Structural Insights Into Pseudomonas aeruginosa Type Six Secretion System Exported Effector 8

Abstract

Recent reports indicate that the Type Six Secretion System exported effector 8 (Tse8) is a cytoactive effector secreted by the Type VI Secretion System (T6SS) of the human pathogen Pseudomonas aeruginosa. The T6SS is a nanomachine that assembles inside the bacteria and injects effectors or toxins into target cells, providing a fitness advantage over competing bacteria and facilitating host colonization. Here we present the first crystal structure of Tse8, showing it conserves a putative catalytic triad Lys84-transSer162-Ser186 found among homologues. Furthermore, we measure the binding of phenylmethylsulfonyl fluoride (PMSF) to Tse8. Remarkably, we observed that PMSF binding is dependent on the putative catalytic residue Ser186, providing evidence of its nucleophilic reactivity. This work demonstrates that Tse8 belongs to the Amidase Signature (AS) superfamily. Furthermore, it highlights Tse8’s similarity to two family members: the Stenotrophomonas maltophilia Peptide Amidase and the Glutamyl-tRNA^Gln amidotransferase subunit A from Staphylococcus aureus.

Introduction

Nearly a quarter of sequenced Gram-negative bacteria encode the T6SS. The T6SS assembles inside the bacteria, and upon cell contact, this molecular machine is propelled towards the target cell, puncturing their membranes and injecting effector molecules using a contractile spike-like device. T6SSs are distantly related to the tail of bacteriophages, which also use a puncturing mechanism to inject their DNA into bacteria, suggesting that bacteria acquired the T6SS by horizontal gene transfer. Known T6SS-dependent effectors mainly target competing bacterial cells, providing a fitness advantage that allows them to thrive and facilitates host colonization. Nonetheless, in chronic infections, such as in Cystic Fibrosis (CF) patients, the T6SS seems to modulate the immune response and pathogenicity. One of the most common chronic infections in CF patients is caused by Pseudomonas aeruginosa, which contains in its genome three T6SS clusters (H1, H2, and H3-T6SS), each encoding the thirteen components required to assemble a functional T6SS.

Pseudomonas aeruginosa is a Gram-negative opportunistic nosocomial pathogen and one of the most typical causes of pneumonia in immunocompromised patients and those with lung diseases. Resistance to carbapenem increases the risk of mortality in patients with bloodstream infections, and there are minimal treatment options. As a consequence, the World Health Organization ranked this pathogen in critical need for the development of novel antibiotics.

In P. aeruginosa, known effectors delivered by the H1-T6SS have antiprokaryotic activity by targeting the cell wall peptidoglycan (Tse1: peptidase activity and Tse3: muramidase activity), the cytoplasmic membrane (Tse4: ion pore-forming activity), NAD(P)+ (Tse6: glycohydrolase activity), and DNA (Tse7: DNase activity). The T6SS secretes effectors either encapsulated within the inner tube that assembles by stacking of Haemolysin-coregulated proteins (Hcp) or associated with the spike complex, formed by trimerization of Valine-glycine repeat proteins G (VgrG). Some effectors can associate through non-covalent interactions to the T6SS VgrG and Hcp components, while others exist as extension domains on VgrG (evolved-VgrG) and Hcp (Hcp-C-terminal extension toxins – Hcp-ET). Furthermore, some effectors contain a small extension domain, defined by a signature sequence Pro-Ala-Ala-Arg (the PAAR domain), that associates with some VgrG spike complexes to sharpen their ends and allow membrane puncturing. Importantly, associated with each effector, there is a cognate immune protein, encoded adjacent to the effector gene, that binds to it for self-protection.

Although remarkable progress has been achieved in understanding the role of the P. aeruginosa T6SS in bacterial competition and virulence, the reduced number of T6SS-dependent effectors identified to date was hampering this achievement. In an attempt to overcome this limitation, a recent report identified novel effectors delivered by the H1-T6SS using a global genomic-based approach. One of these novel effectors and its cognate immunity protein are Tse8 and Tsi8 (the annotated gene numbers in the P. aeruginosa PAO1 reference strain correspond to PA4163 and PA4164, respectively). This report also proposed that Tse8 targets the transamidosome, which is a dynamic ribonucleoprotein particle dedicated to tRNA-dependent amino acid biosynthesis. Furthermore, bacterial competition assays led the authors to suggest that Tse8 does not utilize amidase activity to elicit toxicity in vivo. The same report demonstrated the VgrG1a spike complex delivers Tse8 into prey cells, and the expression of its cognate immunity protein, Tsi8, can overcome the deleterious effect of Tse8. Based on sequence similarity, Tse8 was predicted to belong to the Amidase Signature (AS) superfamily. Enzymes of this family catalyze the hydrolysis of amide bonds; however, the family has diverged widely regarding substrate specificity and function. Even though bioinformatic analysis predicted that Tse8 conserves the catalytic residues Lys84-Ser162-Ser186 found in AS family enzymes, the authors were unable to measure hydrolytic activity on carbon-nitrogen bonds of two molecules, epinecidin-1 and glutamine, which are substrates of two Tse8 homologues: Stenotrophomonas maltophilia Peptide Amidase (SmPAM) enzyme and Staphylococcus aureus Glutamyl-tRNA^Gln amidotransferase subunit A (SaGatA) of the transamidosome, respectively.

