Acinetobacter type VI secretion system comprises a non-canonical membrane complex

A. baumannii can rapidly acquire new resistance mechanisms and persist on abiotic surface, enabling the colonization of asymptomatic human host. In Acinetobacter the type VI secretion system (T6SS) is involved in twitching, surface motility and is used for interbacterial competition allowing the bacteria to uptake DNA. A. baumannii possesses a T6SS that has been well studied for its regulation and specific activity, but little is known concerning its assembly and architecture. The T6SS nanomachine is built from three architectural sub-complexes. Unlike the baseplate (BP) and the tail-tube complex (TTC), which are inherited from bacteriophages, the membrane complex (MC) originates from bacteria. The MC is the most external part of the T6SS and, as such, is subjected to evolution and adaptation. One unanswered question on the MC is how such a gigantesque molecular edifice is inserted and crosses the bacterial cell envelope. The A. baumannii MC lacks an essential component, the TssJ lipoprotein, which anchors the MC to the outer membrane. In this work, we studied how A. baumannii compensates the absence of a TssJ. We have characterized for the first time the A. baumannii’s specific T6SS MC, its unique characteristic, its membrane localization, and assembly dynamics. We also defined its composition, demonstrating that its biogenesis employs three Acinetobacter-specific envelope-associated proteins that define an intricate network leading to the assembly of a five-proteins membrane super-complex. Our data suggest that A. baumannii has divided the function of TssJ by (1) co-opting a new protein TsmK that stabilizes the MC and by (2) evolving a new domain in TssM for homo-oligomerization, a prerequisite to build the T6SS channel. We believe that the atypical species-specific features we report in this study will have profound implication in our understanding of the assembly and evolutionary diversity of different T6SSs, that warrants future investigation.This work was funded by the Centre National de la Recherche Scientifique, the Aix-Marseille Université, and grants from the Agence Nationale de la Recherche (ANR-18-CE11-0023-01) and European Society of Clinical Microbiology and Infectious Diseases (ESCMID) to ED. ED is supported by the Institut National de la Santé et de la Recherche Médicale (INSERM). YC is funded by a Doctoral school PhD fellowship from the FRM (ECO20160736014 & FDT201904008052). OK is funded by a Doctoral school PhD fellowship from DGA and Aix-Marseille University and by the FRM (01D19024292-A AID & FDT202204014851). PS post-doctoral fellowship was supported by the European Respiratory Society under the ERS Long-Term Fellowship grant agreement LTRF - 202101-00862. IFM is funded by ANR-17-CE11-0039. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer ReviewedPostprint (published version

Acinetobacter type VI secretion system comprises a non-canonical membrane complex

A. baumannii can rapidly acquire new resistance mechanisms and persist on abiotic surface, enabling the colonization of asymptomatic human host. In Acinetobacter the type VI secretion system (T6SS) is involved in twitching, surface motility and is used for interbacterial competition allowing the bacteria to uptake DNA. A. baumannii possesses a T6SS that has been well studied for its regulation and specific activity, but little is known concerning its assembly and architecture. The T6SS nanomachine is built from three architectural sub-complexes. Unlike the baseplate (BP) and the tail-tube complex (TTC), which are inherited from bacteriophages, the membrane complex (MC) originates from bacteria. The MC is the most external part of the T6SS and, as such, is subjected to evolution and adaptation. One unanswered question on the MC is how such a gigantesque molecular edifice is inserted and crosses the bacterial cell envelope. The A. baumannii MC lacks an essential component, the TssJ lipoprotein, which anchors the MC to the outer membrane. In this work, we studied how A. baumannii compensates the absence of a TssJ. We have characterized for the first time the A. baumannii's specific T6SS MC, its unique characteristic, its membrane localization, and assembly dynamics. We also defined its composition, demonstrating that its biogenesis employs three Acinetobacter-specific envelope-associated proteins that define an intricate network leading to the assembly of a five-proteins membrane super-complex. Our data suggest that A. baumannii has divided the function of TssJ by (1) co-opting a new protein TsmK that stabilizes the MC and by (2) evolving a new domain in TssM for homo-oligomerization, a prerequisite to build the T6SS channel. We believe that the atypical species-specific features we report in this study will have profound implication in our understanding of the assembly and evolutionary diversity of different T6SSs, that warrants future investigation.ISSN:1553-7374ISSN:1553-736

Characterization of TssM location.

