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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 37 records · Page 2
All sourcesNASA NTRSDOE OSTI
DOE OSTI2018

Skeletal muscle secretion of IL-6 is muscle type specific: Ex vivo evidence

Highlights: • Ex vivo incubation of freshly isolated mouse skeletal in a physiological condition showed inducible secretion of myokines. • IL-6 secretion was only induced from slow oxidative muscle soleus but not from fast glycolytic muscle EDL. • EDL has secretory function with different secretome pattern from soleus. • This is the first evidence that skeletal muscle secretion of myokines is muscle type specific. Emerging evidence indicates that skeletal muscle possesses endocrine function to secret myokines. Interleukin 6 (IL-6) is a well-characterized myokine that is involved in regulation of metabolism and muscle function. Metabolism type and contractile dynamics vary in different muscle types. It is not clear, however, if IL-6 secretion differs in different muscle types. In this study, we first established an ex vivo approach to test the inducible muscle secretion. Freshly isolated muscles were incubated in Krebs solution at 37 °C with oxygen supply. Secreted IL-6 in the incubation media was measure using Western blot and ELISA assay. We first confirmed that the IL-6 release was inducible by treating the incubated muscle with a cytokine stimulant. We demonstrated that physiological temperature (37 °C) and O{sub 2} supply were essential for the induction of IL-6 release from the incubated muscle, suggesting it is a controlled secretion rather than a spontaneous leak. Using this approach, we found that IL-6 release was only inducible from soleus muscle but not EDL muscle. We further showed that IL-6 protein level was higher in slow oxidative muscle fibers. Moreover, we showed that EDL, although lacks of IL-6 release, surely has inducible secretory function that had different secretory pattern from soleus.

60 APPLIED LIFE SCIENCES↗
NASA NTRS1984

Peptides and neurotransmitters that affect renin secretion

Substance P inhibits renin secretion. This polypeptide is a transmitter in primary afferent neurons and is released from the peripheral as well as the central portions of these neurons. It is present in afferent nerves from the kidneys. Neuropeptide Y, which is a cotransmitter with norepinephrine and epinephrine, is found in sympathetic neurons that are closely associated with and presumably innervate the juxtagolmerular cells. Its effect on renin secretion is unknown, but it produces renal vasoconstriction and natriuresis. Vasoactive intestinal polypeptide (VIP) is a cotransmitter with acetylocholine in cholinergic neurons, and this polypeptide stimulates renin secretion. We cannot find any evidence for its occurence in neurons in the kidneys, but various stimuli increase plasma VIP to levels comparable to those produced by doses of exogenous VIP which stimulated renin secretion. Neostigmine increases plasma VIP and plasma renin activity, and the VIP appears to be responsible for the increase in renin secretion, since the increase is not blocked by renal denervation or propranolol. Stimulation of various areas in the brain produces sympathetically mediated increases in plasma renin activity associated with increases in blood pressure. However, there is pharmacological evidence that the renin response can be separated from the blood pressure response. In anaesthetized dogs, drugs that increase central serotonergic discharge increase renin secretion without increasing blood pressure. In rats, activation of sertonergic neurons in the dorsal raphe nucleus increases renin secretion by a pathway that projects from this nucleus to the ventral hypothalamus, and from there to the kidneys via the sympathetic nervous system. The serotonin releasing drug parachloramphetamine also increases plasma VIP, but VIP does not appear to be the primary mediator of the renin response. There is preliminary evidence that the serotonergic neurons in the dorsal raphe nucleus are part of the pathway by which psychosocial stimuli increase renin secretion.

Review↗
DOE OSTI2023

The Missing Pieces: The Role of Secretion Systems in Campylobacter jejuni Virulence

