scholarly journals LeishIF4E1 Deletion Affects the Promastigote Proteome, Morphology, and Infectivity

mSphere ◽  
2019 ◽  
Vol 4 (6) ◽  
Author(s):  
Nitin Tupperwar ◽  
Rohit Shrivastava ◽  
Michal Shapira
Keyword(s):  
Gene Expression ◽  
Life Cycle ◽  
Expression Profile ◽  
Morphological Changes ◽  
Binding Activity ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Sand Fly ◽  
Null Mutant ◽  
Sand Fly Vectors

ABSTRACT Leishmania parasites cycle between sand-fly vectors and mammalian hosts, adapting to changing environmental conditions by driving a stage-specific program of gene expression, which is tightly regulated by translation processes. Leishmania encodes six eIF4E orthologs (LeishIF4Es) and five eIF4G candidates, forming different cap-binding complexes with potentially varying functions. Most LeishIF4E paralogs display temperature sensitivity in their cap-binding activity, except for LeishIF4E1, which maintains its cap-binding activity under all conditions. We used the CRISPR-Cas9 system to successfully generate a null mutant of LeishIF4E1 and examine how its elimination affected parasite physiology. Although the LeishIF4E1–/– null mutant was viable, its growth was impaired, in line with a reduction in global translation. As a result of the mutation, the null LeishIF4E1–/– mutant had a defective morphology, as the cells were round and unable to grow a normal flagellum. This was further emphasized when the LeishIF4E1–/– cells failed to develop the promastigote morphology once they shifted from conditions that generate axenic amastigotes (33°C, pH 5.5) back to neutral pH and 25°C, and they maintained their short flagellum and circular structure. Finally, the LeishIF4E1–/– null mutant displayed difficulty in infecting cultured macrophages. The morphological changes and reduced infectivity of the mutant may be related to differences in the proteomic profile of LeishIF4E1–/– cells from that of controls. All defects monitored in the LeishIF4E1–/– null mutant were reversed in the add-back strain, in which expression of LeishIF4E1 was reconstituted, establishing a strong link between the cellular defects and the absence of LeishIF4E1 expression. IMPORTANCE Leishmania parasites are the causative agents of a broad spectrum of diseases. The parasites migrate between sand-fly vectors and mammalian hosts, adapting to changing environments by driving a regulated program of gene expression, with translation regulation playing a key role. The leishmanias encode six different paralogs of eIF4E, the cap-binding translation initiation factor. Since these vary in function, expression profile, and assemblage, it is assumed that each is assigned a specific role throughout the life cycle. Using the CRISPR-Cas9 system for Leishmania, we generated a null mutant of LeishIF4E1, eliminating both alleles. Although the mutant cells were viable, their morphology was altered and their ability to synthesize the flagellum was impaired. Elimination of LeishIF4E1 affected their protein expression profile and decreased their ability to infect cultured macrophages. Restoring LeishIF4E1 expression restored the affected features. This study highlights the importance of LeishIF4E1 in diverse cellular events during the life cycle of Leishmania.

scholarly journals Distinct features of the Leishmania cap-binding protein LeishIF4E2 revealed by CRISPR-Cas9 mediated heterozygous deletion

2020 ◽  
Author(s):  
Nofar Baron ◽  
Nitin Tupperwar ◽  
Geula Davidov ◽  
Raz Zarivach ◽  
Michal Shapira
Keyword(s):  
Gene Expression ◽  
Translation Initiation ◽  
Ribosomal Proteins ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Sand Fly ◽  
Control Of Gene Expression ◽  
Mutant Cells ◽  
Sand Fly Vectors ◽  
General Translation

