Role of the ESCRT‐III complex in controlling integrity of the Salmonella ‐containing vacuole

Abstract

Intracellular pathogens need to establish specialised niches for survival and proliferation in host cells. The enteropathogen Salmonella enterica accomplishes this by extensive reorganisation of the host endosomal system deploying the SPI2-encoded type III secretion system (SPI2-T3SS). Fusion events of endosomal compartments with the Salmonella-containing vacuole (SCV) form elaborate membrane networks within host cells enabling intracellular nutrition. However, which host compartments exactly are involved in this process and how the integrity of Salmonellamodified membranes is accomplished are not fully resolved. An RNA interference knockdown screen of host factors involved in cellular logistics identified the ESCRT (endosomal sorting complex required for transport) system as important for proper formation and integrity of the SCV in infected epithelial cells. We demonstrate that subunits of the ESCRT-III complex are specifically recruited to the SCV and membrane network. To investigate the role of ESCRT-III for the intracellular lifestyle of Salmonella, a CHMP3 knockout cell line was generated. Infected CHMP3 knockout cells formed amorphous, bulky SCV. Salmonella within these amorphous SCV were in contact with host cell cytosol, and the attenuation of an SPI2-T3SS-deficient mutant strain was partially abrogated. ESCRT-dependent endolysosomal repair mechanisms have recently been described for other intracellular pathogens, and we hypothesise that minor damages of the SCV during bacterial proliferation are repaired by the action of ESCRT-III recruitment in Salmonellainfected host cells.

Ca2+-activated sphingomyelin scrambling and turnover mediate ESCRT-independent lysosomal repair

“…Expression constructs encoding cytoplasmic mKate-tagged EqtSM and EqtSol were created by PCR amplification followed by cloning into NheI and AgeI sites of mKate-LifeAct-7 (Addgene #54697), thereby replacing the LifeAct ORF. Expression constructs encoding mCherry-tagged human Galactin3 (pLX304-mCherry-hGalectin3), GFP-tagged human LAMP1 (pCMV-hLAMP1-GFP) and mCherry-tagged human LAMP1 (pCMV-hLAMP1-mCherry) were kindly provided by Michael Hensel (University of Osnabrück, DE) and have been described in ( 41,42 ). Expression constructs encoding bacterial SMase fused to GFP-tagged LAMP1 were created by PCR amplification of DNA encoding residues 28-333 of Bacillus cereus SMase using pEF6-bSMase-V5-His and pEF6-bSMase D322A/H323A -V5-His 43 as templates, followed by cloning into BamHI and AgeI sites of pCMV-hLAMP1-GFP to yield LAMP1-bSMase-V5-GFP and LAMP1-bSMase dead -V5-GFP.…”

From vacuole to cytosol – Disruptive invasion triggers cytosolic release ofSalmonellaParatyphi A and subsequent cytosolic motility favors evasion of xenophagy

“…Previous work revealed that the SCV is prone to rupture, and damages compartments are targeted by membrane damage sensors and repair mechanisms such as galectins, sphingomyelinases, or the ESCRT machinery (Ellison et al, 2020; Göser et al, 2020; Paz et al, 2010; Thurston et al, 2012). We used an engineered version of equinatoxin II (EqtSM) as rapid membrane damage reporter that is binding cytosol-exposed sphingomyelin in damages endosomal membranes (Deng et al, 2016; Niekamp et al, 2022).…”

“…Previous work revealed that the SCV is prone to rupture, and damages compartments are targeted by membrane damage sensors and repair mechanisms such as galectins, sphingomyelinases, or the ESCRT machinery (Ellison et al, 2020;Göser et al, 2020;Paz et al, 2010;Thurston et al, 2012).…”

The Use of Star Anise-Cinnamon Essential Oil as an Alternative Antibiotic in Prevention of Salmonella Infections in Yellow Chickens

“…Intestinal colonization by Salmonella is usually followed by invasion to the intestinal epithelium and macrophages. Salmonella has evolved a highly specialized membrane-bound compartment ( Salmonella -containing vacuole, SCV), which enables Salmonella to avoid the phagosome/lysosome of macrophage fusion for survival and multiplication [ 28 ]. Survival of Salmonella within macrophages plays an important role in the dissemination of Salmonella to the internal organs [ 29 ].…”