Double Deletion Research Articles

The Gcn5-Ada2-Ada3 histone acetyltransferase module has divergent roles in pathogenesis of Candida glabrata.

Candida glabrata is an opportunistic fungal pathogen and the second most prevalent species isolated from candidiasis patients. C. glabrata has intrinsic tolerance to antifungal drugs and oxidative stresses and the ability to adhere to mucocutaneous surfaces. However, knowledge about the regulation of its virulence traits is limited. The Spt-Ada-Gcn5 acetyltransferase (SAGA) complex modulates gene transcription by histone acetylation through the histone acetyltransferase (HAT) module comprised of Gcn5-Ada2-Ada3. Previously, we showed that the ada2 mutant was hypervirulent but displayed decreased tolerance to antifungal drugs and cell wall perturbing agents. In this study, we further characterized the functions of Ada3 and Gcn5 in C. glabrata. We found that single, double, or triple deletions of the HAT module, as expected, resulted in a decreased level of acetylation on histone H3 lysine 9 (H3K9) and defective growth. These mutants were more susceptible to antifungal drugs, oxidative stresses, and cell wall perturbing agents compared with the wild-type. In addition, HAT module mutants exhibited enhanced agar invasion and upregulation of adhesin and proteases encoding genes, whereas the biofilm formation of those mutants was impaired. Interestingly, HAT module mutants exhibited enhanced induction of catalases (CTA1) expression upon treatment with H2O2 compared with the wild-type. Lastly, although ada3 and gcn5 exhibited marginal hypervirulence, the HAT double and triple mutants were hypervirulent in a murine model of candidiasis. In conclusion, the HAT module of the SAGA complex plays unique roles in H3K9 acetylation, drug tolerance, oxidative stress response, adherence, and virulence in C. glabrata.

Characterization of isogenic mutants with single or double deletions of four phenolic acid esterases in Lactiplantibacillus plantarum TMW1.460

In plants, hydroxycinnamic and hydroxybenzoic acids occur mainly as esters. This study aimed to determine the contribution of individual phenolic acid esterases in Lp. plantarum TMW1.460, which encodes for four esterases: TanA, Lp_0796, Est_1092 and a homolog of Lj0536 and Lj1228 that was termed HceP. To determine which of the phenolic acid esterases present in Lp plantarum TMW1.460 are responsible for esterase activity, mutants with deletions in lp_0796, est_1092, tanB, hceP, or hceP and est_1092 were constructed. The phenotype of wild type strain and mutants was determined with esters of hydroxycinnamic acids (chlorogenic acid and ethyl ferulate) and of hydroxybenzoic acids (methyl gallate, tannic acid and epigallocatechin-3-gallate). Lp. plantarum TMW1.460 hydrolysed chlorogenic acid, methyl ferulate and methyl gallate but not tannic acid or epigallocatechin gallate. The phenotype of mutant strains during growth in mMRS differed from the wild type as follows: Lp. plantarum TMW1.460ΔhceP did not hydrolyse esters of hydroxycinnamic acids; Lp. plantarum TMW1.460ΔtanB did not hydrolyse esters of hydroxybenzoic acids; disruption of est_1092 or Lp_0796 did not alter the phenotype. The phenotype of Lp. plantarum TMW1.460ΔΔhceP/est_1092 was identical to Lp. plantarum TMW1.460ΔhceP. The metabolism of phenolic acids during growth of the mutant strains in broccoli puree and wheat sourdough did not differ from metabolism of the wild type strain. In conclusion, esters of hydroxycinnamic and hydroxybenzoic acids each are hydrolysed by dedicated enzymes. The hydroxycinnamic acid esterase HceP is not expressed, or not active during growth of Lp. plantarum TMW1.460 in all food substrates.

Probing cytoplasmic peroxide metabolism in Shewanella oneidensis.