AbstractLeishmania parasites cycle between sand-fly vectors and mammalian hosts, adapting to alternating environments by stage-differentiation, accompanied by changes in the proteome profiles. Translation regulation plays a central role in driving the differential program of gene expression, since control of gene regulation in Leishmania is mostly post-transcriptional. The Leishmania genome encodes six eIF4E candidates, each assumed to have specific functions, although overlaps are expected. It is noted that some of them can bind to a dedicated eIF4G candidate partners, and LeishIF4E2 does not bind any known eIF4G ortholog. LeishIF4E2 was previously shown to comigrate in the polysomal fractions of sucrose gradients, whereas initiation factors usually comigrate with pre-initiation and initiation complexes. Using the CRISPR-Cas9 methodology, we deleted one of the two LeishIF4E2 gene copies. The deletion caused severe alterations in the morphology of mutant cells that turned round and equipped with a very short flagellum, but their growth rate and general translation remained unaffected. Proteomic analysis of the LeishIF4E2(+/-) mutant cells compared to wild type controls showed that the number of proteins that were upregulated exceeded the number of downregulated proteins, possibly indicating that a repressor function was eliminated. The upregulated proteins were related mainly to general metabolic processes, DNA repair and replication, signaling, and cellular motor activity. The downregulated proteins included several groups, including cytoskeletal and ribosomal proteins. Despite the fact that only one of the two LeishIF4E2 alleles was deleted, the mutant cells were impaired in their ability to infect cultured macrophages. LeishIF4E2 does not behave like a general translation factor and its function remains elusive. Although it could have a repressive function, we cannot exclude the possibility that it is responsible for translation of a specific set of transcripts. Overall, our results are in line with the possibility that the different LeishIF4Es are assigned unique functions.Author summaryLeishmania parasites cause a broad spectrum of diseases that lead to different pathological symptoms. During their life cycle, the parasites shuffle between sand-fly vectors and mammalian hosts, while adapting to changing environments via a stage specific program of gene expression that assists the survival of Leishmania under the changing conditions. Translation initiation plays a key role in control of gene expression, in Leishmania this is exemplified by the presence of multiple cap-binding complexes that interact with mRNAs. The parasites encode multiple paralogs of the cap-binding translation initiation factor eIF4E, and of its corresponding binding partner eIF4G, forming complexes with different potential functions. Using the CRISPR-Cas9 methodology, we generated a heterozygous mutant of the least studied cap-binding paralog, LeishIF4E2, eliminating one of its two alleles. The mutation caused morphological defects, resulting in short and rounded up cells with a significant reduction in their flagellar length. Although general translation rates and growth of the mutant parasite were not affected, total proteome analysis and translation assay suggested that LeishIF4E2 could possibly function as a translation repressor. Alternatively, LeishIF4E-2 could be responsible for promoting translation of a specific set of transcripts.

scholarly journals Application of Serial Analysis of Gene Expression to the Study of the Gene Expression Profile ofLeishmania infantum chagasiPromastigote

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Adelino Soares Lima Neto ◽  
Osvaldo Pompílio de Melo Neto ◽  
Carlos Henrique Nery Costa
Keyword(s):  
Gene Expression ◽  
Life Cycle ◽  
Gene Expression Profile ◽  
Expression Profile ◽  
Leishmania Infantum ◽  
Open Reading Frames ◽  
Genomic Sequences ◽  
Coding Sequences ◽  
Key Genes ◽  
Reading Frames

This study describes the application of the LongSAGE methodology to study the gene expression profile in promastigotes ofLeishmania infantum chagasi. A tag library was created using the LongSAGE method and consisted of 14,208 tags of 17 bases. Of these, 8,427 (59.3%) were distinct. BLAST research of the 1,645 most abundant tags showed that 12.8% of them identified the coding sequences of genes, while 82% (1,349/1,645) identified one or more genomic sequences that did not correspond with open reading frames. Only 5.2% (84/1,645) of the tags were not aligned to any position in theL. infantum genome. The UTR size ofLeishmaniaand the lack of CATG sites in some transcripts were decisive for the generation of tags in these regions. Additional analysis will allow a better understanding of the expression profile and discovering the key genes in this life cycle.

scholarly journals Identification of Specific Cellular Genes Up-Regulated Late in Adenovirus Type 12 Infection

2005 ◽  
Vol 79 (4) ◽  
pp. 2404-2412 ◽  
Author(s):  
Andreas Dorn ◽  
Hongxing Zhao ◽  
Frederik Granberg ◽  
Marianna Hösel ◽  
Dennis Webb ◽  
...  
Keyword(s):  
Gene Expression ◽  
Host Cell ◽  
Late Phase ◽  
Viral Gene Expression ◽  
Adenovirus Type ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Viral Gene ◽  
Cell Functions ◽  
Cellular Genes

ABSTRACT The infection of human cells by adenoviruses leads to a gradual reduction in the activity of host cell functions while viral gene expression progresses in a regulated way. We used the DNA microarray technique to determine the transcriptional activity profiles of cellular genes upon infection with adenovirus type 12 (Ad12). The microarray data were validated by quantitative real-time PCR for genes which showed significant alterations after Ad12 infection. At 12 h postinfection, there is a striking up-regulation between 10- and 30-fold in the expression of the G1P2, IFIT1, and IFIT2 cellular immune response genes compared to mock-infected cells. At later stages of infection, when the majority of regulated cellular genes has been turned down, a limited number of cellular genes exhibit increased activities by factors of 3 or less. These genes belong to the signal transduction or transcriptional regulator classes or are active in protein degradation, like ANPEP, an aminopeptidase. The SCD and CYP2S1 genes function in lipid metabolism. The eucaryotic translation initiation factor 4 is up-regulated, and one of the major histocompatibility complex genes is diminished in activity. For two of the genes, one up-regulated (CTSF gene) and one down-regulated (CYR61 gene), alterations in gene activity were confirmed at the protein level by Western blotting experiments. Increased genetic activity of cellular genes late in adenovirus infection has not been reported previously and demonstrates that Ad12 has a sustained control of host cell gene expression well into the late phase of infection.

