Viperin Is Induced following Dengue Virus Type-2 (DENV-2) Infection and Has Anti-viral Actions Requiring the C-terminal End of Viperin.

Viperin is an anti-viral protein from host that is primarily interferon stimulated gene (ISG). This protein is up-regulated in a number of viral infections via IFN dependent or IFN independent mechanisms. It is reported that viperin inhibits HIV egress, influenza virus release and protein production in HCMV infection.

Researchers in this study have shown that dengue virus type-2 (DENV-2) infection causes induction of viperin. The mechanism for such process involves retinoic acid-inducible gene I (RIG-I) and along with viperin causes production of viral RNA also. Viperin expressing cells show inhibition of virus release and DENV-2 RNA.

The anti DENV effect of viperin protein is mediated by C-terminal, not N-terminal; although N-terminal includes motifs or structural amphiphatic helical domains that are known to be involved in membrane association, e.g., helix domain, leucine zipper and S-adenosylmethionine. The C-terminal is unstructured but highly conserved and with unknown functions.

Viperin colocalised and interacted with lipid droplet markers (dengue capsid protein) and DENV-2 capsid (CA), NS3 protein and viral RNA respectively. Such interaction ability is associated with anti-viral activity of viperin, which is in contrast with lipid droplet markers.

So overall this manuscript suggests that infection of DENV2 causes viperin induction. Since C-terminal of viperin has anti-viral properties associated with it, hence restrict early DENV-RNA accumulation.

viperin 1 viperin 2 viperin 3

 

Reference:  Helbig KJ, Carr JM, Calvert JK, Wati S, Clarke JN, et al. (2013) Viperin Is Induced following Dengue Virus Type-2 (DENV-2) Infection and Has Anti-viral Actions Requiring the C-terminal End of Viperin. PLoS Negl Trop Dis 7(4): e2178. doi:10.1371/journal.pntd.0002178.

How a Specific Component of Host Innate Immunity Modulates Microbial Evolution Towards Pathogenicity?

Researchers can use bacteria in controlled experimental environments to study evolution in real time. In-fact many bacteria have knack to adapt to abiotic challenges under lab environments, however less is known about affect of biotic challenges on adaptive evolution in bacteria.

Escherichia coli are versatile pathogens and commensals. Since there is evidence in literature that some E. coli that are pathogenic evolved actually from commensal strains, hence this organism becomes ideal for studying commensal to pathogenic switch. Most of laymen consider E. coli as friendly commensal, however when gastrointestinal barrier is disrupted, this commensal turns into pathogenic form. The break away from primary immune barrier or innate system is a critical trait relevant in the acquirement of bacterial virulence.

Since macrophages are defensive in nature, they directly attack pathogenic bacteria and kill them by RNS or ROS and phagocytosis. However, many pathogens have evolved mechanisms to evade such capture processes of macrophages. Such mechanisms include adaptive processes like capsule and biofilm formation.

In this study, researchers allowed E. coli to evolve under selective pressure of macrophages and tried to analyze how quickly and by which mechanism commensal E. coli develops resistance to macrophages. Several combinations of investigational evolution, phenotypic characterization, genome sequencing and mathematical modeling were used to tackle how fast and through how many adaptive steps commensal E. coli can acquire this immune evading virulence trait.

Results from the study indicate that E. coli can evolve and adapt very fast to evade innate immune system. Such pathoadaptive process involves the accumulation of mutations caused by transposon insertions and increasing pathogenicity in vivo. Under selective pressure E. coli can evolve in less than 500 generations using mechanisms;

  1.  Single transposable element insertion into the E. coli yrfF gene promoter.
  2. Insertion of IS186 into an ATP-dependent serine protease encoding Lon gene promoter.

Moreover authors have obtained a mathematical model that illustrates the dynamics of pathoadaptive process where in clones carrying distinct beneficial mutations emerge rapidly and turn virulent.

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Reference: The Genetic Basis of Escherichia coli Pathoadaptation to Macrophages.

Migla Miskinyte, Ana Sousa, Ricardo S. Ramiro, Jorge A. Moura de Sousa, Jerzy Kotlinowski, Iris Caramalho, Sara Magalhães, Miguel P. Soares, Isabel Gordo. DOI: 10.1371/journal.ppat.1003802.

