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

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Maureen J. Donlin ◽  
Rajendra Upadhya ◽  
Kimberly J. Gerik ◽  
Woei Lam ◽  
Laura G. VanArendonk ◽  
...  
Keyword(s):  
Cell Wall ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Protein Kinase A ◽  
Cryptococcus Neoformans ◽  
Signaling Pathway ◽  
Cell Wall Integrity ◽  
Wild Type ◽  
Content Type ◽  
Kinase A

ABSTRACTCryptococcus neoformans is a fungal pathogen of immunocompromised people that causes fatal meningitis. The fungal cell wall is essential to viability and pathogenesis ofC. 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, andMPK1results 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 ofBCK1,MKK2, andMPK1compared 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.IMPORTANCECryptococcus neoformans is a fungal pathogen of immunocompromised people that causes fatal meningitis. The fungal cell wall is essential to viability and pathogenesis ofC. neoformans, and biosynthesis and repair of the wall are primarily controlled by the cell wall integrity (CWI) signaling pathway. In this study, we demonstrate that deletion of any of three core kinases in the CWI pathway impacts not only the cell wall but also the amount of surface capsule. Deletion of any of the kinases results in significantly reduced cellular cyclic AMP (cAMP) levels, and addition of exogenous cAMP rescues the capsule defect and some cell wall defects, supporting a direct role for the CWI pathway in regulation of capsule in conjunction with the cAMP/protein kinase A pathway.

scholarly journals Pde1 Phosphodiesterase Modulates Cyclic AMP Levels through a Protein Kinase A-Mediated Negative Feedback Loop in Cryptococcus neoformans

2005 ◽  
Vol 4 (12) ◽  
pp. 1971-1981 ◽  
Author(s):  
Julie K. Hicks ◽  
Yong-Sun Bahn ◽  
Joseph Heitman
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Protein Kinase A ◽  
Cryptococcus Neoformans ◽  
Mutant Strain ◽  
Negative Feedback ◽  
Feedback Loop ◽  
Negative Feedback Loop ◽  
Camp Levels ◽  
Kinase A

ABSTRACT The virulence of the human pathogenic fungus Cryptococcus neoformans is regulated by a cyclic AMP (cAMP)-dependent protein kinase A (PKA) signaling cascade that promotes mating and the production of melanin and capsule. In this study, genes encoding homologs of the Saccharomyces cerevisiae low- and high-affinity phosphodiesterases, PDE1 and PDE2, respectively, were deleted in serotype A strains of C. neoformans. The resulting mutants exhibited moderately elevated levels of melanin and capsule production relative to the wild type. Epistasis experiments indicate that Pde1 functions downstream of the Gα subunit Gpa1, which initiates cAMP-dependent signaling in response to an extracellular signal. Previous work has shown that the PKA catalytic subunit Pka1 governs cAMP levels via a negative feedback loop. Here we show that a pde1Δ pka1Δ mutant strain exhibits cAMP levels that are dramatically increased (∼15-fold) relative to those in a pka1Δ single mutant strain and that a site-directed mutation in a consensus PKA phosphorylation site reduces Pde1 function. These data provide evidence that fluctuations in cAMP levels are modulated by both Pka1-dependent regulation of Pde1 and another target that comprise a robust negative feedback loop to tightly constrain intracellular cAMP levels.

scholarly journals Involvement of Salmonella Pathogenicity Island 2 in the Up-Regulation of Interleukin-10 Expression in Macrophages: Role of Protein Kinase A Signal Pathway

2004 ◽  
Vol 72 (4) ◽  
pp. 1964-1973 ◽  
Author(s):  
Kei-ichi Uchiya ◽  
Eduardo A. Groisman ◽  
Toshiaki Nikai
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Protein Kinase A ◽  
Signal Transduction Pathway ◽  
Pathogenicity Island ◽  
Interleukin 10 ◽  
Wild Type ◽  
Necrosis Factor Alpha ◽  
Creb Phosphorylation ◽  
Kinase A

ABSTRACT Salmonellae are facultative intracellular bacteria capable of surviving within macrophages. Salmonella pathogenicity island 2 (SPI-2) is required for growth within macrophages and for virulence in mice. In this study, we show the involvement of SPI-2 in a signal transduction pathway that induces cytokine expression in Salmonella-infected macrophages. High levels of interleukin-10 (IL-10) mRNA were induced in macrophages by infection with wild-type salmonellae compared to a strain carrying a mutation in the spiC gene, which is encoded within SPI-2. The two strains had the same effect on the expression of proinflammatory cytokines such as IL-1α, IL-6, and tumor necrosis factor alpha. IL-10 expression was dose dependently blocked by treatment of infected macrophages with the protein kinase A (PKA) inhibitor H-89, while IL-10 expression was increased by the PKA activator dibutyryl cyclic AMP. Cyclic AMP-dependent PKA activity was higher in macrophages infected with wild-type salmonellae compared to the spiC mutant, and Ser132 phosphorylation of cyclic AMP response element-binding protein (CREB), which is an important mediator of PKA activation, correlated with the levels of PKA activity. Taken together, these results indicate that salmonellae cause an SPI-2-dependent increase in PKA activity that leads to CREB phosphorylation, resulting in up-regulation of IL-10 expression in Salmonella-infected macrophages. Suppression of IL-10 expression by an antisense oligonucleotide did not affect the growth of wild-type salmonellae within macrophages, whereas growth was dose dependently inhibited by H-89, suggesting that the PKA signaling pathway plays a significant role in intramacrophage Salmonella survival.