The facultative anaerobe Shewanella oneidensis respires an extensive set of electron acceptors and, as a consequence, can leak electrons to produce reactive oxygen species such as hydrogen peroxide (H2O2). However, the effects of respiration on cytoplasmic redox homeostasis are poorly characterized in comparison. In the present study, the H2O2 sensor HyPer-3 was deployed to interrogate cytoplasmic peroxide levels of both wild-type and gene deletion mutants lacking peroxide scavenging enzymes following exposure to H2O2. HyPer-3 signals were validated in the S. oneidensis wild-type strain and exhibited a dynamic range of 0-250μM H2O2. As reported by the HyPer-3 sensor, the cytoplasm of H2O2-perturbed mutant strains lacking periplasmic glutathione peroxidase (PgpD) and double deletion mutants lacking catalase (KatB) and bifunctional catalase-peroxidases (KatG1 or KatG2) contained high H2O2 concentrations. The high cytoplasmic H2O2 concentrations correlated with impaired H2O2 removal rates displayed by the mutant strains. Results of the present study provide the first in vivo interrogation of the redox environment of the S. oneidensis cytoplasm with HyPer-3 sensors and indicate that proper redox conditions in minimal growth medium are maintained by the concerted action of both well-known (periplasmic PgpD, cytoplasmic KatB and KatG1) and previously overlooked (cytoplasmic KatG2) peroxidases and catalases.

Interaction of novel proteins, centrin4 and protein of centriole in Leishmania parasite and their effects on the parasite growth

Centrins are cytoskeletal proteins associated with the centrosomes or basal bodies in the eukaryotes. We previously reported the involvement of Centrin 1–3 proteins in cell division in the protozoan parasites Leishmania donovani and Trypanosoma brucei. Centrin4 and 5, unique to such parasites, had never been characterized in Leishmania parasite. In the current study, we addressed the function of centrin4 (LdCen4) in Leishmania. By dominant-negative study, the episomal expression of C-terminal truncated LdCen4 in the parasite reduced the parasite growth. LdCen4 double allele gene deletion by either homologous recombination or CRISPR-Cas9 was not successful in L. donovani. However, CRISPR-Cas9-based deletion of the homologous gene was possible in L. mexicana, which attenuated the parasite growth in vitro, but not ex vivo in the macrophages. LdCen4 also interacts with endogenous and overexpressed LdPOC protein, a homolog of centrin reacting human POC (protein of centriole) in a calcium sensitive manner. LdCen4 and LdPOC binding has also been confirmed through in silico analysis by protein structural docking and validated by co-immunoprecipitation. By immunofluorescence studies, we found that both the proteins share a common localization at the basal bodies. Thus, for the first time, this article describes novel centrin4 and its binding protein in the protozoan parasites.

Split-marker-mediated genome editing improves homologous recombination frequency in the CTG clade yeast Candida intermedia.

Genome editing toolboxes are essential for the exploration and exploitation of non-conventional yeast species as cell factories, as they facilitate both genome studies and metabolic engineering. The non-conventional yeast Candida intermedia is a biotechnologically interesting species due to its capacity to convert a wide range of carbon sources, including xylose and lactose found in forestry and dairy industry waste and side-streams, into added-value products. However, possibilities of genetic manipulation have so far been limited due to lack of molecular tools for this species. We describe here the development of a genome editing method for C. intermedia, based on electroporation and gene deletion cassettes containing the Candida albicans NAT1 dominant selection marker flanked by 1,000 base pair sequences homologous to the target loci. Linear deletion cassettes targeting the ADE2 gene originally resulted in <1% targeting efficiencies, suggesting that C. intermedia mainly uses non-homologous end joining for integration of foreign DNA fragments. By developing a split-marker based deletion technique for C. intermedia, we successfully improved the homologous recombination rates, achieving targeting efficiencies up to 70%. For marker-less deletions, we also employed the split-marker cassette in combination with a recombinase system, which enabled the construction of double deletion mutants via marker recycling. Overall, the split marker technique proved to be a quick and reliable method for generating gene deletions in C. intermedia, which opens the possibility to uncover and enhance its cell factory potential.

Double and quadruple deletion mutant of EHV-1 is highly attenuated and induces optimal immune response

