scholarly journals Cell Wall Changes in Amphotericin B-Resistant Strains from Candida tropicalis and Relationship with the Immune Responses Elicited by the Host

2016 ◽  
Vol 60 (4) ◽  
pp. 2326-2335 ◽  
Author(s):  
Ana C. Mesa-Arango ◽  
Cristina Rueda ◽  
Elvira Román ◽  
Jessica Quintin ◽  
María C. Terrón ◽  
...  
Keyword(s):  
Immune Response ◽  
Cell Wall ◽  
Amphotericin B ◽  
Candida Tropicalis ◽  
Galleria Mellonella ◽  
Regular Growth ◽  
Cell Integrity ◽  
Proinflammatory Response ◽  
Content Type ◽  
Resistant Strains

ABSTRACTWe have morphologically characterizedCandida tropicalisisolates resistant to amphotericin B (AmB). These isolates present an enlarged cell wall compared to isolates of regular susceptibility. This correlated with higher levels of β-1,3-glucan in the cell wall but not with detectable changes in chitin content. In line with this, AmB-resistant strains showed reduced susceptibility to Congo red. Moreover, mitogen-activated protein kinases (MAPKs) involved in cell integrity were already activated during regular growth in these strains. Finally, we investigated the response elicited by human blood cells and found that AmB-resistant strains induced a stronger proinflammatory response than susceptible strains. In agreement, AmB-resistant strains also induced stronger melanization ofGalleria mellonellalarvae, indicating that the effect of alterations of the cell wall on the immune response is conserved in different types of hosts. Our results suggest that resistance to AmB is associated with pleiotropic mechanisms that might have important consequences, not only for the efficacy of the treatment but also for the immune response elicited by the host.

scholarly journals Paradoxical Growth of Candida albicans in the Presence of Caspofungin Is Associated with Multiple Cell Wall Rearrangements and Decreased Virulence

2013 ◽  
Vol 58 (2) ◽  
pp. 1071-1083 ◽  
Author(s):  
Cristina Rueda ◽  
Manuel Cuenca-Estrella ◽  
Oscar Zaragoza
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Galleria Mellonella ◽  
Morphological Changes ◽  
Antifungal Drugs ◽  
Proinflammatory Response ◽  
Content Type ◽  
Multiple Cell ◽  
Fungal Adaptation ◽  
Paradoxical Growth

ABSTRACTIn the last decade, echinocandins have emerged as an important family of antifungal drugs because of their fungicidal activity againstCandidaspp. Echinocandins inhibit the enzyme β-1,3-d-glucan synthase, encoded by theFKSgenes, and resistance to echinocandins is associated with mutations in this gene. In addition, echinocandin exposure can produce paradoxical growth, defined as the ability to grow at high antifungal concentrations but not at intermediate concentrations. In this work, we have demonstrated that paradoxical growth ofCandida albicansin the presence of caspofungin is not due to antifungal degradation or instability. Media with high caspofungin concentrations recovered from wells whereC. albicansshowed paradoxical growth inhibited the growth of aCandida kruseireference strain. Cells exhibiting paradoxical growth at high caspofungin concentrations showed morphological changes such as enlarged size, abnormal septa, and absence of filamentation. Chitin content increased from the MIC to high caspofungin concentrations. Despite the high chitin levels, around 23% of cells died after treatment with caspofungin, indicating that chitin is required but not sufficient to protect the cells from the fungicidal effect of caspofungin. Moreover, we found that after paradoxical growth, β-1,3-glucan was exposed at the cell wall surface. Cells grown at high caspofungin concentrations had decreased virulence in the invertebrate hostGalleria mellonella. Cells grown at high caspofungin concentrations also induced a proinflammatory response in murine macrophages compared to control cells. Our work highlights important aspects about fungal adaptation to caspofungin, and although this adaptation is associated with reduced virulence, the clinical implications remain to be elucidated.

scholarly journals Lactoferrin Is Broadly Active against Yeasts and Highly Synergistic with Amphotericin B

2020 ◽  
Vol 64 (5) ◽  
Author(s):  
Kenya E. Fernandes ◽  
Kerry Weeks ◽  
Dee A. Carter
Keyword(s):  
Amphotericin B ◽  
Galleria Mellonella ◽  
Comprehensive Evaluation ◽  
Yeast Species ◽  
Phenotypic Diversity ◽  
Antifungal Drugs ◽  
Fungal Cell ◽  
Content Type ◽  
Iron Saturation ◽  
Capsule Size