Campylobacter jejuni is likely the most common bacterial cause of gastroenteritis worldwide, responsible for millions of cases of inflammatory diarrhea characterized by severe abdominal cramps and blood in the stool. Further, C. jejuni infections are associated with post-infection sequelae in developed countries and malnutrition and growth-stunting in low- and middle-income countries. Despite the increasing prevalence of the disease, campylobacteriosis, and the recognition that this pathogen is a serious health threat, our understanding of C. jejuni pathogenesis remains incomplete. In this review, we focus on the Campylobacter secretion systems proposed to contribute to host-cell interactions and survival in the host. Moreover, we have applied a genomics approach to defining the structural and mechanistic features of C. jejuni type III, IV, and VI secretion systems. Special attention is focused on the flagellar type III secretion system and the prediction of putative effectors, given that the proteins exported via this system are essential for host cell invasion and the inflammatory response. We conclude that C. jejuni does not possess a type IV secretion system and relies on the type III and type VI secretion systems to establish a niche and potentiate disease.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Abundance of Secreted Proteins of Trichoderma reesei Is Regulated by Light of Different Intensities

In Trichoderma reesei light is an important factor in the regulation of glycoside hydrolase gene expression. We therefore investigated the influence of different light intensities on cellulase activity and protein secretion. Differentially secreted proteins in light and darkness as identified by mass spectrometry included members of different glycoside hydrolase families, such as CBH1, Cel3A, Cel61B, XYN2, and XYN4. Several of the associated genes showed light-dependent regulation on the transcript level. Deletion of the photoreceptor genes blr1 and blr2 resulted in a diminished difference of protein abundance between light and darkness. The amount of secreted proteins including that of the major exo-acting beta-1,4-glucanases CBH1 and CBH2 was generally lower in light-grown cultures than in darkness. In contrast, cbh1 transcript levels increased with increasing light intensity from 700 to 2,000 lux but dopped at high light intensity (5,000 lux). In the photoreceptor mutants Δblr1 and Δblr2 cellulase activity in light was reduced compared to activity in darkness, showing a discrepancy between transcript levels and secreted cellulase activity. Furthermore, evaluation of different light sensitivities revealed an increased light tolerance with respect to cellulase expression of QM9414 compared to its parental strain QM6a. Investigation of one of the differentially expressed proteins between light and darkness, CLF1, revealed its function as a factor involved in regulation of secreted protease activity. T. reesei secretes a different set of proteins in light compared to darkness, this difference being mainly due to the function of the major known photoreceptors. Moreover, cellulase regulation is adjusted to light intensity and improved light tolerance was correlated with increased cellulase production. Our findings further support the hypothesis of a light intensity dependent post-transcriptional regulation of cellulase gene expression in T. reesei.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Bacterial metabolite S-equol modulates glucagon-like peptide-1 secretion from enteroendocrine L cell line GLUTag cells via actin polymerization

Highlights: • Receptors for S-equol were expressed in GLUTag cells. • S-equol increased the intracellular Ca{sup 2+} levels via G{sub q} signaling. • S-equol prevented GLP-1 secretion. • S-equol caused cortical actin reorganization via G{sub q} and G{sub 12/13} pathways. S-equol is one of gut bacterial metabolites produced from soybean isoflavone daizein. While S-equol is known to promote glucose-induced insulin secretion from pancreatic β cells, whether S-equol affects glucagon-like peptide-1 (GLP-1) secretion from enteroendoceine L cells remains unclear. Here we assessed the effect of S-equol on GLP-1 secretion from mouse enteroendocrine L cell line GLUTag cells. GLUTag cells expressed GPR30 and estrogen receptors, which are putative S-equol receptors. Application of S-equol induced an increase in intracellular Ca{sup 2+} levels via GPR30. However, S-equol did not enhance GLP-1 exocytosis, and long-term treatment of S-equol suppressed GLP-1 secretion. Moreover, immunocytochemistry revealed that S-equol increased the density of cortical actin filaments via G{sub 12/13} signaling under GPR30. These data suggest that S-equol prevents GLP-1 secretion as a result of competing regulation between Ca{sup 2+} mobilization and actin reorganization.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

CCN3 secreted by prostaglandin E2 inhibits intimal cushion formation in the rat ductus arteriosus

