Role of cysteine residues and disulfide bonds in the activity of a legume root nodule-specific, cysteine-rich peptide

The root nodules of certain legumes including Medicago truncatula produce >300 different nodule-specific cysteine-rich (NCR) peptides. Medicago NCR antimicrobial peptides (AMPs) mediate the differentiation of the bacterium, Sinorhizobium meliloti into a nitrogen-fixing bacteroid within the legume root nodules. In vitro, NCR AMPs such as NCR247 induced bacteroid features and exhibited antimicrobial activity against S. meliloti. The bacterial BacA protein is critical to prevent S. meliloti from being hypersensitive toward NCR AMPs. NCR AMPs are cationic and have conserved cysteine residues, which form disulfide (S-S) bridges. However, the natural configuration of NCR AMP S-S bridges and the role of these in the activity of the peptide are unknown. In this study, we found that either cysteine replacements or S-S bond modifications influenced the activity of NCR247 against S. meliloti. Specifically, either substitution of cysteines for serines, changing the S-S bridges from cysteines 1-2, 3-4 to 1-3, 2-4 or oxidation of NCR247 lowered its activity against S. meliloti. We also determined that BacA specifically protected S. meliloti against oxidized NCR247. Due to the large number of different NCRs synthesized by legume root nodules and the importance of bacterial BacA proteins for prolonged host infections, these findings have important implications for analyzing the function of these novel peptides and the protective role of BacA in the bacterial response toward these peptides.     

Antimicrobial nodule-specific cysteine-rich peptides disturb the integrity of bacterial outer and inner membranes and cause loss of membrane potential

Background

Certain legume plants produce a plethora of AMP-like peptides in their symbiotic cells. The cationic subgroup of the nodule-specific cysteine-rich (NCR) peptides has potent antimicrobial activity against gram-negative and gram-positive bacteria as well as unicellular and filamentous fungi.

Findings

It was shown by scanning and atomic force microscopies that the cationic peptides NCR335, NCR247 and Polymyxin B (PMB) affect differentially on the surfaces of Sinorhizobium meliloti bacteria. Similarly to PMB, both NCR peptides caused damages of the outer and inner membranes but at different extent and resulted in the loss of membrane potential that could be the primary reason of their antimicrobial activity.

Conclusions

The primary reason for bacterial cell death upon treatment with cationic NCR peptides is the loss of membrane potential.

     

Direct Recognition of Fusobacterium nucleatum by the NK Cell Natural Cytotoxicity Receptor NKp46 Aggravates Periodontal Disease

Periodontitis is a common human chronic inflammatory disease that results in the destruction of the tooth attachment apparatus and tooth loss. Although infections with periopathogenic bacteria such as Porphyromonas gingivalis (P. gingivalis) and Fusobacterium nucleatum (F. nucleatum) are essential for inducing periodontitis, the nature and magnitude of the disease is determined by the host''s immune response. Here, we investigate the role played by the NK killer receptor NKp46 (NCR1 in mice), in the pathogenesis of periodontitis. Using an oral infection periodontitis model we demonstrate that following F. nucleatum infection no alveolar bone loss is observed in mice deficient for NCR1 expression, whereas around 20% bone loss is observed in wild type mice and in mice infected with P. gingivalis. By using subcutaneous chambers inoculated with F. nucleatum we demonstrate that immune cells, including NK cells, rapidly accumulate in the chambers and that this leads to a fast and transient, NCR1-dependant TNF-α secretion. We further show that both the mouse NCR1 and the human NKp46 bind directly to F. nucleatum and we demonstrate that this binding is sensitive to heat, to proteinase K and to pronase treatments. Finally, we show in vitro that the interaction of NK cells with F. nucleatum leads to an NCR1-dependent secretion of TNF-α. Thus, the present study provides the first evidence that NCR1 and NKp46 directly recognize a periodontal pathogen and that this interaction influences the outcome of F. nucleatum-mediated periodontitis.     

