scholarly journals Wsp1, a GBD/CRIB Domain-Containing WASP Homolog, Is Required for Growth, Morphogenesis, and Virulence of Cryptococcus neoformans

2011 ◽  
Vol 10 (4) ◽  
pp. 521-529 ◽  
Author(s):  
Gui Shen ◽  
Amy Whittington ◽  
Ping Wang
Keyword(s):  
Cryptococcus Neoformans ◽  
Actin Polymerization ◽  
Divergent Evolution ◽  
Phagocytic Index ◽  
Fungal Virulence ◽  
Actin Reorganization ◽  
Content Type ◽  
Lethal Infection ◽  
Endocytic Protein

ABSTRACTHuman endocytic protein ITSN1 regulates actin reorganization by activating Rho family GTPases, such as Cdc42. The process is enhanced by ITSN binding of WASP, an effector of Cdc42 and a potent activator of actin polymerization. In the human pathogenCryptococcus neoformans, endocytic protein Cin1 also interacts with Cdc42 and Wsp1, an uncharacterized WASP homolog, but the significance of these interactions remains unknown. Wsp1 contains several conserved domains, including a WASP homology 1 domain (WH1), a GTPase binding/Cdc42 and Rac interactive binding domain (GBD/CRIB), and a C-terminal domain composed of verprolin-like, central, and acidic motifs (VCA). Thus, Wsp1 exhibits domain compositions more similar to human WASP proteins thanSaccharomyces cerevisiaeLas17/Bee1, a WASP homolog lacking the GDB/CRIB domain. Wsp1 is not an essential protein; however, thewsp1mutant exhibited defects in growth, cytokinesis, chitin distribution, and endocytosis and exocytosis. Thewsp1mutant was also unable to undergo genetic cross, produce the polysaccharide capsule, or secrete the enzyme urease. Anin vitrophagocytosis assay showed a higher phagocytic index for thewsp1mutant, whose ability to cause lethal infection in a murine model of cryptococcosis was also attenuated. Our studies reveal divergent evolution of WASP proteins in the fungal phylum and suggest that the conserved function of WASP proteins in the actin cytoskeleton may also impact fungal 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 DAP12 Inhibits Pulmonary Immune Responses to Cryptococcus neoformans

2016 ◽  
Vol 84 (6) ◽  
pp. 1879-1886 ◽  
Author(s):  
Lena J. Heung ◽  
Tobias M. Hohl
Keyword(s):  
Immune Response ◽  
Nk Cells ◽  
Cryptococcus Neoformans ◽  
Molecular Mechanisms ◽  
Content Type ◽  
Effector Cells ◽  
Opportunistic Fungal Pathogen ◽  
Efficient Uptake ◽  
Bacteria And Fungi

Cryptococcus neoformansis an opportunistic fungal pathogen that is inhaled into the lungs and can lead to life-threatening meningoencephalitis in immunocompromised patients. Currently, the molecular mechanisms that regulate the mammalian immune response to respiratory cryptococcal challenge remain poorly defined. DAP12, a signaling adapter for multiple pattern recognition receptors in myeloid and natural killer (NK) cells, has been shown to play both activating and inhibitory roles during lung infections by different bacteria and fungi. In this study, we demonstrate that DAP12 plays an important inhibitory role in the immune response toC. neoformans. Infectious outcomes in DAP12−/−mice, including survival and lung fungal burden, are significantly improved compared to those in C57BL/6 wild-type (WT) mice. We find that eosinophils and macrophages are decreased while NK cells are increased in the lungs of infected DAP12−/−mice. In contrast to WT NK cells, DAP12−/−NK cells are able to repressC. neoformansgrowthin vitro. Additionally, DAP12−/−macrophages are more highly activated than WT macrophages, with increased production of tumor necrosis factor (TNF) and CCL5/RANTES and more efficient uptake and killing ofC. neoformans. These findings suggest that DAP12 acts as a brake on the pulmonary immune response toC. neoformansby promoting pulmonary eosinophilia and by inhibiting the activation and antifungal activities of effector cells, including NK cells and macrophages.