Highlights: • CCN3 is secreted from smooth muscle cells of the ductus arteriosus by prostaglandin E{sub 2}. • Nano-flow LC-MS/MS analysis is a powerful tool for detecting a novel secreted protein. • CCN3 inhibits intimal cushion formation in the ductus arteriosus. The ductus arteriosus (DA), an essential fetal shunt between the pulmonary trunk and the descending aorta, changes its structure during development. Our previous studies have demonstrated that prostaglandin E{sub 2} (PGE{sub 2})-EP4 signaling promotes intimal cushion formation (ICF) by activating the migration of DA smooth muscle cells via the secretion of hyaluronan. We hypothesized that, in addition to hyaluronan, PGE{sub 2} may secrete other proteins that also regulate vascular remodeling in the DA. In order to detect PGE{sub 2} stimulation-secreted proteins, we found that CCN3 protein was increased in the culture supernatant in the presence of PGE{sub 2} in a dose-dependent manner by nano-flow liquid chromatography coupled with tandem mass spectrometry analysis and enzyme-linked immunosorbent assay. Quantitative RT-PCR analysis revealed that PGE{sub 2} stimulation tended to increase the expression levels of CCN3 mRNA in DA smooth muscle cells. Immunohistochemical analysis revealed that CCN3 was highly localized in the entire smooth muscle layers and the endothelium of the DA. Furthermore, exogenous CCN3 inhibited PGE{sub 2}-induced ICF in the ex vivo DA tissues. These results suggest that CCN3 is a secreted protein of the DA smooth muscle cells induced by PGE{sub 2} to suppress ICF of the DA. The present study indicates that CCN3 could be a novel negative regulator of ICF in the DA to fine-tune the PGE{sub 2}-mediated DA remodeling.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Dual Targeting Factors Are Required for LXG Toxin Export by the Bacterial Type VIIb Secretion System

Bacterial type VIIb secretion systems (T7SSb) are multisubunit integral membrane protein complexes found in Firmicutes that play a role in both bacterial competition and virulence by secreting toxic effector proteins. The majority of characterized T7SSb effectors adopt a polymorphic domain architecture consisting of a conserved N-terminal Leu-X-Gly (LXG) domain and a variable C-terminal toxin domain. Recent work has started to reveal the diversity of toxic activities exhibited by LXG effectors; however, little is known about how these proteins are recruited to the T7SSb apparatus. In this work, we sought to characterize genes encoding domains of unknown function (DUFs) 3130 and 3958, which frequently cooccur with LXG effector-encoding genes. Using coimmunoprecipitation-mass spectrometry analyses, in vitro copurification experiments, and T7SSb secretion assays, we found that representative members of these protein families form heteromeric complexes with their cognate LXG domain and in doing so, function as targeting factors that promote effector export. Additionally, an X-ray crystal structure of a representative DUF3958 protein, combined with predictive modeling of DUF3130 using AlphaFold2, revealed structural similarity between these protein families and the ubiquitous WXG100 family of T7SS effectors. Interestingly, we identified a conserved FxxxD motif within DUF3130 that is reminiscent of the YxxxD/E “export arm” found in mycobacterial T7SSa substrates and mutation of this motif abrogates LXG effector secretion. Overall, our data experimentally link previously uncharacterized bacterial DUFs to type VIIb secretion and reveal a molecular signature required for LXG effector export.

24 antibacterial toxins↗
DOE OSTI2020

Metabolic Feedback Inhibition Influences Metabolite Secretion by the Human Gut Symbiont Bacteroides thetaiotaomicron

ABSTRACT Microbial metabolism and trophic interactions between microbes give rise to complex multispecies communities in microbe-host systems. Bacteroides thetaiotaomicron ( B. theta ) is a human gut symbiont thought to play an important role in maintaining host health. Untargeted nuclear magnetic resonance metabolomics revealed B. theta secretes specific organic acids and amino acids in defined minimal medium. Physiological concentrations of acetate and formate found in the human intestinal tract were shown to cause dose-dependent changes in secretion of metabolites known to play roles in host nutrition and pathogenesis. While secretion fluxes varied, biomass yield was unchanged, suggesting feedback inhibition does not affect metabolic bioenergetics but instead redirects carbon and energy to CO 2 and H 2 . Flux balance analysis modeling showed increased flux through CO 2 -producing reactions under glucose-limiting growth conditions. The metabolic dynamics observed for B. theta , a keystone symbiont organism, underscores the need for metabolic modeling to complement genomic predictions of microbial metabolism to infer mechanisms of microbe-microbe and microbe-host interactions. IMPORTANCE Bacteroides is a highly abundant taxon in the human gut, and Bacteroides thetaiotaomicron ( B. theta ) is a ubiquitous human symbiont that colonizes the host early in development and persists throughout its life span. The phenotypic plasticity of keystone organisms such as B. theta is important to understand in order to predict phenotype(s) and metabolic interactions under changing nutrient conditions such as those that occur in complex gut communities. Our study shows B. theta prioritizes energy conservation and suppresses secretion of “overflow metabolites” such as organic acids and amino acids when concentrations of acetate are high. Secreted metabolites, especially amino acids, can be a source of nutrients or signals for the host or other microbes in the community. Our study suggests that when metabolically stressed by acetate, B. theta stops sharing with its ecological partners.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Heparan sulfate in pancreatic β-cells contributes to normal glucose homeostasis by regulating insulin secretion

