Flp/FRT-mediated disruption of ptex150 and exp2 in Plasmodium falciparum sporozoites inhibits liver-stage development.

Plasmodium falciparum causes severe malaria and assembles a protein translocon (PTEX) complex at the parasitophorous vacuole membrane (PVM) of infected erythrocytes, through which several hundred proteins are exported to facilitate growth. The preceding liver stage of infection involves growth in a hepatocyte-derived PVM; however, the importance of protein export during P. falciparum liver infection remains unexplored. Here, we use the FlpL/FRT system to conditionally excise genes in P. falciparum sporozoites for functional liver-stage studies. Disruption of PTEX members ptex150 and exp2 did not affect sporozoite development in mosquitoes or infectivity for hepatocytes but attenuated liver-stage growth in humanized mice. While PTEX150 deficiency reduced fitness on day 6 postinfection by 40%, EXP2 deficiency caused 100% loss of liver parasites, demonstrating that PTEX components are required for growth in hepatocytes to differing degrees. To characterize PTEX loss-of-function mutations, we localized four liver-stage Plasmodium export element (PEXEL) proteins. P. falciparum liver specific protein 2 (LISP2), liver-stage antigen 3 (LSA3), circumsporozoite protein (CSP), and a Plasmodium berghei LISP2 reporter all localized to the periphery of P. falciparum liver stages but were not exported beyond the PVM. Expression of LISP2 and CSP but not LSA3 was reduced in ptex150-FRT and exp2-FRT liver stages, suggesting that expression of some PEXEL proteins is affected directly or indirectly by PTEX disruption. These results show that PTEX150 and EXP2 are important for P. falciparum development in hepatocytes and emphasize the emerging complexity of PEXEL protein trafficking.

Novel Ion Channel Genes in Malaria Parasites.

Ion channels serve many cellular functions including ion homeostasis, volume regulation, signaling, nutrient acquisition, and developmental progression. Although the complex life cycles of malaria parasites necessitate ion and solute flux across membranes, the whole-genome sequencing of the human pathogen Plasmodium falciparum revealed remarkably few orthologs of known ion channel genes. Contrasting with this, biochemical studies have implicated the channel-mediated flux of ions and nutritive solutes across several membranes in infected erythrocytes. Here, I review advances in the cellular and molecular biology of ion channels in malaria parasites. These studies have implicated novel parasite genes in the formation of at least two ion channels, with additional ion channels likely present in various membranes and parasite stages. Computational approaches that rely on homology to known channel genes from higher organisms will not be very helpful in identifying the molecular determinants of these activities. Given their unusual properties, novel molecular and structural features, and essential roles in pathogen survival and development, parasite channels should be promising targets for therapy development.

The balance between nuclear import and export of NLRC5 regulates MHC class I transactivation.

Major histocompatibility complex (MHC) class I molecules play an essential role in regulating the adaptive immune system by presenting antigens to CD8 T cells. CITA (MHC class I transactivator), also known as NLRC5 (NLR family, CARD domain-containing 5), regulates the expression of MHC class I and essential components involved in the MHC class I antigen presentation pathway. While the critical role of the nuclear distribution of NLRC5 in its transactivation activity has been known, the regulatory mechanism to determine the nuclear localization of NLRC5 remains poorly understood. In this study, a comprehensive analysis of all domains in NLRC5 revealed that the regulatory mechanisms for nuclear import and export of NLRC5 coexist and counterbalance each other. Moreover, GCN5 (general control non-repressed 5 protein), a member of HATs (histone acetyltransferases), was found to be a key player to retain NLRC5 in the nucleus, thereby contributing to the expression of MHC class I. Therefore, the balance between import and export of NLRC5 has emerged as an additional regulatory mechanism for MHC class I transactivation, which would be a potential therapeutic target for the treatment of cancer and virus-infected diseases.

Identification of a new export signal that targets early subunits to the flagellar type III secretion export machinery.