Equid alphaherpesvirus 1 (EHV-1) infection causes significant health problems in equines. The EHV-1 infection leads to abortion storm in mares, respiratory disease and myeloencephalopathy. Despite the wide use of vaccines, the outbreaks of EHV-1 infections keep occurring globally, suggesting the need for the development of improved vaccines. Gene deletion attenuated mutant viruses could be a good candidate for the development of modified live vaccines. Here, we report the generation of mutant EHV-1 by deleting virulence (glycoprotein E & internal repeat 6; IR6) and immune evasive (pUL43 & pUL56) associated genes either individually or in combinations; and comprehensive evaluation of mutants through in vitro characterization followed by in vivo study in murine model to adjudge the attenuation of the virus and immune responses generated by mutants vis-à-vis wild type (wt) virus. The EHV-1 mutants with deletion of IR6 and gE genes (vToH-DMV) and four genes (i.e., gE, IR6, pUL43 and pUL56) (vToH-QMV) revealed a significant reduction in plaque size with minimal loss in replication efficiency in comparison to the wt virus. Further, in vivo studies showed virus attenuation adjudged through significant reduction in clinical signs, weight loss, gross and histopathological lesions in comparison to wt virus also revealed improved immune responses estimated through serum neutralization and flow cytometric analysis of CD4 + and CD8 + cell populations. Thus it can be concluded that EHV-1 mutants viz. vToH-DMV and vToH-QMV (novel combination) are promising vaccine candidates and qualify to be studied for adjudging the protective efficacy with wt virus challenge.

Effect of Exogenous Spermine on Biofilm Formation in Mycobacteria by Stimulating the Synthesis of Glycopeptidolipids

Biofilm formation is of great interest by its ability to increase bacterial tolerance to antibiotics that represent a serious problem for modern medicine. Among mycobacteria, which are also capable of forming biofilms, there are pathogens of socially dangerous infections, including tuberculosis. Basing on these data, the strains of Mycolicibacterium smegmatis mc2 155 were chosen as the objects of this study, including the parent strain without deletions and its mutants with one (ΔrelMsm) and double (ΔrelMsmΔrelZ) chromosomal deletions of the genes responsible for the synthesis of alarmone synthetase enzymes. Biofilms of mutant strains exhibited defects in biofilm formation. We have shown that the integrity, hydrophobicity, and the level of biomass of surface mycobacterial biofilms are dependent on the amount of glycopeptidolipids (GPL) in cells. The level of GPL depends on the activity of alarmone synthetases. The biogenic polyamine spermine is able to enhance the production of GPLs, restoring the integrity of biofilms of mutant strains. It is possible that this effect of spermine is caused by the influence on the activity of mycobacterial alarmone synthetases, which makes promising the further studying the molecular mechanisms of its action.

The Effect of the Knockout of Major Transsulfuration Genes on the Pattern of Protein Synthesis in D. melanogaster

The enzymes involved in the transsulfuration pathway and hydrogen sulfide production-cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST) - play an important cytoprotective role in the functioning of the organism. Using CRISPER/Cas9 technology, we obtained Drosophila strains with deleted cbs, cse, and mst genes as well as with double deletion of cbs and cse genes. We analyzed the effect of these mutations on the pattern of protein synthesis in the salivary glands of third instar larvae and in the ovaries of mature flies. In the salivary glands of strains with cbs and cse deletions, a decrease was found in the accumulation of the FBP2 storage protein containing 20% methionine amino acid residues. In the ovaries, changes were detected in the level of expression and isofocusing points of proteins involved in cell protection against oxidative stress, hypoxia, and protein degradation. It was shown that in the strains with deletions of transsulfuration enzymes the proteins have a similar degree of oxidation to that of the control strain. A decrease in the total number of proteasomes and their activity was found in the strains with deletions of the cbs and cse genes.

Plasmodium falciparum pfhrp2 and pfhrp3 Gene Deletions in Malaria-Hyperendemic Region, South Sudan

Pfhrp2 and pfhrp3 gene deletions threaten the use of Plasmodium falciparum malaria rapid diagnostic tests globally. In South Sudan, deletion frequencies were 15.6% for pfhrp2, 20.0% for pfhrp3, and 7.5% for double deletions. Deletions were approximately twice as prevalent in monoclonal infections than in polyclonal infections.

Comparison of Immunogenicity Between a Candidate Live Attenuated Vaccine and an Inactivated Vaccine for Cache Valley Virus.

