Candida parapsilosis: Heterogeneous and strain-specific expression of secreted aspartic proteases (Sapp1 and Sapp2).

The increasing prevalence of Candida parapsilosis as a causative agent of fungal infections underscores the need to comprehensively understand its virulence factors. Secreted aspartic proteases (Saps) play a significant role in adhesion events, promoting biofilm formation, causing tissue damage and evading the host's immune response. In C. parapsilosis, three Saps have been identified: Sapp1, Sapp2 and Sapp3. The present study investigates the production dynamics of Sapp1 and Sapp2 across 10 clinical isolates of C. parapsilosis using various approaches. Each fungal isolate demonstrated the capability to utilize bovine serum albumin (BSA) as the sole nitrogen source, as evidenced by its degradation in a cell-free culture medium, forming low molecular mass polypeptides. Interestingly, the degradation of different proteinaceous substrates, such as BSA, human serum albumin (HSA), gelatin and hemoglobin, was typically isolate-dependent. Notably, higher proteolysis of HSA compared to BSA, gelatin and hemoglobin was observed. A quantitative assay revealed that the cleavage of a peptide fluorogenic substrate (cathepsin D) was isolate-specific, ranging from 44.15 to 270.61 fluorescence arbitrary units (FAU), with a mean proteolysis of 150.7 FAU. The presence of both Sapp1 and Sapp2 antigens on the cell surface of these fungal isolates was confirmed through immunological detection employing specific anti-Sapp1 and anti-Sapp2 antibodies. The surface levels of Sapp1 were consistently higher, up to fourfold, compared to Sapp2. Similarly, higher levels of Sapp1 than Sapp2 were detected in fungal secretions. This study provides insights into the dynamic expression and regulation of Sapps in C. parapsilosis, highlighting a known virulence factor that is considered a potential target for drug development against this increasingly prominent pathogen.

The fungal pathogen Candida parapsilosis can secrete aspartic proteases (Sapps) as part of its arsenal of virulence factors. We demonstrated that Sapps were able to cleave key host proteins, and the production of Sapp1 and Sapp2 antigens was typically dependent on the fungal isolate when grown in both planktonic- and biofilm-forming cells.

Litchi aspartic protease LcAP1 enhances plant resistance via suppressing cell death triggered by the pectate lyase PlPeL8 from Peronophythora litchii.

Plant cell death is regulated in plant-pathogen interactions. While some aspartic proteases (APs) participate in regulating programmed cell death or defense responses, the defense functions of most APs remain largely unknown. Here, we report on a virulence factor, PlPeL8, which is a pectate lyase found in the hemibiotrophic pathogen Peronophythora litchii. Through in vivo and in vitro assays, we confirmed the interaction between PlPeL8 and LcAP1 from litchi, and identified LcAP1 as a positive regulator of plant immunity. PlPeL8 induced cell death associated with NbSOBIR1 and NbMEK2. The 11 conserved residues of PlPeL8 were essential for inducing cell death and enhancing plant susceptibility. Twenty-three LcAPs suppressed cell death induced by PlPeL8 in Nicotiana benthamiana depending on their interaction with PlPeL8. The N-terminus of LcAP1 was required for inhibiting PlPeL8-triggered cell death and susceptibility. Furthermore, PlPeL8 led to higher susceptibility in NbAPs-silenced N. benthamiana than the GUS-control. Our results indicate the crucial roles of LcAP1 and its homologs in enhancing plant resistance via suppression of cell death triggered by PlPeL8, and LcAP1 represents a promising target for engineering disease resistance. Our study provides new insights into the role of plant cell death in the arms race between plants and hemibiotrophic pathogens.

Characterization of a novel aspartic protease from Trichoderma asperellum for the preparation of duck blood peptides.