Parasitic Infections and Mutilation of T-Cell Function

Recently a review has been published in PLOS Neglected Tropical Diseases (http://www.plosntds.org/). This review focuses mainly on the development of the four intracellular parasite species (Plasmodium spp., Trypanosoma cruzi, Toxoplasma gondii and Leishmania spp.) in the mammalian hosts they infect, with special emphasis on T lymphocyte function. These parasites after invading the host, blight T cell function and augment their apoptosis. Such impairments lead the host unresponsive for the parasite because of the collapse of the T cell number. This weakening of T-cells aids the parasites to survive throughout the infection or become persistent. All of such process follows a particular tier system as:

1: Invading/breaching the host barriers and integument or epidermis.

2: Down-regulate T cell function and lead to their exhaustion.

3: Apoptosis of T-cells or T-cell contraction.

4: Stay persistently in the host.

These parasites can accomplish apoptosis of host T-cells by activation-induced cell death (AICD) involving death ligands and caspase-8 or activated T cell autonomous death (ACAD) involving Bcl-2 family. This course of action respectively results in the formation of the death-inducing signalling complex (DISC) or apoptosome. Several studies have shown that patients infected with Plasmodium falciparum, Trypanosoma cruzi and Leishmania donovani have elevated levels of death ligand FasL. Also some studies have proven that engulfment of apoptotic cells stimulates expansion of parasites like Trypanosoma cruzi and Leishmania major inside host macrophages.

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This work is Licensed under Creative Commons Licenses.

Reference: Vasco Rodrigues,  Anabela Cordeiro-da-Silva, Mireille Laforge, Ali Ouaissi, Khadija Akharid, Ricardo Silvestre mail, Jérôme Estaquier mail.. DOI: 10.1371/journal.pntd.0002567

rK39-ICT Is Less Sensitive in Africa as Compared to Asia.

Leishmaniasis is a fatal systemic disease that occurs due to bite of Leishmania donovani carrier sandfly.  In Asia, Africa and Brazil Viseral Leishmaniasis is most prevalent and is caused by Leishmania donovani complex (L.infantum+L.donovani).

An immunochromatographic test, rK39 (an antibody based test) is used to detect the disease. This test is based on dual 39 amino acid repeats of a Sudanese L. donovani-obtained kinesin homologue of rK39, flanked by HASPB sequences.  However this test has less sensitivity in Africa than in Asia. Possible reasons for such variation could be:

  1. Molecular diversity that is continent specific
  2. Variable immunological response due to different IgG anti-Leish patient levels.

Immunoglobulin G or IgG titers were determined for VL patients from India and Sudan. It was found that IgG titers of VL patients from Sudan were less than Indian VL patients. About 46-61 folds higher mean ELISA titers were found for Indian VL patients as compared to Sudanese patients. Higher titers occurred in adults (both sexes) and children less than 16 years old. Possible cause for such lower titers could be Zinc deficiency or either variable antigencity. Malnutrition of zinc, iron and protein are known to trim down immune responses in experimental models.  Extensive research on zinc deficient models has established earlier that reduced B-cell responses and impairment of memory cells occurs. This could explain as why rK39-ICT has lower sensitivity in African areas or a lower titer of IgG.Picture1 Picture2

Since VL is not the only version of disease caused by Leishmania, other types like (post kala-azar dermal leishmaniasis) PKDL could be of curiosity. Assessment of PKDL patient antibody levels might be the future study of the authors. Such efforts could really speed up the process of disease detection in future and save many lives by earlier and easier diagnosis.

Reference:  Significantly Lower Anti-Leishmania IgG Responses in Sudanese versus Indian Visceral Leishmaniasis. Tapan Bhattacharyya, Duncan E. Bowes, Sayda El-Safi, Shyam Sundar, Andrew K. Falconar, Om Prakash Singh, Rajiv Kumar, Osman Ahmed, Marleen Boelaert, Michael A. Miles. (2014) DOI: 10.1371/journal.pntd.0002675

Lundep increases leishmania parasite survival inside neutrophils

Leishmaniasis or kala azar is a disease caused by the parasite Leishmania, which belongs to lower eukaryotes. The bite of an insect Lutzomyia longipalpis aids in the transmission of the parasite to complete its life cycle.

In earlier studies it has been shown that the components of saliva (like hyaluronidase) from this arthropod aid in the transmission of Leishmania in host systems. However authors here have shown that another protein Lundep is also an active constituent of saliva for enhancing the parasite infection.

