Structural Insights into the Effector – Immunity System Tae4/Tai4 from <i>Salmonella typhimurium</i>

<div><p>Type-6-secretion systems of Gram-negative bacteria are widely distributed needle-like multi-protein complexes that are involved in microbial defense mechanisms. During bacterial competition these injection needles dispense effector proteins into the periplasm of competing bacteria where they induce degradation of the peptidoglycan scaffold and lead to cell lysis. Donor cells co-produce immunity proteins and shuttle them into their own periplasm to prevent accidental toxication by siblings. Recently, a plethora of previously unidentified hydrolases have been suggested to be peptidoglycan degrading amidases. These hydrolases are part of effector/immunity pairs that have been associated with bacterial warfare by type-6-secretion systems. The <i>Tae4</i> and <i>Tai4</i> operon encoded by <i>Salmonella typhimurium</i> is one of these newly discovered effector/immunity pairs. The Tae4 effector proteins induce cell lysis by cleaving the γ-D-glutamyl-L-<i>meso</i>-diaminopimelic acid amide bond of acceptor stem muropeptides of the Gram-negative peptidoglycan. Although homologues of the Tae4/Tai4 system have been identified in various different pathogens, little is known about the functional mechanism of effector protein activity and their inhibition by the cognate immunity proteins. Here, we present the high-resolution crystal structure of the effector Tae4 of <i>S. typhimurium</i> in complex with its immunity protein Tai4. We show that Tae4 contains a classical NlpC/P60-peptidase core which is common to other effector proteins of the type-6-secretion system. However, Tae4 has unique structural features that are exclusively conserved within the family of Tae4 effectors and which are important for the substrate specificity. Most importantly, we show that although the overall structure of Tai4 is different to previously described immunity proteins, the essential mode of enzyme inhibition is conserved. Additionally, we provide evidence that inhibition in the Tae4/Tai4 heterotetramer relies on a central Tai4 dimer in order to acquire functionality.</p></div

Cyrstal structure of the Tae4/Tai4 heteroteramer.

<p>(A) The two symmetry related Tai4 molecules (red transparence and opaque) form a heterotetramer with two Tae4 molecules (orange and green) in our crystal structure. (B) Close up of the interaction between Tae4 and Tai4 at the Tae4 active site. Tai4 inserts the loop region between α-helix d and e (d-e-loop) into the active site of Tae4. This loop harbors Ser98 that forms a hydrogen bond to the catalytic important His126 residue and thereby prevents deprotonation of Cys44. (C) Molecular surface representation of the Tai4 dimer (one monomer colored in white and the second in gray) bound to Tae4 with amino acid sequence conservation mapped onto the molecular surface and colored according to 2E and 4C. Ser98 in Tai4 which has been mutated in this study and its interacting residue in Tae4, His126, are colored in red. The second mutated residue in Tai4, Glu71, and its interacting residues in Tae4, Val80 and Asn81, are highlighted in the red as well. These residues are part of the hydrogen bond network between Tae4 and Tai4. (D) “Open-Book view” of the Tae4/Tai4 heterotetramer interface (as indicated by a dashed line in (C)) with amino acid sequence conservation mapped onto the molecular surface.</p

Data collection and refinement statistics.

<p>Values in parentheses are those for the highest resolution shell.</p

Schematic representation showing the peptidoglycan cleavage specificities of the type-6-secretion amidase (Tae) effector families.

<p>Cleavage specificity of the Tae1–4 effector proteins on Gram-negative tetrapeptide stems (left) or Gram-positive pentapeptide stems (right) is depicted based on previous reports <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067362#pone.0067362-Russell1" target="_blank">[3]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067362#pone.0067362-Russell2" target="_blank">[4]</a>. Tae2 and Tae3 effector proteins hydrolyze the amide bond between the <i>m</i>DAP-D-Ala cross-link in Gram-negative as well as Gram-positive peptidoglycan. In contrast, members of the Tae1 (including Tse1 from <i>P. aeruginosa</i>) and the Tae4 effector family degrade the peptidoglycan scaffold by cleaving the γ-D-glutamyl-<i>m</i>DAP bond. Whereas, Tae4 exclusively hydrolyzes the acceptor stem of cross-linked as well as non-cross-linked Gram-negative peptidoglycan, Tse1 specifically cleaves the cross-linked donor peptide stem. Furthermore, Gram-positive peptidoglycan is a poor substrate for Tae4 family members.</p

Affinity and association/dissociation kinetics of the Tae4/Tai4 complex.