Type III secretion systems (T3SSs) are essential for motility and virulence in many bacterial pathogens. Proteins destined for the flagellar T3SS contain at least two export signals in their N-terminal D0 domain. Here, we describe a third carboxy (C)-terminal signal in early flagellar subunits that facilitates subunit targeting to the export machinery. Mutational analysis identified critical residues within the flagellar hook subunit C-terminal export signal. The flagellar ATPase and cytoplasmic ring components were not required for this targeting, indicating that core export machinery components facilitate substrate targeting via the C-terminal export signal. More broadly, these results demonstrate that multiple distinct export signals within type III secretion substrates facilitate distinct export events at the T3SS export machinery. Our data establish key events in the export mechanism of type III secretion systems: targeting of subunits to and their sequential interactions with key components of the export machinery. IMPORTANCE: Many bacterial pathogens utilize T3SS to inject virulence proteins (effectors) into host cells or to assemble flagella on the bacterial cell surface. Bacterial flagella present a paradigm for how cells build and operate complex cell-surface "nanomachines." Efficient subunit targeting from the bacterial cytosol to type III secretion systems is essential for rapid assembly and secretion by T3SSs. Subunits are thought to dock at the export machinery before being unfolded and translocated into the export channel. However, little is known about how subunits dock at the export machinery and the events that occur post docking. Here, we identified a new export signal within the C-termini of subunits that is essential for targeting of subunits to the type III export machinery. We show that this new export signal and previously identified export signals are recognized separately and sequentially, revealing a pathway for subunit transit through the type III export machinery in which sequential recognition events carry out different roles at major steps in the export pathway.

Structure of a tripartite protein complex that targets toxins to the type VII secretion system.

Type VII secretion systems are membrane-embedded nanomachines used by Gram-positive bacteria to export effector proteins from the cytoplasm to the extracellular environment. Many of these effectors are polymorphic toxins comprised of an N-terminal Leu-x-Gly (LXG) domain of unknown function and a C-terminal toxin domain that inhibits the growth of bacterial competitors. In recent work, it was shown that LXG effectors require two cognate Lap proteins for T7SS-dependent export. Here, we present the 2.6 Å structure of the LXG domain of the TelA toxin from the opportunistic pathogen Streptococcus intermedius in complex with both of its cognate Lap targeting factors. The structure reveals an elongated α-helical bundle within which each Lap protein makes extensive hydrophobic contacts with either end of the LXG domain. Remarkably, despite low overall sequence identity, we identify striking structural similarity between our LXG complex and PE-PPE heterodimers exported by the distantly related ESX type VII secretion systems of Mycobacteria implying a conserved mechanism of effector export among diverse Gram-positive bacteria. Overall, our findings demonstrate that LXG domains, in conjunction with their cognate Lap targeting factors, represent a tripartite secretion signal for a widespread family of T7SS toxins.

Sequence elements within the PEXEL motif and its downstream region modulate PTEX-dependent protein export in Plasmodium falciparum.

The parasite Plasmodium falciparum causes the most severe form of malaria and to invade and replicate in red blood cells (RBCs), it exports hundreds of proteins across the encasing parasitophorous vacuole membrane (PVM) into this host cell. The exported proteins help modify the RBC to support rapid parasite growth and avoidance of the human immune system. Most exported proteins possess a conserved Plasmodium export element (PEXEL) motif with the consensus RxLxE/D/Q amino acid sequence, which acts as a proteolytic cleavage recognition site within the parasite's endoplasmic reticulum (ER). Cleavage occurs after the P1 L residue and is thought to help release the protein from the ER so it can be putatively escorted by the HSP101 chaperone to the parasitophorous vacuole space surrounding the intraerythrocytic parasite. HSP101 and its cargo are then thought to assemble with the rest of a Plasmodium translocon for exported proteins (PTEX) complex, that then recognises the xE/D/Q capped N-terminus of the exported protein and translocates it across the vacuole membrane into the RBC compartment. Here, we present evidence that supports a dual role for the PEXEL's conserved P2 ' position E/Q/D residue, first, for plasmepsin V cleavage in the ER, and second, for efficient PTEX mediated export across the PVM into the RBC. We also present evidence that the downstream 'spacer' region separating the PEXEL motif from the folded functional region of the exported protein controls cargo interaction with PTEX as well. The spacer must be of a sufficient length and permissive amino acid composition to engage the HSP101 unfoldase component of PTEX to be efficiently translocated into the RBC compartment.