scholarly journals Protein Kinase A Regulatory Subunit Isoforms Regulate Growth and Differentiation in Mucor circinelloides: Essential Role of PKAR4

2012 ◽  
Vol 11 (8) ◽  
pp. 989-1002 ◽  
Author(s):  
J. Ocampo ◽  
B. McCormack ◽  
E. Navarro ◽  
S. Moreno ◽  
V. Garre ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Germ Tube ◽  
Mucor Circinelloides ◽  
Wild Type ◽  
Dimorphic Fungus ◽  
Content Type ◽  
Tube Emergence ◽  
Kinase A

ABSTRACTThe protein kinase A (PKA) signaling pathway plays a role in regulating growth and differentiation in the dimorphic fungusMucor circinelloides. PKA holoenzyme is comprised of two catalytic (C) and two regulatory (R) subunits. InM. circinelloides, four genes encode the PKAR1, PKAR2, PKAR3, and PKAR4 isoforms of R subunits. We have constructed null mutants and demonstrate that each isoform has a different role in growth and differentiation. The most striking finding is thatpkaR4is an essential gene, because only heterokaryons were obtained in knockout experiments. Heterokaryons with low levels of wild-type nuclei showed an impediment in the emission of the germ tube, suggesting a pivotal role of this gene in germ tube emergence. The remaining null strains showed different alterations in germ tube emergence, sporulation, and volume of the mother cell. ThepkaR2null mutant showed an accelerated germ tube emission and was the only mutant that germinated under anaerobic conditions when glycine was used as a nitrogen source, suggesting thatpkaR2participates in germ tube emergence by repressing it. From the measurement of the mRNA and protein levels of each isoform in the wild-type and knockout strains, it can be concluded that the expression of each subunit has its own mechanism of differential regulation. The PKAR1 and PKAR2 isoforms are posttranslationally modified by ubiquitylation, suggesting another regulation point in the specificity of the signal transduction. The results indicate that each R isoform has a different role inM. circinelloidesphysiology, controlling the dimorphism and contributing to the specificity of cyclic AMP (cAMP)-PKA pathway.

scholarly journals Protein Kinase A-Dependent Suppression of Reactive Oxygen Species in Transient Focal Ischemia in Adrenomedullin-Deficient Mice

2009 ◽  
Vol 29 (11) ◽  
pp. 1769-1779 ◽  
Author(s):  
Nobukazu Miyamoto ◽  
Ryota Tanaka ◽  
Tatsuo Shimosawa ◽  
Yutaka Yatomi ◽  
Toshiro Fujita ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Protein Kinase A ◽  
Infarct Volume ◽  
Focal Ischemia ◽  
Neurologic Deficit ◽  
Oxygen Species ◽  
Wild Type ◽  
Transient Focal Ischemia ◽  
Kinase A

This study was designed to examine the effect of adrenomedullin deficiency on cerebral infarction and the relationship between adrenomedullin and cyclic AMP–protein kinase A pathway in regulating reactive oxygen species (ROS). Adrenomedullin heterozygous and wild-type mice were subjected to 60-mins focal ischemia. We used adrenomedullin heterozygous mice because adrenomedullin homozygotes die in utero. Infarct volume, neurologic deficit scores, and immunohistochemical analyses were evaluated at several time points after ischemia. The infarct volume and neurologic deficit scores were significantly worse in adrenomedullin heterozygous mice. Significant accumulation of inducible nitric oxide, oxidative DNA damage, and lipid peroxidation was noted after reperfusion in adrenomedullin heterozygous mice. Treatment of wild-type mice with H89, a protein kinase A inhibitor, resulted in increased infarct size, and worsening of neurologic deficit score and other parameters to levels comparable to those of adrenomedullin heterozygous mice. In contrast, cilostazol, which increases cyclic AMP, rescued neurologic deficit and ROS accumulation in adrenomedullin heterozygous mice. This study showed that adrenomedullin downregulation results in increase in ROS after transient focal ischemia in mice. The results also indicated that adrenomedullin has an important function against ischemic injury through the cyclic AMP–protein kinase A pathway.

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