Unraveling the Self-Assembly of the Pseudomonas aeruginosa XcpQ Secretin Periplasmic Domain Provides New Molecular Insights into Type II Secretion System Secreton Architecture and Dynamics

Abstract

The type II secretion system (T2SS) releases large folded exoproteins across the envelope of many Gram-negative pathogens. This secretion process therefore requires specific gating, interacting, and dynamics properties mainly operated by a bipartite outer membrane channel called secretin. We have a good understanding of the structure-function relationship of the pore-forming C-terminal domain of secretins. In contrast, the high flexibility of their periplasmic N-terminal domain has been an obstacle in obtaining the detailed structural information required to uncover its molecular function. In Pseudomonas aeruginosa, the Xcp T2SS plays an important role in bacterial virulence by its capacity to deliver a large panel of toxins and degradative enzymes into the surrounding environment. Here, we revealed that the N-terminal domain of XcpQ secretin spontaneously self-assembled into a hexamer of dimers independently of its C-terminal domain. Furthermore, and by using multidisciplinary approaches, we elucidate the structural organization of the XcpQ N domain and demonstrate that secretin flexibility at interdimer interfaces is mandatory for its function.IMPORTANCE Bacterial secretins are large homooligomeric proteins constituting the outer membrane pore-forming element of several envelope-embedded nanomachines essential in bacterial survival and pathogenicity. They comprise a well-defined membrane-embedded C-terminal domain and a modular periplasmic N-terminal domain involved in substrate recruitment and connection with inner membrane components. We are studying the XcpQ secretin of the T2SS present in the pathogenic bacterium Pseudomonas aeruginosa Our data highlight the ability of the XcpQ N-terminal domain to spontaneously oligomerize into a hexamer of dimers. Further in vivo experiments revealed that this domain adopts different conformations essential for the T2SS secretion process. These findings provide new insights into the functional understanding of bacterial T2SS secretins.

Keywords: Pseudomonas aeruginosa; dynamics; protein structure-function; secretin; stoichiometry; type II secretion system.

FIG 1

XcpQ_N012 forms homomultimers in solution. (A) Schematic representation of XcpQ secretin subdomains and their boundaries (amino acid number on the pre-XcpQ protein). The signal peptide (SP) and the XcpQ_N012 and XcpQ_N01 variants are also represented (3, 8). (B) Size exclusion chromatography (SEC) of the purified XcpQ_N012. The elution volume (from a HiLoad 16/600 Superdex 200 column) is plotted on the x axis, and the 280-nm absorbance is plotted on the y axis. mAU, milli-absorbance units. (C) Coomassie blue-stained SDS-PAGE and 3 to 12% Tris-glycine blue-native PAGE (BN-PAGE) analysis of the high (H)- and low (L)-molecular weight complexes. The excised band from BN-PAGE corresponding to the identified H complex was analyzed by 15% SDS-PAGE. The electrophoretic profile shows that XcpQ_N012 is the only protein forming the H complex compared to the purified XcpQ_N012 loaded in the neighboring lane. For SDS-PAGE and BN-PAGE, molecular mass markers (in kilodaltons) are indicated on the left.

FIG 2

High-resolution 3D structures of XcpQ_N012 and vHH04-XcpQ_N012 determined by X-ray crystallography. (A) Ribbon view of the XcpQ_N012 homodimer. The N0, N1, and N2 subdomains of each monomer are colored in cyan, light blue, and pink, respectively. (B) Zooming in on a top view of the dimer interface highlights the face-to-face assembly driven by the N2-N2* interface. The S210 residues selected for cysteine point substitution at the N2-N2* interface are shown in stick form. (C) Cartoon representation of XcpQ_N012 with the N-terminal domain of GspD (PDB ID 3EZJ) superimposed. (D) Cartoon representation in side view of the XcpQ_N012 structures: PDB ID 5NGI (purple) (present study) versus PDB ID 4E9J (gray) (8). The higher packing of the present dimer is indicated by arrows. (E and F) Side view (E) and top view (F) of the vHH04-XcpQ_N012 complex structure. The vHH04 nanobodies are colored in hot pink. (G) Superimposition of the XcpQ_N012 structure in free state (see panel A legend for color code) or in complex with vHH04 nanobody (green).

FIG 3

Low-resolution EM model of XcpQ_N012 dodecamers. (A) Representative micrograph of the data set used for image processing. White circles indicate isolated Trx-XcpQ_N012 dodecamers. (B) Gallery of representative class averages generated by EMAN2 after 2D classification. (C) Top, side, and bottom views of the three-dimensional reconstruction model of the XcpQ_N012 dodecamer obtained by electron microscopy (accession number EMD-3641). (D) Side and bottom cutout views of the cryo-EM structure of GspD from E. coli K-12 (EMD-6675) colored in black with the low-resolution structure of XcpQ_N012 colored in light gray superimposed. Bar, 5 nm.