Identification of a non-exported Plasmepsin V substrate that functions in the parasitophorous vacuole of malaria parasites.

IMPORTANCE: In the manuscript, the authors investigate the role of the protease Plasmepsin V in the parasite-host interaction. Whereas processing by Plasmepsin V was previously thought to target a protein for export into the host cell, the authors now show that there are proteins cleaved by this protease that are not exported but instead function at the host-parasite interface. This changes the view of this protease, which turns out to have a much broader role than anticipated. The result shows that the protease may have a function much more similar to that of related organisms. The authors also investigate the requirements for protein export by analyzing exported and non-exported proteins and find commonalities between the proteins of each set that further our understanding of the requirements for protein export.

Time-resolved proximity biotinylation implicates a porin protein in export of transmembrane malaria parasite effectors.

The malaria-causing parasite, Plasmodium falciparum completely remodels its host red blood cell (RBC) through the export of several hundred parasite proteins, including transmembrane proteins, across multiple membranes to the RBC. However, the process by which these exported membrane proteins are extracted from the parasite plasma membrane for export remains unknown. To address this question, we fused the exported membrane protein, skeleton binding protein 1 (SBP1), with TurboID, a rapid, efficient and promiscuous biotin ligase (SBP1TbID). Using time-resolved proximity biotinylation and label-free quantitative proteomics, we identified two groups of SBP1TbID interactors - early interactors (pre-export) and late interactors (post-export). Notably, two promising membrane-associated proteins were identified as pre-export interactors, one of which possesses a predicted translocon domain, that could facilitate the export of membrane proteins. Further investigation using conditional mutants of these candidate proteins showed that these proteins were essential for asexual growth and localize to the host-parasite interface during early stages of the intraerythrocytic cycle. These data suggest that they might play a role in ushering membrane proteins from the parasite plasma membrane for export to the host RBC.

The mature N-termini of Plasmodium effector proteins confer specificity of export.

IMPORTANCE: Malaria parasites export hundreds of proteins to the cytoplasm of the host red blood cells for their survival. A five amino acid sequence, called the PEXEL motif, is conserved among many exported proteins and is thought to be a signal for export. However, the motif is cleaved inside the endoplasmic reticulum of the parasite, and mature proteins starting from the fourth PEXEL residue travel to the parasite periphery for export. We showed that the PEXEL motif is dispensable for export as long as identical mature proteins can be efficiently produced via alternative means in the ER. We also showed that the exported and non-exported proteins are differentiated at the parasite periphery based on their mature N-termini; however, any discernible export signal within that region remained cryptic. Our study resolves a longstanding paradox in PEXEL protein trafficking.

Export of Diverse and Bioactive Small Proteins through a Type I Secretion System.

Small proteins perform a diverse array of functions, from microbial competition, to endocrine signaling, to building biomaterials. Microbial systems that can produce recombinant small proteins enable discovery of new effectors, exploration of sequence activity relationships, and have the potential for in vivo delivery. However, we lack simple systems for controlling small-protein secretion from Gram-negative bacteria. Microcins are small-protein antibiotics secreted by Gram-negative bacteria that inhibit the growth of neighboring microbes. They are exported from the cytosol to the environment in a one-step process through a specific class of type I secretion systems (T1SSs). However, relatively little is known about substrate requirements for small proteins exported through microcin T1SSs. Here, we investigate the prototypic microcin V T1SS from Escherichia coli and show that it can export a remarkably wide range of natural and synthetic small proteins. We demonstrate that secretion is largely independent of the cargo protein's chemical properties and appears to be constrained only by protein length. We show that a varied range of bioactive sequences, including an antibacterial protein, a microbial signaling factor, a protease inhibitor, and a human hormone, can all be secreted and elicit their intended biological effect. Secretion through this system is not limited to E. coli, and we demonstrate its function in additional Gram-negative species that can inhabit the gastrointestinal tract. Our findings uncover the highly promiscuous nature of small-protein export through the microcin V T1SS, which has implications for native-cargo capacity and the use of this system in Gram-negative bacteria for small-protein research and delivery. IMPORTANCE Type I secretion systems for microcin export in Gram-negative bacteria transport small antibacterial proteins from the cytoplasm to the extracellular environment in a single step. In nature, each secretion system is generally paired with a specific small protein. We know little about the export capacity of these transporters and how cargo sequence influences secretion. Here, we investigate the microcin V type I system. Remarkably, our studies show that this system can export small proteins of diverse sequence composition and is only limited by protein length. Furthermore, we demonstrate that a wide range of bioactive small proteins can be secreted and that this system can be used in Gram-negative species that colonize the gastrointestinal tract. These findings expand our understanding of secretion through type I systems and their potential uses in a variety of small-protein applications.