A novel aspartic protease gene (TaproA1) from Trichoderma asperellum was successfully expressed in Komagataella phaffii (Pichia pastoris). TaproA1 showed 52.8% amino acid sequence identity with the aspartic protease PEP3 from Coccidioides posadasii C735. TaproA1 was efficiently produced in a 5 L fermenter with a protease activity of 4092 U/mL. It exhibited optimal reaction conditions at pH 3.0 and 50 °C and was stable within pH 3.0-6.0 and at temperatures up to 45 °C. The protease exhibited broad substrate specificity with high hydrolysis activity towards myoglobin and hemoglobin. Furthermore, duck blood proteins (hemoglobin and plasma protein) were hydrolyzed by TaproA1 to prepare bioactive peptides with high ACE inhibitory activity. The IC50 values of hemoglobin and plasma protein hydrolysates from duck blood proteins were 0.105 mg/mL and 0.091 mg/mL, respectively. Thus, the high yield and excellent biochemical characterization of TaproA1 presented here make it a potential candidate for the preparation of duck blood peptides. KEY POINTS: • An aspartic protease (TaproA1) from Trichoderma asperellum was expressed in Komagataella phaffii. • TaproA1 exhibited broad substrate specificity and the highest activity towards myoglobin and hemoglobin. • TaproA1 has great potential for the preparation of bioactive peptides from duck blood proteins.

Cocaprins, ß-Trefoil Fold Inhibitors of Cysteine and Aspartic Proteases from Coprinopsis cinerea.

We introduce a new family of fungal protease inhibitors with ß-trefoil fold from the mushroom Coprinopsis cinerea, named cocaprins, which inhibit both cysteine and aspartic proteases. Two cocaprin-encoding genes are differentially expressed in fungal tissues. One is highly transcribed in vegetative mycelium and the other in the stipes of mature fruiting bodies. Cocaprins are small proteins (15 kDa) with acidic isoelectric points that form dimers. The three-dimensional structure of cocaprin 1 showed similarity to fungal ß-trefoil lectins. Cocaprins inhibit plant C1 family cysteine proteases with Ki in the micromolar range, but do not inhibit the C13 family protease legumain, which distinguishes them from mycocypins. Cocaprins also inhibit the aspartic protease pepsin with Ki in the low micromolar range. Mutagenesis revealed that the ß2-ß3 loop is involved in the inhibition of cysteine proteases and that the inhibitory reactive sites for aspartic and cysteine proteases are located at different positions on the protein. Their biological function is thought to be the regulation of endogenous proteolytic activities or in defense against fungal antagonists. Cocaprins are the first characterized aspartic protease inhibitors with ß-trefoil fold from fungi, and demonstrate the incredible plasticity of loop functionalization in fungal proteins with ß-trefoil fold.

Cryptococcal Protease(s) and the Activation of SARS-CoV-2 Spike (S) Protein.

In this contribution, we report on the possibility that cryptococcal protease(s) could activate the SARS-CoV-2 spike (S) protein. The S protein is documented to have a unique four-amino-acid sequence (underlined, SPRRAR↓S) at the interface between the S1 and S2 sites, that serves as a cleavage site for the human protease, furin. We compared the biochemical efficiency of cryptococcal protease(s) and furin to mediate the proteolytic cleavage of the S1/S2 site in a fluorogenic peptide. We show that cryptococcal protease(s) processes this site in a manner comparable to the efficiency of furin (p > 0.581). We conclude the paper by discussing the impact of these findings in the context of a SARS-CoV-2 disease manifesting while there is an underlying cryptococcal infection.

Re-emerging Aspartic Protease Targets: Examining Cryptococcus neoformans Major Aspartyl Peptidase 1 as a Target for Antifungal Drug Discovery.

Cryptococcosis is an invasive infection that accounts for 15% of AIDS-related fatalities. Still, treating cryptococcosis remains a significant challenge due to the poor availability of effective antifungal therapies and emergence of drug resistance. Interestingly, protease inhibitor components of antiretroviral therapy regimens have shown some clinical benefits in these opportunistic infections. We investigated Major aspartyl peptidase 1 (May1), a secreted Cryptococcus neoformans protease, as a possible target for the development of drugs that act against both fungal and retroviral aspartyl proteases. Here, we describe the biochemical characterization of May1, present its high-resolution X-ray structure, and provide its substrate specificity analysis. Through combinatorial screening of 11,520 compounds, we identified a potent inhibitor of May1 and HIV protease. This dual-specificity inhibitor exhibits antifungal activity in yeast culture, low cytotoxicity, and low off-target activity against host proteases and could thus serve as a lead compound for further development of May1 and HIV protease inhibitors.

