LaXp180, a mammalian ActA-binding protein, identified with the yeast two-hybrid system, co-localizes with intracellular Listeria monocytogenes.
The Listeria monocytogenes surface protein ActA is an important virulence factor required for listerial intracellular movement by inducing actin polymerization. The only host cell protein known that directly interacts with ActA is the phosphoprotein VASP, which binds to the central proline-rich repeat region of ActA. To identify additional ActA-binding proteins, we applied the yeast two-hybrid system to search for mouse proteins that interact with ActA. A mouse cDNA library was screened for ActA-interacting proteins (AIPs) using ActA from strain L. monocytogenes EGD as bait. Three different AIPs were identified, one of which was identical to the human protein LaXp180 (also called CC1). Binding of LaXp180 to ActA was also demonstrated in vitro using recombinant histidine-tagged LaXp180 and recombinant ActA. Using an anti-LaXp180 antibody and fluorescence microscopy, we showed that LaXp180 co-localizes with a subset of intracellular, ActA-expressing L. monocytogenes but was never detected on intracellularly growing but ActA-deficient mutants. Furthermore, LaXp180 binding to intracellular L. monocytogenes was asymmetrical and mutually exclusive with F-actin polymerization on the bacterial surface. LaXp180 is a putative binding partner of stathmin, a protein involved in signal transduction pathways and in the regulation of microtubule dynamics. Using immunofluorescence, we showed that stathmin co-localizes with intracellular ActA-expressing L. monocytogenes.
Pfeuffer
T
T
Lehrstuhl für Mikrobiologie, Theodor-Boveri-Institut für Biowissenschaften der Universität Würzburg, Am Hubland, Germany.
Goebel
W
W
Laubinger
J
J
Bachmann
M
M
Kuhn
M
M
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000071183:Autophagy-Related Proteins; D001426:Bacterial Proteins / Q000378:metabolism*; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D005455:Fluorescent Antibody Technique; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008089:Listeria monocytogenes / Q000378:metabolism* / Q000472:pathogenicity*; D008088:Listeriosis / Q000382:microbiology; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020798:Two-Hybrid System Techniques
D000199:Actins; D000071183:Autophagy-Related Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D011506:Proteins; C428743:Rb1cc1 protein, mouse; C072980:actA protein, Listeria monocytogenes