Dissecting EXP2 sequence requirements for protein export in malaria parasites.

Apicomplexan parasites that reside within a parasitophorous vacuole harbor a conserved pore-forming protein that enables small-molecule transfer across the parasitophorous vacuole membrane (PVM). In Plasmodium parasites that cause malaria, this nutrient pore is formed by EXP2 which can complement the function of GRA17, an orthologous protein in Toxoplasma gondii. EXP2, however, has an additional function in Plasmodium parasites, serving also as the pore-forming component of the protein export machinery PTEX. To examine how EXP2 can play this additional role, transgenes that encoded truncations of EXP2, GRA17, hybrid GRA17-EXP2, or EXP2 under the transcriptional control of different promoters were expressed in EXP2 knockdown parasites to determine which could complement EXP2 function. This revealed that EXP2 is a unique pore-forming protein, and its protein export role in P. falciparum cannot be complemented by T. gondii GRA17. This was despite the addition of the EXP2 assembly strand and part of the linker helix to GRA17, which are regions necessary for the interaction of EXP2 with the other core PTEX components. This indicates that the body region of EXP2 plays a critical role in PTEX assembly and/or that the absence of other T. gondii GRA proteins in P. falciparum leads to its reduced efficiency of insertion into the PVM and complementation potential. Altering the timing and abundance of EXP2 expression did not affect protein export but affected parasite viability, indicating that the unique transcriptional profile of EXP2 when compared to other PTEX components enables it to serve an additional role in nutrient exchange.

Activation mechanism of the bacterial flagellar dual-fuel protein export engine.

Bacteria employ the flagellar type III secretion system (fT3SS) to construct flagellum, which acts as a supramolecular motility machine. The fT3SS of Salmonella enterica serovar Typhimurium is composed of a transmembrane export gate complex and a cytoplasmic ATPase ring complex. The transmembrane export gate complex is fueled by proton motive force across the cytoplasmic membrane and is divided into four distinct functional parts: a dual-fuel export engine; a polypeptide channel; a membrane voltage sensor; and a docking platform. ATP hydrolysis by the cytoplasmic ATPase complex converts the export gate complex into a highly efficient proton (H+)/protein antiporter that couples inward-directed H+ flow with outward-directed protein export. When the ATPase ring complex does not work well in a given environment, the export gate complex will remain inactive. However, when the electric potential difference, which is defined as membrane voltage, rises above a certain threshold value, the export gate complex becomes an active H+/protein antiporter to a considerable degree, suggesting that the export gate complex has a voltage-gated activation mechanism. Furthermore, the export gate complex also has a sodium ion (Na+) channel to couple Na+ influx with flagellar protein export. In this article, we review our current understanding of the activation mechanism of the dual-fuel protein export engine of the fT3SS. This review article is an extended version of a Japanese article, Membrane voltage-dependent activation of the transmembrane export gate complex in the bacterial flagellar type III secretion system, published in SEIBUTSU BUTSURI Vol. 62, p165-169 (2022).

The PTEX Pore Component EXP2 Is Important for Intrahepatic Development during the Plasmodium Liver Stage.