Different expression levels of ALS and SAP genes contribute to recurrent vulvovaginal candidiasis by Candida albicans.

Aim: To study the behavior of Candida albicans in women with vulvovaginal candidiasis (VVC), recurrent VVC (RVVC) and asymptomatic (AS), regarding adhesion on HeLa cells and their ability to express secreted aspartic proteinases (SAP) genes, agglutinin-like sequence (ALS) genes and HWP1. Materials & methods: The adhesion of Candida albicans to HeLa cells was evaluated by colony-forming units, and the expressed genes were evaluated by qRT-PCR. Results: AS and VVC isolates showed greater ability to adhere HeLa cells when compared with RVVC isolate. Nevertheless, RVVC isolate exhibited upregulation of a large number of genes of ALS and SAP gene families and HWP1 gene. Conclusion: The results demonstrated that RVVC isolate expressed significantly important genes for invasion and yeast-host interactions.

An Opaque Cell-Specific Expression Program of Secreted Proteases and Transporters Allows Cell-Type Cooperation in Candida albicans.

An unusual feature of the opportunistic pathogen Candida albicans is its ability to switch stochastically between two distinct, heritable cell types called white and opaque. Here, we show that only opaque cells, in response to environmental signals, massively upregulate a specific group of secreted proteases and peptide transporters, allowing exceptionally efficient use of proteins as sources of nitrogen. We identify the specific proteases [members of the secreted aspartyl protease (SAP) family] needed for opaque cells to proliferate under these conditions, and we identify four transcriptional regulators of this specialized proteolysis and uptake program. We also show that, in mixed cultures, opaque cells enable white cells to also proliferate efficiently when proteins are the sole nitrogen source. Based on these observations, we suggest that one role of white-opaque switching is to create mixed populations where the different phenotypes derived from a single genome are shared between two distinct cell types.

Akkermansia muciniphila Aspartic Protease Amuc_1434* Inhibits Human Colorectal Cancer LS174T Cell Viability via TRAIL-Mediated Apoptosis Pathway.

Mucin2 (Muc2) is the main component of the intestinal mucosal layer and is highly expressed in mucous colorectal cancer. Previous studies conducted by our lab found that the recombinant protein Amuc_1434 (expressed in Escherichia coli prokaryote cell system, hereinafter termed Amuc_1434*), derived from Akkermansia muciniphila, can degrade Muc2. Thus, the main objective of this study was to explore the effects of Amuc_1434* on LS174T in colorectal cancer cells expressing Muc2. Results from this study demonstrated that Amuc_1434* inhibited the proliferation of LS174T cells, which was related to its ability to degrade Muc2. Amuc_1434* also blocked the G0/G1 phase of the cell cycle of LS174T cells and upregulated the expression of tumor protein 53 (p53), which is a cell cycle-related protein. In addition, Amuc_1434* promoted apoptosis of LS174T cells and increased mitochondrial ROS levels in LS174T cells. The mitochondrial membrane potential of LS174T cells was also downregulated by Amuc_1434*. Amuc_1434* can activate the death receptor pathway and mitochondrial pathway of apoptosis by upregulating tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL). In conclusion, our study was the first to demonstrate that the protein Amuc_1434* derived from Akkermansia muciniphila suppresses LS174T cell viability via TRAIL-mediated apoptosis pathway.

Molecular characterization of a Trichinella spiralis aspartic protease and its facilitation role in larval invasion of host intestinal epithelial cells.

