scholarly journals Fungal Virulence in a Lepidopteran Model Is an Emergent Property with Deterministic Features

mBio ◽  
2013 ◽  
Vol 4 (3) ◽  
Author(s):  
Monica A. Garcia-Solache ◽  
David Izquierdo-Garcia ◽  
Cameron Smith ◽  
Aviv Bergman ◽  
Arturo Casadevall
Keyword(s):  
Cryptococcus Neoformans ◽  
Virulence Factors ◽  
Galleria Mellonella ◽  
Emergent Property ◽  
Deterministic System ◽  
Pathogenic Potential ◽  
Fungal Virulence ◽  
Content Type ◽  
Cryptococcal Infection ◽  
Predictable Pattern

ABSTRACTVirulence has been proposed to be an emergent property, which by definition implies that it is not reducible to its components, but this application of a philosophical concept to the host-microbe interaction has not been experimentally tested. The goals of our study were to analyze the correlation of the phenotype with the ability to cause disease and to determine the dynamics of an experimental cryptococcal infection inGalleria mellonellaandAcanthamoeba castellanii. By studying the outcome of infection as host death, we showed that the dynamics of virulence in theG. mellonella/Cryptococcus neoformansinteraction follow a predictable pattern. We also found that the experimental temperature and not the presence of virulence factors was a critical parameter defining the pathogenic potential of cryptococcal species. Our results established that cryptococcal species not considered pathogenic could be pathogens given suitable conditions. Our results support the idea that virulence is an emergent property that cannot be easily predicted by a reductionist approach and yet it behaves as a deterministic system in a lepidopteran cryptococcal infection. These findings provide a road map for evaluating whether host-microbe interactions in other systems are chaotic, deterministic, or stochastic, including those with public health importance.IMPORTANCEVirulence is a complex phenotype that cannot be easily studied by analyzing its individual components in isolation. By studying the outcome of infection as the death of the host, we found that a given microbial phenotype does not necessarily correlate with its ability to cause disease and that the presence of so-called virulence factors does not predict pathogenicity, consistent with the notion that virulence is an emergent property. This paper reports that the dynamics of virulence inGalleria mellonellalarvae infected with the fungusCryptococcus neoformansfollows a predictable pattern. Establishing that virulence is an emergent property is important because it implies that it is not reducible to its components, and consequently, this phenomenon needs to be studied by a holistic approach.

scholarly journals Temperature-Dependent Production of Various PlcR-Controlled Virulence Factors in Bacillus weihenstephanensis Strain KBAB4

2012 ◽  
Vol 78 (8) ◽  
pp. 2553-2561 ◽  
Author(s):  
A. Réjasse ◽  
N. Gilois ◽  
I. Barbosa ◽  
E. Huillet ◽  
C. Bevilacqua ◽  
...  
Keyword(s):  
Low Temperature ◽  
Virulence Factors ◽  
Growth Temperature ◽  
Galleria Mellonella ◽  
Exponential Growth Phase ◽  
Mrna Levels ◽  
Temperature Dependent ◽  
Phylogenetic Groups ◽  
Pathogenic Potential ◽  
Content Type

ABSTRACTTheBacillus cereussensu lato complex has recently been divided into several phylogenetic groups with clear differences in growth temperature range. However, only a few studies have investigated the actual pathogenic potential of the psychrotolerant strains of theB. cereusgroup at low temperature, and little information is available concerning gene expression at low temperature. We found that vegetative cells of the psychrotolerantB. weihenstephanensisstrain KBAB4 were pathogenic against the model insectGalleria mellonellaat 15°C but not at 30°C. A similar temperature-dependent difference also was observed for the supernatant, which was cytotoxic to Vero epithelial cell lines and to murine macrophage J774 cells at 15°C but not at 30°C. We therefore determined the effect of low temperature on the production of various proteins putatively involved in virulence using two-dimensional protein gel electrophoresis, and we showed that the production of the Hbl enterotoxin and of two proteases, NprB and NprP2, was greater at a growth temperature of 15°C than at 30°C. The quantification of the mRNA levels for these virulence genes by real-time quantitative PCR at both temperatures showed that there was also more mRNA present at 15°C than at 30°C. We also found that at 15°C,hblmRNA levels were maximal in the mid- to late exponential growth phase. In conclusion, we found that the higher virulence of theB. cereusKBAB4 strain at low temperature was accompanied by higher levels of the production of various known PlcR-controlled virulence factors and by a higher transcriptional activity of the corresponding genes.

