Development of a Highly Sensitive Luciferase-Based Reporter System To Study Two-Step Protein Secretion in Cyanobacteria.

Cyanobacteria, ubiquitous oxygenic photosynthetic bacteria, interact with the environment and their surrounding microbiome through the secretion of a variety of small molecules and proteins. The release of these compounds is mediated by sophisticated multiprotein complexes, also known as secretion systems. Genomic analyses indicate that protein and metabolite secretion systems are widely found in cyanobacteria; however, little is known regarding their function, regulation, and secreted effectors. One such system, the type IVa pilus system (T4aPS), is responsible for the assembly of dynamic cell surface appendages, type IVa pili (T4aP), that mediate ecologically relevant processes such as phototactic motility, natural competence, and adhesion. Several studies have suggested that the T4aPS can also act as a two-step protein secretion system in cyanobacteria akin to the homologous type II secretion system in heterotrophic bacteria. To determine whether the T4aP are involved in two-step secretion of nonpilin proteins, we developed a NanoLuc (NLuc)-based quantitative secretion reporter for the model cyanobacterium Synechocystis sp. strain PCC 6803. The NLuc reporter presented a wide dynamic range with at least 1 order of magnitude more sensitivity than traditional immunoblotting. Application of the reporter to a collection of Synechocystis T4aPS mutants demonstrated that the two-step secretion of NLuc is independent of T4aP. In addition, our data suggest that secretion differences typically observed in T4aPS mutants are likely due to a disruption of cell envelope homeostasis. This study opens the door to exploring protein secretion in cyanobacteria further. IMPORTANCE Protein secretion allows bacteria to interact and communicate with the external environment. Secretion is also biotechnologically relevant, where it is often beneficial to target proteins to the extracellular space. Due to a shortage of quantitative assays, many aspects of protein secretion are not understood. Here, we introduce an NLuc-based secretion reporter in cyanobacteria. NLuc is highly sensitive and can be assayed rapidly and in small volumes. The NLuc reporter allowed us to clarify the role of type IVa pili in protein secretion and identify mutations that increase secretion yield. This study expands our knowledge of cyanobacterial secretion and offers a valuable tool for future studies of protein secretion systems in cyanobacteria.

Membrane Vesicles of Pectobacterium as an Effective Protein Secretion System.

Bacteria of genus Pectobacterium are Gram-negative rods of the family Pectobacteriaceae. They are the causative agent of soft rot diseases of crops and ornamental plants. However, their virulence mechanisms are not yet fully elucidated. Membrane vesicles (MVs) are universally released by bacteria and are believed to play an important role in the pathogenicity and survival of bacteria in the environment. Our study investigates the role of MVs in the virulence of Pectobacterium. The results indicate that the morphology and MVs production depend on growth medium composition. In polygalacturonic acid (PGA) supplemented media, Pectobacterium produces large MVs (100-300 nm) and small vesicles below 100 nm. Proteomic analyses revealed the presence of pectate degrading enzymes in the MVs. The pectate plate test and enzymatic assay proved that those enzymes are active and able to degrade pectates. What is more, the pathogenicity test indicated that the MVs derived from Pectobacterium were able to induce maceration of Zantedeschia sp. leaves. We also show that the MVs of ß-lactamase producing strains were able to suppress ampicillin activity and permit the growth of susceptible bacteria. Those findings indicate that the MVs of Pectobacterium play an important role in host-pathogen interactions and niche competition with other bacteria. Our research also sheds some light on the mechanism of MVs production. We demonstrate that the MVs production in Pectobacterium strains, which overexpress a green fluorescence protein (GFP), is higher than in wild-type strains. Moreover, proteomic analysis revealed that the GFP was present in the MVs. Therefore, it is possible that protein sequestration into MVs might not be strictly limited to periplasmic proteins. Our research highlights the importance of MVs production as a mechanism of cargo delivery in Pectobacterium and an effective secretion system.

Modulation of heterologous protein secretion in the thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC 656 by CRISPR-Cas9 system.

The thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC 656 is a potential host strain for industrial protein production. Heterologous proteins are often retained intracellularly in yeast resulting in endoplasmic reticulum (ER) stress and poor secretion, and despite efforts to engineer protein secretory pathways, heterologous protein production is often lower than expected. We hypothesized that activation of genes involved in the secretory pathway could mitigate ER stress. In this study, we created mutants defective in protein secretory-related functions using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) tools. Secretion of the model protein xylanase was significantly decreased in loss of function mutants for oxidative stress (sod1Δ) and vacuolar and protein sorting (vps1Δ and ypt7Δ) genes. However, xylanase secretion was unaffected in an autophagy related atg12Δ mutant. Then, we developed a system for sequence-specific activation of target gene expression (CRISPRa) in O. thermomethanolica and used it to activate SOD1, VPS1 and YPT7 genes. Production of both non-glycosylated xylanase and glycosylated phytase was enhanced in the gene activated mutants, demonstrating that CRISPR-Cas9 systems can be used as tools for understanding O. thermomethanolica genes involved in protein secretion, which could be applied for increasing heterologous protein secretion in this yeast.

Minimal peptide length required to span the mitochondrial protein translocases in Trypanosoma brucei.

Mitochondrial protein import depends on heterooligomeric translocases in the outer and inner membranes. Using import substrates consisting of various lengths of the N-terminal part of mitochondrial dihydrolipoamide dehydrogenase (LDH) fused to dihydrofolate reductase we present an in vivo analysis showing that in Trypanosoma brucei at least 96 aa of mature LDH are required to efficiently produce an import intermediate that spans both translocases. This is different to yeast, where around 50 aa are sufficient to achieve the same task and likely reflects the different arrangement and architecture of the trypanosomal mitochondrial translocases. Furthermore, we show that formation of the stuck import intermediate leads to a strong growth inhibition suggesting that, depending on the length of the LDH, the import channels in the translocases are quantitatively blocked.

The extracellular contractile injection system is enriched in environmental microbes and associates with numerous toxins.

The extracellular Contractile Injection System (eCIS) is a toxin-delivery particle that evolved from a bacteriophage tail. Four eCISs have previously been shown to mediate interactions between bacteria and their invertebrate hosts. Here, we identify eCIS loci in 1,249 bacterial and archaeal genomes and reveal an enrichment of these loci in environmental microbes and their apparent absence from mammalian pathogens. We show that 13 eCIS-associated toxin genes from diverse microbes can inhibit the growth of bacteria and/or yeast. We identify immunity genes that protect bacteria from self-intoxication, further supporting an antibacterial role for some eCISs. We also identify previously undescribed eCIS core genes, including a conserved eCIS transcriptional regulator. Finally, we present our data through an extensive eCIS repository, termed eCIStem. Our findings support eCIS as a toxin-delivery system that is widespread among environmental prokaryotes and likely mediates antagonistic interactions with eukaryotes and other prokaryotes.

Hypoxia-induced amniotic fluid stem cell secretome augments cardiomyocyte proliferation and enhances cardioprotective effects under hypoxic-ischemic conditions.

Secretome derived from human amniotic fluid stem cells (AFSC-S) is rich in soluble bioactive factors (SBF) and offers untapped therapeutic potential for regenerative medicine while avoiding putative cell-related complications. Characterization and optimal generation of AFSC-S remains challenging. We hypothesized that modulation of oxygen conditions during AFSC-S generation enriches SBF and confers enhanced regenerative and cardioprotective effects on cardiovascular cells. We collected secretome at 6-hourly intervals up to 30 h following incubation of AFSC in normoxic (21%O2, nAFSC-S) and hypoxic (1%O2, hAFSC-S) conditions. Proliferation of human adult cardiomyocytes (hCM) and umbilical cord endothelial cells (HUVEC) incubated with nAFSC-S or hAFSC-S were examined following culture in normoxia or hypoxia. Lower AFSC counts and richer protein content in AFSC-S were observed in hypoxia. Characterization of AFSC-S by multiplex immunoassay showed higher concentrations of pro-angiogenic and anti-inflammatory SBF. hCM demonstrated highest proliferation with 30h-hAFSC-S in hypoxic culture. The cardioprotective potential of concentrated 30h-hAFSC-S treatment was demonstrated in a myocardial ischemia-reperfusion injury mouse model by infarct size and cell apoptosis reduction and cell proliferation increase when compared to saline treatment controls. Thus, we project that hypoxic-generated AFSC-S, with higher pro-angiogenic and anti-inflammatory SBF, can be harnessed and refined for tailored regenerative applications in ischemic cardiovascular disease.

