Interaction of MxiG with the cytosolic complex of the type III secretion system controls Shigella virulence
ABSTRACT Gram-negative bacteria use the type 3 secretion system (T3SS) to colonize host cells. T3SSs are ring-shaped macromolecular complexes specific for the transport of effector molecules into host cells. It was recently suggested that a cytosolic ring-shaped protein complex delivers effector molecules to the T3SS. However, how transport of effector proteins is regulated is not known. Here, we report the high-resolution X-ray crystal structure of the whole cytosolic domain of MxiG (MxiG(1-126)), a major component of the inner T3SS rings in Shigella flexneri. MxiG(1-126) folds as an FHA domain, which specifically binds phosphorylated threonines. Indeed, MxiG(1-126) binds to Spa33, a cytoplasmic-ring component of Shigella, as revealed in pulldown studies. Surface plasmon resonance analysis showed specific interaction of MxiG with a Spa33 peptide only if phosphorylated. In total, 24 copies of the MxiG(1-126) crystal structure were fitted into the cryo-EM map of the Shigella T3SS. The phosphoprotein binding site of each MxiG molecule faces the channel of the T3SS, allowing interaction with cytosolic binding partners. Secretion assays and host cell invasion studies of complemented Shigella knockout cells indicated that the phosphoprotein binding of MxiG is essential for bacterial virulence. Our findings suggest that MxiG is involved in T3SS regulation.
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- "Its cytoplasmic domain includes a forkhead-associated (FHA) phosphothreoninebinding domain, which binds phosphorylated peptides from Spa33 bearing a FHA recognition motif (Barison et al., 2012), but not phosphothreonine amino acid (McDowell et al., 2011). Indeed, it was recently demonstrated that mutations in MxiG that affect phosphoprotein binding reduce secretion of IpaB and impair invasion of HeLa cells (Barison et al., 2012). These findings suggest that the interaction of phosphorylated proteins, possibly SP components like Spa33, with the T3SS proximal ring is required for translocator secretion (Fig. 2E). "
ABSTRACT: Many Gram-negative pathogens utilize Type 3 Secretion Systems (T3SSs) for a successful infection. The T3SS is a large macromolecular complex which spans both bacterial membranes and delivers effector proteins into the host cell. The infection requires spatio-temporal control of diverse sets of secreted effectors and various mechanisms have evolved to regulate T3SS in response to external stimuli. This review will describe mechanisms that may control T3 secretion, revealing a multi-step regulatory strategy. We then propose an updated model of T3SS that illustrates different stages of secretion and integrates the most recent structural and functional data.Cellular Microbiology 08/2013; DOI:10.1111/cmi.12178 · 4.82 Impact Factor
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ABSTRACT: Flagellar and translocation-associated type III secretion (T3S) systems are present in most gram-negative plant- and animal-pathogenic bacteria and are often essential for bacterial motility or pathogenicity. The architectures of the complex membrane-spanning secretion apparatuses of both systems are similar, but they are associated with different extracellular appendages, including the flagellar hook and filament or the needle/pilus structures of translocation-associated T3S systems. The needle/pilus is connected to a bacterial translocon that is inserted into the host plasma membrane and mediates the transkingdom transport of bacterial effector proteins into eukaryotic cells. During the last 3 to 5 years, significant progress has been made in the characterization of membrane-associated core components and extracellular structures of T3S systems. Furthermore, transcriptional and posttranscriptional regulators that control T3S gene expression and substrate specificity have been described. Given the architecture of the T3S system, it is assumed that extracellular components of the secretion apparatus are secreted prior to effector proteins, suggesting that there is a hierarchy in T3S. The aim of this review is to summarize our current knowledge of T3S system components and associated control proteins from both plant- and animal-pathogenic bacteria.Microbiology and molecular biology reviews: MMBR 06/2012; 76(2):262-310. DOI:10.1128/MMBR.05017-11 · 15.26 Impact Factor
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ABSTRACT: The virulence of a large number of Gram-negative bacterial pathogens depends on the type III secretion (T3S) system, which transports select bacterial proteins into host cells. An essential component of the Yersinia T3S system is YscD, a single-pass inner membrane protein. We report here the 2.52-Å resolution structure of the cytoplasmic domain of YscD, called YscDc. The structure confirms that YscDc consists of a forkhead-associated (FHA) fold, which in many but not all cases specifies binding to phosphothreonine. YscDc, however, lacks the structural properties associated with phosphothreonine binding and thus most likely interacts with partners in a phosphorylation-independent manner. Structural comparison highlighted two loop regions, L3 and L4, as potential sites of interactions. Alanine substitutions at L3 and L4 had no deleterious effects on protein structure or stability but abrogated T3S in a dominant negative manner. To gain insight into the function of L3 and L4, we identified proteins associated with YscD by affinity purification coupled to mass spectrometry. The lipoprotein YscJ was found associated with wild-type YscD, as was the effector YopH. Notably, the L3 and L4 substitution mutants interacted with more YopH than did wild-type YscD. These substitution mutants also interacted with SycH (the specific chaperone for YopH), the putative C-ring component YscQ, and the ruler component YscP, whereas wild-type YscD did not. These results suggest that substitutions in the L3 and L4 loops of YscD disrupted the dissociation of SycH from YopH, leading to the accumulation of a large protein complex that stalled the T3S apparatus.Journal of bacteriology 08/2012; 194(21):5949-58. DOI:10.1128/JB.00513-12 · 2.69 Impact Factor