ABSTRACT Lactoferrin (LF) is a multifunctional milk protein with antimicrobial activity against a range of pathogens. While numerous studies report that LF is active against fungi, there are considerable differences in the level of antifungal activity and the capacity of LF to interact with other drugs. Here we undertook a comprehensive evaluation of the antifungal spectrum of activity of three defined sources of LF across 22 yeast and 24 mold species and assessed its interactions with six widely used antifungal drugs. LF was broadly and consistently active against all yeast species tested (MICs, 8 to 64 μg/ml), with the extent of activity being strongly affected by iron saturation. LF was synergistic with amphotericin B (AMB) against 19 out of 22 yeast species tested, and synergy was unaffected by iron saturation but was affected by the extent of LF digestion. LF-AMB combination therapy significantly prolonged the survival of Galleria mellonella wax moth larvae infected with Candida albicans or Cryptococcus neoformans and decreased the fungal burden 12- to 25-fold. Evidence that LF directly interacts with the fungal cell surface was seen via scanning electron microscopy, which showed pore formation, hyphal thinning, and major cell collapse in response to LF-AMB synergy. Important virulence mechanisms were disrupted by LF-AMB treatment, which significantly prevented biofilms in C. albicans and C. glabrata, inhibited hyphal development in C. albicans, and reduced cell and capsule size and phenotypic diversity in Cryptococcus. Our results demonstrate the potential of LF-AMB as an antifungal treatment that is broadly synergistic against important yeast pathogens, with the synergy being attributed to the presence of one or more LF peptides.

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 Neutrophils Are Essential for Containment of Vibrio cholerae to the Intestine during the Proinflammatory Phase of Infection

2012 ◽  
Vol 80 (8) ◽  
pp. 2905-2913 ◽  
Author(s):  
Jessica Queen ◽  
Karla J. Fullner Satchell
Keyword(s):  
Immune Response ◽  
Vibrio Cholerae ◽  
Diarrheal Disease ◽  
Innate Immune ◽  
Infection Model ◽  
Proinflammatory Response ◽  
Necrosis Factor Alpha ◽  
Live Attenuated Vaccine ◽  
Content Type

ABSTRACTCholera is classically considered a noninflammatory diarrheal disease, in comparison to invasive enteric organisms, although there is a low-level proinflammatory response during early infection withVibrio choleraeand a strong proinflammatory reaction to live attenuated vaccine strains. Using an adult mouse intestinal infection model, this study examines the contribution of neutrophils to host defense to infection. Nontoxigenic El Tor O1V. choleraeinfection is characterized by the upregulation of interleukin-6 (IL-6), IL-10, and macrophage inflammatory protein 2 alpha in the intestine, indicating an acute innate immune response. Depletion of neutrophils from mice with anti-Ly6G IA8 monoclonal antibody led to decreased survival of mice. The role of neutrophils in protection of the host is to limit the infection to the intestine and control bacterial spread to extraintestinal organs. In the absence of neutrophils, the infection spread to the spleen and led to increased systemic levels of IL-1β and tumor necrosis factor alpha, suggesting the decreased survival in neutropenic mice is due to systemic shock. Neutrophils were found not to contribute to either clearance of colonizing bacteria or to alter the local immune response. However, when genes for secreted accessory toxins were deleted, the colonizing bacteria were cleared from the intestine, and this clearance is dependent upon neutrophils. Thus, the requirement for accessory toxins in virulence is negated in neutropenic mice, which is consistent with a role of accessory toxins in the evasion of innate immune cells in the intestine. Overall, these data support that neutrophils impact disease progression and suggest that neutrophil effectiveness can be manipulated through the deletion of accessory toxins.