(A) TssM location determined by sucrose gradient. A. baumannii ATCC 17978 strains were grown, and the outer (OM) and inner (IM) membranes were separated by a continuous sucrose gradient (35%-60%). After centrifugation, 750ul samples were taken from the top to the bottom of the gradient. The numbers represent the number of the fraction and the Whole Cell Lysate (WCL). The samples were subjected to denaturing 12.5%- polyacrylamide gel electrophoresis (PAGE) and immunodetected with the appropriate antibody (including synthetics polyclonal TssM antibody). Immunodetected proteins are indicated on the right. Molecular weight markers (in kDa) are indicated in the left. Tags: GspCsfGFP (T2SS IM protein), Omp28mCherry (OM porin). (B) Dynamics of fluorescent clusters of the TssM protein. Cells were recorded in TIRF illumination during 10 min each 10 seconds (a). The column in panel (a) shows the corresponding average image of the 4 cells. Panel (b) shows 6 time points of these 4 cells. The arrows point to the cluster fluctuations (line 2) or their lateral displacement (line 3). The lower panels (c) are kymographs that illustrate 4 types of movements: fixed clusters (F), intermittent clusters (I), lateral displacement (L), cell without clusters (WC). The kymographs were constructed from the intensity profile of the cell contour. Scale: 2 μm (C) In A. baumannii ATCC 17978, the fusion of sfGFP to TssM C-terminal (TssM-sfGFP) and mCherry to a majority porin (Omp28-mCherry) was used to observe the location of the TssM C-terminal. The porin was used as a proxy to label the outer membrane. (D) Structured illumination microscopy (SIM) images of the TssMsfGFP regarding the Omp28mCherry label. The scale bar is 1μm. (E) Box plot representing the distances measured between the TssM foci and the mCherry label in a WT, a clpV mutant and a tssB-tssC mutant. The difference in position is equivalent to the distance between the two observed fluorophores and was measured in n = 60 bacteria (WT_ ΔclpV: t 3.1331, df = 105.39, p-value = 0.00224; WT_ ΔtssBΔtssC: t = 0.024481, df = 100.92, p-value = 0.9805; ΔclpV_ΔtssBΔtssC: t = -2.6278, df = 95.461, p-value = 0.01002).</p

Structural prediction study of TsmK an <i>Acinetobacter</i>-specific protein related to Ketoacyl synthases.

Analysis of the TsmK predicted structure. (A) AlphaFold2 confident score (predicted LDDT per position) mapped on the model. (B) Topology of the secondary elements of the predicted model for TsmK. The diagram depicts the secondary structure organization of the A. baumannii TsmK model generated with AlphaFold2. The major and minor β-sheets, as well as the long loop, are highlighted with colored squares. Inconsistent regions are represented with pale colors. Five antiparallel β-strands, β1, β7, β6, β4, and β5 assemble into a β-sheet (referred to as the major β-sheet). The α-helices are present at the loops formed between each pair of consecutive β-strand and decorate both sides of the β-sheet. AlphaFold2 predicted a second β-sheet (referred to as minor β-sheet) not present in TrRosetta and RaptorX predictions. This β-sheet is formed with the two β-strands of the converging hairpin (β9 and β10) and the β11, β12, and β8 (C) Structural superimposition of the three structural models of A. baumannii TsmK. The major and minor β-sheets as well as the long loop are highlighted with colored squares. Structural precision ranges between 0 and 15 Å (blue to red) (D) Topology of the secondary elements of the Ketoacyl synthase domain from Acyltransferase type I polyketide synthase (PKS) (PDB 4TKT). The homologous regions between TsmK and the KS domain of the polyketide synthase are highlighted in dashed colors.</p

Sequence and structural analysis of <i>A</i>. <i>baumannii</i> TssM.

(A) MSA of A. baumannii and other proteobacterial species TssM C-terminal domain (from T987 to P1274 of A. baumannii TssM). Conserved regions were highlighted with a red square. The region of interaction between EAEC TssM and TssJ (PDB 4Y7O) was highlighted with a purple square. Residue conservation of the GxxGxxxGxxG motif found in the C-terminal domain of TssM for Acinetobacter was highlighted with grey dotted lines. (B) Multiple sequence alignment of A. baumannii TssM with the EAEC TssM (PDB 6hs7) to generate the structural model of A. baumannii TssM periplasmic domain. (C) Structural models for the TssM-CTD. (a) the structure of EAEC sequence. The region corresponding to the GS-linker of the Ab sequences is highlighted in green and boxed. (b,c) the structure of the A. baumannii sequence computed with AlphaFold (AF) 2.3.1 (cyan), and the A. baumannii-sequence with GS-linker deletion (blue) is aligned on the EAEC structure (gray), the GS-linker is colored in green and boxed. (d,e) 5 AF models for the Ab sequence colored using pLDDT score (red>90, blue(D) Lower panel, western blot assays probing for TssM (WT and mutatants) production in whole cell lysates. The samples were subjected to denaturing 10%-polyacrylamide gel electrophoresis (PAGE) and immunodetected with synthetics polyclonal A. baumannii-TssM antibody. (TIF)</p

<i>A</i>. <i>baumannii tssM-sfGFP</i> clusters are shows very dynamic.

Movie illustrating the dynamics of clusters in tirf. tssM-sfGFP clusters appear and disappear rapidly and some cells where clusters are not well visible. On the left the fluorescent signal and on the right the FFT band pass filtered image 5–1 pixels (Fiji/imageJ processing). Time = 10 sec / frame accelerated 200X. (MP4)</p

Monitoring T6SS MC assembly and the function of accessory proteins.