Genomic insights into cyanobacterial protein translocation systems.

Cyanobacteria are ubiquitous oxygenic photosynthetic bacteria with a versatile metabolism that is highly dependent on effective protein targeting. Protein sorting in diderm bacteria is not trivial and, in cyanobacteria, even less so due to the presence of a complex membrane system: the outer membrane, the plasma membrane and the thylakoid membrane. In cyanobacteria, protein import into the thylakoids is essential for photosynthesis, export to the periplasm fulfills a multifunctional role in maintaining cell homeostasis, and secretion mediates motility, DNA uptake and environmental interactions. Intriguingly, only one set of genes for the general secretory and the twin-arginine translocation pathways seem to be present. However, these systems have to operate in both plasma and thylakoid membranes. This raises the question of how substrates are recognized and targeted to their correct, final destination. Additional complexities arise when a protein has to be secreted across the outer membrane, where very little is known regarding the mechanisms involved. Given their ecological importance and biotechnological interest, a better understanding of protein targeting in cyanobacteria is of great value. This review will provide insights into the known knowns of protein targeting, propose hypotheses based on available genomic sequences and discuss future directions.

Phosphoproteomics of Acute Cell Stressors Targeting Exercise Signaling Networks Reveal Drug Interactions Regulating Protein Secretion.

Exercise engages signaling networks to control the release of circulating factors beneficial to health. However, the nature of these networks remains undefined. Using high-throughput phosphoproteomics, we quantify 20,249 phosphorylation sites in skeletal muscle-like myotube cells and monitor their responses to a panel of cell stressors targeting aspects of exercise signaling in vivo. Integrating these in-depth phosphoproteomes with the phosphoproteome of acute aerobic exercise in human skeletal muscle suggests that co-administration of ß-adrenergic and calcium agonists would activate complementary signaling relevant to this exercise context. The phosphoproteome of cells treated with this combination reveals a surprising divergence in signaling from the individual treatments. Remarkably, only the combination treatment promotes multisite phosphorylation of SERBP1, a regulator of Serpine1 mRNA stability, a pro-fibrotic secreted protein. Secretome analysis reveals that the combined treatments decrease secretion of SERPINE1 and other deleterious factors. This study provides a framework for dissecting phosphorylation-based signaling relevant to acute exercise.

Role of BGS13 in the Secretory Mechanism of Pichia pastoris.

The methylotrophic yeast Pichia pastoris has been utilized for heterologous protein expression for over 30 years. Because P. pastoris secretes few of its own proteins, the exported recombinant protein is the major polypeptide in the extracellular medium, making purification relatively easy. Unfortunately, some recombinant proteins intended for secretion are retained within the cell. A mutant strain isolated in our laboratory, containing a disruption of the BGS13 gene, displayed elevated levels of secretion for a variety of reporter proteins. The Bgs13 peptide (Bgs13p) is similar to the Saccharomyces cerevisiae protein kinase C 1 protein (Pkc1p), but its specific mode of action is currently unclear. To illuminate differences in the secretion mechanism between the wild-type (wt) strain and the bgs13 strain, we determined that the disrupted bgs13 gene expressed a truncated protein that had reduced protein kinase C activity and a different location in the cell, compared to the wt protein. Because the Pkc1p of baker's yeast plays a significant role in cell wall integrity, we investigated the sensitivity of the mutant strain's cell wall to growth antagonists and extraction by dithiothreitol, determining that the bgs13 strain cell wall suffered from inherent structural problems although its porosity was normal. A proteomic investigation of the bgs13 strain secretome and cell wall-extracted peptides demonstrated that, compared to its wt parent, the bgs13 strain also displayed increased release of an array of normally secreted, endogenous proteins, as well as endoplasmic reticulum-resident chaperone proteins, suggesting that Bgs13p helps regulate the unfolded protein response and protein sorting on a global scale.IMPORTANCE The yeast Pichia pastoris is used as a host system for the expression of recombinant proteins. Many of these products, including antibodies, vaccine antigens, and therapeutic proteins such as insulin, are currently on the market or in late stages of development. However, one major weakness is that sometimes these proteins are not secreted from the yeast cell efficiently, which impedes and raises the cost of purification of these vital proteins. Our laboratory has isolated a mutant strain of Pichia pastoris that shows enhanced secretion of many proteins. The mutant produces a modified version of Bgs13p. Our goal is to understand how the change in the Bgs13p function leads to improved secretion. Once the Bgs13p mechanism is illuminated, we should be able to apply this understanding to engineer new P. pastoris strains that efficiently produce and secrete life-saving recombinant proteins, providing medical and economic benefits.

