scholarly journals Contribution of the gas1 Gene of the Entomopathogenic Fungus Beauveria bassiana, Encoding a Putative Glycosylphosphatidylinositol-Anchored β-1,3-Glucanosyltransferase, to Conidial Thermotolerance and Virulence

2011 ◽  
Vol 77 (8) ◽  
pp. 2676-2684 ◽  
Author(s):  
Shizhu Zhang ◽  
Yuxian Xia ◽  
Nemat O. Keyhani
Keyword(s):  
Cell Wall ◽  
Heat Shock ◽  
Beauveria Bassiana ◽  
Galleria Mellonella ◽  
Outer Cell ◽  
Wild Type ◽  
Biological Pest Control ◽  
Content Type ◽  
Carbohydrate Epitopes ◽  
Wild Type Parent

ABSTRACTBeauveria bassianais a mycoinsecticide alternative to chemicals for use in biological pest control. The fungus-insect interaction is also an emerging model system to examine unique aspects of the development, pathogenesis, and diversity of fungal lifestyles. The glycoside hydrolase 72 (GH72) family includes β-1,3-glucanosyltransferases that are glycosylphosphatidylinositol (GPI)-anchored cell wall-modeling enzymes affecting fungal physiology. A putativeB. bassianaGPI-anchored β-1,3-glucanosyltransferase (Bbgas1) was isolated and characterized.B. bassianatargeted gene knockouts lackingBbgas1were affected in Congo red and salt sensitivity but displayed minor growth defects in the presence of sorbitol, SDS, or calcofluor white. Lectin and antibody mapping of surface carbohydrates revealed increased exposure of carbohydrate epitopes, including β-1,3-glucans, in the ΔBbgas1strain. Transmission electron micrographs revealed localized destabilization of the cell wall in ΔBbgas1conidia, in which fraying of the outer cell wall was apparent. Heat shock temperature sensitivity profiling showed that in contrast to the wild-type parent, ΔBbgas1conidial spores displayed decreased germination after 1 to 4 h of heat shock at temperatures >40°C, and propidium iodide exclusion assays revealed decreased membrane stability in the knockout strain at temperatures >50°C. The ΔBbgas1knockout showed reduced virulence inGalleria mellonellainsect bioassays in both topical and intrahemocoel-injection assays.B. bassianaΔBbgas1strains complemented with the completeBbgas1open reading frame were indistinguishable from the wild-type parent in all phenotypes examined. TheBbgas1gene did not complement the phenotype of aSaccharomyces cerevisiaeβ-1,3-glucanosyltransferaseΔgas1mutant, indicating that this family of enzymes likely posses discrete cellular functions.

scholarly journals SlyA Is a Transcriptional Regulator Involved in the Virulence of Enterococcus faecalis

2011 ◽  
Vol 79 (7) ◽  
pp. 2638-2645 ◽  
Author(s):  
Charlotte Michaux ◽  
Maurizio Sanguinetti ◽  
Fany Reffuveille ◽  
Yanick Auffray ◽  
Brunella Posteraro ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Galleria Mellonella ◽  
Peritoneal Macrophages ◽  
Bacterial Virulence ◽  
Transcriptional Analysis ◽  
Infection Model ◽  
Wild Type ◽  
Content Type ◽  
Wild Type Parent

ABSTRACTPhylogenetic analysis of the crystal structure of theEnterococcus faecalisSlyA (EF_3002) transcriptional factor places it between the SlyA and MarR regulator subfamilies. Proteins of these families are often involved in the regulation of genes important for bacterial virulence and stress response. To gather evidence for the role of this putative regulator inE. faecalisbiology, we dissected the genetic organization of theslyA-EF_3001 locus and constructed aslyAdeletion mutant as well as complemented strains. Interestingly, compared to the wild-type parent, the ΔslyAmutant is more virulent in an insect infection model (Galleria mellonella), exhibits increased persistence in mouse kidneys and liver, and survives better inside peritoneal macrophages. In order to identify a possible SlyA regulon, global microarray transcriptional analysis was performed. This study revealed that theslyA-EF_3001 locus appears to be autoregulated and that 117 genes were differentially regulated in the ΔslyAmutant. In the mutant strain, 111 were underexpressed and 6 overexpressed, indicating that SlyA functions mainly as an activator of transcription.

