Components of the endocytic and recycling trafficking pathways interfere with the integrity of theLegionella-containing vacuole

Abstract

SummaryLegionella pneumophila requires the Dot/Icm translocation system to replicate in a vacuolar compartment within host cells. Strains lacking the translocated substrate SdhA form a permeable vacuole during residence in the host cell, exposing bacteria to the host cytoplasm. In primary macrophages, mutants are defective for intracellular growth, with a pyroptotic cell death response mounted due to bacterial exposure to the cytosol. To understand how SdhA maintains vacuole integrity during intracellular growth, we performed high-throughput RNAi screens against host membrane trafficking genes to identify factors that antagonize vacuole integrity in the absence of SdhA. Depletion of host proteins involved in endocytic uptake and recycling resulted in enhanced intracellular growth and lower levels of permeable vacuoles surrounding the ΔsdhA mutant. Of interest were three different Rab GTPases involved in these processes: Rab11b, Rab8b and Rab5 isoforms, that when depleted resulted in enhanced vacuole integrity surrounding the sdhA mutant. Proteins regulated by these Rabs are responsible for interfering with proper vacuole membrane maintenance, as depletion of the downstream effectors EEA1, Rab11FIP1, or VAMP3 rescued vacuole integrity and intracellular growth of the sdhA mutant. To test the model that specific vesicular components associated with these effectors could act to destabilize the replication vacuole, EEA1 and Rab11FIP1 showed enhanced colocalization with the vacuole surrounding the sdhA mutant compared with the WT vacuole. Depletion of Rab5 isoforms or Rab11b reduced this aberrant colocalization. These findings are consistent with SdhA interfering with both endocytic and recycling membrane trafficking events that act to destabilize vacuole integrity during infection.

SdhA interferes with disruption of theLegionella-containing vacuole by hijacking the OCRL phosphatase

“…For EEA1 and CIMPR, there was a significant increase in LCV association for each marker after infection with a DsdhA strain for 3hrs when compared to WT. The increase was particularly striking with EEA1, supporting our previous analysis of the behavior of DsdhA strains with murine bone marrow-derived macrophages (Anand et al, 2020) (Fig. 7A).…”

“…RNAi depletion of Rab5, Rab11, and Rab8, all GTPases involved in endocytic and recycling pathways, partially reverses loss of vacuole integrity observed in sdhA mutants. Consistent with these results, the absence of SdhA results in LCV accumulation of EEA1 and Rab11FIP1, downstream effectors of these GTPases (Anand et al, 2020;Christoforidis et al, 1999;Hales et al, 2001). Therefore, it is likely that SdhA interferes with components of the early endocytic network that are likely to disrupt vacuole integrity.…”

“…In previous work, we presented evidence that SdhA likely acts by preventing access of endosome-derived compartments to the LCV (Anand et al, 2020). This model was based on RNAi screens demonstrating that disruption of ∆sdhA mutant-containing vacuoles can be reversed by depletion of endocytic Rab GTPases.…”

“…1A). Such association could modulate endocytic processes that threaten the integrity of the Legionella-containing vacuole (LCV) (Anand et al, 2020).…”

VAMP3 and VAMP8 regulate the development and functionality of parasitophorous vacuoles housingLeishmania amazonensis

“…Alternatively, VAMP3 may mediate the delivery of an anti-microbial cargo to PV harboring L. amazonensis, thereby limiting parasite replication and PV expansion. Clearly, our study revealed a novel role for VAMP3 in the control of communal PV expansion and L. amazonensis replication, highlighting the fact that depending of the pathogen, a molecule involved in regulating vesicular trafficking and membrane fusion events may either favor or impair pathogen growth, as recently described for Rab11 in the context of L. pneumophila infection (65). Further studies will be aimed at elucidating the underlying mechanism(s).…”