Control of Larval Northern Corn Rootworm. (Diabrotica barberi) with Two Steinernematid Nematode Species

The entomogenous nematodes Steinerema feltiae and S. bibionis did not penetrate the roots of corn, Zea mays, to infect larval northern corn rootworm (NCR), Diabrotica barberi, feeding within. Laboratory bioassays against first instar NCR indicated that S. feltiae, Mexican strain (LD₅₀ = 49 nematodes/insect) is more virulent than S. bibionis (LD₅₀ = 100). Numbers of NCR larvae in a grain corn crop were reduced by both nematode species applied at corn seeding time at the rate of 10,000 infective-stage juveniles per linear meter of corn row. The chemical insecticide fonofos provided significantly better control than either nematode species.     

The involvement of Medicago truncatula non-specific lipid transfer protein N5 in the control of rhizobial infection

Cysteine-rich proteins seem to play important regulatory roles in Medicago truncatula/Sinorhizobium meliloti symbiosis. In particular, a large family of nodule-specific cysteine-rich (NCR) peptides is crucial for the differentiation of nitrogen-fixing bacteroids. The Medicago truncatula N5 protein (MtN5) is currently the only reported non-specific lipid transfer protein necessary for successful rhizobial symbiosis; in addition, MtN5 shares several characteristics with NCR peptides: a small size, a conserved cysteine-rich motif, an N-terminal signal peptide for secretion and antimicrobial activity. Unlike NCR peptides, MtN5 expression is not restricted to the root nodules and is induced during the early phases of symbiosis in root hairs and nodule primordia. Recently, MtN5 was determined to be involved in the regulation of root tissue invasion; while, it was dispensable for nodule primordia formation. Here, we discuss the hypothesis that MtN5 participates in linking the progression of bacterial invasion with restricting the competence of root hairs for infection.     

Complete mtDNA of Meretrix lusoria (Bivalvia: Veneridae) reveals the presence of an atp8 gene, length variation and heteroplasmy in the control region

The complete nucleotide sequence of the mitochondrial genome of the clam Meretrix lusoria (Bivalvia: Veneridae) was determined. It comprises 20,268 base pairs (bp) and contains 13 protein-coding genes, including ATPase subunit 8 (atp8), two ribosomal RNAs, 22 transfer RNAs, and a non-coding control region. The atp8 encodes a protein of 39 amino acids. All genes are encoded on the same strand. A putative control region (CR or D-loop) was identified in the major non-coding region (NCR) between the tRNA^Gly and tRNA^Gln. A 1087 bp tandem repeat fragment was identified that comprises nearly 11 copies of a 101 bp motif and accounts for approximately 41% of the NCR. The 101 bp tandem repeat motif of the NCR can be folded into a stem–loop secondary structure. Samples of eight individuals from Hainan and Fujian provinces were collected and their NCR regions were successfully amplified and sequenced. The data revealed a highly polymorphic VNTR (variable number of tandem repeats) associated with high levels of heteroplasmy in the D-loop region. The size of the CR ranged from 1942 to 3354 bp depending upon the copy number of the repeat sequence.     

Analysis of Replication Intermediates Indicates That Drosophila melanogaster Mitochondrial DNA Replicates by a Strand-Coupled Theta Mechanism

Mitochondrial DNA synthesis is necessary for the normal function of the organelle and for the eukaryotic organism as a whole. Here we demonstrate, using two-dimensional agarose gel electrophoresis to analyse replication intermediates, that unidirectional, strand-coupled DNA synthesis is the prevalent mode of mtDNA replication in Drosophila melanogaster. Commencing within the single, extended non-coding region (NCR), replication proceeds around the circular genome, manifesting an irregular rate of elongation, and pausing frequently in specific regions. Evidence for a limited contribution of strand-asynchronous DNA synthesis was found in a subset of mtDNA molecules, but confined to the ribosomal RNA gene region, just downstream of the NCR. Our findings imply that strand-coupled replication is widespread amongst metazoans, and should inform future research on mtDNA metabolism in D. melanogaster.     