FIG 4

Atomic model of the XcpQ_N012 dodecameric complex. (A) Model of XcpQ_N012 dodecamer generated by SymmDock by imposing a C6 symmetry. The six XcpQ_N012 dimers are shown in cartoon form with a specific color. (B) The residues Thr54 and Gln86 in the N0ⁿ⁺²-N0ⁿ⁺³ interface are shown in stick and mesh presentations. (C) Top, side, and bottom views of the dodecameric XcpQ_N012 model docked into the EM map of the complex. (D) The dodecameric XcpQ_N012 model is organized into two peripheral and internal rings. The peripheral ring is colored in blue and presents the denoted subunits of each of the six dimers. The inner ring is colored in pink and presents the un-stranded subunits of each of the six dimers. (E) Model of vHH04-XcpQ_N012 complex. The crystal structure of the vHH04-XcpQ_N012 complex is overlaid with the dodecameric model of XcpQ_N012. Only six vHH04s presented in hot pink are able to bind the XcpQ_N012 complex. (F) Side view of the 6:12 vHH04-XcpQ_N012 complex docked into the EM map.

FIG 5

In vivo cysteine cross-linking and functionality of full-length XcpQ cysteine variants. (A) Immunoblotting analysis of protein samples from P. aeruginosa strains obtained under denaturing and nonreducing (top panel), denaturing and reducing (middle panel), or nondenaturing and reducing (bottom panel) conditions. Molecular mass markers (in kilodaltons) are indicated on the left. (B) Coomassie blue-stained gel of extracellular protein samples from various P. aeruginosa strains grown in the presence (+) or not (-) of 1 mM reducing agent dithiothreitol (DTT). The five Xcp T2SS effectors PrpL, elastase LasB, chitin binding protein D (CbpD), aminopeptidase PaAP, and metalloprotease ImpA (43) are indicated by black diamonds; the secreted effectors of the Hxc T2SS alkaline phosphatase LapA (44) and the T1SS alkaline protease AprA (45) are indicated in gray by a dot and a star, respectively. Molecular mass markers (in kilodaltons) are indicated on the left. (C) Extracellular elastase LasB activity of various P. aeruginosa strains producing or not producing wild-type or cysteine variants of XcpQ measured on elastin agar plates in the presence (+) or not (-) of 5 mM DTT. The halo of elastin degradation visible around the colony revealed the functionality of the corresponding secretin. (D) Extracellular elastase LasB activity under normal oxidative conditions of various P. aeruginosa strains producing or not producing wild-type or cysteine substitutions of XcpQ measured on elastin agar plates. The halo of elastin degradation visible around the XcpQ T54C and Q86C variants revealed their functionality in contrast to the double T54C-Q86C XcpQ variant.

FIG 6

In vivo vHH04 production and interference. (A) Immunoblot detection with antihistidine antibody of the histidine-tagged vHH04 in the cellular samples (top panel) or T2SS effector identification on Coomassie blue-stained SDS-PAGE of supernatant protein samples (middle panel) from P. aeruginosa strains producing or not producing vHH04. When produced, vHH04 inhibits the specific secretion of the T2SS effectors PaAP, LasB, and the protease PrpL (black diamonds) but not the T1SS effector AprA (green star). The bottom panel shows elastase activity assay on P. aeruginosa strains producing or not producing vHH04. The vHH04-mediated interference with Xcp T2SS extracellular protease activity is characterized by the isopropyl-β-d-thiogalactopyranoside (IPTG)-dependent inhibition of the extracellular halo of protease degradation around the colony on skim milk plates. Molecular mass markers (in kilodaltons) are indicated on the left. (B and C) Bilayer interferometry recordings representing binding of XcpQ_N012 alone (blue) or the XcpQ_N012-vHH04 complex (red) to a sensor coupled to CbpD (B) or to XcpPp (C). The response (in nanometers) is plotted versus the time (in seconds). The response measured in the red sensorgram (B) shows that vHH04 does not interfere with the binding of XcpQ_N012 to CbpD, whereas the absence of response in the red sensorgram (C) revealed that vHH04 prevents the binding of XcpQ_N012 to XcpPp. (D) Superimposition of GspC-GspD complex (PDB ID 3OSS) with the vHH04-XcpQ structure revealing that the vHH04 binding site overlaps GspC (HR) binding to GspD. The structures of XcpQ_N012, vHH04, GspC (HR), and GspD_N01 are colored in gray, yellow, green, and hot pink, respectively. (E) Side and bottom views of the XcpPp-XcpQ_N012 assembly model where only 6 XcpPps (green structures) are bound to the XcpQ_N012 dodecameric complex (gray structure).