During vertebrate infection, obligate intracellular malaria parasites develop within a parasitophorous vacuole, which constitutes the interface between the parasite and its hepatocyte or erythrocyte host cells. To traverse this barrier, Plasmodium spp. utilize a dual-function pore formed by EXP2 for nutrient transport and, in the context of the PTEX translocon, effector protein export across the vacuole membrane. While critical to blood-stage survival, less is known about EXP2/PTEX function in the liver stage, although major differences in the export mechanism are suggested by absence of the PTEX unfoldase HSP101 in the intrahepatic vacuole. Here, we employed the glucosamine-activated glmS ribozyme to study the role of EXP2 during Plasmodium berghei liver-stage development in hepatoma cells. Insertion of the glmS sequence into the exp2 3' untranslated region (UTR) enabled glucosamine-dependent depletion of EXP2 after hepatocyte invasion, allowing separation of EXP2 function during intrahepatic development from a recently reported role in hepatocyte invasion. Postinvasion EXP2 knockdown reduced parasite size and largely abolished expression of the mid- to late-liver-stage marker LISP2. As an orthogonal approach to monitor development, EXP2-glmS parasites and controls were engineered to express nanoluciferase. Activation of glmS after invasion substantially decreased luminescence in hepatoma monolayers and in culture supernatants at later time points corresponding to merosome detachment, which marks the culmination of liver-stage development. Collectively, our findings extend the utility of the glmS ribozyme to study protein function in the liver stage and reveal that EXP2 is important for intrahepatic parasite development, indicating that PTEX components also function at the hepatocyte-parasite interface. IMPORTANCE After the mosquito bite that initiates a Plasmodium infection, parasites first travel to the liver and develop in hepatocytes. This liver stage is asymptomatic but necessary for the parasite to transition to the merozoite form, which infects red blood cells and causes malaria. To take over their host cells, avoid immune defenses, and fuel their growth, these obligately intracellular parasites must import nutrients and export effector proteins across a vacuole membrane in which they reside. In the blood stage, these processes depend on a translocon called PTEX, but it is unclear if PTEX also functions during the liver stage. Here, we adapted the glmS ribozyme to control expression of EXP2, the membrane pore component of PTEX, during the liver stage of the rodent malaria parasite Plasmodium berghei. Our results show that EXP2 is important for intracellular development in the hepatocyte, revealing that PTEX components are also functionally important during liver-stage infection.

Importance of aspartyl protease 5 in the establishment of the intracellular niche during acute and chronic infection of Toxoplasma gondii.

Virulence and persistence of the obligate intracellular parasite Toxoplasma gondii involve the secretion of effector proteins belonging to the family of dense granule proteins (GRAs) that act notably as modulators of the host defense mechanisms and participate in cyst wall formation. The subset of GRAs residing in the parasitophorous vacuole (PV) or exported into the host cell, undergo proteolytic cleavage in the Golgi upon the action of the aspartyl protease 5 (ASP5). In tachyzoites, ASP5 substrates play central roles in the morphology of the PV and the export of effectors across the translocon complex MYR1/2/3. Here, we used N-terminal amine isotopic labeling of substrates to identify novel ASP5 cleavage products by comparing the N-terminome of wild-type and Δasp5 lines in tachyzoites and bradyzoites. Validated substrates reside within the PV or PVM in an ASP5-dependent manner. Remarkably, Δasp5 bradyzoites are impaired in the formation of the cyst wall in vitro and exhibit a considerably reduced cyst burden in chronically infected animals. More specifically two-photon serial tomography of infected mouse brains revealed a comparatively reduced number and size of the cysts throughout the establishment of persistence in the absence of ASP5.

Exported J domain proteins of the human malaria parasite.