scholarly journals The Investigational Fungal Cyp51 Inhibitor VT-1129 Demonstrates PotentIn VitroActivity against Cryptococcus neoformans and Cryptococcus gattii

2016 ◽  
Vol 60 (4) ◽  
pp. 2528-2531 ◽  
Author(s):  
Shawn R. Lockhart ◽  
Annette W. Fothergill ◽  
Naureen Iqbal ◽  
Carol B. Bolden ◽  
Nina T. Grossman ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Geometric Mean ◽  
Cryptococcus Gattii ◽  
The Novel ◽  
Content Type ◽  
Large Panel

ABSTRACTThein vitroactivities of the novel fungal Cyp51 inhibitor VT-1129 were evaluated against a large panel ofCryptococcus neoformansandCryptococcus gattiiisolates. VT-1129 demonstrated potent activities against bothCryptococcusspecies as demonstrated by low MIC50and MIC90values. ForC. gattii, thein vitropotency was maintained against all genotypes. In addition, significantly lower geometric mean MICs were observed for VT-1129 than for fluconazole againstC. neoformans, including isolates with reduced fluconazole susceptibility.

scholarly journals Cationic Liposomes Extend the Immunostimulatory Effect of CpG Oligodeoxynucleotide against Burkholderia pseudomallei Infection in BALB/c Mice

2012 ◽  
Vol 19 (5) ◽  
pp. 675-683 ◽  
Author(s):  
Apichaya Puangpetch ◽  
Robert Anderson ◽  
Yan Y. Huang ◽  
Rasana W. Sermswan ◽  
Wanpen Chaicumpa ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Severe Disease ◽  
Antimicrobial Activities ◽  
Cationic Liposomes ◽  
Phagocytic Index ◽  
Cpg Odn ◽  
Content Type ◽  
Cpg Oligodeoxynucleotide ◽  
Alternative Approach ◽  
Lethal Infection

ABSTRACTMelioidosis is a severe disease caused by the Gram-negative bacteriumBurkholderia pseudomallei. Previously we showed that pretreatment of mice with CpG oligodeoxynucleotide (CpG ODN) 2 to 10 days prior toB. pseudomalleichallenge conferred as high as 90% protection, but this window of protection was rather short. In the present study, we therefore aimed to prolong this protective window and to gain further insight into the mechanisms underlying the protection induced by CpG ODN againstB. pseudomalleiinfection. It was found that the CpG ODN incorporated with cationic liposomes (DOTAP) but not zwitterionic liposomes (DOPC) provided complete protection against bacterial challenge. Although marked elevation of gamma interferon (IFN-γ) was found in the infected animals 2 days postinfection, it was significantly lowered by the DOTAP-plus-CpG ODN pretreatment. When appropriately activated, the phagocytic index and oxidative burst responses of neutrophils appeared not to be elevated. However, macrophages from stimulated mice showed higher levels of nitric oxide production and exhibited higher levels of antimicrobial activities, judging from lower numbers of viable intracellular bacteria. Taken together, our results demonstrate that DOTAP can enhance the protective window period of CpG ODN to at least 30 days and provide 100% protection againstB. pseudomalleiinfection. The protective effect of DOTAP plus CpG ODN could provide an alternative approach to preventing this lethal infection, for which no vaccine is yet available.

scholarly journals In Vitro Activity of a Novel Antifungal Compound, MYC-053, against Clinically Significant Antifungal-Resistant Strains of Candida glabrata, Candida auris, Cryptococcus neoformans, and Pneumocystis spp.