Metabolic Engineering of the Regulators in Nitrogen Catabolite Repression To Reduce the Production of Ethyl Carbamate in a Model Rice Wine System

Rice wine has been one of the most popular traditional alcoholic drinks in China. However, the presence of potentially carcinogenic ethyl carbamate (EC) in rice wine has raised a series of food safety issues. During rice wine production, the key reason for EC formation is urea accumulation, which occurs because of nitrogen catabolite repression (NCR) in Saccharomyces cerevisiae. NCR represses urea utilization by retaining Gln3p in the cytoplasm when preferred nitrogen sources are present. In order to increase the nuclear localization of Gln3p, some possible phosphorylation sites on the nuclear localization signal were mutated and the nuclear localization regulation signal was truncated, and the disruption of URE2 provided an additional method of reducing urea accumulation. By combining these strategies, the genes involved in urea utilization (DUR1,2 and DUR3) could be significantly activated in the presence of glutamine. During shake flask fermentations of the genetically modified strains, very little urea accumulated in the medium. Furthermore, the concentrations of urea and EC were reduced by 63% and 72%, respectively, in a model rice wine system. Examination of the normal nutrients in rice wine indicated that there were few differences in fermentation characteristics between the wild-type strain and the genetically modified strain. These results show that metabolic engineering of the NCR regulators has great potential as a method for eliminating EC during rice wine production.     

Association of CD247 Polymorphisms with Rheumatoid Arthritis: A Replication Study and a Meta-Analysis

Given the role of CD247 in the response of the T cells, its entailment in autoimmune diseases and in order to better clarify the role of this gene in RA susceptibility, we aimed to analyze CD247 gene variants previously associated with other autoimmune diseases (rs1052237, rs2056626 and rs864537) in a large independent European Caucasian population. However, no evidence of association was found for the analyzed CD247 single-nucleotide polymorphisms (SNPs) with RA and with the presence/absence of anti-cyclic citrullinated polypeptide. We performed a meta-analysis including previously published GWAS data from the rs864537 variant, revealing an overall genome-wide significant association between this CD247 SNP and RA with anti-CCP (OR = 0.90, CI 95% = 0.87–0.93, P_overall = 2.1×10⁻¹⁰). Our results show for first time a GWAS-level association between this CD247 polymorphism and RA risk.     

Targeting Natural Killer Cell Reactivity by Employing Antibody to NKp46: Implications for Type 1 Diabetes

Natural killer (NK) cells belong to the innate lymphoid cells. Their cytotoxic activity is regulated by the delicate balance between activating and inhibitory signals. NKp46 is a member of the primary activating receptors of NK cells. We previously reported that the NKp46 receptor is involved in the development of type 1 diabetes (T1D). Subsequently, we hypothesized that blocking this receptor could prevent or hinder disease development. To address this goal, we developed monoclonal antibodies for murine NKp46. One mAb, named NCR1.15, recognizes the mouse homologue protein of NKp46, named Ncr1, and was able to down-regulate the surface expression of NKp46 on primary murine NK cells following antibody injection in vivo. Additionally, NCR1.15 treatments were able to down-regulate cytotoxic activity mediated by NKp46, but not by other NK receptors. To test our primary assumption, we examined T1D development in two models, non-obese diabetic mice and low-dose streptozotocin. Our results show a significantly lower incidence of diabetic mice in the NCR1.15-treated group compared to control groups. This study directly demonstrates the involvement of NKp46 in T1D development and suggests a novel treatment strategy for early insulitis.     

The Functional Roles of the His247 and His281 Residues in Folate and Proton Translocation Mediated by the Human Proton-coupled Folate Transporter SLC46A1

