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Rab5a GTPase regulates fusion between pathogen-containing phagosomes and cytoplasmic organelles in human neutrophils

Nasrin Perskvist1,*, Karin Roberg2, Agné Kulyté1 and Olle Stendahl1

1 Department of Medical Microbiology, Faculty of Health Science, Linköping University, SE-581 85 Linköping, Sweden
2 Department of Pathology II, Faculty of Health Science, Linköping University, SE-581 85 Linköping, Sweden



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  		Fig. 1. Transmission electron microscopy (TEM) of neutrophils with ingested bacteria and of isolated phagosomes. Neutrophils were allowed to phagocytose mycobacteria of the H37Rv strain or S. aureus, and the bacteria-laden phagosomes (MCPv and SCP, respectively) were subsequently isolated. The micrographs show a neutrophil after internalisation of H37Rv for 60 minutes (A); isolated MCPv devoid of contamination with other cellular organelles (B); individual MCPv (C); and individual SCP (D). N, stands for nucleus and the scale bars represent 1 µm (A,B) and 0.5 µm (C,D).

 


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  		Fig. 2. Comparison of the protein composition of lysates of intact neutrophils (A), isolated MCPv (B) and isolated MCPa (C). Neutrophils (4x108 cells) were allowed to phagocytose H37Rv or H37Ra for 60 minutes and were then homogenized and the phagosomes were isolated on 12% sucrose. Equivalent amounts of proteins from the whole lysates of neutrophils and isolated phagosomes were separated according to their isoelectric points on immobilized pH gradient 3-10 gels and then by standard SDS-PAGE. The letters a-c in (B) (MCPv gel) and d-g in (C) (MCPa gel) indicate as yet unidentified proteins (the kDa and pI values of these proteins are given in the text). The molecular weigh markers are indicated to the left in (A). The images shown are representative of silver stained gels from three separate experiments.

 


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  		Fig. 3. Association of endosomal/granule markers and cytosolic proteins with the MCPv, MCPa and SCP. Neutropils (4x108 cells) were allowed to ingest H37Rv, H37Ra or S. aureus for 60 minutes. Thereafter, the phagosomes were isolated and equal amounts of the protein associated with the phagosomes were assayed for hck, lactoferrin and LAMP-1 and for annexin I, III and V by western blotting with appropriate antibodies. The illustrated immunoblots are representative of five independent experiments.

 


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  		Fig. 4. Recruitment of Rab5a and syntaxin-4 to the MCPv, MCPa and SCP. Neutrophils (4x108 cells) were allowed to ingest H37Rv, H37Ra or S. aureus for the indicated periods of time. Thereafter, the phagosomes were isolated and equivalent amounts of proteins were separated by SDS-PAGE and immunoblotted with anti-Rab5a or anti-syntaxin-4 antibodies. LAMP-1 was used as an internal control for the amounts of protein loaded. The blots shown are one representative of five separate experiments.

 


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  		Fig. 5. Interaction between Rab5a GTP and syntaxin-4 on neutrophil phagosomes during phagocytosis. Neutrophils ingested H37Rv, H37Ra or S. aureus for the indicated periods of time, after which the proteins associated with the isolated phagosomes (750 µg of protein for each phagosome fraction) were solubilized and Rab5a or syntaxin-4 was immunoprecipitated as described in the Materials and Methods. The immunoprecipitates (IP) were separated by SDS-PAGE transferred and blotted on the membranes. The GTP-binding state of Rab5a was detected on MCPv, MCPa and SCP by an [{alpha}-32P] GTP overlay assay and visualized by autoradiography. The same membrane was stripped and analysed for the presence of syntaxin-4. Interaction of GTP-bound Rab5a and syntaxin-4 on the MCPv (A) and MCPa (B) during 120 minutes and SCP (C) during 60 minutes of phagocytosis are shown. The immunoblots are representative of three separate experiments. (D) The kinetics of association of Rab5a-GTP on the MCPv ({blacksquare}) and on SCP ([UNK]) and that of the synatxin-4 on the MCPv ({square}) and SCP ({circ}) are illustrated. Data are expressed as mean±s.e.m of three experiments. (E) Syntaxin-4 immunoprecipitated from the MCPv, MCPa and SCP fractions isolated after 30 minutes phagocytosis and Rab5a-GTP co-immunoprecipitated with anti-syntaxin-4. The blot is representative of three independent experiments.

 


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  		Fig. 6. Downregulation of Rab5a impaired the capacity of the phagosomes to fuse with other organelles. Neutrophils were treated with Rab5a-antisense (AS) or -sense (S) oligonucleotides as described in Materials and Methods. (A) The cells were subsequently lysed and equal amounts of the proteins were separated by SDS-PAGE and immunoblotted with the antibodies raised against Rab5a, actin, hck (azurophil granules), lactoferrin (secondary granules) and LAMP-1 (late endosomes) proteins. (B) Antisense- or sense-treated neutrophils (1x108 cells) were allowed to ingest H37Rv at the ratio of one cell to 20 H37Rv or to 10 S. aureus for 30 minutes. Thereafter, the phagosomes were isolated and equivalent amounts of the phagosomal proteins were separated by SDS-PAGE and immunoblotted for the expression of Rab5a, syntaxin-4, hck, lactoferrin and LAMP-1 using appropriate antibodies. The blots shown are representative of three experiments.

 


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  		Fig. 7. The effect of down-regulation of Rab5a on the phagocytic capacity of neutrophils. Neutrophils either treated with Rab5a antisense and then allowed to ingest ({square}) H37Rv or ({blacksquare}) S. aureus, or with Rab5a sense and then phagocytosed (stripes) H37Rv or (grey) S. aureus as described in Materials and Methods. The percentage of the phagocytosed cells with >=1 bacteria was counted by trypan blue exclusion. Data represent mean¶s.e.m. of four experiments.

 

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