Since the first line of defense is by neutrophils and it has been established in case of Leishmaniasis that the parasites dodge this line of defense by entering into the compartments that are nonlytic or by dodging neutrophil extracellular traps (NETs).  Salivary Gland Extracts (SGE) from Lutzomyia longipalpis are known to support parasite survival inside the neutrophils, however still less is known about the NETosis or NET formation in respose to Leishmania parasite.

Lundep (Lutzomyia NET destroying protein), a female specific endonuclease has been shown in this study to have enhancing effect on the infectivity as well as inhibitory effect on intrinsic coagulation pathway. It has been demonstrated that catalytic activity of the salivary endonuclease is accountable for enhancing infectivity or in aiding parasites escape from NETs.

One Two

This study has shown that Lundep:-

  1. Aids in degradation of the DNA scaffold of NETs. [Lundep has a DNase activity of about 300000 Kunitz units per mg of protein and can hydrolise both single stranded and double-stranded DNA.]
  2. Protects parasites from leishmanicidal activity of NETs.
  3. Promotes survival of promastigotes.
  4. Prevents blood coagulation while insect biting. [ Lundep is shown to have DNase activity that promotes antithrombotic effects]
  5. Assists in taking blood meal by decreasing the viscosity at the site of bite. [At the site of bite viscosity augments due to host DNA release]

So far no vaccine is available for Leishmaniasis (kala azar) and the authors consider Lundep as a potential target for vaccine generation.

Reference Used:

Lundep, a Sand Fly Salivary Endonuclease Increases Leishmania Parasite Survival in Neutrophils and Inhibits XIIa Contact Activation in Human Plasma. Andrezza C. Chagas, Fabiano Oliveira, Alain Debrabant, Jesus G. Valenzuela, José M. C. Ribeiro, Eric Calvo.  DOI: 10.1371/journal.ppat.1003923

Elucidation of localization of long non coding RNA in different subcellular compartments

Research has proven the role of noncoding RNA transcripts in various cellular processes which includes telomeric maintenance and chromosome silencing. Although in past decade researchers have identified many long non protein coding RNA transcripts, but the functional role of most of them is still in its infancy.

The sub-cellular localization of lncRNAs is of utmost importance in order to elucidate or account for their functional utilities. Researchers have tried to explore lncRNAs abundance in various compartments within the cellular systems. About 17 percent of lncRNAs are known to occur in nuclear compartment, were as only 4 percent occur in cytosol. This observation is for some of the RNAs that are thought to be involved in nuclear structural organization and gene-expression regulation, e.g., MALAT1 and NEAT1. Moreover ribosomal association of cytosolic lncRNAs is also known. Despite having all this knowledge about lncRNAs, there is a lack of data that could really support this relative lncRNA species abundance in compartments like nucleus, cytosol and ribosomes.

A team of researches at the Netherlands has used sub-cellular RNA-seq and ribosomal fractionation methods in combination with microarray technique to elucidate the exact compartmental locations of the lncRNAs.

From the different sub-cellular sample fractions the authors have considered three transcript types only: sncRNAs, lncRNAs and protein-coding transcripts and excluded miRNAs. In overall the expressed transcript set contained 7734 number of genes, out of which 7206 genes were protein-coding, 152 genes were lncRNAs and 376 genes were sncRNAs. However despite having different compositions of sub-cellular samples, the authors conclude that lncRNAs are existent in each of them. Despite these findings some questions raised, that how the individual transcripts distribute among different sub-cellular sample fractions? And how these lncRNA species behave in a different way as judged against normal coding transcripts? In order to find answers to these queries an investigation was done to find the correlation of the distribution of every lncRNA across the sub-cellular fractions and every coding transcript. Experimentation revealed that the association is complex. However authors have tried to simplify it by using a clustering model wherein a total of eleven individual clusters were made. The first ten clusters (I-X) contained genes showing specific sub-cellular localization, but cluster XI did not demonstrate enrichment anywhere in any of the samples. Nuclear enrichment was found for clusters I, II and III; ribosome-free cytosol compartmentalization for clusters IV and V; and ribosome enrichment for clusters VI to X. Cluster III contained majority of sncRNAs that suggested shuttling of these RNAs between cytosol and nucleus. In terms of percentage about 30% of the lncRNAs were found in ribosome-free cytosol and 38% in ribosome-enriched clusters.

