Biology subjects

Holden, D. W.

Publications and source records attributed to Holden, D. W..

4 recordsLinked to original sources

bioRxiv2025

Salmonella SteD and mammalian SUSD6 use TMEM127 to co-disinhibit the WWP2 E3 ubiquitin ligase

The NEDD4-like E3 ubiquitin ligase, WWP2, is involved in a range of host processes from cell differentiation to T cell immunity. Ligase activity is tightly regulated with WWP2 being held in an autoinhibited state. Binding of a PY motif containing adaptor, an Ndfip, via the WW domains of NEDD4-like E3 ubiquitin ligases leads to their disinhibition. Here, we show that the canonical Ndfip, NDFIP2, requires multiple PY motifs for interaction with and activation of WWP2. In contrast, the single PY-motif containing Ndfips TMEM127 and SUSD6 function as a co-disinhibitory pair. TMEM127 and the Salmonella protein SteD also function as a co-disinhibitory pair. However, SteD interacts with a different region of the WWP2, the C2 domain, and this interaction results in disinhibition of WWP2. These findings demonstrate a range of ways that Ndfips can disinhibit WWP2. To our knowledge, these are the first examples of two Ndfips functioning as co-disinhibitory pairs, and of a bacterial effector that disinhibits an E3 ubiquitin ligase.

cell biology↗

bioRxiv2023

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

The injectisome encoded by Salmonella pathogenicity island 2 (SPI-2) had been thought to translocate 28 effectors. Here, we used a proteomic approach to characterise the secretome of a clinical strain of invasive non-typhoidal Salmonella enterica 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. sseM is widely distributed between the five subspecies of Salmonella enterica, is found in many clinically-relevant serovars, and is co-transcribed with pipB2, a SPI-2 effector gene. 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 {beta}-2-syntrophin, utrophin/ dystrophin, -catulin, -dystrobrevin and {beta}-dystrobrevin. The interaction between SseM and {beta}-2-syntrophin and -dystrobrevin was verified in S. Typhimurium-infected cells and relied on the PDZ domain of {beta}-2-syntrophin and a sequence corresponding to a PDZ-binding motif (PBM) in SseM. A {Delta}sseM mutant strain had a small competitive advantage over the wild-type strain in the S. Typhimurium/mouse model of systemic disease. This phenotype was complemented by a plasmid expressing wild type SseM from S. Typhimurium or S. Enteritidis and was dependent on the PBM of SseM. Therefore, a PBM within a Salmonella effector mediates interactions with the DAPC and modulates systemic growth of bacteria in mice. ImportanceIn Salmonella enterica, the injectisome machinery encoded by Salmonella pathogenicity island 2 (SPI-2) is conserved among the five subspecies and delivers proteins (effectors) into host cells that are required for Salmonella virulence. The identification and functional characterisation of SPI-2 injectisome effectors advances our understanding of the interplay between Salmonella and its host(s). Using an optimised method for preparing secreted proteins and a clinical isolate of the invasive non-typhoidal (iNTS) Salmonella enterica 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 PDZ-binding motif that is essential for interaction with the PDZ-containing host protein {beta}-2-syntrophin and other components of the dystrophin-associated protein complex (DAPC). To our knowledge, SseM is unique among Salmonella effectors in containing a functional PDZ-binding motif and is the first bacterial protein to target the DAPC.

microbiology↗

bioRxiv2022

The Salmonella transmembrane effector SteD hijacks AP1-mediated vesicular trafficking for delivery to antigen-loading MHCII compartments

SteD is a transmembrane effector of the Salmonella SPI-2 type III secretion system that inhibits T cell activation by reducing the amounts of at least three proteins - major histocompatibility complex II (MHCII), CD86 and CD97 - from the surface of antigen-presenting cells. SteD specifically localises at the trans-Golgi network (TGN) and MHCII compartments; however, the targeting, membrane integration and trafficking of SteD are not understood. Using systematic mutagenesis, we identify distinct regions of SteD that are required for these processes. We show that SteD integrates into membranes of the ER/Golgi through a two-step mechanism of membrane recruitment from the cytoplasm followed by integration. SteD then migrates to and accumulates within the TGN. From here it hijacks the host adaptor protein (AP)1-mediated trafficking pathway from the TGN to MHCII compartments. AP1 binding and post-TGN trafficking require a short sequence in the N-terminal cytoplasmic tail of SteD that resembles the AP1-interacting dileucine sorting signal, but in inverted orientation, suggesting convergent evolution.

microbiology↗

bioRxiv2020

Global mapping of Salmonella enterica-host protein-protein interactions during infection

Intracellular bacterial pathogens inject effector proteins into host cells to hijack diverse cellular processes and promote their survival and proliferation. To systematically map effector-host protein-protein interactions (PPIs) during infection, we generated a library of 32 Salmonella enterica serovar Typhimurium (STm) strains expressing chromosomally encoded affinity-tagged effector proteins, and quantified PPIs in macrophages and epithelial cells by Affinity-Purification Quantitative Mass-Spectrometry. Thereby, we identified 25 previously described and 421 novel effector-host PPIs. While effectors converged on the same host cellular processes, most had multiple targets, which often differed between cell types. Using reciprocal co-immunoprecipitations, we validated 13 out of 22 new PPIs. We then used this host-pathogen physical interactome resource to demonstrate that SseJ and SseL collaborate in redirecting cholesterol to the Salmonella Containing Vacuole (SCV) via NPC1, PipB directly recruits the organelle contact site protein PDZD8 to the SCV, and SteC promotes actin bundling by directly phosphorylating formin-like proteins.

microbiology↗