Cache Valley virus (CVV) is a mosquito-borne bunyavirus that is enzootic throughout the new world. Although CVV is known as an important agricultural pathogen, primarily associated with embryonic lethality and abortions in ruminants, it has recently been recognized for its expansion as a zoonotic pathogen. With the increased emergence of bunyaviruses with human and veterinary importance, there have been significant efforts dedicated to the development of bunyavirus vaccines. In this study, the immunogenicity of a candidate live-attenuated vaccine (LAV) for CVV, which contains the deletion of the nonstructural small (NSs) and nonstructural medium (NSm) genes (2delCVV), was evaluated and compared with an autogenous candidate vaccine created through the inactivation of CVV using binary ethylenimine (BEI) with an aluminum hydroxide adjuvant (BEI-CVV) in sheep. Both 2delCVV and BEI-CVV produced a neutralizing antibody response that exceeds the correlate of protection, that is, plaque reduction neutralization test titer >10. However, on day 63 postinitial immunization, 2delCVV was more immunogenic than BEI-CVV. These results warrant further development of 2delCVV as a candidate LAV and demonstrate that the double deletion of the NSs and NSm genes can be applied to the development of vaccines and as a common attenuation strategy for orthobunyaviruses.

Transcriptomic and enzymatic analysis reveals the roles of glutamate dehydrogenase in Corynebacterium glutamicum

Glutamate dehydrogenase (Gdh), catalyzing the reversible conversion between 2-oxoglutarate and glutamate, plays an important role in the connection of nitrogen and carbon metabolism. Yet little is known about these enzymes in the amino acid-manufacturing Corynebacterium glutamicum. In the present study, we firstly identified the enzymatic characteristics of two Gdhs (GdhA and GdhB). The results showed that both GdhA and GdhB prefers NADPH as a coenzyme and have higher affinity for 2-OG than glutamate. The growth characteristics of gdhAΔ mutant and gdhBΔ mutant, gdhABΔ mutant showed GdhA serves as the main conduit for ammonium assimilation, and GdhB is the main glutamate- metabolizing enzyme in C. glutamicum. The full-genome transcriptomic analysis was used to investigate physiological response of C. glutamicum to the glutamate as nitrogen source, and gdh deletion. The results showed that the nitrogen starvation response was elicited when glutamine served as the sole nitrogen source. gdhAΔBΔ double deletion trigger a partially deregulated nitrogen starvation response, in which genes involved in nitrogen assimilation showed obviously upregulated in a certain extent. On the other hand, the genes of phosphotransferase system (PTS) and glycolysis pathway, most genes in pentose phosphate pathway were significantly upregulated, indicating that gdh deficiency initiated the enhancement of the absorption and metabolism of carbon sources. We believed that our results in this study will give new insights on the molecular mechanism of Gdh activity cross-talks with carbon and nitrogen metabolism, also setting a new background for further flux redistribution applied research of biotechnological interest.

Reduction of Ethyl Carbamate in an Alcoholic Beverage by CRISPR/Cas9-Based Genome Editing of the Wild Yeast.

Ethyl carbamate (EC) is a naturally occurring substance in alcoholic beverages from the reaction of ethanol with urea during fermentation and storage. EC can cause dizziness and vomiting when consumed in small quantities and develop kidney cancer when consumed in excess. Thus, the reduction of EC formation in alcoholic beverages is important for food safety and human health. One of the strategies for reducing EC contents in alcoholic beverages is developing a new yeast starter strain to enable less formation of EC during fermentation. In this study, we isolated a polyploid wild-type yeast Saccharomyces cerevisiae strain from the Nuruk (Korean traditional grain-based inoculum of wild yeast and mold) and developed a starter culture by genome engineering to reduce EC contents in alcoholic beverages. We deleted multiple copies of the target genes involved in the EC formation in the S. cerevisiae by a CRISPR/Cas9-based genome editing tool. First, the CAR1 gene encoding for the arginase enzyme responsible for the formation of urea was completely deleted in the genome of S. cerevisiae. Additionally, the GZF3 gene encoding the transcription factor controlling expression levels of several genes (DUR1, 2, and DUR3) related to urea absorption and degradation was deleted in S. cerevisiae to further reduce the EC formation. The effects of gene deletion were validated by RT-qPCR to confirm changes in transcriptional levels of the EC-related genes. The resulting strain of S. cerevisiae carrying a double deletion of CAR1 and GZF3 genes successfully reduced the EC contents in the fermentation medium without significant changes in alcohol contents and fermentation profiles when compared to the wild-type strain. Finally, we brewed the Korean traditional rice wine Makgeolli using the double deletion strain of S. cerevisiae dCAR1&GZF3, resulting in a significant reduction of the EC content in Makgeolli up to 41.6% when compared to the wild-type strain. This study successfully demonstrated the development of a starter culture to reduce the EC formation in an alcoholic beverage by CRISPR/Cas9 genome editing of the wild yeast.

