A novel connection between the Cell Wall Integrity and the PKA pathways regulates cell wall stress response in yeast

Abstract

Fungal cells trigger adaptive mechanisms to survive in situations that compromise cell wall integrity. We show here that the global transcriptional response elicited by inhibition of the synthesis of β-1,3-glucan by caspofungin, encompasses a set of genes that are dependent on Slt2, the MAPK of the Cell Wall Integrity (CWI) pathway, and a broad group of genes regulated independently of Slt2. Genes negatively regulated by the cyclic AMP/Protein Kinase A (PKA) signaling pathway were overrepresented in the latter group. Moreover, cell wall stress mediated by inhibition of β-1,3-glucan synthesis, but not by other cell wall interfering compounds, negatively regulated PKA signaling as indicated by the nuclear localisation of Msn2, cellular glycogen accumulation, a decrease of intracellular cAMP levels and a severe decrease in both the activation of the small GTPase Ras2 and the phosphorylation of known substrates of PKA. All these effects relied on the plasma membrane-spanning sensor of the CWI pathway Wsc1. In addition, caspofungin induced a reduction in the cytosolic pH, which was dependent on the extracellular region of Wsc1. Therefore, alterations of the β-1,3-glucan network in the fungal cell wall, induce, through Wsc1, the activation of the CWI pathway and parallel inhibition of PKA signaling.Fungi cause a large number of infections worldwide and the incidence of fungi-related infections, primarily in hosts with impaired immunity, has risen sharply over the last few decades. The most significant fungal infections, accounting for approximately 90% of human mortality cases, are caused by species of Aspergillus, Candida, Cryptococcus, and Pneumocystis 1 . The most common classes of antifungals that are currently in use are polyenes, azoles, and echinocandins 2 . Echinocandins are synthetically modified lipopeptides that specifically target the fungal cell wall, a structure that is essential in fungi but absent in animal cells, and they represent the most recent group of antifungal agents introduced into the clinical practice. Echinocandins include micafungin, anidulafungin, and caspofungin (CAS), of which, CAS was the first agent of this group to be approved for therapeutic use 3 . Echinocandin drugs are non-competitive inhibitors of the β-1,3-glucan synthase (GS), an enzyme that is required for the formation of the essential polymer β-1,3-glucan found in the cell wall of several fungal pathogens 4 . Echinocandin resistance has been increasing in recent years 5 , which is of great concern because echinocandin drugs are the main course of therapy for patients with invasive candidiasis or aspergillosis. The resistance to echinocandins fungal pathogens exhibit has been attributed to two mechanisms: mutations that alter the target of the enzyme, the Fks1 catalytic subunit of the GS; and the activation of the signalling pathways required to maintain the cell wall integrity, which leads to an increase in the synthesis of the cell wall components, particularly chitin, which compensates for the inhibition of β-1,3-glucan s...

CX-4945 Induces Methuosis in Cholangiocarcinoma Cell Lines by a CK2-Independent Mechanism

“…We measured CK2 activity indirectly by using an antibody against the phospho-CK2 substrate (motif pS/pTDXE) to measure the amount of phosphorylated CK2 substrates. This method is widely used in literature to measure kinase activity, such as activity of the following kinases CK2 [ 16 , 49 , 50 , 51 , 52 ], AMPK [ 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 ], AMT/ATR [ 64 , 65 , 66 , 67 , 68 , 69 , 70 ], PKA [ 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 ], and PKC [ 77 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 , 88 ]. Both CK2 knockdown and CX-4945 treatment inhibited the proliferation of CCA cells.…”

Physiological responses of Saccharomyces cerevisiae to industrially relevant conditions: Slow growth, low pH, and high CO2 levels

“…While the present data do not enable elucidation of the precise nature of the cross‐talks between pH and CO 2 signaling, in S. cerevisiae , two interactions between the abovementioned signaling pathways could provide further leads of investigation. First, CWI is sensed by the GPI‐anchored nano‐spring Wsc1 (Dupres et al, ), ultimately activating CWI and PKA pathways (García et al, ). The kinases Pkh1 and Pkh2 that relay the CO 2 signal to Sch9 are also essential for Pkc1 activation of the CWI pathway (Inagaki et al, ; Levin, ; Pohlers et al, ) and phosphorylate the kinases Ypk1 and Ypk2 that in turn phosphorylate the CWI MAP Kinase Mpk1/Stl2 (Roelants, Torrance, Bezman, & Thorner, ; Schmelzle, Helliwell, & Hall, ).…”

“…While the present data do not enable elucidation of the precise nature of the cross-talks between pH and CO 2 signaling, in S. cerevisiae, two interactions between the abovementioned signaling pathways could provide further leads of investigation. First, CWI is sensed by the GPI-anchored nano-spring Wsc1 (Dupres et al, 2009), ultimately activating CWI and PKA pathways (García et al, 2017).…”

Stress-induced translation inhibition through rapid displacement of scanning initiation factors

“…A decrease in intracellular pH has been suggested to underlie the response of yeast cells to multiple stresses (Dechant et al, 2014;Garcia et al, 2017;Munder et al, 2016). We therefore tested the effects of sorbic acid, a protonophore which equilibrates the cytosolic and extracellular pH.…”