scholarly journals Three Related Enzymes in Candida albicans Achieve Arginine- and Agmatine-Dependent Metabolism That Is Essential for Growth and Fungal Virulence

mBio ◽  
2020 ◽  
Vol 11 (4) ◽  
Author(s):  
Katja Schaefer ◽  
Jeanette Wagener ◽  
Ryan M. Ames ◽  
Stella Christou ◽  
Donna M. MacCallum ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Amino Acid ◽  
Galleria Mellonella ◽  
Fungal Growth ◽  
Open Reading Frames ◽  
Mammalian Host ◽  
No Production ◽  
Triple Mutant ◽  
Fungal Virulence ◽  
Content Type

ABSTRACT Amino acid metabolism is crucial for fungal growth and development. Ureohydrolases produce amines when acting on l-arginine, agmatine, and guanidinobutyrate (GB), and these enzymes generate ornithine (by arginase), putrescine (by agmatinase), or GABA (by 4-guanidinobutyrase or GBase). Candida albicans can metabolize and grow on arginine, agmatine, or guanidinobutyrate as the sole nitrogen source. Three related C. albicans genes whose sequences suggested that they were putative arginase or arginase-like genes were examined for their role in these metabolic pathways. Of these, Car1 encoded the only bona fide arginase, whereas we provide evidence that the other two open reading frames, orf19.5862 and orf19.3418, encode agmatinase and guanidinobutyrase (Gbase), respectively. Analysis of strains with single and multiple mutations suggested the presence of arginase-dependent and arginase-independent routes for polyamine production. CAR1 played a role in hyphal morphogenesis in response to arginine, and the virulence of a triple mutant was reduced in both Galleria mellonella and Mus musculus infection models. In the bloodstream, arginine is an essential amino acid that is required by phagocytes to synthesize nitric oxide (NO). However, none of the single or multiple mutants affected host NO production, suggesting that they did not influence the oxidative burst of phagocytes. IMPORTANCE We show that the C. albicans ureohydrolases arginase (Car1), agmatinase (Agt1), and guanidinobutyrase (Gbu1) can orchestrate an arginase-independent route for polyamine production and that this is important for C. albicans growth and survival in microenvironments of the mammalian host.

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 The F-Box Protein Fbp1 Regulates Sexual Reproduction and Virulence in Cryptococcus neoformans

2011 ◽  
Vol 10 (6) ◽  
pp. 791-802 ◽  
Author(s):  
Tong-Bao Liu ◽  
Yina Wang ◽  
Sabriya Stukes ◽  
Qing Chen ◽  
Arturo Casadevall ◽  
...  
Keyword(s):  
Sexual Reproduction ◽  
Cryptococcus Neoformans ◽  
Stress Responses ◽  
Membrane Integrity ◽  
Infection Model ◽  
Calcofluor White ◽  
Fungal Virulence ◽  
Content Type ◽  
F Box Protein