Highlights: • We examined the roles of heparan sulfate (HS) in pancreatic β-cells. • Inhibition of HS synthesis impaired a broad range of β-cell functions. • HS in β-cells contributed to normal insulin secretion and glucose homeostasis. Heparan sulfate (HS), a linear polysaccharide, is involved in diverse biological functions of various tissues. HS is expressed in pancreatic β-cells and may be involved in β-cell functions. However, the importance of HS for β-cell function remains unknown. Here, we generated mice with β-cell-specific deletion of Ext1 (βExt1CKO), which encodes an enzyme essential for HS synthesis, to investigate the detailed roles of HS in β-cell function. βExt1CKO mice decreased body weights compared with control mice, despite increased food intake. Additionally, βExt1CKO mice showed impaired glucose tolerance associated with decreased insulin secretion upon glucose challenge. Glucose-induced insulin secretion (GIIS) from isolated βExt1CKO islets was also significantly reduced, highlighting the contribution of HS to insulin secretion and glucose homeostasis. The gene expression essential for GIIS was decreased in βExt1CKO islets. Pdx1 and MafA were downregulated in βExt1CKO islets, indicating that HS promoted β-cell development and maturation. BrdU- or Ki67-positive β-cells were reduced in βExt1CKO pancreatic sections, suggesting the involvement of HS in the proliferation of β-cells. Moreover, insufficient vascularization in βExt1CKO islets may contribute to central distribution of α-cells. These data demonstrate HS plays diverse roles in β-cells, and that loss of HS leads to insufficient insulin secretion and dysregulation of glucose homeostasis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Improving the secretion of designed protein assemblies through negative design of cryptic transmembrane domains

Computationally designed protein nanoparticles have recently emerged as a promising platform for the development of new vaccines and biologics. For many applications, secretion of designed nanoparticles from eukaryotic cells would be advantageous, but in practice, they often secrete poorly. Here we show that designed hydrophobic interfaces that drive nanoparticle assembly are often predicted to form cryptic transmembrane domains, suggesting that interaction with the membrane insertion machinery could limit efficient secretion. We develop a general computational protocol, the Degreaser, to design away cryptic transmembrane domains without sacrificing protein stability. The retroactive application of the Degreaser to previously designed nanoparticle components and nanoparticles considerably improves secretion, and modular integration of the Degreaser into design pipelines results in new nanoparticles that secrete as robustly as naturally occurring protein assemblies. Both the Degreaser protocol and the nanoparticles we describe may be broadly useful in biotechnological applications.

77 NANOSCIENCE AND NANOTECHNOLOGY↗
DOE OSTI2020

PE5–PPE4–EspG 3 heterotrimer structure from mycobacterial ESX-3 secretion system gives insight into cognate substrate recognition by ESX systems

