Quantitative proteomic screen identifies annexin A2 as a host target for Salmonella pathogenicity island-2 effectors SopD2 and PipB2

Abstract

Intracellular pathogens need to establish an intracellular replicative niche to promote survival and replication within the hostile environment inside the host cell. Salmonella enterica serovar Typhimurium (S. Typhimurium) initiates formation of the unique Salmonella-containing vacuole and an extensive network of Salmonella-induced tubules in order to survive and thrive within host cells. At least six effectors secreted by the type III secretion system encoded within Salmonella pathogenicity island-2 (SPI-2), namely SifA, SopD2, PipB2, SteA, SseJ, and SseF, purportedly manipulate host cell intracellular trafficking and establish the intracellular replicative niche for S. Typhimurium. The phenotypes of these effectors are both subtle and complex, complicating elucidation of the mechanism underpinning host cell manipulation by S. Typhimurium. In this work we used stable isotope labeling of amino acids in cell culture (SILAC) and a S. Typhimurium mutant that secretes increased amounts of effectors to identify cognate effector binding partners during infection. Using this method, we identified the host protein annexin A2 (AnxA2) as a binding partner for both SopD2 and PipB2 and were able to confirm its binding to SopD2 and PipB2 by reciprocal pull down, although there was a low level of non-specific binding of SopD2-2HA and PipB2-2HA to the Ni-Sepharose beads present. We further showed that knockdown of AnxA2 altered the intracellular positioning of the Salmonella containing vacuole (SCV). This suggests that AnxA2 plays a role in the subcellular positioning of the SCV which could potentially be mediated through protein–protein interactions with either SopD2 or PipB2. This demonstrates the value of studying effector interactions using proteomic techniques and natural effector delivery during infection rather than transfection.

Salmonella enterica serovar Typhi uses two type 3 secretion systems to replicate in human macrophages and colonize humanized mice

“…For example, SopD2, which is pseudogenized in S . Typhi, modulates the trafficking of the SCV and has recently been shown to cooperate with SteA and PipB2 ( 11 , 25 , 44 , 45 ). Therefore, a possible explanation for why some individual effectors, such as PipB2, play a more significant role in intramacrophage replication of S .…”

Salmonella entericaserovar Typhi uses two type 3 secretion systems to replicate in human macrophages and to colonize humanized mice

“…On the other hand, S. Typhi has a significantly reduced T3SS effector repetoire. For example, SopD2, which is pseudogenized in S. Typhi, modulates trafficking of the SCV and has recently been shown to cooperate with SteA and PipB2 (11,25,44,45). Therefore, a possible explanation for why some individual effectors, such as PipB2, play a more significant role for intramacrophage replication of S. Typhi compared to S. Typhimurium, is that the functionally redundant effectors are missing in S. Typhi (11).…”

Speaking the host language: how Salmonella effector proteins manipulate the host

“…For example, PipB, recruits the ER-tethered protein PDZD8 to the SCV, and also interacts with the effector SifA [24], but the significance of these interactions is not clear. PipB2 interacts with several additional host proteins including kinesin light chain, KIF5B and more recently annexin A2, which also interacts with SopD2 [24,244,290]. However, how PipB2 impacts host PTMs and how these interaction partners support bacterial virulence requires further investigation.…”

“…SopD2 is thought to have arisen through gene duplication of SopD, given sequence similarity of 43 % and significant structural similarity of the two effectors [243]. SopD2 is an SPI-2 effector which is associated with the localization and dynamics of the SCV and the obstruction of endosomal movement [243][244][245]. The N-terminal domain of SopD2, which is not homologous with SopD, is essential for its localization at SCVs and endosomes [246].…”

“…Rab7 activity is inhibited by direct SopD2 binding and GAP activity and leads to a halt of endosomal trafficking and dissociation with Rab7 effector proteins, potentially preventing the fusion of the SCV with lysosomes and promoting the cell-to-cell spread of bacteria [ 243 ]. A proteomic screen revealed that annexin A2 (AnxA2) interacts with SopD2 during infection [ 244 ]. Given the ability of AnxA2 to interact with Rab7 and modulate cytoskeletal rearrangement in the host, the authors speculated that AnxA2 assisted in positioning the SCV inside the cell and altered the endocytic pathways to promote intracellular growth; however, this needs validation.…”