The heat shock protein 40 (Hsp40) family, also called J domain proteins (JDPs), regulate their Hsp70 partners by ensuring that they are engaging the right substrate at the right time and in the right location within the cell. A number of JDPs can serve as co-chaperone for a particular Hsp70, and so one generally finds many more JDPs than Hsp70s in the cell. In humans there are 13 Hsp70s and 49 JDPs. The human malaria parasite, Plasmodium falciparum, has dedicated an unusually large proportion of its genome to molecular chaperones, with a disproportionately high number of JDPs (PfJDPs) of 49 members. Interestingly, just under half of the PfJDPs are exported into the host cell during the asexual stage of the life cycle, when the malaria parasite invades mature red blood cells. Recent evidence suggests that these PfJDPs may be functionalizing both host and parasite Hsp70s within the infected red blood cell, and thereby driving the renovation of the host cell towards pathological ends. PfJDPs have been found to localize to the host cytosol, mobile structures within the host cytosol (so called "J Dots"), the host plasma membrane, and specialized structures associated with malaria pathology such as the knobs. A number of these exported PfJDPs are essential, and there is growing experimental evidence that they are important for the survival and pathogenesis of the malaria parasite. This review critiques our understanding of the important role these exported PfJDPs play at the host-parasite interface.

Pharmacological chaperone-rescued cystic fibrosis CFTR-F508del mutant overcomes PRAF2-gated access to endoplasmic reticulum exit sites.

The endoplasmic reticulum exit of some polytopic plasma membrane proteins (PMPs) is controlled by arginin-based retention motifs. PRAF2, a gatekeeper which recognizes these motifs, was shown to retain the GABAB-receptor GB1 subunit in the ER. We report that PRAF2 can interact on a stoichiometric basis with both wild type and mutant F508del Cystic Fibrosis (CF) Transmembrane Conductance Regulator (CFTR), preventing the access of newly synthesized cargo to ER exit sites. Because of its lower abundance, compared to wild-type CFTR, CFTR-F508del recruitment into COPII vesicles is suppressed by the ER-resident PRAF2. We also demonstrate that some pharmacological chaperones that efficiently rescue CFTR-F508del loss of function in CF patients target CFTR-F508del retention by PRAF2 operating with various mechanisms. Our findings open new therapeutic perspectives for diseases caused by the impaired cell surface trafficking of mutant PMPs, which contain RXR-based retention motifs that might be recognized by PRAF2.

Analysis of Slow-Cycling Variants of the Light-Inducible Nuclear Protein Export System LEXY in Mammalian Cells.

The optogenetic tool LEXY consists of the second light oxygen voltage (LOV) domain of Avena sativa phototropin 1 mutated to contain a nuclear export signal. It allows exporting from the nucleus with blue light proteins of interest (POIs) genetically fused to it. Mutations slowing the dark recovery rate of the LOV domain within LEXY were recently shown to allow for better depletion of some POIs from the nucleus in Drosophila embryos and for the usage of low light illumination regimes. We investigated these variants in mammalian cells and found they increase the cytoplasmic localization of the proteins we tested after illumination, but also during the dark phases, which corresponds to higher leakiness of the system. These data suggest that, when aiming to sequester into the nucleus a protein with a cytoplasmic function, the original LEXY is preferable. The iLEXY variants are, instead, advantageous when wanting to deplete the nucleus of the POI as much as possible.

Holin-assisted bacterial recombinant protein export.

A simple generic method for enhancing extracellular protein yields in engineered bacteria is still lacking. Here, we demonstrated that phage-encoded holin can be used to export proteins to the extracellular medium in both Gram-negative Escherichia coli and -positive Lactococcus lactis. When a putative holin gene LLNZ_RS10380 annotated in the genome of L. lactis NZ9000 (hol380) was recombinantly expressed in E. coli BL21(DE3), the Hol380 oligomerized up to hexamer in the cytoplasmic membrane, yielding membrane pore to allow the passage of cytosolic ß-galatosidase (116 kDa), whose extracellular production reached 54.59 U/µl, accounting for 76.37% of the total activity. However, the overexpressed Hol380 could not release cytosolic proteins across the membrane in L. lactis NZ9000, but increased the secretory production of staphylococcal nuclease to 2.55-fold and fimbrial adhesin FaeG to 2.40-fold compared with those guided by signal peptide Usp45 alone. By using a combination of proteomics and transcriptional level analysis, we found that overexpression of the Hol380 raised the accumulation of Ffh and YidC involved in the signal recognition particle pathway in L. lactis, suggesting an alternative road participating in protein secretion. This study proposed a new approach by expressing holin in bacterial cell factories to export target proteins of economic or medical interest.