<p>(A) Isothermal titration calorimetry experiment. The binding constant (<i>K</i>_D), as well as the stoichiometric ratio (N) are determined by fitting a single binding-site model to the data. Original titrations are shown above the fit. (B) Kinetic traces of Tae4-ATTO 488 and wild-type as well as mutated Tai4(E71A_S98A) binding have been recorded at different Tai4 concentration (0.3–1.5 µM from light gray to black). The time has been plotted against the relative fluorescence signal and single exponential functions have been fitted to the data (red). The time traces and the fits are exemplarily shown for binding of the Tai4(E71A_S98A) protein (C) The observed rate constants k_obs from (B) have been plotted against the concentrations of Tai4wt (filled black circles) and Tai4(E71A_S98A) (empty gray circles) and fitted using a linear regression (Tai4wt:black, Tai4(E71A_S98A):gray). The association rate constants of Tai4 binding could be extracted from the slopes of the linear functions. Although the slope of the linear functions is just slightly increased for Tai4(E71A_S98A), the y-axis intercept (corresponding to the off-rate) is significantly lower for wild-type Tai4. (D) The dissociation rate constants were determined separately by dissociation experiments using unlabelled Tae4 protein to chase off the Tae4-ATTO 488/Tai4 complex. Shown are the time traces recorded from 1 µM Tae4 to chase off the effector/immunity complex. The time trace for wild-type Tae4/Tai4 complex is shown in blue. The traces for the mutated Tae4/Tai4 protein complex is shown in black with the corresponding exponential fit in red. Simulated time traces using the exponential fit values of the Tai4(E71A_S98A) experiment with the dissociation rate constant divided by up to a factor of 10⁴ are depicted in gray. Simulated time traces suggested an off-rate between 0.7 ×10⁻³ and 0.7 ×10⁻⁴ s⁻¹for wild-type Tai4; although, it can not be excluded to be lower. All values obtained from the stopped-flow experiments are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067362#pone-0067362-t002" target="_blank">Table 2</a>.</p

Affinity constants for wild-type and mutant Tai4 protein binding to Tae4.

<p>Affinity constants for wild-type and mutant Tai4 protein binding to Tae4.</p

Crystal structure and conservation of the Tai4 immunity protein from <i>S. typhimurium</i>.

<p>(A) Ribbon representation of Tai4. Tai4 is a purely α-helical protein forming a head-to-tail dimer in the crystal structure. The disulfide bond (DSB) formed by Cys48 and Cys108 that links the α-helices b and e with each other is shown as stick representation. Ser98 which is important for effector inhibition is depicted as stick representation as well. (B) Dimeric arrangement of the Tai4 inhibitor showing the molecular surface of one of the immunity proteins. Amino acid sequence conservation (C) is mapped on the molecular surface. (C) Amino acid sequence alignment of Tai4 immunity proteins from <i>Salmonella typhimurium</i> (NP_459276.1), <i>Escherichia coli</i> B354 (ZP_06652155.1), <i>Enterobacter cloacae</i> (YP_006478115.1) and <i>Pantoea sp. Sc1</i> (ZP_09929550.1) going from dark green (identical residues) to light green, orange and yellow (with decreasing conservation). Secondary structure elements above the sequence alignment are colored according to (A). Ser98, located in the inhibition loop between the α-helices d and e as well as the conserved Glu71 residue which makes extensive contacts to Tae4 are marked with a red star. Residues which interact with Tae4 are marked with a green pentagon. Residues that are involved in Tai4 dimer formation by either hydrophobic interactions (blue rectangles) or hydrogen bonds (black dots) are also indicated below the sequence alignment. DSB formation between Cys48 and Cys108 is represented as a dashed line. Cleavage site of the periplasmic leader sequence in Tai4 is indicated with a black arrow above the sequence.</p

Growth of <i>Escherichia coli</i> expressing wild-type and mutated variants of Tae4 and Tai4 proteins.

