Activation of Cph1 causes ß(1,3)-glucan unmasking in Candida albicans and attenuates virulence in mice in a neutrophil-dependent manner

Abstract

Masking the immunogenic cell wall epitope ß(1,3)-glucan under an outer layer of mannosylated glycoproteins is an important virulence factor deployed by Candida albicans during infection. Consequently, increased ß(1,3)-glucan exposure (unmasking) reveals C. albicans to the host’s immune system and attenuates its virulence. We have previously shown that activation of the Cek1 MAPK pathway via expression of a hyperactive allele of an upstream kinase (STE11ΔN467) induces unmasking. It also increased survival of mice in a murine disseminated candidiasis model and attenuated kidney fungal burden by ≥33 fold. In this communication, we utilized cyclophosphamide-induced immunosuppression to test if the clearance of the unmasked STE11ΔN467 mutant was dependent on the host immune system. Suppression of the immune response by cyclophosphamide reduced the attenuation in fungal burden caused by the STE11ΔN467 allele. Moreover, specific depletion of neutrophils via 1A8 antibody treatment also reduced STE11ΔN467-dependent fungal burden attenuation, but to a lesser extent than cyclophosphamide, demonstrating an important role for neutrophils in mediating fungal clearance of unmasked STE11ΔN467 cells. In an effort to understand the mechanism by which Ste11ΔN467 causes unmasking, transcriptomics were used to reveal that several components in the Cek1 MAPK pathway were upregulated, including the transcription factor CPH1 and the cell wall sensor DFI1. In this report we show that a cph1ΔΔ mutation restored ß(1,3)-glucan exposure to wild-type levels in the STE11ΔN467 strain, confirming that Cph1 is the transcription factor mediating Ste11ΔN467-induced unmasking. Furthermore, Cph1 is shown to induce a positive feedback loop that increases Cek1 activation. In addition, full unmasking by STE11ΔN467 is dependent on the upstream cell wall sensor DFI1. However, while deletion of DFI1 significantly reduced Ste11ΔN467-induced unmasking, it did not impact activation of the downstream kinase Cek1. Thus, it appears that once stimulated by Ste11ΔN467, Dfi1 activates a parallel signaling pathway that is involved in Ste11ΔN467-induced unmasking.

When Is It Appropriate to Take Off the Mask? Signaling Pathways That Regulate ß(1,3)-Glucan Exposure in Candida albicans

“…Although it is usually considered to be more basal, large additions of chitin seem to cause substantial unmasking. This has been observed for regions damaged on hyphae by neutrophils, cell wall changes induced by caspofungin treatment, clinical isolates from symptomatic vulvovaginal candidiasis patients, and mutants like a hyperactive STE11 N467 mutant (a MAP3K of the Cek1 MAPK pathway) and a cho1 / mutant (Wheeler et al, 2008;Davis et al, 2014;Hopke et al, 2016;Pericolini et al, 2018;Wagner et al, 2021). This latter mutant is of particular interest, as the cell wall in the cho1 / mutant is accompanied by a loss of the smooth outer structure of the cell and an increase in surface rigidity (Hasim et al, 2017).…”

“…In fact, chitin production increases in response to caspofungin treatment (Badrane et al, 2012;Davis et al, 2014;Hasim et al, 2017). As increased chitin levels have been correlated with increased ß(1,3)-glucan unmasking (see above), the septins may play an upstream role in unmasking as well (Wheeler et al, 2008;Davis et al, 2014;Hopke et al, 2016;Hasim et al, 2017;Pericolini et al, 2018;Valotteau et al, 2019;Wagner et al, 2021).…”

“…The canonical Cek1 MAPK pathway consists of a core kinase cascade of Ste11-Hst7-Cek1/Cek2 (Figure 2) that regulates the activation of the downstream transcription factors Cph1 and Ace2 (Liu et al, 1994;Csank et al, 1998;Ramirez-Zavala et al, 2013;Van Wijlick et al, 2016;Wagner et al, 2021). This pathway is heavily involved in mediating cell wall organization, and has consequently been shown to play an active role in inducing morphogenic changes from yeast to hyphae (through Cph1) (Liu et al, 1994;Csank et al, 1998), from white to opaque phase in mating-competent C. albicans strains (through Cph1) (Ramirez-Zavala et al, 2013), mating signaling itself (Chen et al, 2002), and in responding to glycostructure damage in the cell wall (through Ace2) (Cantero et al, 2007;Roman et al, 2009;Van Wijlick et al, 2016).…”

“…Due to the direct impact that the Cek1 MAPK pathway plays in regulating cell wall architecture, it is unsurprising that genetic alterations within the pathway also induce changes in immunogenic epitope exposure. The impact that deletion of CEK1 itself has on unmasking has been the subject of conflicting reports, with some analyses showing increased unmasking upon deletion of CEK1 (Galan-Diez et al, 2010;Correia et al, 2019) and others showing no impact on epitope exposure (Wagner et al, 2021). However, it has been shown that expression of a single hyperactive allele of the MAP3K STE11, in which the Nterminal 467 amino acid autoinhibitory domain has been deleted (STE11 N467 ), causes hyperactivation of the downstream MAPK Cek1 and induces increased ß(1,3)-glucan unmasking, as well as increases in both total and exposed chitin levels within the cell wall (Chen et al, 2019a,b;Wagner et al, 2021).…”

“…The impact that deletion of CEK1 itself has on unmasking has been the subject of conflicting reports, with some analyses showing increased unmasking upon deletion of CEK1 (Galan-Diez et al, 2010;Correia et al, 2019) and others showing no impact on epitope exposure (Wagner et al, 2021). However, it has been shown that expression of a single hyperactive allele of the MAP3K STE11, in which the Nterminal 467 amino acid autoinhibitory domain has been deleted (STE11 N467 ), causes hyperactivation of the downstream MAPK Cek1 and induces increased ß(1,3)-glucan unmasking, as well as increases in both total and exposed chitin levels within the cell wall (Chen et al, 2019a,b;Wagner et al, 2021). Hyperactive STE11 N467 expression in turn enhances survival in mice and attenuates fungal colonization in the kidneys, spleen and brain (Chen et al, 2019b;Wagner et al, 2021).…”