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. ========= Abstracts ========= 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 In addition, cells in which the Dictyostelium Arp2 has been replaced by the Entamoeba protein are potential targets for drug screens.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] -------------------------------------------------------------------------------- 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 � the flotillin homologue vacuolin and p80, a predicted copper transporter � 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] ============================================================== [End dictyNews, volume 28, number 15]