Tropheryma whipplei, the agent of Whipple's disease, affects the early to late phagosome transition and survives in a Rab5- and Rab7-positive compartment

Abstract

Tropheryma whipplei, the agent of Whipple's disease, inhibits phago-lysosome biogenesis to create a suitable niche for its survival and replication in macrophages. To understand the mechanism by which it subverts phagosome maturation, we used biochemical and cell biological approaches to purify and characterise the intracellular compartment where Tropheryma whipplei resides using mouse bone-marrow-derived macrophages. We showed that in addition to Lamp-1, the Tropheryma whipplei phagosome is positive for Rab5 and Rab7, two GTPases required for the early to late phagosome transition. Unlike other pathogens, inhibition of PI(3)P production was not the mechanism for Rab5 stabilisation at the phagosome. Overexpression of the inactive, GDP-bound form of Rab5 bypassed the pathogen-induced blockade of phago-lysosome biogenesis. This suggests that Tropheryma whipplei blocks the switch from Rab5 to Rab7 by acting on the Rab5 GTPase cycle. A bio-informatic analysis of the Tropheryma whipplei genome revealed a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) homologous with the GAPDH of Listeria monocytogenes, and this may be the bacterial protein responsible for blocking Rab5 activity. To our knowledge, Tropheryma whipplei is the first pathogen described to induce a "chimeric" phagosome stably expressing both Rab5 and Rab7, suggesting a novel and specific mechanism for subverting phagosome maturation.

Figure 1. T. whipplei survives and replicates in a Lamp-1- but not cathepsin D-positive compartment within BMDMs.

(A) BMDMs were incubated with T. whipplei (bacterium-to-cell ratio of 50∶1) for 4 hours (day 0). Cells were then washed to remove free bacteria and incubated for additional periods (days). The copy number of bacterial DNA was determined by qRT-PCR. The results are expressed as the mean ± SD from 3 experiments (*p<0.05). (B–D) The colocalisation of T whipplei with either (B) Lamp-1 or (C) cathepsin D was analysed in BMDMs by immunofluorescence and confocal microscopy. The percentage of T whipplei colocalising with either Lamp-1 or cathepsin D was quantified over the time (D). The results are expressed as the mean ± SD from 3 experiments (*p<0.05). The scale bars indicate 5 µm.

Figure 2. Purification of the T. whipplei-containing compartment from BMDMs.

(A) Schematic view of the purification procedure. The compartments containing latex beads (LBC) or T. whipplei (TwC) were recovered in fractions I1 and I4 of the sucrose gradient, respectively. (B) All the sucrose gradient fractions (fractions I1 to I4) were analysed by immunofluorescence (n = 4) for the presence of T. whipplei (red), EEA1 (blue) or Lamp-1 (green). The scale bars indicate 5 µm. (C) Purified LBC and TwC fractions were analysed by western blot for the presence of Lamp-1 and cathepsin D. Immunoblot representative of 3 experiments.

Figure 3. T. whipplei phagosomes continuously harboured both Rab5 and Rab7.

(A) Purified LBC and TwC fractions were analysed by western blot for the presence of Rab7 and Rab5. Immunoblot representative of 3 experiments. (B–C) The colocalisation of T whipplei with Rab5 (B) or Rab7 (C) was analysed by immunofluorescence and confocal microscopy in BMDMs at day 12 after infection (n = 3). The scale bars indicate 5 µm. (D) The percentage of T whipplei colocalising with either Rab5 or Rab7 was quantified over time. The results are expressed as the mean ± SD from 3 experiments (*p<0.05).

Figure 4. T. whipplei phagosomes transiently expressed PI(3)P.

BMDMs were transfected with 2xFYVE-GFP using lentiviral vector and were infected with T. whipplei. The colocalisation of T. whipplei with 2xFYVE was analysed by immunofluorescence and confocal microscopy at 15 minutes (A) or 8 hours (B) after infection. (C) The percentage of T whipplei colocalising with 2xFYVE-GFP was quantified over time. The results are expressed as the mean ± SD from 3 experiments. The scale bars indicate 5 µm.

Figure 5. Overexpression of Rab5:S34N bypassed the pathogen-induced blockade of phago-lysosome biogenesis.

BMDMs were incubated with T. whipplei for 4 hours and then washed to remove free bacteria. Infected BMDMs were transfected with GFP-Rab5:S34N or GFP (control) and incubated for additional periods. (A) At day 12 post-infection, the copy number of bacterial DNA was evaluated by qRT-PCR. The results are expressed as the mean ± SD from 3 experiments. (B) The colocalisation of T whipplei with cathepsin D was analysed in BMDMs expressing GFP (left panel) or GFP-Rab5:S34N (right panel) by immunofluorescence and confocal microscopy (at day 3 post-infection). (i) and (ii) show the expression patterns of GFP and GFP-Rab5:S34N. The scale bars indicate 5 µm. (C) The percentage of T whipplei colocalising with cathepsin D was quantified (at day 3 post infection). The results are expressed as the mean ± SD from 3 experiments (*p<0.05).

Figure 6. T. whipplei codes for an orthologue of GAPDH-LM.

(A) Results of the Protein BLAST between glyceraldehyde-3-phosphate dehydrogenase from L. monocytogenes (GAPDH-LM, NP465982) and the genome of T. whipplei strain Twist (taxid:203267). (B) Protein-protein alignment between glyceraldehyde-3-phosphate dehydrogenase (GAPDH-LM, NP465982) from L. monocytogenes and GAPDH from T. whipplei (GAPDH-TW). The results are colour-coded for amino acid conservation. The conservation scoring was performed by PRALINE (http://www.ibi.vu.nl/programs/pralinewww/). The scoring scheme works from 0, for the least conserved alignment position, to 10, for the most conserved alignment position.