The murine lung as a factory to produce secreted intrapulmonary and circulatory proteins.

We have shown that a lentiviral vector (rSIV.F/HN) pseudotyped with the F and HN proteins from Sendai virus generates high levels of intracellular proteins after lung transduction. Here, we evaluate the use of rSIV.F/HN for production of secreted proteins. We assessed whether rSIV.F/HN transduction of the lung generates therapeutically relevant levels of secreted proteins in the lung and systemic circulation using human α1-anti-trypsin (hAAT) and factor VIII (hFVIII) as exemplars. Sedated mice were transduced with rSIV.F/HN carrying either the secreted reporter gene Gaussia luciferase or the hAAT or hFVIII cDNAs by nasal sniffing. rSIV.F/HN-hAAT transduction lead to therapeutically relevant hAAT levels (70 µg/ml) in epithelial lining fluid, with stable expression persisting for at least 19 months from a single application. Secreted proteins produced in the lung were released into the circulation and stable expression was detectable in blood. The levels of hFVIII in murine blood approached therapeutically relevant targets. rSIV.F/HN was also able to produce secreted hAAT and hFVIII in transduced human primary airway cells. rSIV.F/HN transduction of the murine lungs leads to long-lasting and therapeutically relevant levels of secreted proteins in the lung and systemic circulation. These data broaden the use of this vector platform for a large range of disease indications.

Alpha-linolenic acid and linoleic acid differentially regulate the skeletal muscle secretome of obese Zucker rats.

Evidence shows that proteins secreted from skeletal muscle influence a broad range of metabolic signaling pathways. We previously reported that essential polyunsaturated fatty acids (PUFA) improved whole-body glucose homeostasis in obese Zucker rats; however, the mechanisms underlying these benefits remain enigmatic. While PUFA and obesity influence skeletal muscle function, their effects on the secretome are unknown. The aim of this work was to determine if improvements in whole-body glucose homeostasis in obese Zucker rats fed diets supplemented with either linoleic acid (LA) or alpha-linolenic acid (ALA) for 12 wk are related to changes in the skeletal muscle secretome. Secreted proteins were identified with a predictive bioinformatic analysis of microarray gene expression from red tibialis anterior skeletal muscle. Approximately 130 genes were differentially expressed (false discovery rate = 0.05) in obese rats compared with lean controls. The expression of 15 genes encoding secreted proteins was differentially regulated in obese controls, obese LA-supplemented, and obese ALA-supplemented rats compared with lean controls. Five secreted proteins ( Col3a1, Col15a1, Pdgfd, Lyz2, and Angptl4) were differentially regulated by LA and ALA. Most notably, ALA supplementation reduced Angptl4 gene expression compared with obese control and obese-LA supplemented rats and reduced circulating ANGPTL4 serum concentrations. ALA also influenced Angptl4 gene expression and ANGPTL4 secretion from differentiated rat L6 myotubes. Altogether, the present data indicate that obesity has a greater global impact on skeletal muscle gene expression than either essential PUFA; however, LA and ALA may exert their metabolic benefits in part by regulating the skeletal muscle secretome.

