Mitochondrial Endosymbiosis: Phylogenetically Reconstructing the Presence of protein HSP70

Presence of a mitochondrial-type 70-kDa heat shock protein in Trichomonas vaginalis

suggests a very early mitochondrial endosymbiosis in eukaryotes

Agnès Germot*†, Hervé Philippe‡, and Hervé Le Guyader‡

Hervé Philippe – Phylogenist from Université de Montréal – “"Phylogenetic structural modeling of molecular evolution".

Terms to Keep in Mind:

Heat-Shock Proteins (HSP70)- among the most highly conserved proteins present in all organisms, Archaebacteria, Eubacteria, and Eukaryota

Trichomonas vaginalis - flagellated protists, anaerobic, lack mitochondria and peroxisomes Hydrogenosomes - double membrane-bounded organelles, lacking DNA, which have no tricarboxylic acid cycle enzymes and cytochromes and do not carry out oxidative phosphorylation

GDAWV sequence signature - exclusively foundin mitochondrial HSP70 and in proteobacterial dnaK.

The Symbiosis In Questions: HSP70 and T.vaginalis

Established knowledge: three independent species of protists have consistently have held a base position in the eukaryotic tree, before mitochondrial endosymbiosis

Symbiotic Question: How to empirically prove HSP70 is responsible for a phylogenetically earlier than anticipated mitochondrial endosymbiosis?

HSP70: Function and Relevance to T.vaginalis

• Boorstein W R • Ziegelhoffer T 

• Craig E A(1994) J Mol Evol 38:1–17.

Methods and Materials - Isolation to Analysis

1) DNA Isolation - axenic culture

2) Cloning of T.vaginalis HSP70 - fragment amplificationand via primer selectionv+ PCR

http://rbg-web2.rbge.org.uk/bbs/learning/exsitu/exsitu.htm

3) Southern Blotting - Enzyme Digestion via EcoRV, HindIII, Sspl to obtain fragments

4) Phylogenetic Analyses -  MUST Package & PROTML program - Genetic analysis for Maximum Likelihood, Maximum Parsimony, and Neighbor Joining

http://www.molecularstation.com/images/southern-blot.jpg

Significant Results

Significant Results: Findings to Keep In Mind

- High G+C and low G+C Gram-positive bacteria were monophyletic.- Position of T. vaginalis within this mitochondrial group was supported by high bootstrap values, whatever the tree reconstruction method used: maximum likelihood, 100%; maximum parsimony, 80%; and neighbor joining, 94%.- Two distinct signatures - GDAWV sequence & YSPSQI sequence. Confirm phylogenetic affinity between spirochetes and monophyly of mitochondri. 

Germot et Al: Implications and Discussion

- The consistencies in phylogenetic analysis suggest that trichomonad hydrogenosomes may have in fact involved from mitochondria."Phylogenetic analyses located T. vaginalis sequence inside the mitochondrial group. In addition, this HSP70 presented mitochondrial signatures shared only by α-Proteobacteria or the whole group of purple bacteria."

Potential Problems:

1) Lack of current studies peer reviewing the evidence behind the Montreal team’s work. - Secondary mitochondrial endosymbiosis has not been researched past phylogenetic trees."our data provide evidence that T. vaginalis may have had mitochondria during its evolution as it is the case for other extant amitochondriate protists such as Entamoeba histolytica"

 "More generally, the absence of some characteristics in these lineages (for example, peroxisomes) has led others to propose scenarii of progressive construction of the eukaryotic cell"