Vkuehn Week 11

Journal Club Preparation: Leishmania Major

Genome Reference Paper: The Genome of the Kinetoplastid Parasite, Leishmania major (Reference Genome)

Article Outline

Introduction

• It is important to study the genome of Leishmania major because of the various human diseases that this parasite is capable of causing. If infected by a leishmania parasite a number of diseases can form. Annually there are 2 million cases in 88 tropical and subtropical countries.
• How it infects:
  1. Parasite transmitted by sand flies as proliferative promastigote
  2. Differentiate into nondividing forms before inoculation into vertebrate host
  3. In host macrophages, phagocytose metacyclics --> differentiate into amastigotes (proliferate in phagolysosome)
  4. Leads to host macrophage lysis and infection of other macrophages
  5. Outcome of infection depends on species, host immune system and host genetics
• Interesting to look at genome because of the unique mechanism of regulating transcription which is atypical for eukaryotes

  Leishmania major is considered an "Old World Leishmania" species, meaning it contains 36 chromosome pairs. There are approximately 30 Leishmania species who's gene order is highly conserved.

  Ways in which it differs:

  1. Organization of protein coding genes: long, strand-specific polycistronic clusters
  2. No transcription factors

• This article determined the genome sequence of Leishmania major on a chromosome by chromosome basis. Present the structure and content based on molecular processes such as:
  • chromatin remodeling
  • transcription
  • RNA processing
  • Translation
  • posttranslational modification
  • protein turnover

  Also discuss essential host parasite interface developmental processes

Genome Structure and Content

• 32,816,678 base pairs obtained by shotgun sequencing insert colonies and purified chromosomal DNA
• Genome is partially aneuploid
• L. major sequence analysis yielded 911 RNA genes, 39 pseudogenes, 8272 protein coding genes
• L. major telomeres distinct from other Trityps and have heterogeneous structure
• The end of Leishmania major chromosomes have tripartite "repeat-repeat" structure
• "Leichmania restricted" genes: responsible for metablic differences from T. brucei and T. cruzi found randomly distributed in genome
• Two genes of interest: LmjF33.1740 and LmjF33.1750
  • Because resulting proteins contain macrophage migration inhibition factor (MIF)
  • Homologues found in other Leishmania species
  • L. major MIFs thought to retain tautomerase activity, but dies not have oxidoreductase activity.

    Interesting because this ties it to eukaryotic similarities but also ties genes to bacteria

  • Suggests that L. major MIFs could use eukaryotic similarities to modulate host macrophage response and help them survive in the host

RNA Genes

• RNAs participate in many cellular processes:

  RNA replication, splicing, RNA processing and modification, translation, translation regulation, protein translocation across membranes

• Differences in organization of RNA genes in genomes of L. major and the other trypanosomes.

  All 3 tritryp genomes have different numbers of genes and location differs as well.

Chromatin Remodeling

• Trypanosomatids have multiple copies of 4 core histone genes

  package chromosomal DNA into nucleosomes in eukaryotes and the access is also regulated by the RNA polytranscription complexes.

• Most genes are clustered in discrete single tandem arrays. L. major is different in this sense because these gene types occur in 2 or more separate loci, which is not the case for the other tritryps.
• Some variants in histone complexes in L. major may play roles in:

  gene slicing, gene expression, DNA repair, and centromere function

• Trytrip parasites have typical chromatin remodeling activities of eukaryotes, but also have some significan differences.

Transcription

• Little is known about the mechanisms of transcription initiation and few promoters have been analyzed in trypanosomatids
• The chromosome is characterized by the unique arrangement of directional gene clusters:
  • Polycistronic transcription by RNA polymerase II initiates bidirectionality within divergent strand-switch regions
  • Terminates within convergent strand switch regions
• Tritryps have conserved protein subunits. The difference between the species is that in L. major many of the homologues for RNA polymerase specific subunits are absent.
• Few potential homologues of RNA polymerase II basal transcription factors were found in L. major that were present in other eukaryotes.
• Findings show that primary determinants of tritryp gene expression is via posttranscriptional control mechanisms.

RNA Processing

• Tritryp RNA processing is distinctive because the site of polyadenylation is determined by trans-splicing of downstream mRNA
• Identified many putative tritryp splicing regulatory proteins and proteins implicated in alternative splicing. These suggest that regulation of splicing may have arisen early in eukaryotic evolution
• There is an absence of an RNA polymerase II C-terminal domain which may have a distinct functional role in transcription
• Degradation of mRNAs in regulating gene expression is similar to the process in mammals (the exosome plays a dominant role)
• The number of RNA recognition motifs (RRMs) is similar in Tritryps and yeast proteins

Translation and co-/posttranslational modification

• Major components of translational machinery found in L. major aslo found in other lower eukaryotes
• There is a higher number of potential translation factors in Tritryps which suggests that there is a high degree of specialization
• Most protein modification within tritryps involves usual eukaryotic processes. But there are some essential modificationsin L. major:

  glycosylphosphatidylinositol anchor addition, acylation, and prenylation

  all facilitate membrane attachment and/or protein-protein interactions

• Enzymes that catalyze these modifications may be promising drug targets

Surface Molecules

• Surface molecules of Leishmania is important because of its role in the infectious cycle in the host.

  Many of the anchored proteins contain similar posttranslational modifications but vary in other ways both within the Leishmania species and between Tritryps

• Many of the functions of the identified genes have not been determined
• Genes that result in nucleotide sugar transporters and their roles have been found to be unique in L. major
• Sphingolipids= essential membrane components in eukaryotic cells, contribute to intracellular function
  • Primary sphingolipid in Tritryps is IPC -->could be a drug target because of its role in intracellular function

Proteolysis

• Some peptidase protein-coding genes have been found to be virulence factors in Tritryps

  Potential vaccine and drug targets

• No representatives of mammalian peptidase inhibitors were found

  But have IPCs that mammals lack, suggesting these play important role in host-parasite interaction

• Tritryps also contain inhibitors of serine peptidases (ISPs) that are normally only found in bacteria

  ISPs also likely play an important role in host-parasite interactions

Concluding Remarks

• Comparing genomic sequences of tritryps helps gain insight into possible locations for drug targeting
• Its similarities, and divergences in genome organization and replication to both bacterial and eukaryotic cells also provides information regarding eukaryotic evolution
• The availability of the entire L. major genome and the subsequent analysis of the protein-coding genes is important in further researching their role in virulence
• This brings up possibilities for drug intervention and a better understanding of the mechanisms of the parasites' entrance into the host macrophage and its disease pathology

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