2019 ◽  
Vol 63 (4) ◽  
Author(s):  
G. Tetz ◽  
M. Collins ◽  
D. Vikina ◽  
V. Tetz
Keyword(s):  
Cryptococcus Neoformans ◽  
Candida Glabrata ◽  
Fungal Infections ◽  
Antifungal Compound ◽  
Candida Auris ◽  
Content Type ◽  
Treatment And Prevention ◽  
Resistant Strains ◽  
Clinically Significant

ABSTRACT An urgent need exists for new antifungal compounds to treat fungal infections in immunocompromised patients. The aim of the current study was to investigate the potency of a novel antifungal compound, MYC-053, against the emerging yeast and yeast-like pathogens Candida glabrata, Candida auris, Cryptococcus neoformans, and Pneumocystis species. MYC-053 was equally effective against the susceptible control strains, clinical isolates, and resistant strains, with MICs of 0.125 to 4.0 μg/ml. Notably, unlike other antifungals such as azoles, polyenes, and echinocandins, MYC-053 was effective against Pneumocystis isolates, therefore being the only synthetic antifungal that may potentially be used against Pneumocystis spp., Candida spp., and Cryptococcus spp. MYC-053 was highly effective against preformed 48-h-old C. glabrata and C. neoformans biofilms, with minimal biofilm eradication concentrations equal to 1 to 4 times the MIC. Together, these data indicated that MYC-053 may be developed into a promising antifungal agent for the treatment and prevention of invasive fungal infections caused by yeasts and yeast-like fungi.

scholarly journals New Triazole NT-a9 Has Potent Antifungal Efficacy against Cryptococcus neoformans In Vitro and In Vivo

2019 ◽  
Vol 64 (2) ◽  
Author(s):  
Ren-Yi Lu ◽  
Ting-Jun-Hong Ni ◽  
Jing Wu ◽  
Lan Yan ◽  
Quan-Zhen Lv ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Amphotericin B ◽  
Pathogenic Fungi ◽  
Control Group ◽  
Survival Times ◽  
Content Type ◽  
Fungal Burden ◽  
Wide Range

ABSTRACT In the past decades, the incidence of cryptococcosis has increased dramatically, which poses a new threat to human health. However, only a few drugs are available for the treatment of cryptococcosis. Here, we described a leading compound, NT-a9, an analogue of isavuconazole, that showed strong antifungal activities in vitro and in vivo. NT-a9 showed a wide range of activities against several pathogenic fungi in vitro, including Cryptococcus neoformans, Cryptococcus gattii, Candida albicans, Candida krusei, Candida tropicalis, Candida glabrata, and Candida parapsilosis, with MICs ranging from 0.002 to 1 μg/ml. In particular, NT-a9 exhibited excellent efficacy against C. neoformans, with a MIC as low as 0.002 μg/ml. NT-a9 treatment resulted in changes in the sterol contents in C. neoformans, similarly to fluconazole. In addition, NT-a9 possessed relatively low cytotoxicity and a high selectivity index. The in vivo efficacy of NT-a9 was assessed using a murine disseminated-cryptococcosis model. Mice were infected intravenously with 1.8 × 106 CFU of C. neoformans strain H99. In the survival study, NT-a9 significantly prolonged the survival times of mice compared with the survival times of the control group or the isavuconazole-, fluconazole-, or amphotericin B-treated groups. Of note, 4 and 8 mg/kg of body weight of NT-a9 rescued all the mice, with a survival rate of 100%. In the fungal-burden study, NT-a9 also significantly reduced the fungal burdens in brains and lungs, while fluconazole and amphotericin B only reduced the fungal burden in lungs. Taken together, these data suggested that NT-a9 is a promising antifungal candidate for the treatment of cryptococcosis infection.

scholarly journals Inhibition of LpxC Protects Mice from Resistant Acinetobacter baumannii by Modulating Inflammation and Enhancing Phagocytosis

mBio ◽  
2012 ◽  
Vol 3 (5) ◽  
Author(s):  
Lin Lin ◽  
Brandon Tan ◽  
Paul Pantapalangkoor ◽  
Tiffany Ho ◽  
Beverlie Baquir ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Gram Negative ◽  
Content Type ◽  
Novel Treatments ◽  
New Class ◽  
Virulent Strains ◽  
Lethal Infection ◽  
Tlr4 Activation ◽  
Opsonophagocytic Killing

