Insights on genomic diversity of Vibrio spp. through Pan-genome analysis

Purpose

The aquaculture sector is a major contributor to the economic and nutritional security for a number of countries. India’s total seafood exports for the year 2017–2018 accounted for US$ Million 7082. One of the major setbacks in this sector is the frequent outbreaks of diseases often due to bacterial pathogens. Vibriosis is one of the major diseases caused by bacteria of Vibrio spp., causing significant economic loss to the aquaculture sector. The objective of this study was to understand the genetic composition of Vibrio spp.

Methods

Thirty-five complete genomes were downloaded from GenBank comprising seven vibrio species, namely, Vibrio alginolyticus, V. anguillarum, V. campbellii, V. harveyi, V. furnissii, V. parahaemolyticus, and V. vulnificus. Pan-genome analysis was carried out with coding sequences (CDS) generated from all the Vibrio genomes. In addition, genomes were mined for genes coding for toxin-antitoxin systems, antibiotic resistance, genomic islands, and virulence factors.

Results

Results revealed an open pan-genome comprising of 2004 core, 8249 accessory, and 6780 unique genes. Downstream analysis of genomes and the identified unique genes resulted in 312 antibiotic resistance genes, 430 genes coding for toxin and antitoxin systems along with 4802, and 4825 putative virulent genes from genomic island regions and unique gene sets, respectively.

Conclusion

Pan-genome and other downstream analytical procedures followed in this study have the potential to predict strain-specific genes and their association with habitat and pathogenicity.

     

Temporal changes in stress and tissue-specific metabolic responses to municipal wastewater effluent exposure in rainbow trout

Sub-chronic exposure to municipal wastewater effluent (MWWE) in situ was recently shown to impact the acute response to a secondary stressor in rainbow trout (Oncorhynchus mykiss). However, little is known about whether MWWE exposure in itself is stressful to the animal. To address this, we carried out a laboratory study to examine the organismal and cellular stress responses and tissue-specific metabolic capacity in trout exposed to MWWE. Juvenile rainbow trout were exposed to 0, 20 and 90% MWWE (from a tertiary wastewater treatment plant), that was replenished every 2d, for 14 d. Fish were sampled 2, 8 or 14 d post-exposure. Plasma cortisol, glucose and lactate levels were measured as indicators of organismal stress response, while inducible heat shock protein 70 (hsp70), constitutive heat shock protein 70 (hsc70) and hsp90 expression in the liver were used as markers of cellular stress response. Impact of MWWE on cortisol signaling was ascertained by determining glucocorticoid receptor protein (GR) expression in the liver, brain and, heart, and metabolic capacity was evaluated by measuring liver glycogen content and tissue-specific activities of key enzymes in intermediary metabolism. Plasma glucose and lactate levels were unaffected by exposure to MWWEs, whereas cortisol showed a transient increase in the 20% group at 8d. Liver hsc70 and hsp90, but not hsp70 expression, were higher in the 90% MWWE group after 8d. There was a temporal change in GR expression in the liver and heart, but not brain of trout exposed to MWWE. Liver glycogen content and activities liver gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK), and alanine aminotransferase (AlaAT) were significantly affected by MWWE exposure. The glycolytic enzymes pyruvate kinase (PK) and hexokinase (HK) activities were significantly higher temporally by MWWE exposure in the gill and heart, but not in the liver and brain. Overall, a 14 d exposure to MWWE evokes a cellular stress response and perturbs the cortisol stress response in rainbow trout. The tissue-specific temporal changes in the metabolic capacity suggest enhanced energy demand in fish exposed to MWWE, which may eventually lead to reduced fitness.     

Vaccine efficacy against malaria by the combination of porcine parvovirus-like particles and vaccinia virus vectors expressing CS of Plasmodium

With the aim to develop an efficient and cost-effective approach to control malaria, we have generated porcine parvovirus-like particles (PPV-VLPs) carrying the CD8(+) T cell epitope (SYVPSAEQI) of the circumsporozoite (CS) protein from Plasmodium yoelii fused to the PPV VP2 capsid protein (PPV-PYCS), and tested in prime/boost protocols with poxvirus vectors for efficacy in a rodent malaria model. As a proof-of concept, we have characterized the anti-CS CD8(+) T cell response elicited by these hybrid PPV-VLPs in BALB/c mice after immunizations with the protein PPV-PYCS administered alone or in combination with recombinant vaccinia virus (VACV) vectors from the Western Reserve (WR) and modified virus Ankara (MVA) strains expressing the entire P. yoelii CS protein. The results of different immunization protocols showed that the combination of PPV-PYCS prime/poxvirus boost was highly immunogenic, inducing specific CD8+ T cell responses to CS resulting in 95% reduction in liver stage parasites two days following sporozoite challenge. In contrast, neither the administration of PPV-PYCS alone nor the immunization with the vectors given in the order poxvirus/VLPs was as effective. The immune profile induced by VLPs/MVA boost was associated with polyfunctional and effector memory CD8+ T cell responses. These findings highlight the use of recombinant parvovirus PPV-PYCS particles as priming agents and poxvirus vectors, like MVA, as booster to enhance specific CD8+ T cell responses to Plasmodium antigens and to control infection. These observations are relevant in the design of T cell-inducing vaccines against malaria.     