In this article, we present the first crystal structure of Tse8 at atomic resolution. We also employ the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) to probe the role of residues found in the peptide-binding site. The data shown here demonstrate that Tse8 belongs to the Amidase Signature superfamily, identify the pocket containing the conserved putative Lys84-Ser162-Ser186 catalytic triad, and provide insight into the nucleophilic reactivity of the S186 residue.

Materials and Methods

Tse8 Cloning, Protein Expression and Purification

The Pseudomonas aeruginosa tse8 gene (PA4163) was synthesized by GenScript. The construct contains a 5’ tag coding for a 9xHis tag and a tobacco etch virus protease cleavage site. The construct was cloned into a pET29a(+) vector between the NdeI and HindIII restriction sites. For protein expression, Escherichia coli BL21(DE3) cells were transformed with the resulting pET29a::his-tag-tse8 plasmid and grown in 50 µg/mL kanamycin-supplemented Luria Broth (LB) medium at 37°C. When the culture reached an OD600 value of approximately 0.7, Tse8 expression was induced by adding 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), and the temperature was dropped to 18°C. After approximately 18 hours, cells were harvested and frozen for later use. The cell pellet obtained from a 2-liter culture was resuspended in 40 mL of 50 mM Tris-HCl pH 8.0, 500 mM NaCl, 20 mM imidazole, 0.5 mM EDTA, and 2 µL of benzonase endonuclease. Cells were then disrupted by sonication, and the suspension was centrifuged for 40 minutes at 56,000 xg. The supernatant was filtered with a 0.2 µm syringe filter and subjected to immobilized metal affinity chromatography using a HisTrap HP column of 1 mL on a fast protein liquid chromatography system (ÄKTA FPLC) equilibrated with 5 mL of 50 mM Tris-HCl pH 8, 500 mM NaCl, and 20 mM imidazole. The column was washed with this buffer at 1 mL/min until no change in absorbance at 280 nm was detected. Elution was performed with a linear gradient between 0% and 50% of 50 mM Tris-HCl pH 8, 500 mM NaCl, and 500 mM imidazole in 30 mL at 1 mL/min. Fractions containing Tse8 protein were pooled, and protein concentration was measured. Tse8 protein was injected into a HiLoad Superdex 200 column, previously equilibrated with 20 mM Tris-HCl pH 7.5 and 125 mM NaCl. Eluted protein was then concentrated using Centricon centrifugal filter units of 30 kDa molecular mass cut-off to a final concentration of approximately 17 mg/mL, followed by flash-freezing under liquid nitrogen for later crystallization trials. The purity of the protein was verified by SDS-PAGE and mass spectrometry analysis.

Mass Spectrometry Analysis of Tse8

Intact mass spectrometry analysis of Tse8, following expression and purification from E. coli, was carried out at the Functional Genomics Centre Zurich. The sample was desalted using C4 ZipTips and analyzed in methanol:2-propanol:0.2% formic acid (30:20:50). The solutions were infused through a fused silica capillary at a flow rate of 1 µL/min and sprayed through a PicoTip. Nano electrospray ionization mass spectrometry (ESI-MS) analyses of the samples were performed on a Synapt G2_Si mass spectrometer, and the data were recorded with the MassLynx 4.2 software. Mass spectra were acquired in the positive-ion mode by scanning an m/z range from 100 to 5000 Da with a scan duration of one second and an interscan delay of 0.1 seconds. The spray voltage was set to 3 kV, source temperature at 80°C, and the cone voltage to 50 V. The recorded m/z data were then deconvoluted into mass spectra by applying the maximum entropy algorithm with a resolution of the output mass 0.5 Da/channel and a Uniform Gaussian Damage Model at the half-height of 0.7 Da. The expected peptide was detected unaltered, and the molecular weight was confirmed. The results verified the calculated molecular weight from the primary sequence and identified two peptides in high abundance. Based on their masses, these two peptides might result from cleavage of the C-terminal part of Tse8.

