The cytoplasmic domain of MxiG interacts with MxiK and directs assembly of the sorting platform in the Shigella type III secretion system

Abstract

Many Gram-negative bacteria use type III secretion systems (T3SSs) to inject virulence effector proteins into eukaryotic cells. The T3SS apparatus (T3SA) is structurally conserved among diverse bacterial pathogens and consists of a cytoplasmic sorting platform, an envelope-spanning basal body, and an extracellular needle with tip complex. The sorting platform is essential for effector recognition and powering secretion. Studies using bacterial "minicells" have revealed an unprecedented level of structural detail of the sorting platform; however, many of the structure-function relationships within this complex remain enigmatic. Here, we report on improved cryo-electron tomographic approaches to enhance the resolution of the Shigella T3SA sorting platform (at ≤2 nm resolution) done in concert with biochemical and genetic methods to define the sorting platform interactome and interactions with the T3SA inner membrane ring (IR). We observed that the sorting platform consists of "pods" with 6-fold symmetry that interact with the Spa47 ATPase via radial extensions comprising MxiN. Most importantly, MxiK maintained an interaction with the IR via specific interactions with the cytoplasmic domain of the IR protein MxiG (MxiG^C), which is a noncanonical forkhead-associated domain, and MxiK has an elongated structure that interacts with the IR via MxiG^C T4 lysozyme-mediated insertional mutagenesis of MxiK revealed its orientation within the sorting platform and enabled disruption of interactions with its binding partners, which abolished sorting platform assembly. Finally, a comparison with the homologous interactions in the Salmonella T3SS sorting platform revealed clear differences in their IR-sorting platform interfaces that have possible mechanistic implications.

Keywords: MxiG; MxiK; Shigella; bacteria; cryo-electron tomography; effector protein; protein complex; protein-protein interaction; secretion; sorting platform; type III secretion system (T3SS); virulence factor.

Figure 1.

In situ structure of the S. flexneri T3SA with a focus on the interface between the IR and the pods of the sorting platform. A, the entire T3SA at 2-nm resolution. The IR component was locally refined to reveal an apparent 24-fold symmetry. B, the unrolled map of the locally refined IR component (unrolled along the long axis of the T3SA). The tightly packed densities for the outer IR component (MxiG) are shown following opening of the entire ring. All 24 densities of the MxiG periplasmic domain (MxiG^P) can be seen. The densities of the cytoplasmic domain of MxiG (MxiG^C) are less dense due to the smaller size of this domain relative to the periplasmic domain. C, apparent 24-fold symmetry of the MxiG periplasmic domain (indicated in A). In D, the region just below the MxiG cytoplasmic domain (indicated in A and B) shows that the SP component interfacing with the IR has changed to an evenly spaced 6-fold symmetry that has a somewhat elongated structure. The large density seen in the middle is the structure formed by the cytoplasmic portions of MxiA.

Figure 2.

MxiK is required for formation of the sorting platform and forms an interface with MxiG. A, cryo-ET image of the S. flexneri T3SA for a mxiK null mutant. Relative to the apparatus from WT bacteria (B), the entire sorting platform and the extracellular needle are absent, and the central channel is blocked within the basal body. The cytoplasmic MxiA export gate is still visible in this structure. C, zoom view of the single “pod” located in the boxed region of B (WT T3SA). The boundaries between the domains of the pod are not well-defined, partly because of the flexibility of the pod. In D, the pod is shown after further refinement with several densities becoming apparent. To better define the pod structure, a perpendicular view of this pod is shown in E, in which additional boundaries and protein-protein interfaces become visible. F, a surface rendering of the image shown in B with the darkened region representing a single pod. That pod's rendering is enlarged in G (equivalent to D), where a single small density interfaces with the bacterial inner membrane (containing the IR). A larger multidomain density makes up the bulk of the pod. H then shows a perpendicular representation of the pod (equivalent to the image in E). When bacteriophage T4L was fused to the C terminus of MxiK, it retained its WT activity, and the resulting added density could then be seen on the inside of the SP density nearest the IR (shown as the green structure in I, which is equivalent to the rendering in G). MxiK is thus indicated by the orange structure shown here. J then is a perpendicular image of the rendering shown in I with the T4L density masked by the outer face of MxiK. Based on the SP interactome reported in Table 1, the larger density shown in yellow is a complex of multiple Spa33 proteins. CM, cytoplasmic membrane; OM, outer membrane.

