Summary

RMgm-4636
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0417700; Gene model (P.falciparum): PF3D7_0903700; Gene product: alpha tubulin 1
Phenotype Oocyst; Sporozoite;
Last modified: 10 June 2019, 15:13
  *RMgm-4636
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption
Reference (PubMed-PMID number) Reference 1 (PMID number) : 31126959
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherSpreng B, Frischknecht F
Name Group/DepartmentIntegrative Parasitology, Center for Infectious Diseases
Name InstituteHeidelberg University Medical School
CityHeidelberg
CountryGermany
Name of the mutant parasite
RMgm numberRMgm-4636
Principal namea1-tubulin(-)
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNot different from wild type
OocystBoth a1-tubulin(-) parasite lines showed no defect in blood-stage growth or initial mosquito infection. However, no sporozoites were found in the midgut or salivary glands of the mosquito; thus, a main defect seems to occur at the oocyst stage. This defect could be completely rescued by complementing the gene in the a1-tubulin(-) parasite line.
Deletion of a1-tubulin affects Plasmodium sporozoite formation during budding.
SporozoiteBoth a1-tubulin(-) parasite lines showed no defect in blood-stage growth or initial mosquito infection. However, no sporozoites were found in the midgut or salivary glands of the mosquito; thus, a main defect seems to occur at the oocyst stage. This defect could be completely rescued by complementing the gene in the a1-tubulin(-) parasite line.
Deletion of a1-tubulin affects Plasmodium sporozoite formation during budding.
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of alpha tubulin 1

Protein (function)
Microtubules are cytoskeletal filaments formed as hollow cylinders from dimers of a-tubulin and b-tubulin. Eukaryotic cells can arrange microtubules into different assemblies, including those forming axonemes of flagella and cilia, spindles for genome segregation during cell division, or cytoplasmic asters originating from the microtubule organizing centre and mediating intracellular transport and force distribution.
In some organisms, different isoforms of tubulin are expressed in different cells or tissues, suggesting that the different physical and biological properties of microtubules can be encoded in the subtle variations of the protein sequence of these isoforms.
Work on spindle microtubules shows that their number depends on the numbers of kinetochores. Both, number and length of spindle microtubules, can be fixed and are likely important for spindle function, as shown in fission yeast.

Sporozoites of P. berghei contain a fixed number of 16 subpellicular microtubules that extend from an apical polar ring at the front of the sporozoite to the nucleus at the centre of the cell. These microtubules are arranged in a typical 1 + 15 pattern and have been suggested to be important for vesicular trafficking, morphogenesis, cellular mechanics, polarity and motility of sporozoites.

All sequenced Plasmodium genomes contain one gene encoding b-tubulin and two genes encoding a-tubulins. The two a-tubulins likely arose by gene duplication from an ancestral a-tubulin. Such duplications can allow for a change in either the level or the timing of gene expression and/or to generate different functionalities arising from differences within the two coding sequences.
Expression analysis suggested that the a2-tubulin gene is expressed between one and two orders of magnitude higher than the a1- tubulin gene in blood stages. Indeed, the a2-tubulin gene appears essential and cannot be deleted, while we could readily delete the a1-tubulin gene in a wild type (WT) background and in a parasite expressing GFP and mCherry as cytoplasmic markers at different life cycle stages (see below).

Phenotype
We could readily delete the a1-tubulin gene in a wild type (WT) background and in a parasite expressing GFP and mCherry as cytoplasmic markers at different life cycle stages. Both a1-tubulin(-) parasite lines showed no defect in blood-stage growth or initial mosquito infection. However, no sporozoites were found in the midgut or salivary glands of the mosquito; thus, a main defect seems to occur at the oocyst stage. This defect could be completely rescued by complementing the gene in the a1-tubulin(-) parasite line.
Deletion of a1-tubulin affects Plasmodium sporozoite formation during budding.