Mycobacterium tuberculosis has evolved numerous type VII secretion (ESX) systems to secrete multiple factors important for both growth and virulence across their cell envelope. ESX-1, ESX-3, and ESX-5 systems have been shown to each secrete a distinct set of substrates, including PE and PPE families of proteins, named for conserved Pro-Glu and Pro-Pro-Glu motifs in their N termini. Proper secretion of the PE–PPE proteins requires the presence of EspG, with each system encoding its own unique copy. There is no cross-talk between any of the ESX systems, and how each EspG recognizes its subset of PE–PPE proteins is currently unknown. The only current structural characterization of PE–PPE–EspG heterotrimers is from the ESX-5 system. Here we present the crystal structure of the PE5 mt –PPE4 mt –EspG 3mm heterotrimer from the ESX-3 system. Our heterotrimer reveals that EspG 3mm interacts exclusively with PPE4 mt in a similar manner to EspG 5 , shielding the hydrophobic tip of PPE4 mt from solvent. The C-terminal helical domain of EspG 3mm is dynamic, alternating between “open” and “closed” forms, and this movement is likely functionally relevant in the unloading of PE–PPE heterodimers at the secretion machinery. In contrast to the previously solved ESX-5 heterotrimers, the PE–PPE heterodimer of our ESX-3 heterotrimer is interacting with its chaperone at a drastically different angle and presents different faces of the PPE protein to the chaperone. We conclude that the PPE–EspG interface from each ESX system has a unique shape complementarity that allows each EspG to discriminate among noncognate PE–PPE pairs.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Biology of extracellular vesicles secreted from senescent cells as senescence‐associated secretory phenotype factors

The increase of the morbidity rate in age‐related diseases, such as cancer, Alzheimer’s disease, arteriosclerosis and pulmonary fibrosis, has become a profound social problem. Recent reports have pointed out that senescent cells accumulated in the body with aging might cause these aged‐related pathologies. Cellular senescence is known as an irreversible cell cycle arrest induced by various stresses, and can function as an important tumor suppression mechanism to exclude the premalignant cells. In contrast, senescent cells provoke the phenomenon, termed the senescence‐associated secretory phenotype, which causes the secretion of various inflammatory proteins, and it is at risk of facilitating chronic inflammation and oncogenic transformation to surrounding cells. We have previously reported that senescent cells secrete not only inflammatory proteins, but also extracellular vesicles (EV). EV include various cellular components, such as proteins, lipids and nucleic acids, which are proven to be important factors for cell‐to‐cell communication. Recent evidence suggests that EV secreted from senescent cells might contribute to tumorigenesis and age‐associated pathologies as new senescence‐associated secretory phenotype factors. In addition, we also showed that the EV secretion pathway is one of the essential defense mechanisms to maintain cellular homeostasis by excretion of intercellular toxic substances into extracellular space. Herein, this review shows the biological functions of EV secreted from senescent cells. Geriatr Gerontol Int 2020; ••: ••–•• .

Misawa, Tomoka↗
DOE OSTI2024

Bactericidal effectors of the Stenotrophomonas maltophilia type IV secretion system: functional definition of the nuclease TfdA and structural determination of TfcB

ABSTRACT Stenotrophomonas maltophilia expresses a type IV protein secretion system (T4SS) that promotes contact-dependent killing of other bacteria and does so partly by secreting the effector TfcB. Here, we report the structure of TfcB, comprising an N-terminal domain similar to the catalytic domain of glycosyl hydrolase (GH-19) chitinases and a C-terminal domain for recognition and translocation by the T4SS. Utilizing a two-hybrid assay to measure effector interactions with the T4SS coupling protein VirD4, we documented the existence of five more T4SS substrates. One of these was protein 20845, an annotated nuclease. A S. maltophilia mutant lacking the gene for 20845 was impaired for killing Escherichia coli , Klebsiella pneumoniae , and Pseudomonas aeruginosa . Moreover, the cloned 20845 gene conferred robust toxicity, with the recombinant E. coli being rescued when 20845 was co-expressed with its cognate immunity protein. The 20845 effector was an 899 amino-acid protein, comprised of a GHH-nuclease domain in its N-terminus, a large central region of indeterminant function, and a C-terminus for secretion. Engineered variants of the 20845 gene that had mutations in the predicted catalytic site did not impede E. coli , indicating that the antibacterial effect of 20845 involves its nuclease activity. Using flow cytometry with DNA staining, we determined that 20845, but not its mutant variants, confers a loss in DNA content of target bacteria. Database searches revealed that uncharacterized homologs of 20845 occur within a range of bacteria. These data indicate that the S. maltophilia T4SS promotes interbacterial competition through the action of multiple toxic effectors, including a potent, novel DNase. IMPORTANCE Stenotrophomonas maltophilia is a multi-drug-resistant, Gram-negative bacterium that is an emerging pathogen of humans. Patients with cystic fibrosis are particularly susceptible to S. maltophilia infection. In hospital water systems and various types of infections, S. maltophilia co-exists with other bacteria, including other pathogens such as Pseudomonas aeruginosa . We previously demonstrated that S. maltophilia has a functional VirB/D4 type VI protein secretion system (T4SS) that promotes contact-dependent killing of other bacteria. Since most work on antibacterial systems involves the type VI secretion system, this observation remains noteworthy. Moreover, S. maltophilia currently stands alone as a model for a human pathogen expressing an antibacterial T4SS. Using biochemical, genetic, and cell biological approaches, we now report both the discovery of a novel antibacterial nuclease (TfdA) and the first structural determination of a bactericidal T4SS effector (TfcB).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Widespread signatures of selection for secreted peptidases in a fungal plant pathogen

