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In vitro
reconstitution of Salmonella phagosome-endosome fusion |
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Principal Investigator : Amitabha Mukhopadhyay Research Associates/Assistants Ph D Students Pathogenesis of typhoid fever is related to the
survival of Salmonella sp. in macrophages. The main goal of this project is to
understand the mechanism of survival of Salmonella in macrophages in the
pathogenesis. Phagocytosis is an important process in host defense and is
mediated by complex interactions between defined intracellular compartments.
The final fate of the nascent phagosomes usually culminates with the fusion of
lysosomes. But some invading microorganisms modulate this central process for
their survival in the phagocytic cells. The major objectives of the present
investigations are i) reconstitution of in vitro fusion of
Salmonella-containing phagosomes with early endosome to understand the
interaction between these two compartments, ii) modulation of Salmonella
phagosome and endosome fusion by GTP binding proteins and iii) determination
of the role of various signal transduction intermediates in the phagosome
endosome fusion. We have investigated the intracellular route of
Salmonella in macrophages to determine a plausible mechanism for their
survival in phagocytes. Western blot analysis of isolated phagosomes at
different times of their maturation, using specific antibodies revealed that
by 5 min after internalization dead Salmonella-containing phagosomes (DSP)
acquire transferrin receptors (a marker for early endosomes) while by 30 min
the dead bacteria are found in vesicles carrying the late endosomal markers
cation-dependent mannose-6-phosphate receptors (M6PR), rab7 and rab9. In
contrast, live Salmonella-containing phagosomes (LSP) retain significant
amount of rab5 and transferrin receptor till 30 min, selectively deplete rab7
and rab9, and never acquire M6PR even 90 min after internalization. Retention
of rab5, rab18 and selective depletion of rab7 and rab9 presumably enable the
LSP to avoid transport to lysosomes through late endosomes. Presence of
immature cathepsin D (48 kDa) and selective depletion of the vacuolar ATPase
in LSP presumably contributes to the less acidic pH of LSP. In contrast,
proteolytically processed cathepsin D (Mr 17 kDa) was detected by 30 min on
the DSP. In order to quantify the transport of the live or dead
Salmonella from the early compartment to the lysosomes, cells were preloaded
with avidin-HRP and chased for 90 min to label the lysosomes. Subsequently,
cells were pulsed with live or dead biotinylated bacteria at 37°C for a short
period of time (5 min) to restrict their entry to the early compartment
followed by a chase. At indicated times the formation of bacteria biotin-avidin-HRP
complex was measured to determine the transport of the Salmonella to lysosomes.
The results showed that dead Salmonella co-localized with avidin-HRP preloaded
lysosomes within 45 min and maximum fusion was observed within 90 min. In
contrast, live Salmonella did not form complexes with avidin-HRP even after 90
min. We concluded that live Salmonella modulate the
expression of various rabs (e.g., rab5, rab7, rab9 and rab18) on the
phagosomes to reside in a specialized low acidity compartment devoid of active
lysosomal enzymes and transferrin receptors. Publications Original
peer-reviewed article 1. *Hashim S, Mukherjee K, Raje M, Basu SK and Mukhopadhyay A (2000) Live Salmonella modulate expression of rab proteins to persist in a specialized compartment and escape transport to lysosomes. J Biol Chem 275:16281-16288 (*in press last year, since published). |