Modulation of virulence by a novel SPI-2 injectisome effector that interacts with the dystrophin-associated protein complex.

Unlabelled: The injectisome encoded by pathogenicity island 2 (SPI-2) had been thought to translocate 28 effectors. Here, we used a proteomic approach to characterize the secretome of a clinical strain of invasive non-typhoidal serovar Enteritidis that had been mutated to cause hyper-secretion of the SPI-2 injectisome effectors. Along with many known effectors, we discovered the novel SseM protein. is widely distributed among the five subspecies of is found in many clinically relevant serovars, and is co-transcribed with , a SPI-2 effector gene. The translocation of SseM required a functional SPI-2 injectisome. Following expression in human cells, SseM interacted with five components of the dystrophin-associated protein complex (DAPC), namely, β-2-syntrophin, utrophin/dystrophin, α-catulin, α-dystrobrevin, and β-dystrobrevin. The interaction between SseM and β-2-syntrophin and α-dystrobrevin was verified in Typhimurium-infected cells and relied on the postsynaptic density-95/discs large/zonula occludens-1 (PDZ) domain of β-2-syntrophin and a sequence corresponding to a PDZ-binding motif (PBM) in SseM. A Δ mutant strain had a small competitive advantage over the wild-type strain in the . Typhimurium/mouse model of systemic disease. This phenotype was complemented by a plasmid expressing wild-type SseM from . Typhimurium or . Enteritidis and was dependent on the PBM of SseM. Therefore, a PBM within a effector mediates interactions with the DAPC and modulates the systemic growth of bacteria in mice. Furthermore, the Δ mutant strain displayed enhanced replication in bone marrow-derived macrophages, demonstrating that SseM restrains intracellular bacterial growth to modulate virulence.

Importance: In , the injectisome machinery encoded by pathogenicity island 2 (SPI-2) is conserved among the five subspecies and delivers proteins (effectors) into host cells, which are required for virulence. The identification and functional characterization of SPI-2 injectisome effectors advance our understanding of the interplay between and its host(s). Using an optimized method for preparing secreted proteins and a clinical isolate of the invasive non-typhoidal serovar Enteritidis strain D24359, we identified 22 known SPI-2 injectisome effectors and one new effector-SseM. SseM modulates bacterial growth during murine infection and has a sequence corresponding to a postsynaptic density-95/discs large/zonula occludens-1 (PDZ)-binding motif that is essential for interaction with the PDZ-containing host protein β-2-syntrophin and other components of the dystrophin-associated protein complex (DAPC). To our knowledge, SseM is unique among effectors in containing a functional PDZ-binding motif and is the first bacterial protein to target the DAPC.