Background: Fungal plant pathogens secrete a large arsenal of hydrolytic enzymes during the course of infection, including peptidases. Secreted peptidases have been extensively studied for their role as effectors. In this study, we combined transcriptomics, comparative genomics and evolutionary analyses to investigate all 39 secreted peptidases in the fungal wheat pathogen Zymoseptoria tritici and its close relatives Z. pseudotritici and Z. ardabiliae. Results: RNA-seq data revealed that a majority of the secreted peptidases displayed differential transcription during the course of Z. tritici infection, indicative of specialization for different stages in the life cycle. Evolutionary analyses detected widespread evidence of adaptive evolution acting on at least 28 of the peptidases. A few peptidases displayed lineage-specific rates of molecular evolution, suggesting altered selection pressure in Z. tritici following host specialization on domesticated wheat. The peptidases belonging to MEROPS families A1 and G1 emerged as a particularly interesting group that may play key roles in host-pathogen co-evolution, host adaptation and pathogenicity. Sister genes in the A1 and G1 families showed accelerated substitution rates after gene duplications. Conclusions: These results suggest widespread evolution of secreted peptidases leading to novel gene functions, consistent with predicted models of “escape from adaptive conflict” and “neo-functionalization”. Our analyses identified candidate genes worthy of functional analyses that may encode effector functions, for example by suppressing plant defenses during the biotrophic phase of infection.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Mathematical modeling of hepatitis C RNA replication, exosome secretion and virus release

Hepatitis C virus (HCV) causes acute hepatitis C and can lead to life-threatening complications if it becomes chronic. The HCV genome is a single plus strand of RNA. Its intracellular replication is a spatiotemporally coordinated process of RNA translation upon cell infection, RNA synthesis within a replication compartment, and virus particle production. While HCV is mainly transmitted via mature infectious virus particles, it has also been suggested that HCV-infected cells can secrete HCV RNA carrying exosomes that can infect cells in a receptor independent manner. In order to gain insight into these two routes of transmission, we developed a series of intracellular HCV replication models that include HCV RNA secretion and/or virus assembly and release. Fitting our models to in vitro data, in which cells were infected with HCV, suggests that initially most secreted HCV RNA derives from intracellular cytosolic plus-strand RNA, but subsequently secreted HCV RNA derives equally from the cytoplasm and the replication compartments. Furthermore, our model fits to the data suggest that the rate of virus assembly and release is limited by host cell resources. Including the effects of direct acting antivirals in our models, we found that in spite of decreasing intracellular HCV RNA and extracellular virus concentration, low level HCV RNA secretion may continue as long as intracellular RNA is available. This may possibly explain the presence of detectable levels of plasma HCV RNA at the end of treatment even in patients that ultimately attain a sustained virologic response.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Rhizobial Secretion of Truncated Exopolysaccharides Severely Impairs the Mesorhizobium-Lotus Symbiosis