Identification of a novel protein complex essential for effector translocation across the parasitophorous vacuole membrane of Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular parasite that can infect virtually all nucleated cells in warm-blooded animals. The ability of Toxoplasma tachyzoites to infect and successfully manipulate its host is dependent on its ability to transport "GRA" proteins that originate in unique secretory organelles called dense granules into the host cell in which they reside. GRAs have diverse roles in Toxoplasma's intracellular lifecycle, including co-opting crucial host cell functions and proteins, such as the cell cycle, c-Myc and p38 MAP kinase. Some of these GRA proteins, such as GRA16 and GRA24, are secreted into the parasitophorous vacuole (PV) within which Toxoplasma replicates and are transported across the PV membrane (PVM) into the host cell, but the translocation process and its machinery are not well understood. We previously showed that TgMYR1, which is cleaved by TgASP5 into two fragments, localizes to the PVM and is essential for GRA transport into the host cell. To identify additional proteins necessary for effector transport, we screened Toxoplasma mutants defective in c-Myc up-regulation for their ability to export GRA16 and GRA24 to the host cell nucleus. Here we report that novel proteins MYR2 and MYR3 play a crucial role in translocation of a subset of GRAs into the host cell. MYR2 and MYR3 are secreted into the PV space and co-localize with PV membranes and MYR1. Consistent with their predicted transmembrane domains, all three proteins are membrane-associated, and MYR3, but not MYR2, stably associates with MYR1, whose N- and C-terminal fragments are disulfide-linked. We further show that fusing intrinsically disordered effectors to a structured DHFR domain blocks the transport of other effectors, consistent with a translocon-based model of effector transport. Overall, these results reveal a novel complex at the PVM that is essential for effector translocation into the host cell.

The major cellulases CBH-1 and CBH-2 of Neurospora crassa rely on distinct ER cargo adaptors for efficient ER-exit.

Filamentous fungi are native secretors of lignocellulolytic enzymes and are used as protein-producing factories in the industrial biotechnology sector. Despite the importance of these organisms in industry, relatively little is known about the filamentous fungal secretory pathway or how it might be manipulated for improved protein production. Here, we use Neurospora crassa as a model filamentous fungus to interrogate the requirements for trafficking of cellulase enzymes from the endoplasmic reticulum to the Golgi. We characterized the localization and interaction properties of the p24 and ERV-29 cargo adaptors, as well as their role in cellulase enzyme trafficking. We find that the two most abundantly secreted cellulases, CBH-1 and CBH-2, depend on distinct ER cargo adaptors for efficient exit from the ER. CBH-1 depends on the p24 proteins, whereas CBH-2 depends on the N. crassa homolog of yeast Erv29p. This study provides a first step in characterizing distinct trafficking pathways of lignocellulolytic enzymes in filamentous fungi.

Cryo-EM Structure of the TOM Core Complex from Neurospora crassa.

The TOM complex is the main entry gate for protein precursors from the cytosol into mitochondria. We have determined the structure of the TOM core complex by cryoelectron microscopy (cryo-EM). The complex is a 148 kDa symmetrical dimer of ten membrane protein subunits that create a shallow funnel on the cytoplasmic membrane surface. In the core of the dimer, the ß-barrels of the Tom40 pore form two identical preprotein conduits. Each Tom40 pore is surrounded by the transmembrane segments of the α-helical subunits Tom5, Tom6, and Tom7. Tom22, the central preprotein receptor, connects the two Tom40 pores at the dimer interface. Our structure offers detailed insights into the molecular architecture of the mitochondrial preprotein import machinery.

Identification of Protein Secretion Systems in Bacterial Genomes Using MacSyFinder.

Protein secretion systems are complex molecular machineries that translocate proteins through the outer membrane, and sometimes through multiple other barriers. They have evolved by co-option of components from other envelope-associated cellular machineries, making them sometimes difficult to identify and discriminate. Here, we describe how to identify protein secretion systems in bacterial genomes using MacSyFinder. This flexible computational tool uses the knowledge stemming from experimental studies to identify homologous systems in genome data. It can be used with a set of predefined models-"TXSScan"-to identify all major secretion systems of diderm bacteria (i.e., with inner and with LPS-containing outer membranes). For this, it identifies and clusters colocalized components of secretion systems using sequence similarity searches with hidden Markov model protein profiles. Finally, it checks whether the genetic content and organization of clusters satisfy the constraints of the model. TXSScan models can be customized to search for variants of known systems. The models can also be built from scratch to identify novel systems. In this chapter, we describe a complete pipeline of analysis, including the identification of a reference set of experimentally studied systems, the identification of components and the construction of their protein profiles, the definition of the models, their optimization, and, finally, their use as tools to search genomic data.