Suppression of high lipid diet induced by atherosclerosis sarpogrelate

Sarpogrelate (SP), a serotonin (5‐HT2A) receptor antagonist, is used as an anti‐platelet agent for the treatment of some vascular diseases. SP has been reported to inhibit 5‐HT induced coronary artery spasm, increase in intracellular calcium and smooth muscle cells proliferation. This study was undertaken to test that SP suppresses the development of atherosclerosis due to high cholesterol diet (HCD) by decreasing blood viscosity and oxidative stress. For this purpose, 29 rabbits were divided into four groups: control group (normal diet); normal diet group with SP at the dose of 5 mg/kg/day; HCD group fed 1% cholesterol; and HCD group with SP at the dose of 5 mg/kg/day. After 90 days of the experiment, blood samples were collected and the animals were killed; the thoracic aorta was stained by the Oil Red O staining method. The results indicate that plasma levels of cholesterol, triglycerides and malondialdehyde were increased in rabbits fed HCD. Plasma viscosity and whole blood viscosity were also higher in the HCD group than that in normal diet group. Treatment with SP prevented these alterations induced by HCD whereas this agent had no significant effect in rabbits fed normal diet. Morphological examination of the aorta revealed that SP treatment prevented the formation of foam cells and atherosclerotic plaque. It is suggested that the beneficial effects of SP in atherosclerosis may be due to actions on blood viscosity, lipid levels and oxidative stress.     

Reversal of subcellular remodelling by losartan in heart failure due to myocardial infarction

This study tested the reversal of subcellular remodelling in heart failure due to myocardial infarction (MI) upon treatment with losartan, an angiotensin II receptor antagonist. Twelve weeks after inducing MI, rats were treated with or without losartan (20 mg/kg; daily) for 8 weeks and assessed for cardiac function, cardiac remodelling, subcellular alterations and plasma catecholamines. Cardiac hypertrophy and lung congestion in 20 weeks MI‐induced heart failure were associated with increases in plasma catecholamine levels. Haemodynamic examination revealed depressed cardiac function, whereas echocardiographic analysis showed impaired cardiac performance and marked increases in left ventricle wall thickness and chamber dilatation at 20 weeks of inducing MI. These changes in cardiac function, cardiac remodelling and plasma dopamine levels in heart failure were partially or fully reversed by losartan. Sarcoplasmic reticular (SR) Ca²⁺‐pump activity and protein expression, protein and gene expression for phospholamban, as well as myofibrillar (MF) Ca²⁺‐stimulated ATPase activity and α‐myosin heavy chain mRNA levels were depressed, whereas β‐myosin heavy chain expression was increased in failing hearts; these alterations were partially reversed by losartan. Although SR Ca²⁺‐release activity and mRNA levels for SR Ca²⁺‐pump were decreased in failing heart, these changes were not reversed upon losartan treatment; no changes in mRNA levels for SR Ca²⁺‐release channels were observed in untreated or treated heart failure. These results suggest that the partial improvement of cardiac performance in heart failure due to MI by losartan treatment is associated with partial reversal of cardiac remodelling as well as partial recovery of SR and MF functions.     

Structural studies on the second Mycobacterium smegmatis Dps: invariant and variable features of structure, assembly and function

A second DNA binding protein from stationary-phase cells of Mycobacterium smegmatis (MsDps2) has been identified from the bacterial genome. It was cloned, expressed and characterised and its crystal structure was determined. The core dodecameric structure of MsDps2 is the same as that of the Dps from the organism described earlier (MsDps1). However, MsDps2 possesses a long N-terminal tail instead of the C-terminal tail in MsDps1. This tail appears to be involved in DNA binding. It is also intimately involved in stabilizing the dodecamer. Partly on account of this factor, MsDps2 assembles straightway into the dodecamer, while MsDps1 does so on incubation after going through an intermediate trimeric stage. The ferroxidation centre is similar in the two proteins, while the pores leading to it exhibit some difference. The mode of sequestration of DNA in the crystalline array of molecules, as evidenced by the crystal structures, appears to be different in MsDps1 and MsDps2, highlighting the variability in the mode of Dps-DNA complexation. A sequence search led to the identification of 300 Dps molecules in bacteria with known genome sequences. Fifty bacteria contain two or more types of Dps molecules each, while 195 contain only one type. Some bacteria, notably some pathogenic ones, do not contain Dps. A sequence signature for Dps could also be derived from the analysis.     

NaCl induced morpho-biochemical and anatomical changes in mulberry (<Emphasis Type="Italic">Morus</Emphasis> spp.)