(A)Western blot assays probing for Hcp secretion and RNA polymerase (RNAP) in whole-cell pellets (P) and supernatants (S), by A. baumannii ATCC 17978 wildtype (WT, parental strain) and several mutants. The RNAP was used as control. The supernatants of each strain of A. baumannii are isolated, concentrated and then analyzed by denatured 12.5%-polyacrylamide gel electrophoresis (PAGE). Immunodetected proteins are indicated on the right. Molecular weight markers (in kDa) are indicated in the left. (B) Fluorescence microscopy recordings showing different constructions aimed at fusing the fluorophore (sfGFP) to a protein of the membrane complex. The images show: the phase contrast (top) and the fluorescence (bottom). The positions of the foci are indicated by arrows. The scale bars are 1 μm. (C) Comparison of fluorescent cluster distribution of TssM in different mutant. Diagram comparing the number of foci in TssM-sfGFP and TssM-mCherry. Left panel, histogram representation of cluster distribution of TssM-mCherry. Right panel, histogram representation of cluster distribution of the main axis of the cell with a preferential accumulation at the poles of the cell in two different mutants. The different mutants do not show very marked differences. The polar accumulation is less important in the d-ClpV mutant (n > 1000 cells for each group from two biological replicates). (D) Row data of the number of fluorescent TssMsfGFP foci in different mutants (E) Non-polar effect of mutation on TssM expression. Western blot assays probing for the TssMH and the EF-Tu expression in whole cell by A. baumannii ATCC 17978. The EF-Tu was used as control. The samples were analyzed by denatured 12.5%-acrylamide polyacrylamide gel electrophoresis (PAGE) and immunodetected with a Alexa Fluor Anti-Fluorescein (FITC) anti ET-Tu (FITC) and a fluorescent antibodies, λexitation = 488 nm. Immunodetected proteins are indicated on the right. Molecular weight markers (in kDa) are indicated in the left. (F) Time-lapse fluorescence microscopy recordings showing localization and dynamics of the mCherryClpV and TssMsfGFP fusions proteins (see Fig 2 for full legend). Two independent colocalization events are shown. (TIF)</p

Determination of the protein-protein interaction network (PPIN) of <i>A</i>. <i>baumannii</i> T6SS membrane complex.

(A) Western blot probing binary interaction between TagX, TssM, TssL and TsmK respectively. (B) Co-purification of the 3-, 4- and 5-protein complexes. (A-D) Detergent-solubilized extract of E. coli BL21(DE3) cells producing the indicated protein were submitted to an affinity purification step on Strep- or His- Trap. The detergent-solubilized total membrane (L), and eluate (E) were subjected to denaturing 12.5%-polyacrylamide gel electrophoresis (PAGE) and immunodetected with the appropriate antibody. The composition of the protein mix is indicated on the top of each panel, the protein that binds to the column is underlined. Immunodetected proteins are indicated on the right. Molecular weight markers (in kDa) are indicated on the left. Tags: H, 6×His; S, Strep-tag; F, FLAG; HA, Hemagglutinin; V, VSVG. (C) Negative controls. (D) Western blot probing the binary interaction between TsmK and TssK (left panel), including negative control (TssK alone, right panel). (E) Native pull-down of TssM from A. baumannii cells. Western blot probing the identified interaction partners (left panel). Mass spectrometry analysis of TssM copurified interactants (right panel).(F) Schematic interaction network determined by co-purification and topology of the A. baumannii T6SS-MC full-length proteins. Interactions between proteins are represented with arrows: TsmK is in the center of the interaction network (red arrows). Indirect interactions (native pull-down are shown by dashed double-head arrows.</p

Model for the assembly of the <i>A</i>. <i>baumannii</i> T6SS MC.

(A) Conditional probability tree of the co-occurrence of “GS-linker motif”, TsmK, and TssJ on genomes containing both a tssB and a tssM gene. Percentages inside the rounded rectangles represent the conditional probabilities after each branching. Percentages reported on the bottom are the joint probabilities of the different GS-linker -TsmK -TssJ configurations in baumannii, non-baumannii and non-Acinetobacter genomes. Rounded red and green rectangles represent the absence and the presence of one of the two genes (tssJ or tsmK) or the GS-linker motif, respectively. (B) A. baumannii T6SS offers a unique glimpse of molecular evolution showing how the absence of a major T6SS component (like TssJ, upper panel) could be compensated by small additional domain or co-opted protein (lower panel). (1) TsmK stabilizes the pillar of the membrane complex (MC), the protein TssM. (2) Helped by the GS-linker, TssM oligomerizes to assemble a mega-membrane complex with TsmK. (3) TssL, TagX and TlsA are recruited to form the Acinetobacter T6SS MC. (4) The molecular mechanism and dynamic of outer membrane anchoring and piercing by the MC is not yet known.</p

Monitoring T6SS MC function of accessory proteins.

(A)Bacterial competition experiments. Table associated with the Fig 1C. survival of E. coli rifampicin-resistant after incubation with ATCC 17978 WT and several mutants. (B) Phenotypic complementation of tsmK mutation measuring the Hcp secretion. Dot blot probing for Hcp secretion in supernatants, by A. baumannii ATCC 17978 WT, ΔtssM and the mutant ΔtsmK, transformed with the plasmid control empty (pVRL1) or the plasmid overexpressing TsmK. The TsmK protein production was induced by 1 mM IPTG. (TIF)</p