BACKGROUND: T. spiralis aspartic protease has been identified in excretion/secretion (ES) proteins, but its roles in larval invasion are unclear. The aim of this study was to characterize T. spiralis aspartic protease-2 (TsASP2) and assess its roles in T. spiralis invasion into intestinal epithelial cells (IECs) using RNAi. METHODOLOGY/PRINCIPAL FINDINGS: Recombinant TsASP2 (rTsASP2) was expressed and purified. The native TsASP2 of 43 kDa was recognized by anti-rTsASP2 serum in all worm stages except newborn larvae (NBL), and qPCR indicated that TsASP2 transcription was highest at the stage of intestinal infective larvae (IIL). IFA results confirmed that TsASP2 was located in the hindgut, midgut and muscle cells of muscle larvae (ML) and IIL and intrauterine embryos of the female adult worm (AW), but not in NBL. rTsASP2 cleaved several host proteins (human hemoglobin (Hb), mouse Hb, collagen and IgM). The proteolytic activity of rTsASP2 was host-specific, as it hydrolyzed mouse Hb more efficiently than human Hb. The enzymatic activity of rTsASP2 was significantly inhibited by pepstatin A. The expression levels of TsASP2 mRNA and protein were significantly suppressed by RNAi with 5 µM TsASP2-specific siRNA. Native aspartic protease activity in ML crude proteins was reduced to 54.82% after transfection with siRNA. Larval invasion of IECs was promoted by rTsASP2 and inhibited by anti-rTsASP2 serum and siRNA. Furthermore, cell monolayer damage due to larval invasion was obviously alleviated when siRNA-treated larvae were used. The adult worm burden, length of adult worms and female fecundity were clearly reduced in mice challenged using siRNA-treated ML relative to the PBS group. CONCLUSIONS: rTsASP2 possesses the enzymatic activity of native aspartic protease and facilitates T. spiralis invasion of host IECs.

MicroRNA-3607 inhibits the tumorigenesis of colorectal cancer by targeting DDI2 and regulating the DNA damage repair pathway.

Mutations in the DNA damage repair (DDR) pathway are frequently detected in colorectal cancer (CRC). The dysregulation of miRNAs, such as oncogenes or tumor suppressors, participates in CRC tumorigenesis. A previous study showed that low miR-3607 expression correlated with poor survival in prostate cancer patients, but its role in CRC remains unclear. In this study, we analyzed miR-3607 expression Pan-Cancer data from the NCI's Genomic Data Commons (GDC) and found that miR-3607 was downregulated in lymphatic invasion patients and in recurrent cancer and correlated with Pan-Cancer patient survival. Functional studies indicated that the overexpression of miR-3607 decreased CRC cell proliferation, migration and invasion. Additionally, we used gene set enrichment analysis (GSEA), Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and a protein-protein interaction network to demonstrate that miR-3607 affects the DDR pathway. Luciferase reporter and apoptosis assays confirmed that DNA damage inducible 1 homolog 2 (DDI2) is the functional target of miR-3607. Therefore, miR-3607 inhibits the tumorigenesis of CRC probably by suppressing the oncogene DDI2, and it might serve as a novel target for CRC prediction and therapy.

A unique aspartyl protease gene expansion in Talaromyces marneffei plays a role in growth inside host phagocytes.

Aspartyl proteases are a widely represented class of proteolytic enzymes found in eukaryotes and retroviruses. They have been associated with pathogenicity in a range of disease-causing microorganisms. The dimorphic human-pathogenic fungus Talaromyces marneffei has a large expansion of these proteases identified through genomic analyses. Here we characterize the expansion of these genes (pop - paralogue of pep) and their role in T. marneffei using computational and molecular approaches. Many of the genes in this monophyletic family show copy number variation and positive selection despite the preservation of functional regions and possible redundancy. We show that the expression profile of these genes differs and some are expressed during intracellular growth in the host. Several of these proteins have distinctive localization as well as both additive and epistatic effects on the formation of yeast cells during macrophage infections. The data suggest that this is a recently evolved aspartyl protease gene family which affects intracellular growth and contributes to the pathogenicity of T. marneffei.

An atypical aspartic protease modulates lateral root development in Arabidopsis thaliana.

