Cross Talk between the Cell Wall Integrity and Cyclic AMP/Protein Kinase A Pathways in Cryptococcus neoformans

Abstract

Cryptococcus neoformans is a fungal pathogen of immunocompromised people that causes fatal meningitis. The fungal cell wall is essential to viability and pathogenesis of C. neoformans, and biosynthesis and repair of the wall is primarily controlled by the cell wall integrity (CWI) signaling pathway. Previous work has shown that deletion of genes encoding the four major kinases in the CWI signaling pathway, namely, PKC1, BCK1, MKK2, and MPK1 results in severe cell wall phenotypes, sensitivity to a variety of cell wall stressors, and for Mpk1, reduced virulence in a mouse model. Here, we examined the global transcriptional responses to gene deletions of BCK1, MKK2, and MPK1 compared to wild-type cells. We found that over 1,000 genes were differentially expressed in one or more of the deletion strains, with 115 genes differentially expressed in all three strains, many of which have been identified as genes regulated by the cyclic AMP (cAMP)/protein kinase A (PKA) pathway. Biochemical measurements of cAMP levels in the kinase deletion strains revealed significantly less cAMP in all of the deletion strains compared to the wild-type strain. The deletion strains also produced significantly smaller capsules than the wild-type KN99 strain did under capsule-inducing conditions, although the levels of capsule they shed were similar to those shed by the wild type. Finally, addition of exogenous cAMP led to reduced sensitivity to cell wall stress and restored surface capsule to levels near those of wild type. Thus, we have direct evidence of cross talk between the CWI and cAMP/PKA pathways that may have important implications for regulation of cell wall and capsule homeostasis.

A fluorogenic C. neoformans reporter strain with a robust expression of m-cherry expressed from a safe haven site in the genome

“…We did not find any RNA reads mapping to this part of the chromosome in a previously published expression dataset employing RNA Seq using RNA isolated from wild-type KN99 cells (Fig. S1B and (Donlin et al, 2014). Successful integration at the SH2 locus results in the replacement of a 139 bp chromosomal region with the desired foreign DNA fragment (Fig.…”

“…To further evaluate the ability of these strains to produce viable spores, we incubated MATα type strains marked at their SH2 locus with hygromycin (JLCN869) and G418 resistance cassettes (JLCN830 and JLCN923; Table 1) with a NAT resistant mpk1 Δ strain generated in KN99 of MATa mating type background (Donlin et al, 2014). We also crossed a strain with the NAT marker at the SH2 locus (JLCN821) with a G418 resistant mkk2 Δ generated in KN99 MATa background (Donlin et al, 2014).…”

“…We also crossed a strain with the NAT marker at the SH2 locus (JLCN821) with a G418 resistant mkk2 Δ generated in KN99 MATa background (Donlin et al, 2014). We randomly chose these deletion strains since they had the compatible drug resistant cassette and were of the appropriate mating type.…”

The cAMP/protein kinase A signaling pathway in pathogenic basidiomycete fungi: Connections with iron homeostasis

“…Ric8 interacts with both Gpa1 and Gpa2 and establishes a connection between cAMP signaling via Gpa1 and pheromone signaling via Gpa1 and a MAP kinase pathway (Gong et al ., 2014). A connection between cAMP signaling and another MAP kinase pathway for cell wall integrity has also been established recently in C. neoformans (Donlin et al ., 2014) (Fig. 1).…”

“…Solid arrows indicate the flow of signals through the pathways with dotted arrows representing the influence on downstream target phenotypes. The representations of the signaling pathways are based on previous reviews and the recent findings (Regenfelder et al ., 1997; Muller et al ., 2004; Arechiga-Carvajal and Ruiz-Herrera, 2005; Klosterman et al ., 2007; Kozubowski et al ., 2009; Wang et al ., 2011, 2014; Kronstad et al ., 2011a; Kozubowski and Heitman, 2012; Donlin et al ., 2014; Roach et al ., 2014). Details on additional signaling components can be found in these reviews.…”