Additional information
Through analysis of a number of mutants expressing different mutated forms of alpha tubulin 1 the following was shown:

From the paper:
'Here, we quantitatively studied the impact of modulating microtubule number and length in Plasmodium, the protozoan parasite causing malaria. Using a gene deletion and replacement strategy targeting one out of two a-tubulin genes, we show that chromosome segregation proceeds in the oocysts even in the absence of microtubules. However, fewer and shorter microtubules severely impaired the formation, motility and infectivity of Plasmodium sporozoites, the forms transmitted by the mosquito, which usually contain 16 microtubules. We found that a-tubulin expression levels directly determined the number of microtubules, suggesting a high nucleation barrier as supported by a mathematical model. Infectious sporozoites were only formed in parasite lines featuring at least 10 microtubules, while parasites with 9 or fewer microtubules failed to transmit'.

- Deletion of a1-tubulin affects Plasmodium sporozoite formation during budding.
It was shown that during late budding in the oocyst microtubules were longest and that they subsequently shrank as the sporozoites matured in the oocysts to again slightly grow as the parasites reside in the salivary gland. This suggests that, contrary to previous suggestions, cytoplasmic microtubules in Plasmodium undergo some level of shrinkage and growth, although much more slowly than in mammalian cells, on the time scale of hours to days rather than seconds.
- a1-tubulin deletion does not affect genome replication and nuclear division.
- Aberrant sporozoites are formed by intermediate numbers of microtubules.
- a2-tubulin can only partially complement a1-tubulin.
- Microtubule length and numbers affect sporozoite curvature and infectivity

Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0417700
Gene Model P. falciparum ortholog PF3D7_0903700
Gene productalpha tubulin 1
Gene product: Alternative name
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(Linear) plasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationPlasmids were designed for double-homologous crossover integration into the Plasmodium genome. The Pb262 vector (Singer et al, 2015) was modified by replacing the chromosome 12 integration sites with a1-tubulin 5UTR and 3'UTR integration sites for all vectors used in this study. Furthermore, the mCherry open reading frame (ORF) and the dhfs 30UTR were replaced with a set of different versions of a1-tubulin or a1/a2-tubulin chimeras and the a1-tubulin 3'UTR, respectively. In the case of the a2+++ vector, the dhfs 30UTR was replaced by a a2-tubulin 3'UTR. In case of the a1-tubulin(-) vector, the mCherry and the dhfs 3'UTR were deleted.
All vectors contained the positive–negative selection marker hdhfr-yfcu and could be used for either the “gene insertion/marker out” technique (Lin et al, 2011) or the standard transfection protocols (Janse et al, 2006a). Final vectors were linearized before transfection by SalI and KpnI in case of the GIMO technique or by SalI and XhoI (or ScaI) for standard transfection.

The linearized a1-tubulin(-) vector was transfected into an unmodified P. berghei strain ANKA or P. berghei strain ANKA expressing mCherry under the CSP and eGFP under the ef1a promoter using standard protocols. Parasites that integrated the desired DNA construct were selected by administration of pyrimethamine (0.07 mg/ml) via the mouse drinking water. An isogenic population was obtained by a dilution series, which was then followed by elimination of the positive–negative selection marker hdhfr-yfcu by applying 5-fluorocytosine (1 mg/ml). Another dilution series was performed to obtain an isogenic population of the selection marker-free a1-tubulin(-) RG parasites, which were used for complementation approaches with WT (a1a1(-) compl), deletion of a1-tubulin introns (a1Δintrons) and a set of a1/a2-tubulin chimera constructs (a2+++, a2++, a2+). The a1-tubulin codon-modified and intron-deleted construct (a1 cm&Δintrons) was transfected into the P. berghei strain ANKA. A receiver parasite line only differing in the dhfs 30UTR was used to generate the C-terminally truncated a1-tubulin parasite line (a1Δc-term) via a “gene insertion/marker out” approach.
Additional remarks selection procedure
Primer information: Primers used for amplification of the target sequences  Click to view information
Primer information: Primers used for amplification of the target sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6