scholarly journals Neisseria gonorrhoeaePBP3 and PBP4 Facilitate NOD1 Agonist Peptidoglycan Fragment Release and Survival in Stationary Phase

2018 ◽  
Vol 87 (2) ◽  
Author(s):  
Ryan E. Schaub ◽  
Krizia M. Perez-Medina ◽  
Kathleen T. Hackett ◽  
Daniel L. Garcia ◽  
Joseph P. Dillard
Keyword(s):  
Molecular Weight ◽  
Cell Wall ◽  
Inflammatory Response ◽  
Neisseria Gonorrhoeae ◽  
Wild Type ◽  
Cross Link ◽  
Content Type ◽  
Lytic Transglycosylases ◽  
Penicillin Binding Proteins ◽  
Proinflammatory Molecules

ABSTRACTNeisseria gonorrhoeaereleases peptidoglycan fragments during growth, and these molecules induce an inflammatory response in the human host. The proinflammatory molecules include peptidoglycan monomers, peptidoglycan dimers, and free peptides. These molecules can be released by the actions of lytic transglycosylases or an amidase. However, >40% of the gonococcal cell wall is cross-linked, where the peptide stem on one peptidoglycan strand is linked to the peptide stem on a neighboring strand, suggesting that endopeptidases may be required for the release of many peptidoglycan fragments. Therefore, we characterized mutants with individual or combined mutations in genes for the low-molecular-mass penicillin-binding proteins PBP3 and PBP4. Mutations in eitherdacB, encoding PBP3, orpbpG, encoding PBP4, did not significantly reduce the release of peptidoglycan monomers or free peptides. A mutation indacBcaused the appearance of a larger-sized peptidoglycan monomer, the pentapeptide monomer, and an increased release of peptidoglycan dimers, suggesting the involvement of this enzyme in both the removal of C-terminald-Ala residues from stem peptides and the cleavage of cross-linked peptidoglycan. Mutation of bothdacBandpbpGeliminated the release of tripeptide-containing peptidoglycan fragments concomitantly with the appearance of pentapeptide and dipeptide peptidoglycan fragments and higher-molecular-weight peptidoglycan dimers. In accord with the loss of tripeptide peptidoglycan fragments, the level of human NOD1 activation by thedacB pbpGmutants was significantly lower than that by the wild type. We conclude that PBP3 and PBP4 overlap in function for cross-link cleavage and that these endopeptidases act in the normal release of peptidoglycan fragments during growth.

scholarly journals ThemtfATranscription Factor Gene Controls Morphogenesis, Gliotoxin Production, and Virulence in the Opportunistic Human Pathogen Aspergillus fumigatus

2014 ◽  
Vol 13 (6) ◽  
pp. 766-775 ◽  
Author(s):  
Timothy D. Smith ◽  
Ana M. Calvo
Keyword(s):  
Aspergillus Fumigatus ◽  
Protease Activity ◽  
Galleria Mellonella ◽  
Molecular Genetic ◽  
Colony Growth ◽  
Infection Model ◽  
Slight Reduction ◽  
Wild Type ◽  
Content Type ◽  
Factor Gene

ABSTRACTAspergillus fumigatusis the leading causative agent of invasive aspergillosis (IA). The number of cases is on the rise, with mortality rates as high as 90% among immunocompromised patients. Molecular genetic studies inA. fumigatuscould provide novel targets to potentially set the basis for antifungal therapies. In the current study, we investigated the role of the transcription factor genemtfAinA. fumigatus. Our results revealed thatmtfAplays a role in the growth and development of the fungus. Deletion or overexpression ofmtfAleads to a slight reduction in colony growth, as well as a reduction in conidiation levels, in the overexpression strain compared to the wild-type strain. Furthermore, production of the secondary metabolite gliotoxin increased whenmtfAwas overexpressed, coinciding with an increase in the transcription levels of the gliotoxin genesgliZandgliPwith respect to the wild type. In addition, our study showed thatmtfAis also necessary for normal protease activity inA. fumigatus; deletion ofmtfAresulted in a reduction of protease activity compared to wild-type levels. Importantly, the absence ofmtfAcaused a decrease in virulence in theGalleria mellonellainfection model, indicating thatmtfAis necessary forA. fumigatuswild-type pathogenesis.

