Poster Presentation Lorne Infection and Immunity 2020

The Salmonella effector SseK3 targets small Rab GTPases and inhibits host protein secretion (#104)

Jiyao Gan 1 2 , Nichollas Scott 2 , Joshua Newson 2 , Tania Wong Fok Lung 3 , Garrett Z Ng 4 , Jaclyn Pearson 1 , Cristina Giogha 1 , Elizabeth Hartland 1 5
  1. Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Melbourne, Victoria, Australia
  2. Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia
  3. Sir Peter MacCallum Department of oncology and Department of Biochemistry and Pharmacology, University of Melbourne, Melbourne, Victoria, Australia
  4. Department of Biochemistry and Molecular Biology, The University of Melbourne at the Bio21 Molecular Science & Biotechnology Institute, Melbourne, Victoria, Australia
  5. Department of Molecular and Translational Science, Monash University, Melbourne, Victoria, Australia

Salmonella species are among the most common foodborne pathogens that incur significant burden on the healthcare system worldwide. As an intracellular pathogen, Salmonella utilises two Type III secretion systems (SPI-1 and SPI-2) to inject virulence effector proteins into host cells to mediate invasion and subsequent intracellular replication via subverting a series of critical host cell biochemical and physiological events. It has become clear that one strategy employed by Salmonella to survive intracellularly is the subversion of key intracellular vesicle transport regulators, the Rab GTPases.

Within the Salmonella SPI-2 effector cohort, SseK1, SseK2 and SseK3 have been identified as three homologues of NleB1, an arginine N-acetylglucosamine (GlcNAc) transferase of enteropathogenic Escherichia coli that blocks protective cell death by modifying death domain containing proteins in host cells. Although these effectors have been studied previously, detailed virulence mechanisms and host targets of these SseK proteins still remain to be explored and verified.

Preliminary work from our laboratory revealed that SseK3 modifies several Rab GTPases during Salmonella infection of murine macrophages. Here, using co-transfection and immunoprecipitation experiments, we confirmed the modification of Rab1, Rab5 and Rab11 by SseK3. Three arginine residues were confirmed as SseK3 modification sites on Rab1; and the modification exerted on Rab1 by SseK3 was independent of the GTP/GDP – nucleotide binding states of Rab1. Rab1 regulates vesicular protein transport from endoplasmic reticulum to the Golgi apparatus. Importantly, a secreted alkaline phosphatase reporter assay indicated that expression of either SseK3 and SseK2 impeded the secretory pathway in HEK293T cells. During Salmonella infection on HeLa229 cells, overexpression of SseK3 resulted in reduced secretion of IL-6, IL-1α and IL-10 from infected host cells. Collectively, these results suggest that SseK3 contributes to Salmonella infection by blocking host protein secretion.