Figure 3.

MxiK peptide binding to MxiG^C measured using FP. A peptide comprised of MxiK residues 51–60 (YDLNCDIEPL) was generated with an additional two lysine residues placed at the N terminus to permit labeling with FITC. Left, the labeled peptide (100 nm) was incubated with increasing concentrations of unlabeled MxiG^C. This is a plot of the resulting change in the FP (given as millipolarization units (mP)) value (n = 6) with S.D. (error bars). Right, the ability of the labeled peptide (100 nm) to bind to 100 nm MxiG^C was monitored in the presence of increasing concentrations of the 10-amino acid peptide representing residues 51–60 of MxiK.

Figure 4.

Mutations in MxiG and MxiK that block T3SS activity result in loss of SP assembly and formation of the external needle. A library of T4L insertion mutants was generated for MxiK, and they were expressed in a Shigella mxiK null mutant. The ability of these bacteria to carry out contact-mediated hemolysis (A) and secretion of the translocator protein IpaB (B) was then tested. In parallel, a mutant mxiG gene encoding Ala in place of residues 61–66 and 81–85 was generated (mxiG^{61–66/81–85Ala}). This gene was then expressed in a Shigella mxiG null mutant, and contact-hemolysis (A) and IpaB secretion were tested (B). In parallel, S. flexneri minicells expressing mxiG^{61–66/81–85Ala} were examined by cryo-ET. These minicells (C) fail to form the external T3SA needle, and SP formation does not occur (E). The same result is seen when S. flexneri minicells are making MxiK-T4L-136 in place of WT MxiK (D and F, respectively). Error bars, S.D.

Figure 5.

Some mutations within the region spanning residues 51–60 of MxiK eliminate its activity. Four mutations were introduced into the region of MxiK corresponding to the MxiK peptide found to associate with MxiG^C by BLI and fluorescence polarization analyses. Three of the mutations completely eliminate MxiK activity in vivo based on loss of Shigella contact-mediated hemolysis activity and secretion of IpaB. In contrast, an Ala substitution at residue 56 had no effect on MixK activity or binding to MxiG^C and Spa33. Error bars, S.D.

Figure 6.

Surface rendering of the proposed placement of the IR and SP components. A, rendering of the sorting platform indicating the positions of MxiG (MxiG^P above the inner membrane and MxiG^C below it), MxiK, Spa33, MxiN, and Spa47. A cross-section of the complex is shown in B. MxiG^C is very close to the inner membrane, where it interacts with MxiK (arrows). This view better depicts MxiN and Spa47 with the export gate (MxiA) found inside the MxiG^C ring. The homologous models of MxiG^P (PDB code 6DUZ), MxiG^C (PDB code 3J1W), basal body (PDB code 6DV3), rod (PDB code 6DWB), and socket (PDB code 6F2D) were fitted into the structure. C, top view of the MxiG^C docking model.

Figure 7.

Summary of MixK interactions with MxiG^C and Spa33. WT MxiK and MxiK-T4L-C are able to restore virulence activities to a mxiK null Shigella mutant. In contrast, mutations that disrupt MxiK association with MxiG^C or Spa33 in vitro fail to restore contact-hemolysis or IpaB secretion activities to Shigella. Interestingly, a single substitution mutation at MxiK position 53 (MxiK^Leu53Ala) eliminated hemolysis and secretion activity, and this was predicted to be due to loss of an interaction with MxiG^C; however, this mutant was also defective in Spa33 binding in vitro.

Figure 8.

Remodeling of MxiG^C ring in S. flexneri. A, a density map of the purified needle complex from Salmonella (gray color, EMD-1875) was fitted into the in situ T3SA structure from Shigella. The corresponding atomic model of the purified needle complex from Salmonella was fitted into the Shigella T3SA structure in a side view (B) and a cross-section view (C), respectively. In C, the cytoplasmic domain of the Salmonella PrgH cytoplasmic domain (PrgH^C) of the inner ring 2 (IR2), equivalent to MxiG^C, is completely embedded in the cytoplasmic membrane of the Shigella in situ T3SA model. In fact, it takes about a 5-nm shift of PrgH^C for it to be relocated underneath the cytoplasmic membrane in D and E. In addition, the original PrgH^C ring (gray) is smaller than the IR2 density actually formed by the MxiG^C density seen in the Shigella in situ model, as shown in blue in F–H. G and H, a central cross-section and a top view of F, respectively.