Few atypical aspartic proteases (APs) present in plants have been functionally studied to date despite having been implicated in developmental processes and stress responses. Here we characterize a novel atypical AP that we name Atypical Aspartic Protease in Roots 1 (ASPR1), denoting its expression in Arabidopsis roots. Recombinant ASPR1 produced by transient expression in Nicotiana benthamiana was active and displayed atypical properties, combining optimum acidic pH, partial sensitivity to pepstatin, pronounced sensitivity to redox agents, and unique specificity preferences resembling those of fungal APs. ASPR1 overexpression suppressed primary root growth and lateral root development, implying a previously unknown biological role for an AP. Quantitative comparison of wild-type and aspr1 root proteomes revealed deregulation of proteins associated with both reactive oxygen species and auxin homeostasis in the mutant. Together, our findings on ASPR1 reinforce the diverse pattern of enzymatic properties and biological roles of atypical APs and raise exciting questions on how these distinctive features impact functional specialization among these proteases.

Atypical and nucellin-like aspartic proteases: emerging players in plant developmental processes and stress responses.

Members of the pepsin-like family (A1) of aspartic proteases (APs) are widely distributed in plants. A large number of genes encoding putative A1 APs are found in different plant genomes, the vast majority of which exhibit distinct features when compared with the so-called typical APs (and, therefore, grouped as atypical and nucellin-like APs). These features include the absence of the plant-specific insert; an unusually high number of cysteine residues; the nature of the amino acids preceding the first catalytic aspartate; and unexpected localizations. The over-representation of atypical and nucellin-like APs in plants is suggestive of greater diversification of protein functions and a more regulatory role for these APs, as compared with the housekeeping function generally attributed to typical APs. New functions have been uncovered for non-typical APs, with proposed roles in biotic and abiotic stress responses, chloroplast metabolism, and reproductive development, clearly suggesting functional specialization and tight regulation of activity. Furthermore, unusual enzymatic properties have also been documented for some of these proteases. Here, we give an overview of the current knowledge on the distinctive features and functions of both atypical and nucellin-like APs, and discuss this emerging pattern of functional complexity and specialization among plant pepsin-like proteases.

The effect of released new synthetic peptide from nanofibrous scaffold of peptide/Poly (Vinyl Alcohol)/Poly l-Lactic Acid on expression of Secretory aspartyl proteinases 4 to 6 genes of Candida albicans.

Secreted aspartyl proteinases (Saps), are gene products that have been shown to directly contribute to Candida albicans pathogenicity. Despite the clear difficulties of systemic C. albicans infections control, Antimicrobial peptides (AMPs) are regarded as one of the most promising alternatives in this regard. Recently, drug-loaded electrospun nanofibers have attracted a great deal of attention. In this study we have established the nanofibrous scaffold of new synthetic peptide/Poly (Vinyl Alcohol)/Poly l-Lactic Acid on expression of Secretory aspartyl proteinases 4 to 6 genes of C. albicans in comparison with free peptide. We designed new synthetic Antimicrobial peptide (AMPs) used bioinformatics tools to predict structure and stabilities. The electrospinning method was used to produce the polymeric nanofibers. Scanning Electron Microscopy (SEM) was used to measure the nanofibers diameters and morphology. To analyze the expression of SAP4, SAP5 and SAP6 genes, RNA was extracted from clinical isolates of C. albicans before and after treatment with subinhibitory concentrations of new synthetic peptide on nanofibrous scaffold of new synthetic peptide/PVA/PLLA and then the cDNA was synthesized and used for Real-time PCR assay. Scanning electron microscopy (SEM) images showed that the morphology, the diameter, are affected from peptide. Reletive quantitative Real-time PCR results revealed that the mRNA levels of SAP4, SAP5 and SAP6 genes significantly decreased after treatment with nanofibrous scaffold of new synthetic peptide/PVA/PLLA (P < .05). Electrospun nanofibrous of new Synthetic Peptide/PVA/PLLA is effective in downregulating of expression SAP4, SAP5 and SAP6 genes of C. albicans strains.

