Archive 22/5/11

Differences in glycan binding specificities among Rotavirus-A, -B, and -C

A group from Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX USA, etc. has reported about folding structures of rotavirus spike protein VP4 and the difference in those glycan binding specificities.

Rotaviruses are classified into ten different species or groups (A–J). Group A, B, C, and H rotaviruses infect both humans and animals. Epidemiologically, groups A, B, and C are the best characterized. Group A rotaviruses (RVA) and to a lesser extent group C rotaviruses (RVC) are the causative agents of most gastroenteric infections worldwide, the group B rotaviruses (RVB) have been associated with large epidemic outbreaks of severe gastroenteritis in China and sporadic infection in several countries.

The viral genome consists of 11 segments of double-stranded RNA that code for 6 structural viral proteins (VP) and 6 nonstructural proteins. The infectious virion is a triple-layered particle consisting of a core layer made of VP2, an intermediate layer made of VP6, and an outer shell made of glycoprotein VP7. Sixty protein spikes made of a protease-sensitive protein, VP4, extend from the VP7 shell.

The proteolytic treatment of VP4 results in two fragments, VP8* and VP5*. Sequence comparison of the structural proteins encoded by different rotavirus groups shows that the VP8* domain of the spike protein VP4 is the most variable. Extensive structural studies have shown that there is a galectin-like domain in human RVA and RVC (VP8*A and VP8*C), and recognize various cellular glycans in a genotype-dependent manner. The most typical glycan binding specificity of VP8*A and VP8*C are known to be H-antigen and A-antigen respectively.

Interestingly, the VP8*B shares no sequence identity with either VP8*A or VP8*C, which could differentially impact not only the structure but also the glycan-binding properties. Authors have found that VP8*B exhibits a fold with a twisted β-sheet clasping an α-helix that is entirely different from VP8*A or VP8*C, and glycan array screening and in silico docking analysis show VP8*B recognizes glycans containing LacNAc.

Wheat Rhizosphere: Effects of ACC deaminase-producing rhizobacteria (Enterobacter cloacae ZNP-4)on wheat growth

A group from Department of Bioengineering and Biotechnology, Birla Institute of Technology, Mesra, Ranchi, Jharkhand, India, etc. has reported about the effects of 1-aminocyclopropane-1-carboxylic acid deaminase (ACCD) producing Plant growth promoting rhizobacterium (PGPR) designated as Enterobacter cloacae ZNP-4 on wheat growth under abiotic stressors such as salt (NaCl) and metal (ZnSO4) stress.

Many of the rhizosphere bacteria produce ACCD, which reduces the level of ‘stress ethylene’ in their associated plants by degrading ACC to ammonia and α-ketobutyrate, thereby minimizing the substrate availability for ethylene generation. It has been shown that micro-organisms with ACCD activity >20 nmol α-ketobutyrate mg-1 h-1 are sufficient to enhance plant growth under stress conditions.

Like many other crops, germination of wheat seed and seedling growth are severely affected by salt and metal stress worldwide. The various conventional methods are in practice for alleviating salt stress, but most of them are costly and deleterious to environments. The micro-organisms residing in the rhizosphere have proved to regulate plant growth under normal and stress conditions. Therefore, this study aimed to investigate the effectiveness of ACCD-producing bacterium Enterobacter cloacae ZNP-4 as a biological tool for alleviating the adverse effects of abiotic stressors and examined for its potential to alleviate stress-induced plant growth inhibition.

As a result, the inoculation of strain ZNP-4 significantly improved the various growth parameters of wheat plant such as shoot length (41%), root length (31%), fresh weight (28%), dry weight (29%), photosynthetic pigments chlorophyll a (62%) and chlorophyll b (34%). Additionally, the strain was found to be efficient for minimizing the imposed Zn stress in terms of improving plant growth, biomass and photosynthetic pigments in pots containing different levels of metal stress of 150 mg kg-1 (treatment T1) and 250 mg kg-1 (treatment T2), where (T0: 0 mM; T1: 150 mM, T2: 200 mM NaCl) are different salinity conditions.

In addition, Effect of bacterial inoculation on generation of abiotic stress-induced reactive oxygen species (ROS) was monitored under salinity and metal stress treatments.
The positive effects of PGPR occurred concurrently with the decrease in abiotic stress-induced reactive oxygen species (ROS) molecules such as hydrogen peroxide (H2O2) and superoxide (O2) contents, which lead to lipid peroxidation, membrane deterioration, metabolic and structural dysfunctions, further leading to cell death.
The bacterial inoculation significantly reduced the H2O2 level under tested salinity stress. The highest significant (p=0.05) decrease of 43.2% was recorded at treatment T1 followed by 32.5% at treatment T2. The salinity induced generation of O2 content was also minimized with 52.7% (p=0.05) and 49% (p=0.05) at treatment T1 and T2 in bacterial treated plants.