Transmembrane transport process and endoplasmic reticulum function facilitate the role of gene cel1b in cellulase production of Trichoderma reesei.

BACKGROUND: A total of 11 ß-glucosidases are predicted in the genome of Trichoderma reesei, which are of great importance for regulating cellulase biosynthesis. Nevertheless, the relevant function and regulation mechanism of each ß-glucosidase remained unknown. RESULTS: We evidenced that overexpression of cel1b dramatically decreased cellulase synthesis in T. reesei RUT-C30 both at the protein level and the mRNA level. In contrast, the deletion of cel1b did not noticeably affect cellulase production. Protein CEL1B was identified to be intracellular, being located in vacuole and cell membrane. The overexpression of cel1b reduced the intracellular pNPGase activity and intracellular/extracellular glucose concentration without inducing carbon catabolite repression. On the other hand, RNA-sequencing analysis showed the transmembrane transport process and endoplasmic reticulum function were affected noticeably by overexpressing cel1b. In particular, some important sugar transporters were notably downregulated, leading to a compromised cellular uptake of sugars including glucose and cellobiose. CONCLUSIONS: Our data suggests that the cellulase inhibition by cel1b overexpression was not due to the ß-glucosidase activity, but probably the dysfunction of the cellular transport process (particularly sugar transport) and endoplasmic reticulum (ER). These findings advance the knowledge of regulation mechanism of cellulase synthesis in filamentous fungi, which is the basis for rationally engineering T. reesei strains to improve cellulase production in industry.

Kinetic Tracking of Plasmodium falciparum Antigens on Infected Erythrocytes with a Novel Reporter of Protein Insertion and Surface Exposure.

Intracellular malaria parasites export many proteins into their host cell, inserting several into the erythrocyte plasma membrane to enable interactions with their external environment. While static techniques have identified some surface-exposed proteins, other candidates have eluded definitive localization and membrane topology determination. Moreover, both export kinetics and the mechanisms of membrane insertion remain largely unexplored. We introduce Reporter of Insertion and Surface Exposure (RISE), a method for continuous nondestructive tracking of antigen exposure on infected cells. RISE utilizes a small 11-amino acid (aa) HiBit fragment of NanoLuc inserted into a target protein and detects surface exposure through high-affinity complementation to produce luminescence. We tracked the export and surface exposure of CLAG3, a parasite protein linked to nutrient uptake, throughout the Plasmodium falciparum cycle in human erythrocytes. Our approach revealed key determinants of trafficking and surface exposure. Removal of a C-terminal transmembrane domain aborted export. Unexpectedly, certain increases in the exposed reporter size improved the luminescence signal, but other changes abolished the surface signal, revealing that both size and charge of the extracellular epitope influence membrane insertion. Marked cell-to-cell variation with larger inserts containing multiple HiBit epitopes suggests complex regulation of CLAG3 insertion at the host membrane. Quantitative, continuous tracking of CLAG3 surface exposure thus reveals multiple factors that determine this protein's trafficking and insertion at the host erythrocyte membrane. The RISE assay will enable study of surface antigens from divergent intracellular pathogens. IMPORTANCE Malaria parasites invade and replicate within red blood cells of their human or animal hosts to avoid immune detection. At the same time, these parasites insert their own proteins into the host membrane to scavenge plasma nutrients, facilitate immune evasion, and perform other essential activities. As there is broad interest in developing vaccines and antimalarial therapies against these surface-exposed antigens, robust methods are needed to examine how and when parasite proteins insert at the host membrane. We therefore developed and used Reporter of Insertion and Surface Exposure (RISE) to track parasite antigen exposure. Using RISE, we followed the time course of membrane insertion for CLAG3, a conserved protein linked to a nutrient uptake channel on infected erythrocytes. We found that CLAG3 insertion occurs at specific parasite stages and that this insertion is required for the formation of the nutrient uptake channel. We also varied the size and charge of the extracellular domain to define constraints on protein insertion at the host membrane. Single-cell imaging revealed that some cells continued to export CLAG3 even with large extracellular loops, suggesting sophisticated strategies used by malaria parasites to control their interactions with host plasma.