Candida albicans Sfl1/Sfl2 regulatory network drives the formation of pathogenic microcolonies

Abstract

Candida albicans is an opportunistic fungal pathogen that can infect oral mucosal surfaces while being under continuous flow from saliva. Under specific conditions, C. albicans will form microcolonies that more closely resemble the biofilms formed in vivo than standard in vitro biofilm models. However, very little is known about these microcolonies, particularly genomic differences between these specialized biofilm structures and the traditional in vitro biofilms. In this study, we used a novel flow system, in which C. albicans spontaneously forms microcolonies, to further characterize the architecture of fungal microcolonies and their genomics compared to non-microcolony conditions. Fungal microcolonies arose from radially branching filamentous hyphae that increasingly intertwined with one another to form extremely dense biofilms, and closely resembled the architecture of in vivo oropharyngeal candidiasis. We identified 20 core microcolony genes that were differentially regulated in flow-induced microcolonies using RNA-seq. These genes included HWP1, ECE1, IHD1, PLB1, HYR1, PGA10, and SAP5. A predictive algorithm was utilized to identify ten transcriptional regulators potentially involved in microcolony formation. Of these transcription factors, we found that Rob1, Ndt80, Sfl1 and Sfl2, played a key role in microcolony formation under both flow and static conditions and to epithelial surfaces. Expression of core microcolony genes were highly up-regulated in Δ sfl1 cells and down-regulated in both Δ sfl2 and Δ rob1 strains. Microcolonies formed on oral epithelium using C. albicans Δ sfl1, Δ sfl2 and Δ rob1 deletion strains all had altered adhesion, invasion and cytotoxicity. Furthermore, epithelial cells infected with deletion mutants had reduced ( SFL2, NDT80, and ROB1) or enhanced ( SFL2) immune responses, evidenced by phosphorylation of MKP1 and c-Fos activation, key signal transducers in the hyphal invasion response. This profile of microcolony transcriptional regulators more closely reflects Sfl1 and Sfl2 hyphal regulatory networks than static biofilm regulatory networks, suggesting that microcolonies are a specialized pathogenic form of biofilm.

Author summary

Candida albicans is an opportunistic fungal pathogen that can infect many parts of the body, including the oral cavity, where saliva continuously flows across exposed surfaces. However, only a handful of studies have looked at growth under flow, and many questions about how C. albicans grows in these conditions remain unanswered. In our previous study, we found that C. albicans grown on plastic slides and with continuous flow will spontaneously form distinct specialized biofilm structures termed microcolonies. The structure of these microcolonies closely resembles C. albicans plaques found within the oral cavity during mucosal infection. Here, we investigated the genes and genetic regulators that play a role in microcolony formation, and which of these genetic factors activate an early response in the host. We determined that 20 C. albicans genes make up the core microcolony genes, with many playing key roles in the formation of these structures. We also discovered six gene regulators of microcolony formation. Each of these regulators also plays a role in the ability of these microcolonies to adhere and invade into oral epithelial cells.