Complicated control of phytobeneficial bacteria in rhizosphere: DAPG, Siderophores

A group from Lab Microbial Ecology of the Rhizosphere (LEMiRE), CEA, CNRS, BIAM, Aix Marseille Univ, Saint-Paul-Lez-Durance, France, etc. has reported about the complexity of the multiple molecular dialogues that take place underground between microorganisms and between plants and its rhizosphere microbiota.

In this report, it was investigated the ability of the plant root-associated Pseudomonas brassicacearum NFM421 known as phytobeneficial bacteria to compete with two other rhizobacteria, Kosakonia sacchari NO9, a root-associated diazotroph and Rhizobium alamii YAS34, known for its ability to produce exopolysaccharide. Especially, the expression of the well known phytobeneficial genes phlD (production of DAPG), hcnA (production of hydrogen cyanide) and acdS (ACC deaminase activity) was evaluated in a variety of situations. The intracellular status of iron in P. brassicacearum NFM421 was also evaluated by analysing the expression of iron-regulatory RNAs prrF in this strain, because when iron is scarce, bacteria produce siderophores that may act as antifungals by depriving fungi of iron. The antagonism of P. brassicacearum NFM421 wt and the knockout mutants ∆phlD and ∆gacA against the soil-borne plant pathogens Fusarium culmorum, Fusarium graminearum and Microdochium nivale was evaluated as well, to compare the antifungal activity of DAPG and HCN.

Pseudomonads promote plant growth and produce antimicrobial secondary metabolites including HCN and DAPG. The expression of phlD was one thousand times higher than that of hcnA, indicating that even if HCN is known to be effective against pathogenic fungi, the levels of its expression by P. brassicacearum NFM421 are too low to inhibit fungal growth. Therefore, the mode of biocontrol of plant pathogenic fungi used in this study, which seems plausible in P. brassicacearum NFM421, most likely involves DAPG.

showing antifungal activity of P. brassicacearum NFM421wt, ∆phlD and ∆gacA towards Fusarium culmorum (Fc), Fusarium graminearum (Fg) or Microdochium nivale (Mn)

On in vitro conditions in CAA medium, the presence of the other two strains, N09 and YAS34, (separately or together) caused significant transcriptional changes in the expression of phlD, hcnA and acdS in P. brassicacearum NFM421 under iron-rich conditions. phlD was positively regulated by iron, an almost fourfold increase under iron-rich conditions, when the bacterium was grown alone. However, the presence of the competitors in CAA medium significantly decreased the phlD expression under iron-rich conditions (twofold), while no significant difference was observed under iron-depleted conditions. However, contrary to what was observed under in vitro conditions, phlD expression in P. brassicacearum NFM421 did not change during their interaction with B. napus root system in response to iron availability when grown alone.

As a conclusion, a scheme targeting the modulation of phl gene expression by other microorganisms and the plant was proposed, as well as the role of iron and its regulation by prrF RNAs. Anyway, the mechanisms by which one bacterial population interferes with the gene expression of another population are not yet fully understood and are future problems.

Pseudomonas strains inhibit Ralstonia solanacearum and its underlying mechanisms: Importance of Secondary metabolites, DAPG and Orfamides

A group from Department of Biology, University of York, York, UK, etc. has reported about Ralstonia solanacearum (the causative agent of bacterial wilt) inhibition by Pseudomonas strains and its underlying mechanisms of potential pathogen suppression.

It was found that Pseudomonas protegens CHA0 was the most inhibitory biocontrol strain against Ralstonia solanacearum.

Secondary metabolite clusters were analyzed by using antiSMASH, and from 11 to 17 metabolic clusters were identified in each of the eight Pseudomonas genomes. Nonribosomal peptide synthetases (NRPS) were the most abundant secondary metabolite clusters. Similarly, DAPG metabolite (belonging to the T3PKS cluster), as well as the pyoverdine siderophore (NRPS cluster) metabolite clusters, were found in all strains. Overall, the highest number of clusters were detected in CHA0 and Pf-5 strains. These strains also harbored some unique metabolite clusters such as the T1PKS metabolic cluster, which encodes pyoluteorin antimicrobial, and the CDPS cluster, which encodes unknown metabolites. CHA0 and Pf-5 also had the greatest number of NRPS clusters and were the only two strains capable of producing the cyclic lipopeptides known as orfamides, and only orfamide A was production by CHA0 strain.