ABSTRACTCryptococcus neoformansis the leading cause of fungal meningitis in immunocomprised populations. Although extensive studies have been conducted on signal transduction pathways important for fungal sexual reproduction and virulence, how fungal virulence is regulated during infection is still not understood. In this study, we identified the F-box protein Fbp1, which contains a putative F-box domain and 12 leucine-rich repeats (LRR). Althoughfbp1mutants showed normal growth and produced normal major virulence factors, such as melanin and capsule, Fbp1 was found to be essential for fungal virulence, asfbp1mutants were avirulent in a murine systemic-infection model. Fbp1 is also important for fungal sexual reproduction. Basidiospore production was blocked in bilateral mating betweenfbp1mutants, even though normal dikaryotic hyphae were observed during mating.In vitroassays of stress responses revealed thatfbp1mutants are hypersensitive to SDS, but not calcofluor white (CFW) or Congo red, indicating that Fbp1 may regulate cell membrane integrity. Fbp1 physically interacts with Skp1 homologues in bothSaccharomyces cerevisiaeandC. neoformansvia its F-box domain, suggesting it may function as part of an SCF (Skp1, Cullins, F-box proteins) E3 ligase. Overall, our study revealed that the F-box protein Fbp1 is essential for fungal sporulation and virulence inC. neoformans, which likely represents a conserved novel virulence control mechanism that involves the SCF E3 ubiquitin ligase-mediated proteolysis pathway.

scholarly journals Fbp1-Mediated Ubiquitin-Proteasome Pathway Controls Cryptococcus neoformans Virulence by Regulating Fungal Intracellular Growth in Macrophages

2013 ◽  
Vol 82 (2) ◽  
pp. 557-568 ◽  
Author(s):  
Tong-Bao Liu ◽  
Chaoyang Xue
Keyword(s):  
Cryptococcus Neoformans ◽  
Time Course ◽  
Ubiquitin Proteasome System ◽  
Phenotypic Analysis ◽  
Fungal Virulence ◽  
Content Type ◽  
Brain Infection ◽  
Virulence Attenuation ◽  
Ubiquitin Proteasome ◽  
F Box Protein

ABSTRACTCryptococcus neoformansis a human fungal pathogen that often causes lung and brain infections in immunocompromised patients, with a high fatality rate. Our previous results showed that an F-box protein, Fbp1, is essential forCryptococcusvirulence independent of the classical virulence factors, suggesting a novel virulence control mechanism. In this study, we show that Fbp1 is part of the ubiquitin-proteasome system, and we further investigated the mechanism of Fbp1 function during infection. Time course studies revealed that thefbp1Δ mutant causes little damage in the infected lung and that the fungal burden in the lung remains at a low but persistent level throughout infection. Thefbp1Δ mutant cannot disseminate to other organs following pulmonary infection in the murine inhalation model of cryptococcosis but still causes brain infection in a murine intravenous injection model, suggesting that the block of dissemination of thefbp1Δ mutant is due to its inability to leave the lung. Thefbp1Δ mutant showed a defect in intracellular proliferation after phagocytosis in aCryptococcus-macrophage interaction assay, which likely contributes to its virulence attenuation. To elucidate the molecular basis of the SCF(Fbp1) E3 ligase function, we analyzed potential Fbp1 substrates based on proteomic approaches combined with phenotypic analysis. One substrate, the inositol phosphosphingolipid-phospholipase C1 (Isc1), is required for fungal survival inside macrophage cells, which is consistent with the role of Fbp1 in regulatingCryptococcus-macrophage interaction and fungal virulence. Our results thus reveal a new determinant of fungal virulence that involves the posttranslational regulation of inositol sphingolipid biosynthesis.

scholarly journals Molecular epidemiology and phylogenomic analysis of Mycobacterium abscessus clinical isolates in an Asian population

2021 ◽  
Vol 7 (11) ◽  
Author(s):  
Ka Lip Chew ◽  
Sophie Octavia ◽  
Roland Jureen ◽  
Oon Tek Ng ◽  
Kalisvar Marimuthu ◽  
...  
Keyword(s):  
Virulence Factors ◽  
Type Species ◽  
Asian Population ◽  
Bioinformatic Analysis ◽  
Mycobacterium Abscessus ◽  
Phylogenomic Analysis ◽  
Pathogenic Potential ◽  
Content Type ◽  
Link Type ◽  
Sequence Types