Citrus Psorosis Virus Movement Protein Contains an Aspartic Protease Required for Autocleavage and the Formation of Tubule-Like Structures at Plasmodesmata.

Plant virus cell-to-cell movement is an essential step in viral infections. This process is facilitated by specific virus-encoded movement proteins (MPs), which manipulate the cell wall channels between neighboring cells known as plasmodesmata (PD). Citrus psorosis virus (CPsV) infection in sweet orange involves the formation of tubule-like structures within PD, suggesting that CPsV belongs to "tubule-forming" viruses that encode MPs able to assemble a hollow tubule extending between cells to allow virus movement. Consistent with this hypothesis, we show that the MP of CPsV (MPCPsV) indeed forms tubule-like structures at PD upon transient expression in Nicotiana benthamiana leaves. Tubule formation by MPCPsV depends on its cleavage capacity, mediated by a specific aspartic protease motif present in its primary sequence. A single amino acid mutation in this motif abolishes MPCPsV cleavage, alters the subcellular localization of the protein, and negatively affects its activity in facilitating virus movement. The amino-terminal 34-kDa cleavage product (34KCPsV), but not the 20-kDa fragment (20KCPsV), supports virus movement. Moreover, similar to tubule-forming MPs of other viruses, MPCPsV (and also the 34KCPsV cleavage product) can homooligomerize, interact with PD-located protein 1 (PDLP1), and assemble tubule-like structures at PD by a mechanism dependent on the secretory pathway. 20KCPsV retains the protease activity and is able to cleave a cleavage-deficient MPCPsV in trans Altogether, these results demonstrate that CPsV movement depends on the autolytic cleavage of MPCPsV by an aspartic protease activity, which removes the 20KCPsV protease and thereby releases the 34KCPsV protein for PDLP1-dependent tubule formation at PD.IMPORTANCE Infection by citrus psorosis virus (CPsV) involves a self-cleaving aspartic protease activity within the viral movement protein (MP), which results in the production of two peptides, termed 34KCPsV and 20KCPsV, that carry the MP and viral protease activities, respectively. The underlying protease motif within the MP is also found in the MPs of other members of the Aspiviridae family, suggesting that protease-mediated protein processing represents a conserved mechanism of protein expression in this virus family. The results also demonstrate that CPsV and potentially other ophioviruses move by a tubule-guided mechanism. Although several viruses from different genera were shown to use this mechanism for cell-to-cell movement, our results also demonstrate that this mechanism is controlled by posttranslational protein cleavage. Moreover, given that tubule formation and virus movement could be inhibited by a mutation in the protease motif, targeting the protease activity for inactivation could represent an important approach for ophiovirus control.

Quantitative proteomics reveals neuronal ubiquitination of Rngo/Ddi1 and several proteasomal subunits by Ube3a, accounting for the complexity of Angelman syndrome.

Angelman syndrome is a complex neurodevelopmental disorder caused by the lack of function in the brain of a single gene, UBE3A. The E3 ligase coded by this gene is known to build K48-linked ubiquitin chains, a modification historically considered to target substrates for degradation by the proteasome. However, a change in protein abundance is not proof that a candidate UBE3A substrate is indeed ubiquitinated by UBE3A. We have here used an unbiased ubiquitin proteomics approach, the bioUb strategy, to identify 79 proteins that appear more ubiquitinated in the Drosophila photoreceptor cells when Ube3a is over-expressed. We found a significantly high number of those proteins to be proteasomal subunits or proteasome-interacting proteins, suggesting a wide proteasomal perturbation in the brain of Angelman patients. We focused on validating the ubiquitination by Ube3a of Rngo, a proteasomal component conserved from yeast (Ddi1) to humans (DDI1 and DDI2), but yet scarcely characterized. Ube3a-mediated Rngo ubiquitination in fly neurons was confirmed by immunoblotting. Using human neuroblastoma SH-SY5Y cells in culture, we also observed that human DDI1 is ubiquitinated by UBE3A, without being targeted for degradation. The novel observation that DDI1 is expressed in the developing mice brain, with a significant peak at E16.5, strongly suggests that DDI1 has biological functions not yet described that could be of relevance for Angelman syndrome clinical research.