Deciphering the global roles of Cold shock proteins in Listeria monocytogenes nutrient metabolism and stress tolerance.

Listeria monocytogenes (Lm) accounts for serious public health and food safety problems owing to its stress resilience and pathogenicity. Based on their regulatory involvement in global gene expression events, cold-shock domain family proteins (Csps) are crucial in expression of various stress fitness and virulence phenotypes in bacteria. Lm possesses three Csps (CspA, CspB, and CspD) whose regulatory roles in the context of the genetic diversity of this bacterium are not yet fully understood. We examined the impacts of Csps deficiency on Lm nutrient metabolism and stress tolerance using a set of csp deletion mutants generated in different genetic backgrounds. Phenotype microarrays (PM) analysis showed that the absence of Csps in ∆cspABD reduces carbon (C-) source utilization capacity and increases Lm sensitivity to osmotic, pH, various chemical, and antimicrobial stress conditions. Single and double csp deletion mutants in different Lm genetic backgrounds were used to further dissect the roles of individual Csps in these phenotypes. Selected PM-based observations were further corroborated through targeted phenotypic assays, confirming that Csps are crucial in Lm for optimal utilization of various C-sources including rhamnose and glucose as well as tolerance against NaCl, β-phenyethylamine (PEA), and food relevant detergent stress conditions. Strain and genetic lineage background-based differences, division of labour, epistasis, and functional redundancies among the Csps were uncovered with respect to their roles in various processes including C-source utilization, cold, and PEA stress resistance. Finally, targeted transcriptome analysis was performed, revealing the activation of csp gene expression under defined stress conditions and the impact of Csps on expression regulation of selected rhamnose utilization genes. Overall, our study shows that Csps play important roles in nutrient utilization and stress responses in Lm strains, contributing to traits that are central to the public health and food safety impacts of this pathogen.

Type 2C Protein Phosphatases MoPtc5 and MoPtc7 Are Crucial for Multiple Stress Tolerance, Conidiogenesis and Pathogenesis of Magnaporthe oryzae.

Protein kinases and phosphatases catalyze the phosphorylation and dephosphorylation of their protein substrates, respectively, and these are important mechanisms in cellular signal transduction. The rice blast fungus Magnaporthe oryzae possesses 6 protein phosphatases of type 2C class, including MoPtc1, 2, 5, 6, 7 and 8. However, only very little is known about the roles of these phosphatases in filamentous fungi. Here in, we deployed genetics and molecular biology techniques to identify, characterize and establish the roles of MoPtc5 and MoPtc7 in M. oryzae development and pathogenicity. We found that during pathogen-host interaction, MoPTC7 is differentially expressed. Double deletion of MoPTC7 and MoPTC5 suppressed the fungal vegetative growth, altered its cell wall integrity and reduced its virulence. The two genes were found indispensable for stress tolerance in the phytopathogen. We also demonstrated that disruption of any of the two genes highly affected appressorium turgor generation and Mps1 and Osm1 phosphorylation levels. Lastly, we demonstrated that both MoPtc5 and MoPtc7 are localized to mitochondria of different cellular compartments in the blast fungus. Taken together, our study revealed synergistic coordination of M. oryzae development and pathogenesis by the type 2C protein phosphatases.

140. Enterococcus faecalis CL Synthases Have Redundant Roles and Play A Major Role in Phospholipid Redistribution Associated with Daptomycin Resistance