Mycobacterium abscessus comprises three subspecies: M. abscessus subsp. abscessus , M. abscessus subsp. bolletii , and M. abscessus subsp. massiliense . These closely related strains are typically multi-drug-resistant and can cause difficult-to-treat infections. Dominant clusters of isolates with increased pathogenic potential have been demonstrated in pulmonary infections in the global cystic fibrosis (CF) population. An investigation was performed on isolates cultured from an Asian, predominantly non-CF population to explore the phylogenomic relationships within our population and compare it to global M. abscessus isolates. Whole-genome-sequencing was performed on M. abscessus isolates between 2017 and 2019. Bioinformatic analysis was performed to determine multi-locus-sequence-type, to establish the phylogenetic relationships between isolates, and to identify virulence and resistance determinants in these isolates. A total of 210 isolates were included, of which 68.5 % (144/210) were respiratory samples. These isolates consisted of 140 (66.6 %) M . abscessus subsp. massiliense , 67 (31.9 %) M . abscessus subsp. abscessus, and three (1.4 %) M . abscessus subsp. bolletii . Dominant sequence-types in our population were similar to those of global CF isolates, but SNP differences in our population were comparatively wider despite the isolates being from the same geographical region. ESX (ESAT-6 secretory) cluster three appeared to occur most commonly in ST4 and ST6 M. abscessus subsp. massiliense , but other virulence factors did not demonstrate an association with isolate subspecies or sample source. We demonstrate that although similar predominant sequence-types are seen in our patient population, cross-transmission is absent. The risk of patient-to-patient transmission appears to be largely limited to the vulnerable CF population, indicating infection from environmental sources remains more common than human-to-human transmission. Resistance and virulence factors are largely consistent across the subspecies with the exception of clarithromycin susceptibility and ESX-3.

scholarly journals Dramatic Improvement of CRISPR/Cas9 Editing in Candida albicans by Increased Single Guide RNA Expression

mSphere ◽  
2017 ◽  
Vol 2 (2) ◽  
Author(s):  
Henry Ng ◽  
Neta Dean
Keyword(s):  
Candida Albicans ◽  
Genome Editing ◽  
Virulence Factors ◽  
Fungal Pathogen ◽  
Fungal Virulence ◽  
Important Conclusion ◽  
Content Type ◽  
Guide Rna ◽  
Improve Efficiency ◽  
Human Fungal Pathogen

ABSTRACT Candida albicans is an important human fungal pathogen. An understanding of fungal virulence factors has been slow because C. albicans is genetically intractable. The recent development of CRISPR/Cas in C. albicans (V. K. Vyas, M. I. Barrasa, G. R. Fink, Sci Adv 1:e1500248, 2015, https://doi.org/10.1126/sciadv.1500248 ) has the potential to circumvent this problem. However, as has been found in other organisms, CRISPR/Cas mutagenesis efficiency can be frustratingly variable. Here, we systematically examined parameters hypothesized to alter sgRNA intracellular levels in order to optimize CRISPR/Cas in C. albicans. Our most important conclusion is that increased sgRNA expression and maturation dramatically improve efficiency of CRISPR/Cas mutagenesis in C. albicans by ~10-fold. Thus, we anticipate that the modifications described here will further advance the application of CRISPR/Cas for genome editing in C. albicans. The clustered regularly interspaced short palindromic repeat system with CRISPR-associated protein 9 nuclease (CRISPR/Cas9) has emerged as a versatile tool for genome editing in Candida albicans. Mounting evidence from other model systems suggests that the intracellular levels of single guide RNA (sgRNA) limit the efficiency of Cas9-dependent DNA cleavage. Here, we tested this idea and describe a new means of sgRNA delivery that improves previously described methods by ~10-fold. The efficiency of Cas9/sgRNA-dependent cleavage and repair of a single-copy yeast enhanced monomeric red fluorescent protein (RFP) gene was measured as a function of various parameters that are hypothesized to affect sgRNA accumulation, including transcriptional and posttranscriptional processing. We analyzed different promoters (SNR52, ADH1, and tRNA), as well as different posttranscriptional RNA processing schemes that serve to generate or stabilize mature sgRNA with precise 5′ and 3′ ends. We compared the effects of flanking sgRNA with self-cleaving ribozymes or by tRNA, which is processed by endogenous RNases. These studies demonstrated that sgRNA flanked by a 5′ tRNA and transcribed by a strong RNA polymerase II ADH1 promoter increased Cas9-dependent RFP mutations by 10-fold. Examination of double-strand-break (DSB) repair in strains hemizygous for RFP demonstrated that both homology-directed and nonhomologous end-joining pathways were used to repair breaks. Together, these results support the model that gRNA expression can be rate limiting for efficient CRISPR/Cas mutagenesis in C. albicans. IMPORTANCE Candida albicans is an important human fungal pathogen. An understanding of fungal virulence factors has been slow because C. albicans is genetically intractable. The recent development of CRISPR/Cas in C. albicans (V. K. Vyas, M. I. Barrasa, G. R. Fink, Sci Adv 1:e1500248, 2015, https://doi.org/10.1126/sciadv.1500248 ) has the potential to circumvent this problem. However, as has been found in other organisms, CRISPR/Cas mutagenesis efficiency can be frustratingly variable. Here, we systematically examined parameters hypothesized to alter sgRNA intracellular levels in order to optimize CRISPR/Cas in C. albicans. Our most important conclusion is that increased sgRNA expression and maturation dramatically improve efficiency of CRISPR/Cas mutagenesis in C. albicans by ~10-fold. Thus, we anticipate that the modifications described here will further advance the application of CRISPR/Cas for genome editing in C. albicans.