scholarly journals The Ser/Thr Kinase PrkC Participates in Cell Wall Homeostasis and Antimicrobial Resistance inClostridium difficile

2019 ◽  
Vol 87 (8) ◽  
Author(s):  
Elodie Cuenot ◽  
Transito Garcia-Garcia ◽  
Thibaut Douche ◽  
Olivier Gorgette ◽  
Pascal Courtin ◽  
...  
Keyword(s):  
Cell Wall ◽  
Type Strain ◽  
Wild Type Strain ◽  
Cell Envelope ◽  
Wild Type ◽  
Hamster Model ◽  
Content Type ◽  
Physiological Processes ◽  
Signal Transduction Networks ◽  
Mean Size

ABSTRACTClostridium difficileis the leading cause of antibiotic-associated diarrhea in adults. During infection,C. difficilemust detect the host environment and induce an appropriate survival strategy. Signal transduction networks involving serine/threonine kinases (STKs) play key roles in adaptation, as they regulate numerous physiological processes. PrkC ofC. difficileis an STK with two PASTA domains. We showed that PrkC is membrane associated and is found at the septum. We observed that deletion ofprkCaffects cell morphology with an increase in mean size, cell length heterogeneity, and presence of abnormal septa. A ΔprkCmutant was able to sporulate and germinate but was less motile and formed more biofilm than the wild-type strain. Moreover, a ΔprkCmutant was more sensitive to antimicrobial compounds that target the cell envelope, such as the secondary bile salt deoxycholate, cephalosporins, cationic antimicrobial peptides, and lysozyme. This increased susceptibility was not associated with differences in peptidoglycan or polysaccharide II composition. However, the ΔprkCmutant had less peptidoglycan and released more polysaccharide II into the supernatant. A proteomic analysis showed that the majority ofC. difficileproteins associated with the cell wall were less abundant in the ΔprkCmutant than the wild-type strain. Finally, in a hamster model of infection, the ΔprkCmutant had a colonization delay that did not significantly affect overall virulence.

scholarly journals Cardiolipin Alters Rhodobacter sphaeroides Cell Shape by Affecting Peptidoglycan Precursor Biosynthesis

mBio ◽  
2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Ti-Yu Lin ◽  
William S. Gross ◽  
George K. Auer ◽  
Douglas B. Weibel
Keyword(s):  
Cell Wall ◽  
Rhodobacter Sphaeroides ◽  
Cell Morphology ◽  
Cell Shape ◽  
Molecular Mechanisms ◽  
Wild Type ◽  
Anionic Phospholipid ◽  
Content Type ◽  
Lipid Ii ◽  
Topological Features

