependent growth inhibition of C. albicans.

Yeast cells of C. albicans (Ca) wt or flo9 mutant were inoculated into the upper chamber, while co-cultures of Ca wt and F. nucleatum (Fn) wt (A1); Ca wt and Fn radD (B1); or Ca flo9 and Fn wt (C1) were inoculated into the lower chamber. Chambers were incubated at 37 °C for 4 hours. Samples in both chambers were taken before or 4 hours after incubation and Ca viability was monitored as described in Materials and Methods. All assays were performed in duplicate and repeated three times on different days. Error bars = SD. A star indicates P < 0.05.

Figure 4. Killing of C. albicans (Ca) cells by RAW macrophages.

The yeast or hyphal Ca was incubated in the presence or absence of RAW cells with or without F. nucleatum(Fn) for 90 minutes. C. albicans cell viability was determined as described in Materials and Methods and expressed as the percentage of viable cells 90 minutes after incubation compared to the starting cell number. Three independent experiments were carried out under each condition. Error bars = SD. A star indicates P < 0.05.

Figure 5. Differential induction of Monocyte Chemotactic Protein 1 (MCP-1) and Tumor Necrosis Factor (TNFα) in RAW macrophages.

RAW cells were incubated with different combinations of C. albicans (Ca) and F. nucleatum (Fn) for 4 hours. Release of MCP-1 (A) and TNFα (B) proteins in the supernatant were quantified by an ELISA-based standard assay (see Materials and Methods). Three independent experiments were carried out under each condition. Error bars = Standard Error of the Mean. A star indicates significant differences (P < 0.05) between values of the two groups linked by lines above them.

Figure 6. Propidium iodide (PI) staining of Macrophages after challenge with C. albicans (Ca) and/or F. nucleatum (Fn).

Overnight seeded macrophages were challenged with different combinations of Ca and/or Fn for 2, 4 and 6 hours. The incubation media contains PI, a fluorescent stain which only incorporates into cells that have a compromised membrane. Experiment was performed in triplicate and representative images are shown. The scale bars are 100 μm.