Abstract Background Daptomycin (DAP) is a lipopeptide antibiotic targeting membrane anionic phospholipids (APLs) at the division septum. DAP resistance (DAP-R) has been associated with activation of the LiaFSR system resulting in redistribution of APL microdomains (likely containing cardiolipin, CL) away from the septum. E. faecalis (Efs) possess two CL synthase genes, (cls1 and cls2) and changes in Cls1 are associated with DAP-R. However, the roles of each enzyme are unknown. Here, we characterize the roles of cls genes in DAP-R in the context of LiaFSR activation. Methods cls1 and/or cls2 were deleted from Efs OG117 and OG117ΔliaX (DAP-R strain with an activated LiaFSR response). qRT-PCR was used to study gene expression of cls1 and cls2 in the cls mutants. Membrane lipid content was analyzed using hydrophilic interaction chromatography-mass spectrometry. Mutants were characterized by DAP minimum inhibitory concentration (MIC) using E-test and localization of APL microdomains with 10-N-nonyl-acridine orange. Results cls1 and cls2 are upregulated in exponential phase of DAP-R Efs OG117ΔliaX relative to DAP-S Efs OG117, with only cls1 upregulated in stationary phase. Deletion of cls1 or cls2 resulted in upregulation of the other cls gene, independent of activation of LiaFSR. Lipidomics analysis confirmed that deletion of both cls resulted in complete absence of cell membrane CL content. When comparing CL profiles of Δcls1 relative to Δcls2 in both DAP-S and DAP-R, both strains produced similar levels and species of CL. However, development of DAP-R caused a change in membrane lipid content, namely, an increase in CL with no significant difference in phosphatidylglycerol compared to DAP-S strain. Evaluation of CL species in DAP-R shows a shift towards species containing longer fatty acid chains and higher saturation. Independent deletions of cls1 or cls2 did not revert the DAP phenotype. In contrast, deletion of both cls genes decreased the DAP MIC (2-3 fold) relative to the parent strain and restored septal localization of APL microdomains. DAP MIC was restored upon trans complementation of either cls1 or cls2 into the double deletion mutant. Conclusion Our results support a major role of Cls in changes in cell membrane architecture and DAP-R in enterococci, with overlapping roles for Cls1 and Cls2. Disclosures Cesar A. Arias, MD, PhD, Entasis Phramceuticals: Grant/Research Support|MeMed Diagnostics: Grant/Research Support|Merck: Grant/Research Support.

Mutations in troABCD against Copper Overload in a copA Mutant of Streptococcus suis.

Streptococcus suis is a major swine pathogen that is increasingly recognized as a porcine zoonotic pathogen that threatens the health of both pigs and humans. Metal homeostasis plays a critical role during the process of bacterial infection. In this study, RNA sequencing was used to identify potential candidate genes involved in the maintenance of intracellular copper homeostasis. CopA was identified as the primary copper exporter in S. suis. The copA deletion mutant strain was found to be more sensitive to copper and accumulated more intracellular copper than the wild-type (WT) parent strain. In addition, adding manganese increased the ability of S. suis to resist copper, and the manganese transporter, TroABCD, was involved in tolerance to copper. The copA deletion mutant strain accumulated less copper when supplemented with manganese. Furthermore, when cultured with copper, the double deletion mutant (ΔcopAΔtroA) exhibited improved growth compared to the copA deletion mutant strain. In addition, the double deletion mutant (ΔcopAΔtroA) accumulated less copper than the copA deletion mutant strain. These data were consistent with a model wherein defective TroABCD resulted in decreased cellular copper accumulation and protected the strain against copper poisoning. IMPORTANCE Metal homeostasis plays a critical role during the process of bacterial infection. We identified three important potential candidate genes involved in maintenance of intracellular copper homeostasis. CopA was demonstrated to be the main copper exporter in Streptococcus suis, and manganese increased the tolerance of S. suis to copper. The double deletion mutant (ΔcopAΔtroA) improved growth ability over the copA deletion mutant strain in the presence of high concentrations of copper and accumulated less copper. These findings are consistent with a model wherein defective TroABCD resulted in decreased cellular accumulation of copper and protected the strain against copper poisoning.

Genetic Interaction of Global Regulators AflatfA and AflatfB Mediating Development, Stress Response and Aflatoxins B1 Production in Aspergillus flavus.