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.

scholarly journals De Novo Pyrimidine Biosynthesis Connects Cell Integrity to Amphotericin B Susceptibility in Cryptococcus neoformans

mSphere ◽  
2016 ◽  
Vol 1 (6) ◽  
Author(s):  
Dithi Banerjee ◽  
Timothy C. Umland ◽  
John C. Panepinto
Keyword(s):  
Cell Wall ◽  
Pyrimidine Biosynthesis ◽  
Structural Defects ◽  
Cell Integrity ◽  
Wild Type ◽  
Nucleotide Sugar ◽  
Nucleotide Biosynthesis ◽  
Content Type ◽  
Uridine Nucleotide ◽  
Precursor Molecules

ABSTRACT Synergy between AmB and nucleotide biosynthetic pathways has been documented, but the mechanism of this interaction has not been delineated. Results from this study suggest a correlation between uridine nucleotide biosynthesis and cell integrity likely mediated through the pool of nucleotide-sugar conjugates, which are precursor molecules for both capsule and cell wall of C. neoformans. Thus, we propose a mechanism by which structural defects in the cell wall resulting from perturbation of pyrimidine biosynthesis allow faster and increased penetration of AmB molecules into the cell membrane. Overall, our work demonstrates that impairment of pyrimidine biosynthesis in C. neoformans could be a potential target for antifungal therapy, either alone or in combination with AmB. The use of amphotericin B (AmB) in conjunction with 5-fluorocytosine (5-FC) is known to be the optimal therapy for treating cryptococcosis, but the mechanism by which 5-FC synergizes with AmB is unknown. In this study, we generated a Cryptococcus neoformans ura1Δ mutant lacking dihydroorotate dehydrogenase (DHODH), which demonstrated temperature-sensitive growth due to a defect in cell integrity and sensitivity to cell wall-damaging agents. In addition, sensitivity to AmB was greatly increased. Inclusion of uracil or uridine in the medium did not suppress the cell wall or AmB phenotype, whereas complementation with the wild-type URA1 gene complemented the mutant phenotype. As a measure of membrane accessibility, we assayed the rate of association of the lipid-binding dye 3,3′-dihexyloxacarbocyanine iodide (DiOC6) and saw more rapid association in the ura1Δ mutant. We likewise saw an increased rate of DiOC6 association in other AmB-sensitive mutants, including a ura − spontaneous URA5 mutant made by 5-fluoroorotic acid (5-FOA) selection and a bck1Δ mutant defective in cell integrity signaling. Similar results were also obtained by using a specific plasma membrane-binding CellMask live stain, with cell integrity mutants that exhibited increased and faster association of the dye with the membrane. Chitin synthase mutants (chs5Δ and chs6Δ) that lack any reported cell wall defects, in turn, demonstrate neither any increased susceptibility to AmB nor a greater accessibility to either of the dyes. Finally, perturbation of the cell wall of the wild type by treatment with the β-1,6-glucan synthase inhibitor caspofungin was synergistic with AmB in vitro. IMPORTANCE Synergy between AmB and nucleotide biosynthetic pathways has been documented, but the mechanism of this interaction has not been delineated. Results from this study suggest a correlation between uridine nucleotide biosynthesis and cell integrity likely mediated through the pool of nucleotide-sugar conjugates, which are precursor molecules for both capsule and cell wall of C. neoformans. Thus, we propose a mechanism by which structural defects in the cell wall resulting from perturbation of pyrimidine biosynthesis allow faster and increased penetration of AmB molecules into the cell membrane. Overall, our work demonstrates that impairment of pyrimidine biosynthesis in C. neoformans could be a potential target for antifungal therapy, either alone or in combination with AmB.

scholarly journals Cell Wall Chitosan Is Necessary for Virulence in the Opportunistic Pathogen Cryptococcus neoformans

2011 ◽  
Vol 10 (9) ◽  
pp. 1264-1268 ◽  
Author(s):  
Lorina G. Baker ◽  
Charles A. Specht ◽  
Jennifer K. Lodge
Keyword(s):  
Cell Wall ◽  
Cryptococcus Neoformans ◽  
Fungal Pathogen ◽  
Slow Growth ◽  
Opportunistic Pathogen ◽  
Mammalian Host ◽  
Cell Integrity ◽  
Content Type ◽  
Opportunistic Fungal Pathogen ◽  
Chitin And Chitosan

ABSTRACTCryptococcus neoformansis an opportunistic fungal pathogen that causes meningoencephalitis. Its cell wall is composed of glucans, proteins, chitin, and chitosan. Multiple genetic approaches have defined a chitosan-deficient syndrome that includes slow growth and decreased cell integrity. Here we demonstrate chitosan is necessary for virulence and persistence in the mammalian host.

