INsPECT, an Open-Source and Versatile Software for Automated Quantification of (Leishmania) Intracellular Parasites

The major health problems of developing countries constitute infectious diseases caused by intracellular parasites. Leishmania, Toxoplasma and Trypanosoma parasites are smart enough to reside inside the host cells.

Researchers working on the aspect of how such parasites proliferate intracellularly, have to do some laborious parasite counting, analyze drug [natural/synthetic] susceptibility or compare virulence properties of modified strains? High Content Screening (HCS) Technology has emerged recently and is a powerful tool for screening new compounds or for testing susceptibility of parasites to drugs. However the laborious procedure and cost of the equipments makes HCS poorly accessible for academics and research.

In this paper novel open-source, Java based software has been introduced that is quite helpful in automating the measurement of infection levels. This method is based on fluorescent DNA staining and has been named as INsPECT (Intracellular ParasitE CounTer). This software is believed to be versatile enough by the authors; to precisely detect intracellular parasites and to be the best one for parasite quantification.

The image based INsPECT is more fast, user friendly and open-source based software for quantification of parasites. The fluorescent images taken are used by the software to calculate the required data automatically by using image processing framework. The software can run based on DNA fluorescent images alone or in combination with phase contrast/Differential Interference Contrast. Such combination makes the intra- and extracellular parasites to be discriminated without any use of fluorescent membrane marker.

The out-put of the software provides one with annotated images, a report table, total cell count, total infected cell count, parasite index etc. The processing pipeline used by INsPECT software can be seen in the image below:

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Citation:

INsPECT, an Open-Source and Versatile Software for Automated Quantification of (Leishmania) Intracellular Parasites. Ehsan Yazdanparast, Antonio Dos Anjos, Deborah Garcia, Corinne Loeuillet,  Hamid Reza Shahbazkia mail, Baptiste Vergnes mail. Published: May 15, 2014 DOI: 10.1371/journal.pntd.0002850

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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

Viral Fusion, Stability and Infectivity Affected by a Single Amino Acid Mutation in H1N1 Influenza Virus.

Flu or Influenza is a seasonal infectious disease caused by an RNA virus that belongs to Orthomyxoviridae family. A seasonal epidemic of this disease causes millions of deaths. Influenza virus mutates to produce a new strain every time. This process occurs by blending of genes of humans and birds mostly. In the year 2009 a novel strain appeared and was named as swine flu or A/H1N1. This strain also caused Spanish flu of 1918. Strain A is most virulent and has many identified serotypes like H1N1, H2N2 (Asian Flu), H3N2, H5N1 (Bird Flu), H7N7, H1N2, H9N2, H7N9 etc.

RNA genome is segmented into seven to eight pieces of RNA and encodes proteins like neuraminidase(NA), hemagglutinin (HA), nucleoprotein(NP), nuclear export protein (NS1 and 2) etc. HA is indispensable for target binding and NA for release of progeny virus. HA has a stalk region and a head region. It specifically binds sialic acid residues on host cell surfaces and is known to be major determinant of viral host tropism and pathogenicity. After viral fusion and endocytosis, the low pH inside endosome is known to be encouraging for irreversible structural changes for HA subunits viz., HA1 and HA2. Scrutiny of data suggests that in the HA2 stalk there is a mutation (E47K). This mutation of H1N1 isolates has been exploited by the authors to contrast two H1N1 viruses that differ at HA2 position 47 of their HA proteins.

Original strain of H1N1 A/California/7/2009 (Cal/09) and a later strain A/Brisbane/10/2010 (Bris/10) were used in this study. Cal/09-like strains contain E47 in the HA2 stalk. However since 2009, an E47K mutation emerged and became dominant globally. Membrane fusion activity as determined by transient expression assay and viral fusion assay revealed that the HA2-47 in H1N1 HA stalk region affects the threshold pH for membrane fusion. Cal/09 HA fusion triggers at pH 5.4 and that of Bris/10 at pH 5, indicating a difference of 0.4 units. Moreover HA2-E47K substitution (glutamic acid to lysine substitution) lowered threshold pH of Cal/09 HA fusion, indicating HA2 47th residues regulatory role. It is to note that higher threshold pH for fusion means a lower viral thermal stability.

Since it is known both for balance of viral acidic stability in invaded tissues and for fusion activation, optimal pH is required. Hence pH affects host-specific replication and pathogenicity even in narrow pH range differences. Thus for transmission and virus host tropism, pH of HA fusion activation could be an important factor. In order to account for such statements vero cells (Verda Reno Cells) were used. Vero cells are known to have higher endosomal pH than MDCK cells; they were exploited for comparing pH and viral replication support. Experiments revealed that replication support for viruses with a fusion pH threshold of 5.4 is more as compared to those with pH 5. Moreover it has been demonstrated that Cal/09 HA2-E47 virus that has low acid stability (high threshold pH for fusion) also displays lower thermal stability compared to HA2-K47 virus. Compared to Cal/09, the H1N1pdm strain of 2012 has lower fusion pH, indicating that lower fusion pH might be coupled with adapting to human environment.

Most effective approach to prevent influenza is vaccination. Vaccinations against flu usually contains antigens from three viral strains (two A strains and one B strain) that are inactivated and is called as trivalent influenza vaccine(TIV). Each year a new vaccine has to be formulated as the virus is mutating rapidly to render previously formulated vaccines as ineffective. For the 2009 pandemic, a monovalent live attenuated vaccine (LAIV) was produced. In this study it has been found that E47 mutation of HA2 in Cal/09 vaccine awards more stability from 4°C to about 57.5°C.

In summary, this study adds to the better understanding of influenza infection and might aid in speedy reaction to future epidemics. In-fact genetic signature identified in the HA stalk has noteworthy implication for production of vaccines for future pandemics.