scholarly journals A Novel Function of the MA-3 Domains in Transformation and Translation Suppressor Pdcd4 Is Essential for Its Binding to Eukaryotic Translation Initiation Factor 4A

2004 ◽  
Vol 24 (9) ◽  
pp. 3894-3906 ◽  
Author(s):  
Hsin-Sheng Yang ◽  
Myung-Haing Cho ◽  
Halina Zakowicz ◽  
Glenn Hegamyer ◽  
Nahum Sonenberg ◽  
...  
Keyword(s):  
Amino Acid ◽  
Translation Initiation ◽  
Binding Activity ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Amino Acid Residues ◽  
Eukaryotic Translation Initiation Factor ◽  
Stem Loop ◽  
Eukaryotic Translation ◽  
Eukaryotic Translation Initiation

ABSTRACT Αn α-helical MA-3 domain appears in several translation initiation factors, including human eukaryotic translation initiation factor 4G (eIF4G) and DAP-5/NAT1/p97, as well as in the tumor suppressor Pdcd4. The function of the MA-3 domain is, however, unknown. C-terminal eIF4G (eIG4Gc) contains an MA-3 domain that is located within the eIF4A-binding region, suggesting a role for eIF4A binding. Interestingly, C-terminal DAP-5/NAT1/p97 contains an MA-3 domain, but it does not bind to eIF4A. Mutation of amino acid residues conserved between Pdcd4 and eIF4Gc but not in DAP-5/NAT1/p97 to the amino acid residues found in the DAP-5/NAT1/p97 indicates that some of these amino acid residues within the MA-3 domain are critical for eIF4A-binding activity. Six Pdcd4 mutants (Pdcd4E249K, Pdcd4D253A, Pdcd4D414K, Pdcd4D418A, Pdcd4E249K,D414K, and Pdcd4D253A,D418A) lost >90% eIF4A-binding activity. Mutation of the corresponding amino acid residues in the eIF4Gc also produced similar results, as seen for Pdcd4. These results demonstrate that the MA-3 domain is important for eIF4A binding and explain the ability of Pdcd4 or eIF4Gc but not DAP-5/NAT1/p97 to bind to eIF4A. Competition experiments indicate that Pdcd4 prevents ca. 60 to 70% of eIF4A binding to eIF4Gc at a Pdcd4/eIF4A ratio of 1:1, but mutants Pdcd4D253A and Pdcd4D253A,D418A do not. Translation of stem-loop structured mRNA is susceptible to inhibition by wild-type Pdcd4 but not by Pdcd4D253A, Pdcd4D418A, or Pdcd4D235A,D418A. Together, these results indicate that not only binding to eIF4A but also prevention of eIF4A binding to the MA-3 domain of eIF4Gc contributes to the mechanism by which Pdcd4 inhibits translation.

scholarly journals The Zebrafish Xenograft Platform – A Novel Tool for Modeling KSHV-Associated Diseases

2019 ◽  
Author(s):  
Eric S. Pringle ◽  
Jaime Wertman ◽  
Nicole Melong ◽  
Andrew J. Coombs ◽  
Andrew L. Young ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Lymphocytes ◽  
Primary Effusion Lymphoma ◽  
Yolk Sac ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Viral Gene ◽  
Kshv Infection ◽  
Hypoxic Environment

Kaposi’s sarcoma associated-herpesvirus (KSHV, also known as human herpesvirus-8) is a gammaherpesvirus that establishes life-long infection in human B lymphocytes. KSHV infection is typically asymptomatic but immunosuppression can predispose KSHV-infected individuals to primary effusion lymphoma (PEL); a malignancy driven by aberrant proliferation of latently infected B lymphocytes, and supported by pro-inflammatory cytokines and angiogenic factors produced by cells that succumb to lytic viral replication. Here, we report the development of the first in vivo model for a virally-induced lymphoma in zebrafish, whereby KSHV-infected PEL tumour cells engraft and proliferate in the yolk sac of zebrafish larvae. Using a PEL cell line engineered to produce the viral lytic switch protein RTA in the presence of doxycycline, we demonstrate drug-inducible reactivation from KSHV latency in vivo, which enabled real-time observation and evaluation of latent and lytic phases of KSHV infection. In addition, we developed a sensitive droplet digital PCR method to monitor latent and lytic viral gene expression and host cell gene expression in xenografts. The zebrafish yolk sac is not well-vascularized and using fluorogenic assays we confirmed that this site provides a hypoxic environment that may mimic the microenvironment of some human tumors. We found that PEL cell proliferation in xenografts was dependent on the host hypoxia-dependent translation initiation factor, eukaryotic initiation factor 4E2 (eIF4E2). This demonstrates that the zebrafish yolk sac is a functionally hypoxic environment and xenografted cells must switch to dedicated hypoxic gene expression machinery to survive and proliferate. The establishment of the PEL xenograft model enables future studies that exploit the innate advantages of the zebrafish as a model for genetic and pharmacologic screens.

Sign in / Sign up

Export Citation Format

Share Document