B-213. Role of Syntaxins in the Maturation Process of Salmonella Containing Phagosomes

R. Madan, S. Parashuraman, A. Mukhopadhyay;
Natl. Inst. of Immunology, New Delhi, INDIA.

Salmonella, after being phagocytosed by host macrophages, survive and replicate in the intracellular niche. Previously, we have shown that Salmonella-containing phagosomes recruit host Rab5 through SopE, a Salmonella effector molecule and promote fusion with early endosomal compartment. Thus, Salmonella survive in a specialized compartment by inhibiting their transport to lysosomes(JBC 2000,JBC 2001). To understand the role of other Rab interacting proteins in intracellular trafficking of live Salmonella during their maturation, we compared the characteristics of in vitro fusion between live Salmonella containing early phagosomes(LSEP) and live Salmonella containing late phagosomes (LSLP) with early endosomes(EE). We found that in vitro fusion of LSEP with EE was significantly inhibited by ATPγS whereas LSLP fusion with EE was insensitive to ATPγS treatment. Moreover, complete depletion of NEM-sensitive factor (NSF) from cytosol and phagosomes in the fusion assay inhibited the fusion between LSEP with EE while fusion of LSLP with EE was not inhibited. Thus, fusion of LSLP with EE is ATPγS insensitive and NSF independent suggesting that during maturation, Salmonella must have modulated some SNAREs in an active unpaired state on their phagosomes. Consequently, we have found significantly enhanced levels of syntaxin 7, syntaxin 8 and syntaxin 13 on LSLP than LSEP. Currently, we are trying to understand the mechanism of recruitment of these syntaxins by LSLP. Our results show that LSLP also recruit significantly more Rabex 5 and Rabaptin 5 than LSEP along with EEA1. It has been shown that EEA1 assembles into a complex with Rabaptin 5, Rabex 5 and provides the necessary connection to recruit different syntaxins, which possibly explains the presence of increased amounts of these syntaxins on LSLP. Moreover, among different syntaxins, only anti-syntaxin 8 specifically inhibited the fusion of LSLP with EE. These results demonstrate that live Salmonella during maturation recruits syntaxin8 in activated form thereby, bypassing the requirement of NSF to promote fusion with EE to inhibit their transport to degradative compartment.