Mulberry is an economically important tree, used for feeding the silkworm Bombyx mori L. Effect of different levels of NaCl on growth and development of mulberry has been studied using five mulberry genotypes selected on the basis of their performance under in vitro salinity. The study while endorsing the efficacy of in vitro screening of axillary buds of mulberry for salt tolerance, showed genotypic variability in its response to salinity. Salinity reduced growth and development of all genotypes. However, the putative tolerant genotypes showed better performance than the putative susceptible genotypes. Under low salinity (<0.5% NaCl) salt tolerant genotypes showed an increase in chlorophyll and protein concentrations, while in susceptible genotypes both were reduced by 3–58% at 0.5% NaCl and 50–64% at 1.00% NaCl. Leaf thickness increased by 16% at 1.00% NaCl in C776 and reduced by 1.0% in Mandalaya. The increase in chlorophyll concentration and leaf thickness under high salinity can be considered as preliminary selection parameters for salt tolerance in mulberry. The study confirmed the efficacy of in vitro method for screening of large number of genotypes for salt tolerance in mulberry.     

Loose shell syndrome of farmed Penaeus monodon in India is caused by a filterable agent

Loose shell syndrome (LSS) of farmed black tiger shrimp Penaeus monodon has been reported from Indian shrimp farms since 1998 and is recognized as a major disease problem causing significant economic loss to the shrimp aquaculture sector. Unlike the rapid mortalities associated with viral pathogens such as white spot syndrome virus and yellow head virus, progression of LSS is gradual, leading to low-level progressive mortalities. The signs of LSS include a flaccid spongy abdomen due to muscular dystrophy, space between the exoskeleton and muscle, and a shrunken hepatopancreas. The feed conversion efficiency is reduced, and shrimp have poor meat quality, caused by impairment of the hepatopancreatic functions such as digestion and absorption as evidenced by the atrophy of the hepatopancreas. Histopathological investigations on LSS-affected shrimp showed shrinkage of extensor and flexor muscles with occasional hemocytic infiltration. The hepatopancreas showed inflammation of hepatopancreatic tubules with enlargement of intertubular spaces, hemocytic infiltration, and low levels of lipid reserves in the R cells. In advanced stages of LSS, many tubules were in highly necrotic condition with a sloughed epithelium, reflecting the dysfunction of the digestive gland. LSS could be induced in healthy tiger shrimp by challenge studies using membrane-filtered LSS-affected shrimp tissues, suggesting involvement of a filterable infectious agent.     

Molecular basis of kainate receptor modulation by sodium

Membrane proteins function in a polarized ionic environment with sodium-rich extracellular and potassium-rich intracellular solutions. Glutamate receptors that mediate excitatory synaptic transmission in the brain show unusual sensitivity to external ions, resulting in an apparent requirement for sodium in order for glutamate to activate kainate receptors. Here, we solve the structure of the Na(+)-binding sites and determine the mechanism by which allosteric anions and cations regulate ligand-binding dimer stability, and hence the rate of desensitization and receptor availability for gating by glutamate. We establish a stoichiometry for binding of 2 Na(+) to 1 Cl(-) and show that allosteric anions and cations bind at physically discrete sites with strong electric fields, that the binding sites are not saturated in CSF, and that the requirement of kainate receptors for Na(+) occurs simply because other cations bind with lower affinity and have lower efficacy compared to Na(+).     

Differences in ischemia-reperfusion-induced endothelial changes in hearts perfused at constant flow and constant pressure

Isolated hearts subjected to ischemia-reperfusion (I/R) exhibit depressed cardiac performance and alterations in subcellular function. Since hearts perfused at constant flow (CF) and constant pressure (CP) show differences in their contractile response to I/R, this study was undertaken to examine mechanisms responsible for these I/R-induced alterations in CF-perfused and CP-perfused hearts. Rat hearts, perfused at CF (10 ml/min) or CP (80 mmHg), were subjected to I/R (30 min global ischemia followed by 60 min reperfusion), and changes in cardiac function as well as sarcolemmal (SL) Na(+)-K(+)-ATPase activity, sarcoplasmic reticulum (SR) Ca(2+) uptake, and endothelial function were monitored. The I/R-induced depressions in cardiac function, SL Na(+)-K(+)-ATPase, and SR Ca(2+)-uptake activities were greater in hearts perfused at CF than in hearts perfused at CP. In hearts perfused at CF, I/R-induced increase in calpain activity and decrease in nitric oxide (NO) synthase (endothelial NO synthase) protein content in the heart as well as decrease in NO concentration of the perfusate were greater than in hearts perfused at CP. These changes in contractile activity and biochemical parameters due to I/R in hearts perfused at CF were attenuated by treatment with l-arginine, a substrate for NO synthase, while those in hearts perfused at CP were augmented by treatment with N(G)-nitro-l-arginine methyl ester, an inhibitor of NO synthase. The results indicate that the I/R-induced differences in contractile responses and alterations in subcellular organelles between hearts perfused at CF and CP may partly be attributed to greater endothelial dysfunction in CF-perfused hearts than that in CP-perfused hearts.