scholarly journals Role of an Expanded Inositol Transporter Repertoire in Cryptococcus neoformans Sexual Reproduction and Virulence

mBio ◽  
2010 ◽  
Vol 1 (1) ◽  
Author(s):  
Chaoyang Xue ◽  
Tongbao Liu ◽  
Lydia Chen ◽  
Wenjun Li ◽  
Iris Liu ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Gene Family ◽  
Sexual Reproduction ◽  
Cryptococcus Neoformans ◽  
Yeast Cells ◽  
Sexual Cycle ◽  
Fungal Virulence ◽  
Content Type ◽  
Fungal Meningitis

ABSTRACTCryptococcus neoformansandCryptococcus gattiiare globally distributed human fungal pathogens and the leading causes of fungal meningitis. Recent studies reveal thatmyo-inositol is an important factor for fungal sexual reproduction. ThatC. neoformanscan utilizemyo-inositol as a sole carbon source and the existence of abundant inositol in the human central nervous system suggest that inositol is important forCryptococcusdevelopment and virulence. In accord with this central importance of inositol, an expandedmyo-inositol transporter (ITR) gene family has been identified inCryptococcus. This gene family contains two phylogenetically distinct groups, with a total of 10 or more members inC. neoformansand at least six members in the sibling speciesC. gattii. These inositol transporter genes are differentially expressed under inositol-inducing conditions based on quantitative real-time PCR analyses. Expression ofITRgenes in aSaccharomyces cerevisiaeitr1 itr2mutant lacking inositol transport can complement the slow-growth phenotype of this strain, confirming thatITRgenes arebona fideinositol transporters. Gene mutagenesis studies reveal that the Itr1 and Itr1A transporters are important formyo-inositol stimulation of mating and that functional redundancies among themyo-inositol transporters likely exist. Deletion of the inositol 1-phosphate synthase geneINO1in anitr1oritr1amutant background compromised virulence in a murine inhalation model, indicating the importance of inositol sensing and acquisition for fungal infectivity. Our study provides a platform for further understanding the roles of inositol in fungal physiology and virulence.IMPORTANCECryptococcus neoformansis an AIDS-associated human fungal pathogen that causes over 1 million cases of meningitis annually and is the leading cause of fungal meningitis in immunosuppressed patients. The initial cryptococcal infection is caused predominantly via inhalation of sexual spores or desiccated yeast cells from the environment. How this fungus completes its sexual cycle and produces infectious spores in nature and why it frequently infects the central nervous system to cause fatal meningitis are critical questions that remain to be understood. In this study, we demonstrate that inositol acquisition is important not only for fungal sexual reproduction but also for fungal virulence. We identified an expanded inositol transporter gene family that contains over 10 members, important for both fungal sexual reproduction and virulence. Our work contributes to our understanding of how fungi respond to the environmental inositol availability and its impact on sexual reproduction and virulence.