scholarly journals Global Screening of Genomic and Transcriptomic Factors Associated with Phenotype Differences between Multidrug-Resistant and -Susceptible Candida haemulonii Strains

mSystems ◽  
2019 ◽  
Vol 4 (6) ◽  
Author(s):  
Hao Zhang ◽  
Yifei Niu ◽  
Jingwen Tan ◽  
Weixia Liu ◽  
Ming-an Sun ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Cell Wall ◽  
Multidrug Resistance ◽  
Multidrug Resistant ◽  
Antifungal Drugs ◽  
Cell Wall Integrity ◽  
Close Relative ◽  
Content Type ◽  
Candida Haemulonii ◽  
Resistant Strains

ABSTRACT Candida haemulonii, a close relative of Candida auris, is an emerging pathogen which frequently shows multidrug resistance especially to triazoles, the most used antifungal drugs. The mechanisms of drug resistance in C. haemulonii, however, are largely unknown. Here, we sequenced and annotated the genomes of two reference strains from the C. haemulonii complex, compared the phenotypes, genomes, and transcriptomes of a triazole-susceptible and two triazole-resistant C. haemulonii strains, and identified triazole susceptibility, morphology, fitness, and the major genetic and gene expression differences between the strains. A multidrug efflux gene, CDR1, was recurrently found to be upregulated for expression in triazole-resistant strains. Blocking the activity of Cdr1 increased the susceptibility to triazoles strikingly. Comparative transcriptome analysis also demonstrated impaired cell wall integrity, filamentous growth, and iron homeostasis in triazole-resistant strains. Finally, we also identified a zinc-binding MHR family transcription regulator gene that mutated in triazole-resistant strains spontaneously, contributing to the changes of morphology and, possibly, cell wall integrity between the strains. The study provided important clues for future studies exploring the mechanisms of multidrug resistance and related phenotypic differences of C. haemulonii strains. IMPORTANCE A comprehensive, multi-omic survey was performed to disclose the genetic backgrounds and differences between multidrug-resistant and -susceptible C. haemulonii strains. Genes were identified with mutations or significant expression differences in multidrug-resistant compared to multidrug-susceptible strains, which were mainly involved in multidrug resistance, stress fitness, and morphology. The Cdr1-encoding gene, C. haemulonii 2486 (CH_2486), was expressed at a significantly increased level in multidrug-resistant strains. Functional inhibition experiments further implicated potential roles of CH_2486 in drug resistance. A gene spontaneously mutated in resistant strains, CH_4347, was experimentally validated to influence the morphology of spores, possibly by controlling cell wall integrity.

scholarly journals Pleiotropic Effects of Cell Wall Amidase LytA on Streptococcus pneumoniae Sensitivity to the Host Immune Response

2014 ◽  
Vol 83 (2) ◽  
pp. 591-603 ◽  
Author(s):  
Elisa Ramos-Sevillano ◽  
Ana Urzainqui ◽  
Susana Campuzano ◽  
Miriam Moscoso ◽  
Fernando González-Camacho ◽  
...  
Keyword(s):  
Immune Response ◽  
Cell Wall ◽  
Streptococcus Pneumoniae ◽  
Complement Activation ◽  
Host Immune Response ◽  
Factor H ◽  
Pleiotropic Effects ◽  
Complement C3 ◽  
Classical Pathway ◽  
Content Type

The complement system is a key component of the host immune response for the recognition and clearance ofStreptococcus pneumoniae. In this study, we demonstrate that the amidase LytA, the main pneumococcal autolysin, inhibits complement-mediated immunity independently of effects on pneumolysin by a complex process of impaired complement activation, increased binding of complement regulators, and direct degradation of complement C3. The use of human sera depleted of either C1q or factor B confirmed that LytA prevented activation of both the classical and alternative pathways, whereas pneumolysin inhibited only the classical pathway. LytA prevented binding of C1q and the acute-phase protein C-reactive protein toS. pneumoniae, thereby reducing activation of the classical pathway on the bacterial surface. In addition, LytA increased recruitment of the complement downregulators C4BP and factor H to the pneumococcal cell wall and directly cleaved C3b and iC3b to generate degradation products. As a consequence, C3b deposition and phagocytosis increased in the absence of LytA and were markedly enhanced for thelytA plydouble mutant, confirming that a combination of LytA and Ply is essential for the establishment of pneumococcal pneumonia and sepsis in a murine model of infection. These data demonstrate that LytA has pleiotropic effects on complement activation, a finding which, in combination with the effects of pneumolysin on complement to assist with pneumococcal complement evasion, confirms a major role of both proteins for the full virulence of the microorganism during septicemia.

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.

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