ABSTRACT Cardiolipin (CL) is an anionic phospholipid that plays an important role in regulating protein biochemistry in bacteria and mitochondria. Deleting the CL synthase gene (Δcls) in Rhodobacter sphaeroides depletes CL and decreases cell length by 20%. Using a chemical biology approach, we found that a CL deficiency does not impair the function of the cell wall elongasome in R. sphaeroides; instead, biosynthesis of the peptidoglycan (PG) precursor lipid II is decreased. Treating R. sphaeroides cells with fosfomycin and d-cycloserine inhibits lipid II biosynthesis and creates phenotypes in cell shape, PG composition, and spatial PG assembly that are strikingly similar to those seen with R. sphaeroides Δcls cells, suggesting that CL deficiency alters the elongation of R. sphaeroides cells by reducing lipid II biosynthesis. We found that MurG—a glycosyltransferase that performs the last step of lipid II biosynthesis—interacts with anionic phospholipids in native (i.e., R. sphaeroides) and artificial membranes. Lipid II production decreases 25% in R. sphaeroides Δcls cells compared to wild-type cells, and overexpression of MurG in R. sphaeroides Δcls cells restores their rod shape, indicating that CL deficiency decreases MurG activity and alters cell shape. The R. sphaeroides Δcls mutant is more sensitive than the wild-type strain to antibiotics targeting PG synthesis, including fosfomycin, d-cycloserine, S-(3,4-dichlorobenzyl)isothiourea (A22), mecillinam, and ampicillin, suggesting that CL biosynthesis may be a potential target for combination chemotherapies that block the bacterial cell wall. IMPORTANCE The phospholipid composition of the cell membrane influences the spatial and temporal biochemistry of cells. We studied molecular mechanisms connecting membrane composition to cell morphology in the model bacterium Rhodobacter sphaeroides. The peptidoglycan (PG) layer of the cell wall is a dominant component of cell mechanical properties; consequently, it has been an important antibiotic target. We found that the anionic phospholipid cardiolipin (CL) plays a role in determination of the shape of R. sphaeroides cells by affecting PG precursor biosynthesis. Removing CL in R. sphaeroides alters cell morphology and increases its sensitivity to antibiotics targeting proteins synthesizing PG. These studies provide a connection to spatial biochemical control in mitochondria, which contain an inner membrane with topological features in common with R. sphaeroides.

scholarly journals Aspergillus fumigatus G-Protein Coupled Receptors GprM and GprJ Are Important for the Regulation of the Cell Wall Integrity Pathway, Secondary Metabolite Production, and Virulence

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Aílton Pereira da Costa Filho ◽  
Guilherme Thomaz Pereira Brancini ◽  
Patrícia Alves de Castro ◽  
Clara Valero ◽  
Jaire Alves Ferreira Filho ◽  
...  
Keyword(s):  
Cell Wall ◽  
G Protein ◽  
Galleria Mellonella ◽  
G Protein Coupled Receptors ◽  
Cell Wall Integrity ◽  
Fungal Development ◽  
Content Type ◽  
Cell Wall Integrity Pathway ◽  
Mycotoxin Biosynthesis ◽  
G Protein Coupled

ABSTRACT G-protein coupled receptors (GPCRs) are extracellular signaling receptors that sense environmental cues. Fungi sense their environment primarily through GPCR-mediated signaling pathways, which, in turn, regulate fungal development, metabolism, virulence, and mycotoxin biosynthesis. Aspergillus fumigatus is an important human pathogen that causes aspergillosis, a heterogeneous group of diseases that present a wide range of clinical manifestations. Here, we investigate in detail the role of the GPCRs GprM and GprJ in growth and gene expression. GprM and GprJ are important for melanin production and the regulation of the cell wall integrity (CWI) pathway. Overexpression of gprM and gprJ causes a 20 and 50% reduction in growth rate compared to the wild-type (WT) strain and increases sensitivity to cell wall-damaging agents. Phosphorylation of the CWI protein kinase MpkA is increased in the ΔgprM and ΔgprJ strains and decreased in the overexpression mutants compared to the WT strain. Furthermore, differences in cell wall polysaccharide concentrations and organization were observed in these strains. Transcriptome sequencing suggests that GprM and GprJ negatively regulate genes encoding secondary metabolites (SMs). Mass spectrometry analysis confirmed that the production of fumagillin, pyripyropene, fumigaclavine C, fumiquinazoline, and fumitremorgin is reduced in the ΔgprM and ΔgprJ strains, at least partially through the activation of MpkA. Overexpression of grpM also resulted in the regulation of many transcription factors, with AsgA predicted to function downstream of GprM and MpkA signaling. Finally, we show that the ΔgprM and ΔgprJ mutants are reduced in virulence in the Galleria mellonella insect model of invasive aspergillosis. IMPORTANCE A. fumigatus is the main etiological agent of invasive pulmonary aspergillosis, a life-threatening fungal disease that occurs in severely immunocompromised humans. Withstanding the host environment is essential for A. fumigatus virulence, and sensing of extracellular cues occurs primarily through G-protein coupled receptors (GPCRs) that activate signal transduction pathways, which, in turn, regulate fungal development, metabolism, virulence, and mycotoxin biosynthesis. The A. fumigatus genome encodes 15 putative classical GPCRs, with only three having been functionally characterized to date. In this work, we show that the two GPCRs GprM and GprJ regulate the phosphorylation of the mitogen-activated protein kinase MpkA and thus control the regulation of the cell wall integrity pathway. GprM and GprJ are also involved in the regulation of the production of the secondary metabolites fumagillin, pyripyropene, fumigaclavine C, fumiquinazoline, melanin, and fumitremorgin, and this regulation partially occurs through the activation of MpkA. Furthermore, GprM and GprJ are important for virulence in the insect model Galleria mellonella. This work therefore functionally characterizes two GPCRs and shows how they regulate several intracellular pathways that have been shown to be crucial for A. fumigatus virulence.

