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dictyNews Volume 28 Number 15
dictyNews
Electronic Edition
Volume 28, number 15
June 1, 2007
Please submit abstracts of your papers as soon as they have been
accepted for publication by sending them to dicty@northwestern.edu
or by using the form at
http://dictybase.org/db/cgi-bin/dictyBase/abstract_submit.
Back issues of dictyNews, the Dicty Reference database and other
useful information is available at dictyBase - http://dictybase.org.
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Abstracts
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Replacement of the essential Dictyostelium Arp2 gene by its Entamoeba
homologue using parasexual genetics
Mehreen Zaki, Jason King, Klaus Fuetterer and Robert H. Insall
BioMed Central, in press
Background
Cell motility is an essential feature of the pathogenesis and morbidity of
amoebiasis caused by Entamoeba histolytica. As motility depends on
cytoskeletal organisation and regulation, a study of the molecular components
involved is key to a better understanding of amoebic pathogenesis. However,
little is known about the physiological roles, interactions and regulation
of the proteins of the Entamoeba cytoskeleton.
Results
We have established a genetic strategy that uses parasexual genetics to allow
essential Dictyostelium discoideum genes to be manipulated and replaced with
modified or tagged homologues. Our results show that actin related protein 2
(Arp2) is essential for survival, but that the Dictyostelium protein can be
complemented by E. histolytica Arp2, despite the presence of an insertion of
16 amino acids in an otherwise highly conserved protein. Replacement of
endogenous Arp2 with myc-tagged Entamoeba or Dictyostelium Arp2 has no
obvious effects on growth and the protein incorporates effectively into the
Arp2/3 complex.
Conclusions
We have established an effective two-step method for replacing genes that
are required for survival. Our protocol will allow such genes to be studied
far more easily, and also allows an unambiguous demonstration that particular
genes are truly essential. In addition, cells in which the Dictyostelium
Arp2 has been replaced by the Entamoeba protein are potential targets for
drug screens.
Submitted by: Mehreen Zaki [M.Zaki@bham.ac.uk]
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Flotillin and RacH modulate the intracellular immunity of Dictyostelium to
Mycobacterium marinum infection
Monica Hagedorn and Thierry Soldati
Department de Biochimie, Faculte des Sciences, Universite de Geneve,
Sciences II, 30 quai Ernest Ansermet, CH-1211-Geneve-4, Switzerland
Cellular Microbiology, in press
Mycobacterium marinum, a close relative of M. tuberculosis, provides a
useful model to study the pathogenesis of tuberculosis in genetically
tractable model organisms. Using the amoeba Dictyostelium discoideum as a
host, we show that expression of the M. marinum protein MAG24-1 is crucial
to interfere with phagosome maturation. We find that two host proteins Ð
the flotillin homologue vacuolin and p80, a predicted copper transporter Ð
accumulate at the vacuole during pathogen replication until it finally
ruptures and the bacteria are released into the host cytosol. Flotillin-1
accumulation at the replication niche and its rupture were also observed
in human peripheral blood monocytes. By infecting various Dictyostelium
mutants, we show that the absence of one of the two Dictyostelium vacuolin
isoforms renders the host more immune to M. marinum. Conversely, the
absence of the small GTPase RacH renders the host more susceptible to
M. marinum proliferation but inhibits its cell-to-cell spreading.
Submitted by: Thierry Soldati [thierry.soldati@biochem.unige.ch]
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[End dictyNews, volume 28, number 15]
