Dysregulated endolysosomal trafficking in cells arrested in the G₁ phase of the host cell cycle impairs Salmonella vacuolar replication

ABSTRACT

Modulation of the host cell cycle has emerged as a common theme among the pathways regulated by bacterial pathogens, arguably to promote host cell colonization. However, in most cases the exact benefit ensuing from such interference to the infection process remains unclear. Previously, we have shown that Salmonella actively induces G₂/M arrest of host cells, and that infection is severely inhibited in cells arrested in G₁. In this study, we demonstrate that Salmonella vacuolar replication is inhibited in host cells blocked in G₁, whereas the cytosolic replication of the closely related pathogen Shigella is not affected. Mechanistically, we show that cells arrested in G₁, but not cells arrested in G₂, present dysregulated endolysosomal trafficking, displaying an abnormal accumulation of vesicles positive for late endosomal and lysosomal markers. In addition, the macroautophagic/autophagic flux and degradative lysosomal function are strongly impaired. This endolysosomal trafficking dysregulation results in sustained activation of the SPI-1 type III secretion system and lack of vacuole repair by the autophagy pathway, ultimately compromising the maturation and integrity of the Salmonella-containing vacuole. As such, Salmonella is released in the host cytosol. Collectively, our findings demonstrate that the modulation of the host cell cycle occurring during Salmonella infection is related to a disparity in the permissivity of cells arrested in G₁ and G₂/M, due to their intrinsic characteristics.

Abbreviations: CDK4: cyclin dependent kinase 4; CDK6: cyclin dependent kinase 6; CDK4-CDK6i: CDK4-CDK6 inhibitor IV; cfu: colony-forming units; CHQ: chloroquine; DMSO: dimethyl sulfoxide; EEA1: early endosome antigen 1; FITC: fluorescein isothiocyanate; GFP: green fluorescent protein; hpi: hours post-infection; LAMP1: lysosomal associated membrane protein 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MOI: multiplicity of infection; RAB7: RAB7, member RAS oncogene family; SCV: Salmonella-containing vacuole; SPI-1: Salmonella pathogenicity island-1; SPI-2: Salmonella pathogenicity island-2; TFEB: transcription factor EB; T3SS: type III secretion system

KEYWORDS:

• Autophagy
• cell cycle
• endolysosomal trafficking
• G₁ arrest
• Salmonella
• Salmonella-containing vacuole
• Salmonella cytosolic replication
• type III secretion system

Acknowledgments

JD and SC are recipients of PhD scholarships from the Portuguese Foundation for Science and Technology (SFRH/BD/144443/2019 and 2020.06572.BD, respectively); RS is a recipient of a PhD scholarship from the Doctoral Programme in Experimental Biology and Biomedicine of the Center for Neuroscience and Cell Biology, University of Coimbra (PD/BD/129294/2017)

Disclosure statement

The authors declare no competing interests.

Supplementary material

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Additional information

Funding

This work was supported by grants from the Bavarian Ministry of Sciences, Research and the Arts in the framework of the Bavarian Molecular Biosystems Research Network (BioSysNet), DFG project BR 4837/1-1 and the ERDF - European Regional Development Fund through COMPETE 2020 and Portuguese Foundation for Science and Technology (POCI-01-0145-FEDER-007440, UIDB/04539/2020, IF/01105/2015).