scholarly journals Implications of the EUCAST Trailing Phenomenon inCandida tropicalisfor theIn VivoSusceptibility in Invertebrate and Murine Models

2018 ◽  
Vol 62 (12) ◽  
Author(s):  
K. M. T. Astvad ◽  
D. Sanglard ◽  
E. Delarze ◽  
R. K. Hare ◽  
M. C. Arendrup
Keyword(s):  
Susceptibility Testing ◽  
Galleria Mellonella ◽  
Growth Control ◽  
Resistant Isolate ◽  
Murine Models ◽  
Wild Type ◽  
Content Type ◽  
Gradual Loss

ABSTRACTCandida tropicalisisolates often display reduced but persistent growth (trailing) over a broad fluconazole concentration range during EUCAST susceptibility testing. Whereas weak trailing (<25% of the positive growth control) is common and found not to impair fluconazole efficacy, we investigated if more pronounced trailing impacted treatment efficacy. Fluconazole efficacy against two weakly (≤25% growth), two moderately (26% to 50% growth), and one heavily (>70% growth) trailing resistant isolate and one resistant (100% growth) isolate were investigatedin vitroandin vivo(in aGalleria mellonellasurvival model and two nonlethal murine models).CDR1expression levels andERG11sequences were characterized. The survival in fluconazole-treatedG. mellonellawas inversely correlated with the degree of trailing (71% to 9% survival in treatment groups). In mice, resistant and heavily trailing isolates responded poorly to fluconazole treatment.CDR1expression was significantly higher in trailing and resistant isolates than in wild-type isolates (1.4-fold to 10-fold higher). All isolates exhibitedERG11wild-type alleles. Heavily trailing isolates were less responsive to fluconazole in allin vivomodels, indicating an impact on fluconazole efficacy.CDR1upregulation may have contributed to the observed differences. Moderately trailing isolates responded less well to fluconazole in larvae only. This confirms clinical data suggesting fluconazole is effective against infections with such isolates in less severely ill patients and supports the current 50% growth endpoint for susceptibility testing. However, it is still unclear if the gradual loss of efficacy observed for moderately trailing isolates in the larva model may be a reason for concern in selected vulnerable patient populations.

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 Landscape of Resistance-Nodulation-Cell Division (RND)-Type Efflux Pumps in Enterobacter cloacae Complex

2016 ◽  
Vol 60 (4) ◽  
pp. 2373-2382 ◽  
Author(s):  
François Guérin ◽  
Claire Lallement ◽  
Christophe Isnard ◽  
Anne Dhalluin ◽  
Vincent Cattoir ◽  
...  
Keyword(s):  
Cell Division ◽  
Antimicrobial Resistance ◽  
Galleria Mellonella ◽  
Acquired Resistance ◽  
Enterobacter Cloacae ◽  
Efflux Pumps ◽  
Wild Type ◽  
Content Type ◽  
Active Efflux