This report addresses the functional role of His residues in the proton-coupled folate transporter (PCFT; SLC46A1), which mediates intestinal folate absorption. Of ten His residues, only H247A and H281A mutations altered function. The folic acid influx K_t at pH 5.5 for H247A was ↓8.4-fold. Although wild type (WT)-PCFT K_i values varied among the folates, K_i values were much lower and comparable for H247-A, -R, -Q, or -E mutants. Homology modeling localized His²⁴⁷ to the large loop separating transmembrane domains 6 and 7 at the cytoplasmic entrance of the translocation pathway in hydrogen-bond distance to Ser¹⁷². The folic acid influx K_t for S172A-PCFT was decreased similar to H247A. His²⁸¹ faces the extracellular region in the seventh transmembrane domain. H281A-PCFT results in loss-of-function due to ∼12-fold↑ in the folic acid influx K_t. When the pH was decreased from 5.5 to 4.5, the WT-PCFT folic acid influx K_t was unchanged, but the K_t decreased 4-fold for H281A. In electrophysiological studies in Xenopus oocytes, both WT-PCFT- and H281A-PCFT-mediated folic acid uptake produced current and acidification, and both exhibited a low level of folate-independent proton transport (slippage). Slippage was markedly increased for the H247A-PCFT mutant. The data suggest that disruption of the His²⁴⁷ to Ser¹⁷² interaction results in a PCFT conformational alteration causing a loss of selectivity, increased substrate access to a high affinity binding pocket, and proton transport in the absence of a folate gradient. The His²⁸¹ residue is not essential for proton coupling but plays an important role in PCFT protonation, which, in turn, augments folate binding to the carrier.     

Cysteine Residues in the Major Capsid Protein,Vp1, of the JC Virus Are Important for Protein Stability and Oligomer Formation

The capsid of the human polyomavirus JC virus (JCV) consists of 72 pentameric capsomeres of a major structural protein, Vp1. The cysteine residues of the related Vp1 of SV40 are known to contribute to Vp1 folding, pentamer formation, pentamer-pentamer contacts, and capsid stabilization. In light of the presence of a slight structural difference between JCV Vp1 and SV40 counterpart, the way the former folds could be either different from or similar to the latter. We found a difference: an important contribution of Vp1 cysteines to the formation of infectious virions, unique in JCV and absent in SV40. Having introduced amino acid substitution at each of six cysteines (C42, C80, C97, C200, C247, and C260) in JCV Vp1, we found that, when expressed in HeLa cells, the Vp1 level was decreased in C80A and C247A mutants, and remained normal in the other mutants. Additionally, the C80A and C247A Vp1-expressing cell extracts did not show the hemagglutination activity characteristic of JCV particles. The C80A and C247A mutant Vp1s were found to be less stable than the wild-type Vp1 in HeLa cells. When produced in a reconstituted in vitro protein translation system, these two mutant proteins were stable, suggesting that some cellular factors were responsible for their degradation. As determined by their sucrose gradient sedimentation profiles, in vitro translated C247A Vp1 formed pentamers, but in vitro translated C80A Vp1 was entirely monomeric. When individually incorporated into the JCV genome, the C80A and C247A mutants, but not the other Vp1 cysteine residues mutants, interfered with JCV infectivity. Furthermore, the C80A, but not the C247A, mutation prevented the nuclear localization of Vp1 in JCV genome transfected cells. These findings suggest that C80 of JCV Vp1 is required for Vp1 stability and pentamer formation, and C247 is involved in capsid assembly in the nucleus.     

Antifungal peptidic compound from the deep-sea bacterium <Emphasis Type="Italic">Aneurinibacillus</Emphasis> sp. YR247

Aneurinibacillus: sp. YR247 was newly isolated from the deep-sea sediment inside the Calyptogena community at a depth of 1171 m in Sagami Bay. The strain exhibited antifungal activity against the filamentous fungus Aspergillus brasiliensis NBRC9455. A crude extract prepared from the YR247 cells by ethanol extraction exhibited broad antimicrobial activities. The antifungal compound is stable at 4–70?°C and pH 2.0–12.0. After treatment with proteinase K, the antifungal activity was not detected, indicating that the antifungal compound of strain YR247 is a peptidic compound. Electrospray ionization mass spectrometry of the purified antifungal compound indicated that the peptidic compound has an average molecular weight of 1167.9. The molecular weight of the antifungal compound from strain YR247 is different from those of antimicrobial peptides produced by the related Aneurinibacillus and Bacillus bacteria. The antifungal peptidic compound from the deep-sea bacterium Aneurinibacillus sp. YR247 may be useful as a biocontrol agent.