ABSTRACT New treatments are needed for extensively drug-resistant (XDR) Gram-negative bacilli (GNB), such as Acinetobacter baumannii. Toll-like receptor 4 (TLR4) was previously reported to enhance bacterial clearance of GNB, including A. baumannii. However, here we have shown that 100% of wild-type mice versus 0% of TLR4-deficient mice died of septic shock due to A. baumannii infection, despite having similar tissue bacterial burdens. The strain lipopolysaccharide (LPS) content and TLR4 activation by extracted LPS did not correlate with in vivo virulence, nor did colistin resistance due to LPS phosphoethanolamine modification. However, more-virulent strains shed more LPS during growth than less-virulent strains, resulting in enhanced TLR4 activation. Due to the role of LPS in A. baumannii virulence, an LpxC inhibitor (which affects lipid A biosynthesis) antibiotic was tested. The LpxC inhibitor did not inhibit growth of the bacterium (MIC > 512 µg/ml) but suppressed A. baumannii LPS-mediated activation of TLR4. Treatment of infected mice with the LpxC inhibitor enhanced clearance of the bacteria by enhancing opsonophagocytic killing, reduced serum LPS concentrations and inflammation, and completely protected the mice from lethal infection. These results identify a previously unappreciated potential for the new class of LpxC inhibitor antibiotics to treat XDR A. baumannii infections. Furthermore, they have far-reaching implications for pathogenesis and treatment of infections caused by GNB and for the discovery of novel antibiotics not detected by standard in vitro screens. IMPORTANCE Novel treatments are needed for infections caused by Acinetobacter baumannii, a Gram-negative bacterium that is extremely antibiotic resistant. The current study was undertaken to understand the immunopathogenesis of these infections, as a basis for defining novel treatments. The primary strain characteristic that differentiated virulent from less-virulent strains was shedding of Gram-negative lipopolysaccharide (LPS) during growth. A novel class of antibiotics, called LpxC inhibitors, block LPS synthesis, but these drugs do not demonstrate the ability to kill A. baumannii in vitro. We found that an LpxC inhibitor blocked the ability of bacteria to activate the sepsis cascade, enhanced opsonophagocytic killing of the bacteria, and protected mice from lethal infection. Thus, an entire new class of antibiotics which is already in development has heretofore-unrecognized potential to treat A. baumannii infections. Furthermore, standard antibiotic screens based on in vitro killing failed to detect this treatment potential of LpxC inhibitors for A. baumannii infections.

scholarly journals Enhancing the Promiscuous Phosphotriesterase Activity of a Thermostable Lactonase (GkaP) for the Efficient Degradation of Organophosphate Pesticides

2012 ◽  
Vol 78 (18) ◽  
pp. 6647-6655 ◽  
Author(s):  
Yu Zhang ◽  
Jiao An ◽  
Wei Ye ◽  
Guangyu Yang ◽  
Zhi-Gang Qian ◽  
...  
Keyword(s):  
Catalytic Efficiency ◽  
Dynamics Simulation ◽  
Saturation Mutagenesis ◽  
Divergent Evolution ◽  
Laboratory Evolution ◽  
Content Type ◽  
Hydrolytic Activities ◽  
Geobacillus Kaustophilus ◽  
Amidohydrolase Superfamily

