Components of the endocytic and recycling trafficking pathways interfere with the integrity of the Legionella ‐containing vacuole

Abstract

Legionella 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 antagonise 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 destabilise the replication vacuole, EEA1 and Rab11FIP1 showed increased density about the sdhA mutant vacuole compared with the wild type (WT) vacuole. Depletion of Rab5 isoforms or Rab11b reduced this aberrant redistribution. These findings are consistent with SdhA interfering with both endocytic and recycling membrane trafficking events that act to destabilise vacuole integrity during infection.

SdhA blocks disruption of the Legionella-containing vacuole by hijacking the OCRL phosphatase

“…RNAi depletion of Rab5, Rab11, and Rab8, all guanosine triphosphatases (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, 2020b ; 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.…”

“…Given the presence of a potential OCRL binding site and the presence of an array of sites uniquely found in SdhA that would direct it toward endocytic transport intermediates, we reasoned that SdhA might associate with OCRL ( Figure 1A ). Such association could modulate endocytic processes that threaten the integrity of the LCV ( Anand et al, 2020b ).…”

Shigella escapes lysosomal degradation through inactivation of Rab31 by IpaH4.5

“…VirA from nonvacuole S. flexneri , EspG from extracellular enteropathogenic Escherichia coli (EPEC) and LepB from intracellular Legionella pneumophila , have been shown to possess specific GAP activity [22, 26]. VirA and EspG exhibit potent RabGAP activities towards Rab1.…”

Members of the Legionella pneumophila Sde family target tyrosine residues for phosphoribosyl-linked ubiquitination

“…1,2 Intracellular growth and biogenesis of the replication vacuole requires the assembly of the Icm/Dot complex, [3][4][5] a type IV secretion system (T4SS) that spans the bacterial envelope and allows translocation of over 300 proteins (called effectors, throughout) across the vacuole. [6][7][8][9][10] The biochemical activities of dozens of these substrates are known to regulate host cell vesicle trafficking, [11][12][13] influence tubular endoplasmic reticulum (ER) dynamics, [14][15][16][17] rewire host ubiquitination, 18,19 as well as inhibit host protein synthesis. [20][21][22] Manipulation of these processes largely results from either enzymatic post-translational modification of host targets or misregulation of specific host cell GTPases by L. pneumophila proteins.…”