Aspergillus flavus produces carcinogenic and mutagenic aflatoxins, which cause economic losses and risk of food safety by contaminating grains, food and feed. In this study, we characterized two bZIP transcription factors, AflatfA and AflatfB, and their genetic interaction. Compared to the wild type (WT), AflatfA deletion and AflatfA and AflatfB double deletion both caused retarded vegetative growth of mycelia. Relative to WT, the AflatfA deletion strain (ΔAflatfA) and AflatfA and AflatfB double deletion strain (ΔAflatfAΔAflatfB) produced more sclerotia, whereas the AflatfB deletion strain (ΔAflatfB) produced less sclerotia. After 4 °C preservation and incubation at 50 °C, conidia viability dramatically decreased in the ΔAflatfA and ΔAflatfAΔAflatfB but ΔAflatfB mutants, whereas conidia viability of the ΔAflatfAΔAflatfB strain was higher after storage at 4 °C than in AflatfA mutant. Conidia of ΔAflatfA, ΔAflatfB and ΔAflatfAΔAflatfB strains significantly increased in sensitivity to H2O2 in comparison with WT. Compared to WT, the mycelium of ΔAflatfA and ΔAflatfB strains were more sensitive to H2O2; conversely, the ΔAflatfAΔAflatfB strain showed less sensitivity to H2O2. ΔAflatfA and ΔAflatfAΔAflatfB strains displayed less sensitivity to the osmotic reagents NaCl, KCl and Sorbitol, in comparison with WT and ΔAflatfB strains. When on YES medium and hosts corn and peanut, ΔAflatfA and ΔAflatfAΔAflatfB strains produced less aflatoxin B1 (AFB1) than ΔAflatfB, and the AFB1 yield of ΔAflatfB was higher than that of WT. When WT and mutants were inoculated on corn and peanut, the ΔAflatfA and ΔAflatfAΔAflatfB but not ΔAflatfB mutants produced less conidia than did WT. Taken together, this study reveals that AflatfA controls more cellular processes, and the function of AflatfA is stronger than that of AflatfB when of the same process is regulated, except the response to H2O2, which might result from the effect of AflatfA on the transcriptional level of AflatfB.

Extension of O-Linked Mannosylation in the Golgi Apparatus Is Critical for Cell Wall Integrity Signaling and Interaction with Host Cells in Cryptococcus neoformans Pathogenesis.

The human-pathogenic yeast Cryptococcus neoformans assembles two types of O-linked glycans on its proteins. In this study, we identified and functionally characterized the C. neoformans CAP6 gene, encoding an α1,3-mannosyltransferase responsible for the second mannose addition to minor O-glycans containing xylose in the Golgi apparatus. Two cell surface sensor proteins, Wml1 (WSC/Mid2-like) and Wml2, were found to be independent substrates of Cap6-mediated minor or Ktr3-mediated major O-mannosylation, respectively. The double deletion of KTR3 and CAP6 (ktr3Δ cap6Δ) completely blocked the mannose addition at the second position of O-glycans, resulting in the accumulation of proteins with O-glycans carrying only a single mannose. Tunicamycin (TM)-induced phosphorylation of the Mpk1 mitogen-activated protein kinase (MAPK) was greatly decreased in both ktr3Δ cap6Δ and wml1Δ wml2Δ strains. Transcriptome profiling of the ktr3Δ cap6Δ strain upon TM treatment revealed decreased expression of genes involved in the Mpk1-dependent cell wall integrity (CWI) pathway. Consistent with its defective growth under several stress conditions, the ktr3Δ cap6Δ strain was avirulent in a mouse model of cryptococcosis. Associated with this virulence defect, the ktr3Δ cap6Δ strain showed decreased adhesion to lung epithelial cells, decreased proliferation within macrophages, and reduced transcytosis of the blood-brain barrier (BBB). Notably, the ktr3Δ cap6Δ strain showed reduced induction of the host immune response and defective trafficking of ergosterol, an immunoreactive fungal molecule. In conclusion, O-glycan extension in the Golgi apparatus plays critical roles in various pathobiological processes, such as CWI signaling and stress resistance and interaction with host cells in C. neoformans. IMPORTANCE Cryptococcus neoformans assembles two types of O-linked glycans on its surface proteins, the more abundant major O-glycans that do not contain xylose residues and minor O-glycans containing xylose. Here, we demonstrate the role of the Cap6 α1,3-mannosyltransferase in the synthesis of minor O-glycans. Previously proposed to be involved in capsule biosynthesis, Cap6 works with the related Ktr3 α1,2-mannosyltransferase to synthesize O-glycans on their target proteins. We also identified two novel C. neoformans stress sensors that require Ktr3- and Cap6-mediated posttranslational modification for full function. Accordingly, the ktr3Δ cap6Δ double O-glycan mutant strain displays defects in stress signaling pathways, CWI, and ergosterol trafficking. Furthermore, the ktr3Δ cap6Δ strain is completely avirulent in a mouse infection model. Together, these results demonstrate critical roles for O-glycosylation in fungal pathogenesis. As there are no human homologs for Cap6 or Ktr3, these fungus-specific mannosyltransferases are novel targets for antifungal therapy.

