Inhibition of Rab5a exchange activity is a key step for Listeria monocytogenes survival.
Listeria monocytogenes (LM) modifies the phagocytic compartment by targeting Rab5a function through an unknown mechanism. Inhibition of Rab5a exchange by LM can be considered the main virulence mechanism as it favours viability of the parasite within the phagosome as well as the exclusion of putative listericidal lysosomal proteases such as cathepsin-D. The significance of this survival mechanism is evidenced by the overexpression of Rab5a mutants in CHO cells that promoted GDP exchange on Rab5a and eliminated pathogenic LM. The following mutants showed listericidal effects: Rab5a:Q79L, a constitutively active mutant with accelerated GDP exchange and Rab5a GEF, Vps9, which overactivates the endogenous protein. Clearance of LM from these phagosomes was controlled by the hydrolytic action of cathepsin-D as suggested by the lysosomal protease inhibitor chloroquine, or the cathepsin-D inhibitor, pepstatin A, which caused a reversion of listericidal activity. Moreover, the effects of LM on Rab5a phagocytic function mimics those reported for the GDP locked dominant negative Rab5a mutant, S34N. Transfection of these mutants into CHO cells increased pathogen survival as they showed higher numbers of viable bacteria, complete inhibition of GDP exchange on Rab5a and impairment of the listericidal action probably exerted by cathepsin-D. We cotransfected functional Rab5a GEF into this dominant negative mutant and restored normal LM intraphagosomal viability, Rab5a exchange and listericidal action of cathepsin-D.
Prada-Delgado
Amaya
A
Servicio de Inmunología, Hospital Universitario 'Marqués de Valdecilla', Servicio Cántabro de Salud, 39008-Santander, Spain.
Carrasco-Marín
Eugenio
E
Peña-Macarro
Carla
C
Del Cerro-Vadillo
Elida
E
Fresno-Escudero
Manuel
M
Leyva-Cobián
Francisco
F
Alvarez-Dominguez
Carmen
C
D000818:Animals; D016466:CHO Cells; D002403:Cathepsins / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D011992:Endosomes / Q000502:physiology; D020690:GTPase-Activating Proteins / Q000502:physiology; D020662:Guanine Nucleotide Exchange Factors / Q000502:physiology; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D008089:Listeria monocytogenes / Q000502:physiology*; D010588:Phagosomes / Q000502:physiology*; D014162:Transfection; D020696:rab5 GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*
D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D002403:Cathepsins; D020696:rab5 GTP-Binding Proteins