Identification of Placental Aspartic Proteinase in the Eurasian Beaver (Castor fiber L.).

Aspartic proteinases (AP) form a multigenic group widely distributed in various organisms and includes pepsins (pep), cathepsins D and E, pregnancy associated glycoproteins (PAGs) as well as plant, fungal, and retroviral proteinases. This study describes the transcript identification and expression localization of the AP within the discoid placenta of the Castor fiber. We identified 1257 bp of the AP cDNA sequence, encoding 391 amino acids (aa) of the polypeptide precursor composed of 16 aa signal peptide, 46 aa pro-piece, and 329 aa of the mature protein. Within the AP precursor, one site of potential N-glycosylation (NPS119–121) and two Asp residues (D) specific for the catalytic cleft of AP were identified (VLFDTGSSNLWV91–102 and GIVDTGTSLLTV277–288). The highest homology of the identified placental AP nucleotide and aa sequence was to mouse pepsinogen C (75.8% and 70.1%, respectively). Identified AP also shared high homology with other superfamily members: PAGs, cathepsins, and napsins. The AP identified in this study was named as pepsinogen/PAG-Like (pep/PAG-L). Diversified pep/PAG-L protein profiles with a dominant 58 kDa isoform were identified. Immune reactive signals of the pep/PAG-L were localized within the trophectodermal cells of the beaver placenta. This is the first report describing the placental AP (pep/PAG-L) in the C. fiber.

Aspartyl proteases in Candida glabrata are required for suppression of the host innate immune response.

A family of 11 cell surface-associated aspartyl proteases (CgYps1-11), also referred as yapsins, is a key virulence factor in the pathogenic yeast Candida glabrata However, the mechanism by which CgYapsins modulate immune response and facilitate survival in the mammalian host remains to be identified. Here, using RNA-Seq analysis, we report that genes involved in cell wall metabolism are differentially regulated in the Cgyps1-11Δ mutant. Consistently, the mutant contained lower ß-glucan and mannan levels and exhibited increased chitin content in the cell wall. As cell wall components are known to regulate the innate immune response, we next determined the macrophage transcriptional response to C. glabrata infection and observed differential expression of genes implicated in inflammation, chemotaxis, ion transport, and the tumor necrosis factor signaling cascade. Importantly, the Cgyps1-11Δ mutant evoked a different immune response, resulting in an enhanced release of the pro-inflammatory cytokine IL-1ß in THP-1 macrophages. Further, Cgyps1-11Δ-induced IL-1ß production adversely affected intracellular proliferation of co-infected WT cells and depended on activation of spleen tyrosine kinase (Syk) signaling in the host cells. Accordingly, the Syk inhibitor R406 augmented intracellular survival of the Cgyps1-11Δ mutant. Finally, we demonstrate that C. glabrata infection triggers elevated IL-1ß production in mouse organs and that the CgYPS genes are required for organ colonization and dissemination in the murine model of systemic infection. Altogether, our results uncover the basis for macrophage-mediated killing of Cgyps1-11Δ cells and provide the first evidence that aspartyl proteases in C. glabrata are required for suppression of IL-1ß production in macrophages.

Removal of RTF2 from Stalled Replisomes Promotes Maintenance of Genome Integrity.

The protection and efficient restart of stalled replication forks is critical for the maintenance of genome integrity. Here, we identify a regulatory pathway that promotes stalled forks recovery from replication stress. We show that the mammalian replisome component C20orf43/RTF2 (homologous to S. pombe Rtf2) must be removed for fork restart to be optimal. We further show that the proteasomal shuttle proteins DDI1 and DDI2 are required for RTF2 removal from stalled forks. Persistence of RTF2 at stalled forks results in fork restart defects, hyperactivation of the DNA damage signal, accumulation of single-stranded DNA (ssDNA), sensitivity to replication drugs, and chromosome instability. These results establish that RTF2 removal is a key determinant for the ability of cells to manage replication stress and maintain genome integrity.