Liver-adipose tissue crosstalk in alcohol-associated liver disease: The role of mTOR

BackgroundAlcohol-associated liver disease (ALD) is a major chronic liver disease around the world without successful treatment. Acute alcoholic hepatitis is one of the most severe forms of ALD with high mortality, which is often associated with binge drinking. Alcohol drinking dysregulates lipid metabolism, increases adipose tissue lipolysis, and induces liver steatosis and adipose tissue atrophy. Increasing evidence implicates that crosstalk of liver and adipose tissue in the pathogenesis of ALD. Mechanistic target of rapamycin (mTOR) is a phosphatidylinositol 3-kinase (PI3K)-like serine/threonine protein kinase that regulates lipid metabolism, cell proliferation and autophagy. However, the role of mTOR in regulating adipose-liver crosstalk in binge drinking-induced organ damage remains unclear. MethodsWe generated liver-specific and adipocyte-specific regulatory-associated protein of mTOR (Rptor) knockout (RptorLKO and RptorAKO) as well as Mtor knockout (MtorLKO and MtorAKO) mice, by crossing Rptorflox and Mtorflox mice with albumin Cre or adiponectin Cre mice, respectively. In addition, we generated liver and adipocyte double deletion of Rptor or Mtor (MtorLAKO and RptorLAKO) mice. The knockout mice with their matched wild-type littermates (RptorWT and MtorWT) were subjected to acute gavage of 7 g/kg ethanol. ResultsMice with adipocyte deletion of Rptor or Mtor developed hepatomegaly and adipose tissue atrophy. Alcohol gavage increased liver injury, hepatic steatosis and inflammation in mouse livers as demonstrated by elevated serum alanine aminotransferase activities, increased hepatic levels of triglyceride and increased hepatic numbers of CD68 positive macrophages in mouse livers after alcohol gavage. Liver injury was further exacerbated by deletion of adipocyte Rptor or Mtor. Serum adipokine array analysis revealed that increased levels of pro-inflammatory cytokines IL-6 and TNFα as well as chemokine MCP-1 following acute alcohol gavage in wild-type mice, which were further increased in adipocyte-specific Mtor or Rptor knockout mice. Conversely, levels of anti-inflammatory cytokine IL-10 decreased in adipocyte-specific Mtor or Rptor knockout mice. The levels of circulating fibroblast growth factor 21 (FGF21) increased whereas levels of circulating adiponectin and fetuin A decreased in wild-type mice after alcohol gavage. Intriguingly, adipocyte-specific Mtor or Rptor knockout mice already had decreased basal level of FGF21 which increased by alcohol gavage. Moreover, adipocyte-specific Mtor or Rptor knockout mice already had increased basal level of adiponectin and decreased fetuin A which were not further changed by alcohol gavage. ConclusionsAdipocyte but not hepatocyte ablation of Mtor pathway contributes to acute alcohol-induced liver injury with increased inflammation. Our results demonstrate the critical role of adipocyte mTOR in regulating the adipose-liver crosstalk in ALD.

Deletion of Double Copies of the US1 Gene Reduces the Infectivity of Recombinant Duck Plague Virus In Vitro and In Vivo.

Duck plague caused by duck plague virus (DPV) is one of the main diseases that seriously endangers the production of waterfowl. DPV possesses a large genome consisting of 78 open reading frames (ORFs), and understanding the function and mechanism of each encoded protein in viral replication and pathogenesis is the key to controlling duck plague outbreaks. US1 is one of the two genes located in the repeat regions of the DPV genome, but the function of its encoded protein in DPV replication and pathogenesis remains unclear. Previous studies found that the US1 gene or its homologs exist in almost all alphaherpesviruses, but the loci, functions, and pathogenesis of their encoded proteins vary among different viruses. Here, we aimed to define the roles of US1 genes in DPV infection and pathogenesis by generating a double US1 gene deletion mutant and its revertant without any mini-F cassette retention. In vitro and in vivo studies found that deletion of both copies of the US1 gene significantly impaired the replication, gene expression, and virulence of DPV, which could represent a potential candidate vaccine strain for the prevention of duck plague. IMPORTANCE Duck plague virus contains nearly 80 genes, but the functions and mechanisms of most of the genes have not yet been elucidated, including those of the newly identified immediate early gene US1. Here, we found that US1 deletion reduces viral gene expression, replication, and virus production both in vitro and in vivo. This insight defines a fundamental role of the US1 gene in DPV infection and indicates its involvement in DPV transcription. These results provide clues for the study of the pathogenesis of the US1 gene and the development of attenuated vaccines targeting this gene.