ABSTRACTThe phosphotriesterase-like lactonase (PLL) enzymes in the amidohydrolase superfamily hydrolyze various lactones and exhibit latent phosphotriesterase activities. These enzymes serve as attractive templates forin vitroevolution of neurotoxic organophosphates (OPs) with hydrolytic capabilities that can be used as bioremediation tools. Here, a thermostable PLL fromGeobacillus kaustophilusHTA426 (GkaP) was targeted for joint laboratory evolution with the aim of enhancing its catalytic efficiency against OP pesticides. By a combination of site saturation mutagenesis and whole-gene error-prone PCR approaches, several improved variants were isolated. The most active variant, 26A8C, accumulated eight amino acid substitutions and demonstrated a 232-fold improvement over the wild-type enzyme in reactivity (kcat/Km) for the OP pesticideethyl-paraoxon. Concomitantly, this variant showed a 767-fold decrease in lactonase activity with δ-decanolactone, imparting a specificity switch of 1.8 × 105-fold. 26A8C also exhibited high hydrolytic activities (19- to 497-fold) for several OP pesticides, including parathion, diazinon, and chlorpyrifos. Analysis of the mutagenesis sites on the GkaP structure revealed that most mutations are located in loop 8, which determines substrate specificity in the amidohydrolase superfamily. Molecular dynamics simulation shed light on why 26A8C lost its native lactonase activity and improved the promiscuous phosphotriesterase activity. These results permit us to obtain further insights into the divergent evolution of promiscuous enzymes and suggest that laboratory evolution of GkaP may lead to potential biological solutions for the efficient decontamination of neurotoxic OP compounds.

scholarly journals Identification of Pathogen Genomic Differences That Impact Human Immune Response and Disease during Cryptococcus neoformans Infection

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Aleeza C. Gerstein ◽  
Katrina M. Jackson ◽  
Tami R. McDonald ◽  
Yina Wang ◽  
Benjamin D. Lueck ◽  
...  
Keyword(s):  
Immune Response ◽  
Cryptococcus Neoformans ◽  
Candidate Genes ◽  
Cryptococcal Meningitis ◽  
Human Disease ◽  
Whole Genome ◽  
Content Type ◽  
Future Studies ◽  
Human Survival

ABSTRACT Patient outcomes during infection are due to a complex interplay between the quality of medical care, host immunity factors, and the infecting pathogen’s characteristics. To probe the influence of pathogen genotype on human survival, immune response, and other parameters of disease, we examined Cryptococcus neoformans isolates collected during the Cryptococcal Optimal Antiretroviral Therapy (ART) Timing (COAT) Trial in Uganda. We measured human participants’ survival, meningitis disease parameters, immunologic phenotypes, and pathogen in vitro growth characteristics. We compared those clinical data to whole-genome sequences from 38 C. neoformans isolates of the most frequently observed sequence type (ST), ST93, in our Ugandan participant population and to sequences from an additional 18 strains of 9 other sequence types representing the known genetic diversity within the Ugandan Cryptococcus clinical isolates. We focused our analyses on 652 polymorphisms that were variable among the ST93 genomes, were not in centromeres or extreme telomeres, and were predicted to have a fitness effect. Logistic regression and principal component analysis identified 40 candidate Cryptococcus genes and 3 hypothetical RNAs associated with human survival, immunologic response, or clinical parameters. We infected mice with 17 available KN99α gene deletion strains for these candidate genes and found that 35% (6/17) directly influenced murine survival. Four of the six gene deletions that impacted murine survival were novel. Such bedside-to-bench translational research identifies important candidate genes for future studies on virulence-associated traits in human Cryptococcus infections. IMPORTANCE Even with the best available care, mortality rates in cryptococcal meningitis range from 20% to 60%. Disease is often due to infection by the fungus Cryptococcus neoformans and involves a complex interaction between the human host and the fungal pathogen. Although previous studies have suggested genetic differences in the pathogen impact human disease, it has proven quite difficult to identify the specific C. neoformans genes that impact the outcome of the human infection. Here, we take advantage of a Ugandan patient cohort infected with closely related C. neoformans strains to examine the role of pathogen genetic variants on several human disease characteristics. Using a pathogen whole-genome sequencing approach, we showed that 40 C. neoformans genes are associated with human disease. Surprisingly, many of these genes are specific to Cryptococcus and have unknown functions. We also show deletion of some of these genes alters disease in a mouse model of infection, confirming their role in disease. These findings are particularly important because they are the first to identify C. neoformans genes associated with human cryptococcal meningitis and lay the foundation for future studies that may lead to new treatment strategies aimed at reducing patient mortality.

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.

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