DAPG suppressed every R. solanacearum strain in a concentration-dependent manner, and all tested strains were unable to grow at the two highest concentrations (500 and 1000 μM).

Orfamide variants “A” and “B” isolated from the Pseudomonas CHA0 strain were tested against R solanacearum strains (only #1 and #7 due to limited quantities of extracted compounds). Orfamide variants suppressed both Ralstonia strains as well.

It is quite likely that secondary metabolites from Pseudomonas strains such as DAPG and Orfamides could be pathogen-suppressing materials against R. solanacearum.

A unique plant lectin from Narcissus tazetta bulb, NTL-125, could effectively inhibit SARS-CoV-2 replication

A group from Division of Plant Biology, Bose Institute, P/12 C.I.T. Scheme VII(M), Kolkata, 700054, India, etc. has reported that a unique mannose binding plant lectin from Narcissus tazetta bulb, NTL-125, could effectively inhibit SARS-CoV-2 replication in Vero-E6 cell line.

A unique mannose binding plant lectin from Narcissus tazetta bulb named NTL-125 could inhibit SARS-CoV-2 replication in Vero-E6 cell line. The inhibitory concentration using Vero-E6 cells that reduced the SARS-CoV-2 viral entry by 50% (IC50) was approximately 0.8 µg/mL (i.e., 50 nM), and the cytotoxicity of NLE-125 on Vero-E6 cells was above 95% viable at 5 µg/mL and 85% viable at 10 µg/mL concentration.

The molecular docking studies revealed that thirty-six residues of RBD and 44 residues of NTL-125 were located within 5Å distance from each other in the complex whereas 27 residues of ACE-2 are in proximity to 32 residues of RBD. There are 17 common residues of RBD that interact with both ACE-2 and NTL-125. Among all these residues, for the RBD:NTL-125 complex, 10 of RBD and 11 of NTL-125 are within 3Å distance in the docked structure. For the RBD:ACE-2 complex, 9 residues of RBD and 9 residues of ACE-2 are within 3Å distance. NTL-125 occupies exactly the same region of the receptor binding motif (RBM) of the spike protein where hACE2 usually binds. As a result, the binding free energy change of NTL125–Spike protein interaction (-13.3 kcal/mol, kd ~0.41nM) is more negative than that between ACE2-Spike protein (-11.2 kcal/mol, kd ~12 nM) which clearly indicates that the former is more stable than the later.

Docking study confirmed further that the interaction between NTL-125-spike protein is also mediated through a S-protein glycan moiety, covalently linked to Asn165 of the spike protein and interacts with Ile137 and Thr138 of NTL-125 protein. Thus, the interaction between NTL-125-Spike is not only through amino acid residues but also through the glycan moieties.

O-glycosylation unique to SARS-CoV-2 Omicron variant: GalNAcGal(NeuAc)2 at Thr376

A group from Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53706, USA has reported existence of O-glycosylation unique to SARS-CoV-2 Omicron variant.

HEK293 expressed S-RBD protein arising from WT (WA1/2020), Delta (T478K), and Omicron (BA.1) variants were analyzed by Fourier transform ion cyclotron (FTICR)-MS and trapped ion mobility spectrometry (TIMS)-MS. To elucidate the molecular sequence and O-glycans of the various S-RBDs, N-glycans from the S-RBD were removed by using a PNGase F treatment to minimize the interference posed by N-glycan heterogeneity.

Detailed top-down MS/MS analysis of the S-RBD O-glycoforms revealed the presence of a new O-glycosite (Thr376) unique to the Omicron variant. Fascinatingly, all detected S-RBD O-glycans for the WT and Delta variants were confidently assigned solely to Thr323. Given the smaller number of mutations present on Delta as compared to Omicron, it is not surprising that the O-glcyosite Thr323 was conserved between Delta and WT variants.
On the other hand, the Omicron variant yielded both the familiar Thr323 O-glycosite and a new Thr376 O-glycosite corresponding to the GalNAcGal(NeuAc)2 O-glycoform that is simultaneously occupied. This Thr376 O-glycosite is conveniently n + 3 adjacent to a proline at residue 373, which is consistent with previous reports of increased O-glycosylation frequency near proline. This particular Pro373 is a site-specific mutation unique to the Omicron variant and likely is the reason for this new O-glycosite. It might be better to note that the site occupancy of the Thr376 site was low(< 30%) relative to the Thr323. However, it is unclear at this moment if Thr376 O-glycan has any relationship with Omicron infectivity and escape from immunological protection.

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