Apart from this some known lncRNAs like MALAT1, NEAT1 and TUG1 were found localized in nuclear fraction; RPPH1, RN7SL1 and DANCR were found in cytosolic compartments and H19 and TUG1 in ribosomal fractions. Although high levels of TUG1 occur in nucleus, but authors have found appreciable levels in ribosomal fractions also. This diverse localization of various lncRNAs in different sub-cellular compartments implies that an extensive array of functions is associated with lncRNAs than is known so far. So in overall for the functional characterization of these individual lncRNAs the data presented in this very study can be of priceless use.

Reference: Extensive localization of long noncoding RNAs to the cytosol and mono- and polyribosomal complexes. Sebastiaan van Heesch, Maarten van Iterson, Jetse Jacobi, Sander Boymans, Paul B Essers, Ewart de Bruijn, Wensi Hao, Alyson W MacInnes, Edwin Cuppen and Marieke Simonis. Genome Biology 2014, 15:R6  doi:10.1186/gb-2014-15-1-r6

Role of WASP and N-WASP in B Cell Receptor Signaling.

After the elimination of infection, termination of immune response is necessary. This process can occur only when B-cell activation is shut down otherwise autoimmunity can occur. Recently researchers from University of Maryland and Harvard Medical School USA tried to analyze the process involved in inactivation of B-cells after infection has been cleared from the body. The main focus was on Wiskott-Aldrich syndrome protein (WASP) and neural WASP (N-WASP). Wiskott-Aldrich syndrome is X-linked disorder and results in immune dysregulation. WASP is entirely expressed in hematopoietic cells and N-WASP in neuronal cells.

Which molecules are involved in activation or inhibition of N-WSAP and how the latter affects B-cell activation were some key areas of enquiry and research in this study. The role of N-WASP in BCR (B-cell receptor) activation was analyzed by devising an experiment that activates human B cells and mouse BCR in a similar way. Results of this experiment revealed that at places where BCRs interact with antigen similar to WASP, transient activation of N-WASP occurs. In other words BCR stimulation causes N-WASP activation following WASP activation. Earlier studies lead this team to hypothesize that N-WASP has a compensatory role in WASP KO B-cells. For investigating this hypothesis KO mice were used. Results revealed that for antigen-induced BCR clustering both N-WASP and WASP are critical. Also B-cell morphology and B-cell spreading are affected.  However N-WASP in the absence of WASP, supports B-cell spreading and in presence of WASP, B-cell contraction.

The effects of N-WASP and WASP KO on B-cell morphology led the team of researchers to dig into the BCR signaling.  Experiments revealed that attenuation as well as stimulation of BCR signaling involves N-WASP. Earlier study from the same authors had shown that BCR signaling and clustering are in a two-phase relationship. Since cNKO had effects on both B-cell contraction and clustering, the researchers thought of N-WASP regulating signaling via cluster modulation of surface BCRs. Data from experiments showed role of N-WASP in promoting growth of BCR micro-clusters into the central cluster by down-regulating the BCR signaling system. Infact authors of this paper have clearly demonstrated that N-WASP has role in both positive and negative regulation of BCR signaling. However negative regulation suggested that B-cell self tolerance could also be affected by N-WASP. For clarification serum levels of anti- dsDNA and anti-nuclear DNA antibody were measured in cKNO (N-WASP deleted) mice and were found to be elevated, suggesting a clear role in self-tolerance. Moreover activation of BCR induces receptor internalization which in turn involves reorganization of actin. To elaborate the role of N-WASP and WASP in internalization and co-localization and immmunoflorescence studies were done with a marker named as LAMP-1 and surface-labeled BCRs respectively.

WASP and N-WASP involvement in activation of BCR led to understanding of their relationship. The experimental data suggests that both of them regulate each other negatively during activation of B-cells. However, BCR signaling inversely regulates both WASP and N-WASP activation.

On the whole the results have shown that B cells lacking N-WASP protein are activated for extended periods of time than the normal B-cells. Also mice with B-cells deficient in making N-WASP show increase in number of self-reactive B cells.

Open Access Article Under Creative Commons Attribution License.

References:

Chaohong Liu, Xiaoming Bai, Junfeng Wu, Shruti Sharma, Arpita Upadhyaya, Carin I. M. Dahlberg, Lisa S. Westerberg, Scott B. Snapper, Xiaodong Zhao, and Wenxia Song. N-WASP Is Essential for the Negative Regulation of B Cell Receptor Signaling. PLoS Biol. doi:  10.1371/journal.pbio.1001704.