scholarly journals Identification of Burkholderia cenocepacia Strain H111 Virulence Factors Using Nonmammalian Infection Hosts

2012 ◽  
Vol 81 (1) ◽  
pp. 143-153 ◽  
Author(s):  
Stephan Schwager ◽  
Kirsty Agnoli ◽  
Manuela Köthe ◽  
Friederike Feldmann ◽  
Michael Givskov ◽  
...  
Keyword(s):  
Virulence Factors ◽  
Galleria Mellonella ◽  
Burkholderia Cenocepacia ◽  
Infection Model ◽  
Extracellular Proteases ◽  
Content Type ◽  
C Elegans ◽  
Content Model ◽  
Genes Encoding ◽  
Insertion Mutants

Burkholderia cenocepaciaH111, a strain isolated from a cystic fibrosis patient, has been shown to effectively kill the nematodeCaenorhabditis elegans. We used theC. elegansmodel of infection to screen a mini-Tn5mutant library ofB. cenocepaciaH111 for attenuated virulence. Of the approximately 5,500B. cenocepaciaH111 random mini-Tn5insertion mutants that were screened, 22 showed attenuated virulence inC. elegans. Except for the quorum-sensing regulatorcepR, none of the mutated genes coded for the biosynthesis of classical virulence factors such as extracellular proteases or siderophores. Instead, the mutants contained insertions in metabolic and regulatory genes. Mutants attenuated in virulence in theC. elegansinfection model were also tested in theDrosophila melanogasterpricking model, and those also attenuated in this model were further tested inGalleria mellonella. Six of the 22 mutants were attenuated inD. melanogaster, and five of these were less pathogenic in theG. mellonellamodel. We show that genes encoding enzymes of the purine, pyrimidine, and shikimate biosynthesis pathways are critical for virulence in multiple host models of infection.

scholarly journals Cryptococcal Phospholipase B1 Is Required for Intracellular Proliferation and Control of Titan Cell Morphology during Macrophage Infection

2015 ◽  
Vol 83 (4) ◽  
pp. 1296-1304 ◽  
Author(s):  
Robert J. Evans ◽  
Zhongming Li ◽  
William S. Hughes ◽  
Julianne T. Djordjevic ◽  
Kirsten Nielsen ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Virulence Factors ◽  
Fold Increase ◽  
Macrophage Infection ◽  
Content Type ◽  
The Central Nervous System ◽  
Opportunistic Fungal Pathogen ◽  
And Control

Cryptococcus neoformansis an opportunistic fungal pathogen and a leading cause of fungal-infection-related fatalities, especially in immunocompromised hosts. Several virulence factors are known to play a major role in the pathogenesis of cryptococcal infections, including the enzyme phospholipase B1 (Plb1). Compared to other well-studiedCryptococcus neoformansvirulence factors such as the polysaccharide capsule and melanin production, very little is known about the contribution of Plb1 to cryptococcal virulence. Phospholipase B1 is a phospholipid-modifying enzyme that has been implicated in multiple stages of cryptococcal pathogenesis, including initiation and persistence of pulmonary infection and dissemination to the central nervous system, but the underlying reason for these phenotypes remains unknown. Here we demonstrate that a Δplb1knockout strain ofC. neoformanshas a profound defect in intracellular growth within host macrophages. This defect is due to a combination of a 50% decrease in proliferation and a 2-fold increase in cryptococcal killing within the phagosome. In addition, we show for the first time that the Δplb1strain undergoes a morphological change duringin vitroandin vivointracellular infection, resulting in a subpopulation of very large titan cells, which may arise as a result of the attenuated mutant's inability to cope within the macrophage.

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