Shigella hijacks the exocyst to cluster macropinosomes for efficient vacuolar escape

Abstract

Shigella flexneri invades host cells by entering within a bacteria-containing vacuole (BCV). In order to establish its niche in the host cytosol, the bacterium ruptures its BCV. Contacts between S. flexneri BCV and infection-associated macropinosomes (IAMs) formed in situ have been reported to enhance BCV disintegration. The mechanism underlying S. flexneri vacuolar escape remains however obscure. To decipher the molecular mechanism priming the communication between the IAMs and S. flexneri BCV, we performed mass spectrometry-based analysis of the magnetically purified IAMs from S. flexneri-infected cells. While proteins involved in host recycling and exocytic pathways were significantly enriched at the IAMs, we demonstrate more precisely that the S. flexneri type III effector protein IpgD mediates the recruitment of the exocyst to the IAMs through the Rab8/Rab11 pathway. This recruitment results in IAM clustering around S. flexneri BCV. More importantly, we reveal that IAM clustering subsequently facilitates an IAM-mediated unwrapping of the ruptured vacuole membranes from S. flexneri, enabling the naked bacterium to be ready for intercellular spread via actin-based motility. Taken together, our work untangles the molecular cascade of S. flexneri-driven host trafficking subversion at IAMs to develop its cytosolic lifestyle, a crucial step en route for infection progression at cellular and tissue level.

Proteomic Analysis of Humoral Immune Components in Bronchoalveolar Lavage of Patients Infected or Colonized by Aspergillus fumigatus

“…The statistical analysis of the proteomics data was performed as described previously ( 6 ). Functional annotation of BALF proteomes was conducted using database for annotation, visualization, and integrated discovery (DAVID) v6.8 ( 7 ).…”

“…Peptides were eluted with a linear gradient from 3 to 22% solvent-B in 160 min, followed by a stepwise increase of solvent-B to 50% in 70 min and finally to 90% in another 5 min. Mass-spectra were acquired with a Top10 datadependent acquisition mode, with a scan range set to 300-1,700 m/z and an AGC target value of 3x10 6 . The fragmentation of precursor ions was performed by HCD (NCE 27) at 17.5 K resolving power (at m/z 200) with an AGC target value of 1x10 6 and a maximum injection time of 60 ms. Precursors with unknown charge state or charge state of <1 and >7 were excluded; dynamic exclusion was set to 35 s.…”

New methods to decrypt emerging macropinosome functions during the host–pathogen crosstalk

“…Due to the rapid maturation of macropinosomes and dynamics of host–pathogen interactions, methodologies were thus under‐equipped to elucidate the molecular players on IAMs. These hurdles have been overcome by a novel method to purify IAMs inspired by a reported magnetic cell fractionation method (Chang et al, 2020 ; Steinhäuser et al, 2014 ; Stévenin et al, 2019 ). This magnetic purification utilises magnetic beads of moderate size (100 nm in diameter), which are readily englobed within IAMs during Salmonella and Shigella invasion when administered to the extracellular medium (Figure 1 ).…”

“…The described method innovations have been crucial to discover the central role of IAMs on intracellular niche formation of two bacterial pathogens, Salmonella and Shigella . Combining CLEM and rapid time‐lapse microscopy, Shigella was found to induce the clustering of IAM around its BCV at the moment of vacuolar rupture (Chang et al, 2020 ; Weiner et al, 2016 ) (Figure 2 ). By applying the above‐mentioned magnetic purification, we mined the proteome of Shigella IAMs and uncovered the molecular players that mediate the BCV–IAM interaction.…”

“…By applying the above‐mentioned magnetic purification, we mined the proteome of Shigella IAMs and uncovered the molecular players that mediate the BCV–IAM interaction. More specifically, statistical analysis of the large datasets identified enrichment of the components of the exocyst complex on the IAMs, a multi‐subunit tethering complex that tethers opposing membranes for interaction (Chang et al, 2020 ). We further validated that the exocyst complex is manipulated by Shigella via the action of its T3SS effector IpgD to hijack Rab8 and Rab11 trafficking.…”