ABSTRACTIn Gram-negative bacteria, the active efflux is an important mechanism of antimicrobial resistance, but little is known about theEnterobacter cloacaecomplex (ECC). It is mediated primarily by pumps belonging to the RND (resistance-nodulation-cell division) family, and only AcrB, part of the AcrAB-TolC tripartite system, was characterized in ECC. However, detailed genome sequence analysis of the strainE. cloacaesubsp.cloacaeATCC 13047 revealed to us that 10 other genes putatively coded for RND-type transporters. We then characterized the role of all of these candidates by construction of corresponding deletion mutants, which were tested for their antimicrobial susceptibility to 36 compounds, their virulence in the invertebrateGalleria mellonellamodel of infection, and their ability to form biofilm. Only the ΔacrBmutant displayed significantly different phenotypes compared to that of the wild-type strain: 4- to 32-fold decrease of MICs of several antibiotics, antiseptics, and dyes, increased production of biofilm, and attenuated virulence inG. mellonella. In order to identify specific substrates of each pump, we individually expressed intransall operons containing an RND pump-encoding gene into the ΔacrBhypersusceptible strain. We showed that three other RND-type efflux systems (ECL_00053-00055, ECL_01758-01759, and ECL_02124-02125) were able to partially restore the wild-type phenotype and to superadd to and even enlarge the broad range of antimicrobial resistance. This is the first global study assessing the role of all RND efflux pumps chromosomally encoded by the ECC, which confirms the major role of AcrB in both pathogenicity and resistance and the potential involvement of other RND-type members in acquired resistance.

scholarly journals Cryptococcus neoformans Phosphoinositide-Dependent Kinase 1 (PDK1) Ortholog Is Required for Stress Tolerance and Survival in Murine Phagocytes

2012 ◽  
Vol 12 (1) ◽  
pp. 12-22 ◽  
Author(s):  
Yeissa Chabrier-Roselló ◽  
Kimberly J. Gerik ◽  
Kristy Koselny ◽  
Louis DiDone ◽  
Jennifer K. Lodge ◽  
...  
Keyword(s):  
Cell Wall ◽  
Stress Tolerance ◽  
Cryptococcus Neoformans ◽  
Nitrosative Stress ◽  
Galleria Mellonella ◽  
Mapk Pathway ◽  
Mitogen Activated Protein Kinase ◽  
Oxidative And Nitrosative Stress ◽  
Independent Manner ◽  
Content Type

ABSTRACTCryptococcus neoformansPKH2-01andPKH2-02are orthologous to mammalian PDK1 kinase genes. Although orthologs of these kinases have been extensively studied inS. cerevisiae, little is known about their function in pathogenic fungi. In this study, we show thatPKH2-02but notPKH2-01is required forC. neoformansto tolerate cell wall, oxidative, nitrosative, and antifungal drug stress. Deletion ofPKH2-02leads to decreased basal levels of Pkc1 activity and, consequently, reduced activation of the cell wall integrity mitogen-activated protein kinase (MAPK) pathway in response to cell wall, oxidative, and nitrosative stress.PKH2-02function also is required for tolerance of fluconazole and amphotericin B, two important drugs for the treatment of cryptococcosis. Furthermore, OSU-03012, an inhibitor of human PDK1, is synergistic and fungicidal in combination with fluconazole. Using aGalleria mellonellamodel of low-temperature cryptococcosis, we found thatPKH2-02is also required for virulence in a temperature-independent manner. Consistent with the hypersensitivity of thepkh2-02Δ mutant to oxidative and nitrosative stress, this mutant shows decreased survival in murine phagocytes compared to that of wild-type (WT) cells. In addition, we show that deletion ofPKH2-02affects the interaction betweenC. neoformansand phagocytes by decreasing its ability to suppress production of tumor necrosis factor alpha (TNF-α) and reactive oxygen species. Taken together, our studies demonstrate that Pkh2-02-mediated signaling inC. neoformansis crucial for stress tolerance, host-pathogen interactions, and both temperature-dependent and -independent virulence.

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