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

Date Published: September 25, 2018

Publisher: Public Library of Science

Author(s): Andrew D. McCall, Rohitashw Kumar, Mira Edgerton, Leah E. Cowen.


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 Δsfl1 cells and down-regulated in both Δsfl2 and Δrob1 strains. Microcolonies formed on oral epithelium using C. albicans Δsfl1, Δsfl2 and Δrob1 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.

Partial Text

Candida albicans is a polymorphic fungal pathogen and is an opportunistic causative agent of oropharyngeal candidiasis (OPC) [1]. In oral mucosal disease, C. albicans cells adhere to epithelium and form surface plaques characterized by dense interlocking hyphae. In vivo plaques recovered from tongues of infected mice consist of discrete patches of cells with multiple hyphae extending from the central mass in a starburst shape [2]. We found that the length and density of hyphae was dependent upon Sap6 expression, and that this phenotype was well reproduced by C. albicans microcolonies formed in vitro on solid surfaces using hyphal-inducing medium (RPMI, serum or Spider) and CO2. Furthermore, in vivo plaques with more extensive filamentation and hyphal length also had higher levels of Mkc1 and Cek1 MAPKinase phosphorylation [2], indicating that morphogenic signaling processes are integral to microcolony development. Thus, C. albicans microcolonies represent a specific, and potentially specialized, form of biofilm growth whose function is not known.

Microcolony formation is excellent example of the genomic and phenotypic plasticity of C. albicans that enable this pathogen to colonize a wide range of surfaces. As a dense mass of hyphae, microcolony formation enables C. albicans to strongly adhere, invade and forage for nutrients as a community of cells. We show for the first time that C. albicans microcolonies express a specific set of virulence and metabolic genes required to colonize surfaces in response to environmental cues. These environmental signals, including flow, initiate a transcriptional regulon of core microcolony genes that are differentially expressed depending on host environment. Involvement of multiple C. albicans TRs within the microcolony transcriptional network might be a strategy for adaptation to diverse host environments such as the oral cavity or gut.




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