The symbiosis between Mesorhizobium japonicum R7A and Lotus japonicus Gifu is an important model system for investigating the role of bacterial exopolysaccharides (EPS) in plant-microbe interactions. Previously, we showed that R7A exoB mutants that are affected at an early stage of EPS synthesis and in lipopolysaccharide (LPS) synthesis induce effective nodules on L. japonicus Gifu after a delay, whereas exoU mutants affected in the biosynthesis of the EPS side chain induce small uninfected nodule primordia and are impaired in infection. The presence of a halo around the exoU mutant when grown on Calcofluor-containing media suggested the mutant secreted a truncated version of R7A EPS. A nonpolar ΔexoA mutant defective in the addition of the first glucose residue to the EPS backbone was also severely impaired symbiotically. Here, we used a suppressor screen to show that the severe symbiotic phenotype of the exoU mutant was due to the secretion of an acetylated pentasaccharide, as both monomers and oligomers, by the same Wzx/Wzy system that transports wild-type exopolysaccharide. We also present evidence that the ΔexoA mutant secretes an oligosaccharide by the same transport system, contributing to its symbiotic phenotype. In contrast, ΔexoYF and polar exoA and exoL mutants have a similar phenotype to exoB mutants, forming effective nodules after a delay. These studies provide substantial evidence that secreted incompatible EPS is perceived by the plant, leading to abrogation of the infection process.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Suppressor Mutations in Type II Secretion Mutants of Vibrio cholerae : Inactivation of the VesC Protease

The type II secretion system (T2SS) is a conserved transport pathway responsible for the secretion of a range of virulence factors by many pathogens, including Vibrio cholerae. Disruption of the T2SS genes in V. cholerae results in loss of secretion, changes in cell envelope function, and growth defects. While T2SS mutants are viable, high-throughput genomic analyses have listed these genes among essential genes. To investigate whether secondary mutations arise as a consequence of T2SS inactivation, we sequenced the genomes of six V. cholerae T2SS mutants with deletions or insertions in either the epsG, epsL, or epsM genes and identified secondary mutations in all mutants. Two of the six T2SS mutants contain distinct mutations in the gene encoding the T2SS-secreted protease VesC. Other mutations were found in genes coding for V. cholerae cell envelope proteins. Subsequent sequence analysis of the vesC gene in 92 additional T2SS mutant isolates identified another 19 unique mutations including insertions or deletions, sequence duplications, and single-nucleotide changes resulting in amino acid substitutions in the VesC protein. Analysis of VesC variants and the X-ray crystallographic structure of wild-type VesC suggested that all mutations lead to loss of VesC production and/or function. One possible mechanism by which V. cholerae T2SS mutagenesis can be tolerated is through selection of vesC-inactivating mutations, which may, in part, suppress cell envelope damage, establishing permissive conditions for the disruption of the T2SS. Other mutations may have been acquired in genes encoding essential cell envelope proteins to prevent proteolysis by VesC.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Long‐term drench of exopolysaccharide from Leuconostoc pseudomesenteroides XG5 protects against type 1 diabetes of NOD mice via stimulating GLP ‐1 secretion

Abstract BACKGROUND Type 1 diabetes is an autoimmune disease that results in the specific destruction of insulin‐producing beta cells in the pancreas. The aim of this study was to investigate the mechanism of exopolysaccharide from Leuconostoc pseudomesenteroides XG5 (XG5 EPS) against type 1 diabetes. RESULTS Long‐term drench of XG5 EPS delayed the onset of autoimmune diabetes and had fewer islets with high‐grade infiltration (an insulitis score of 3 or 4) than untreated NOD mice. Oral administration of 50 mg kg −1 d −1 XG5 EPS increased the insulin and glucagon‐like peptide‐1 (GLP‐1) levels of serum, stimulated GLP‐1 secretion and upregulated gcg mRNA expression of colon in NOD mice. Moreover, oral administration of 50 mg kg −1 d −1 XG5 EPS significantly increased total short‐chain fatty acids levels in the colon contents, especially those of acetic acid and butyric acid. In NCI‐H716 cells, 500 and 1000 μmol L −1 sodium butyrate promoted the secretion of GLP‐1 and upregulated the mRNA expression of gcg and PC3 , while XG5 EPS and sodium acetate did not stimulate the GLP‐1 secretion. Therefore, long‐term drench of XG5 EPS delayed the onset of autoimmune diabetes, which may be directly correlated with the increase of butyrate in the colon of NOD mice. CONCLUSION Long‐term drench of 50 mg kg −1 d −1 XG5 EPS promoted the expression and secretion of GLP‐1 by increasing the production of butyric acid, thereby delaying T1D onset in NOD mice. © 2021 Society of Chemical Industry.

Pan, Lei↗