Native and Foreign Proteins Secreted by the Cupriavidus metallidurans Type II System and an Alternative Mechanism.

The type II secretion system (T2SS), which transports selected periplasmic proteins across the outer membrane, has rarely been studied in nonpathogens or in organisms classified as Betaproteobacteria. Therefore, we studied Cupriavidus metallidurans (Cme), a facultative chemilithoautotroph. Gel analysis of extracellular proteins revealed no remarkable differences between the wild type and the T2SS mutants. However, enzyme assays revealed that native extracellular alkaline phosphatase is a T2SS substrate, because activity was 10-fold greater for the wild type than a T2SS mutant. In Cme engineered to produce three Ralstonia solanacearum (Rso) exoenzymes, at least 95% of their total activities were extracellular, but unexpectedly high percentages of these exoenzymes remained extracellular in T2SS mutants cultured in rich broth. These conditions appear to permit an alternative secretion process, because neither cell lysis nor periplasmic leakage was observed when Cme produced a Pectobacterium carotovorum exoenzyme, and wild-type Cme cultured in minimal medium secreted 98% of Rso polygalacturonase, but 92% of this exoenzyme remained intracellular in T2SS mutants. We concluded that Cme has a functional T2SS despite lacking any abundant native T2SS substrates. The efficient secretion of three foreign exoenzymes by Cme is remarkable, but so too is the indication of an alternative secretion process in rich culture conditions. When not transiting the T2SS, we suggest that Rso exoenzymes are probably selectively packaged into outer membrane vesicles. Phylogenetic analysis of T2SS proteins supports the existence of at least three T2SS subfamilies, and we propose that Cme, as a representative of the Betaproteobacteria, could become a new useful model system for studying T2SS substrate specificity.

Identification of protein secretion systems in bacterial genomes.

Bacteria with two cell membranes (diderms) have evolved complex systems for protein secretion. These systems were extensively studied in some model bacteria, but the characterisation of their diversity has lagged behind due to lack of standard annotation tools. We built online and standalone computational tools to accurately predict protein secretion systems and related appendages in bacteria with LPS-containing outer membranes. They consist of models describing the systems' components and genetic organization to be used with MacSyFinder to search for T1SS-T6SS, T9SS, flagella, Type IV pili and Tad pili. We identified ~10,000 candidate systems in bacterial genomes, where T1SS and T5SS were by far the most abundant and widespread. All these data are made available in a public database. The recently described T6SS(iii) and T9SS were restricted to Bacteroidetes, and T6SS(ii) to Francisella. The T2SS, T3SS, and T4SS were frequently encoded in single-copy in one locus, whereas most T1SS were encoded in two loci. The secretion systems of diderm Firmicutes were similar to those found in other diderms. Novel systems may remain to be discovered, since some clades of environmental bacteria lacked all known protein secretion systems. Our models can be fully customized, which should facilitate the identification of novel systems.

Secretion of dengue virus envelope protein ectodomain from mammalian cells is dependent on domain II serotype and affects the immune response upon DNA vaccination.

Dengue virus (DENV) is currently among the most important human pathogens and affects millions of people throughout the tropical and subtropical regions of the world. Although it has been a World Health Organization priority for several years, there is still no efficient vaccine available to prevent infection. The envelope glycoprotein (E), exposed on the surface on infective viral particles, is the main target of neutralizing antibodies. For this reason it has been used as the antigen of choice for vaccine development efforts. Here we show a detailed analysis of factors involved in the expression, secretion and folding of E ectodomain from all four DENV serotypes in mammalian cells, and how this affects their ability to induce neutralizing antibody responses in DNA-vaccinated mice. Proper folding of E domain II (DII) is essential for efficient E ectodomain secretion, with DIII playing a significant role in stabilizing soluble dimers. We also show that the level of protein secreted from transfected cells determines the strength and efficiency of antibody responses in the context of DNA vaccination and should be considered a pivotal feature for the development of E-based DNA vaccines against DENV.