<p>Inoculi were prepared by serial dilutions from 10⁰ to 10⁻⁶ of overnight cultures and spotted with decreasing optical density from left to the right onto LB-agar plates containing IPTG to induce protein expression. The effector protein Tae4 leads to a significant reduction of bacterial growth upon periplasmic localization. Expression of variants mutated in the active site residues Cys44, H126 and D137 in Tae4 to alanine residues did no more interfere with bacterial growth. A replacement of Cys135 and Cys139 by serine residues did not affect bacterial growth compared to wild-type Tae4. Wild-type Tai4 could rescue the growth defect induced by periplasmic localization of the Tae4. In contrast, a Tai4 variant missing its periplasmic leader sequence (Tai4ΔN26) could no more counteract the growth defect. Additionally, mutations in the effector/immunity interface in Tai4(E71A_S98A) did not rescue the growth phenotype as efficient as the wild-type protein. Proteins which were expressed from pET22b vector constructs contained the pelB leader sequence for artificial periplasmic localization and are labeled in black. Proteins which were expressed from pET28b vector constructs are labeled in red. Vector controls can be found at the bottom of the panel.</p

Biological assembly of Tai4 and the Tae4/Tai4 complex.

<p>(A) Cross-linking experiments of wild-type Tai4 using glutaraldehyde. Aliquots of cross-linked Tai4 have been taken at different time points (0 to 60 min) and separated on Coomassie stained SDS-PAGE. Bands of monomeric Tai4 with an electrophoretic mobility corresponding to 13 kDa as well as increasing amounts of cross-linked dimeric species of Tai4 (26 kDa) were observed. Note, that minor traces of unspecific intermolecular cross-links are observed (marked with a star) which result in apparent trimeric and tetrameric Tai4 species. LMW: low molecular weight marker in kDa. (B) Gel-filtration and static light scattering of the Tae4/Tai4 effector/immunity complex (solid line) as well as of the wild-type Tai4 immunity protein (dotted line). According to gel-filtration experiments using a gel-filtration standard from BioRad the proteins elute at a corresponding molecular weight of approximately 25 kDa for Tai4 and 46 kDa for the Tae4/Tai4 complex. However, using a multi-angle static light detector averaged molecular masses of 25.26±0.07 kDa for the Tai4 protein and 60.38±0.12 kDa for the Tae4/Tai4 protein complex could be determined. Note that the discrepancy between the masses calculated from the gel-filtration and the static light scattering experiments most likely are caused by the elongated shape of the Tae4/Tai4 complex as observed in the crystal structure. (C) Cross-linking experiments of mutated Tai4(E71A_S98A) using glutaraldehyde have been performed similar to wild-type Tai4 (A).</p

Structural Insights into the Effector – Immunity System Tse1/Tsi1 from <em>Pseudomonas aeruginosa</em>

<div><p>During an interbacterial battle, the type-6-secretion-system (T6SS) of the human pathogen <em>Pseudomonas aeruginosa</em> injects the peptidoglycan(PG)-hydrolase Tse1 into the periplasm of Gram-negative enemy cells and induces their lysis. However, for its own benefit, <em>P. aeruginosa</em> produces and transports the immunity-protein Tsi1 into its own periplasm where in prevents accidental exo- and endogenous intoxication. Here we present the high-resolution X-ray crystal structure of the lytic enzyme Tse1 and describe the mechanism by which Tse1 cleaves the γ-D-glutamyl-l-<em>meso</em>-diaminopimelic acid amide bond of crosslinked PG. Tse1 belongs to the superfamily of N1pC/P60 peptidases but is unique among described members of this family of which the structure was described, since it is a single domain protein without any putative localization domain. Most importantly, we present the crystal structure of Tse1 bound to its immunity-protein Tsi1 as well and describe the mechanism of enzyme inhibition. Tsi1 occludes the active site of Tse1 and abolishes its enzyme activity by forming a hydrogen bond to a catalytically important histidine residue in Tse1. Based on our structural findings in combination with a bioinfomatic approach we also identified a related system in <em>Burkholderia phytofirmans</em>. Not only do our findings point to a common catalytic mechanism of the Tse1 PG-hydrolases, but we can also show that it is distinct from other members of this superfamily. Furthermore, we provide strong evidence that the mechanism of enzyme inhibition between Tsi1 orthologues is conserved. This work is the first structural description of an entire effector/immunity pair injected by the T6SS system. Moreover, it is also the first example of a member of the N1pC/P60 superfamily which becomes inhibited upon binding to its cognate immunity protein.</p> </div