Tse8 Crystallization, Data Collection and Processing

Crystals of Tse8 in complex with the peptide were obtained by mixing protein (5 mg/mL) in 20 mM Tris-HCl pH 7.0 and 50 mM NaCl with a mother liquor containing 1.6 M magnesium sulphate heptahydrate and 0.1 M MES buffer pH 6.5. Crystals grew to maximum dimensions in five to seven days as prisms. Single crystals were cryo-cooled in liquid nitrogen by using a cryo-protectant solution containing the mother liquor complemented with 25% glycerol. Preliminary X-ray data were collected at the microfocus I24 beamline (Diamond Light Source, UK), and the best diffracting crystal was collected on a PILATUS3 6M detector at the XALOC beamline (ALBA synchrotron, Spain). X-ray diffraction data were integrated with the autoPROC toolbox, which uses several programs for data processing, scaling, and reduction. Data anisotropy was analyzed with STARANISO, and reciprocal plane plots indicated the data is anisotropic, which can be modeled by an ellipsoid with specific resolution limits. Data quality was assessed and found to be normal, with no signs of translational NCS, twinning, or ice rings.

Crystals of unliganded Tse8 were obtained by random seeding techniques. Seeds were mixed with protein (10 mg/mL; 20 mM Tris-HCl pH 7.0 and 50 mM NaCl) and a mother liquor containing 0.2 M calcium acetate, 0.1 M sodium cacodylate pH 6.5, and 18% PEG8000. Crystals grew to maximum dimension in approximately two weeks and were cryo-cooled with a cryo-protectant solution complemented with 15% ethylene glycol.

Structure Solution and Refinement

The structures of both the peptide-bound and unliganded Tse8 crystals were solved by molecular replacement using the program PHASER. The search model was based on a homologue from the Protein Data Bank with substantial similarity to Tse8. Iterative rounds of model building and refinement were performed using COOT and PHENIX. Initial electron density maps revealed clear density for most of the Tse8 polypeptide chain. In the peptide-bound structure, additional electron density corresponding to the peptide ligand was observed in the catalytic cleft. Hydrogens were placed in riding positions, and anisotropic B-factors were not applied due to the data resolution.

The model geometry was validated with MOLPROBITY, ensuring proper bond angles and Ramachandran statistics. No significant outliers were identified. The final refined structures of Tse8, both liganded and unliganded, exhibited excellent fit to the experimental maps, with R-factors in acceptable ranges.

Structure Analysis

Overall Architecture of Tse8

The overall fold of Tse8 consists of a characteristic α/β/α sandwich typical of Amidase Signature (AS) superfamily members, with a central β-sheet flanked by layers of α-helices. The catalytic site is located in a prominent cleft near the surface of the molecule. The organization of secondary structure elements is highly conserved with previously characterized amidases.

Catalytic Site and Active Residues

A detailed inspection of the catalytic pocket revealed the spatial arrangement of the putative triad Lys84, Ser162, and Ser186, corresponding to conserved residues in other AS family proteins. Side chains of these residues are appropriately oriented to support catalysis, suggesting they contribute to nucleophilic attack and stabilization of transition states during amide bond hydrolysis.

In the peptide-bound structure, the C-terminal serine, Ser186, is positioned adjacent to the substrate, compatible with a nucleophilic mechanism. Hydrogen bond networks involving these residues and bound water molecules further stabilize the active state.

Additional Features and Potential Substrate Recognition

Structural alignments with homologues indicate Tse8 shares overall similarity with the Stenotrophomonas maltophilia Peptide Amidase (SmPAM) and Glutamyl-tRNA^Gln amidotransferase subunit A (SaGatA) from Staphylococcus aureus. However, subtle conformational differences in loop regions surrounding the active site suggest possible adaptations for substrate specificity. The peptide ligand in the Tse8 complex structure adopts an extended conformation within the active site cleft, stabilized by multiple hydrogen bonds and van der Waals contacts.

PMSF Binding and Functional Implications

To probe the nucleophilicity of Ser186, phenylmethylsulfonyl fluoride (PMSF), a classic serine-reactive inhibitor, was introduced and its binding assessed by mass spectrometry and crystallography. Evidence of covalent adduct formation at Ser186 was observed, confirming its accessibility and reactivity as an active serine. Upon PMSF modification, structural changes localized to the active cleft, with minimal perturbation to the global fold, reaffirming the functional importance of Ser186.

Functional Consequences and Biological Implications

The structural elucidation of Tse8 clarifies its classification as an Amidase Signature family member, despite previous failure to detect in vitro enzymatic activity on model substrates. The conserved organization of the catalytic triad and its demonstrated reactivity towards PMSF strongly suggest Tse8 employs classical amidase chemistry, even if its physiological substrate remains elusive. The presence of homologous folds among diverse species further supports the notion that Tse8 may act on a specialized substrate, possibly in the context of bacterial competition or modulation of host processes via the Type VI secretion system.

Discussion

The atomic resolution structures of both liganded and unliganded Tse8 reveal a conserved amidase fold and the presence of the canonical Lys84-Ser162-Ser186 triad. Structural homology to peptide amidases and amidotransferases, along with the observed reactivity towards PMSF, reinforce the functional assignment of Tse8 within the AS superfamily.

Intriguingly, despite conservation of the active site, in vitro activity assays have yet to identify a native substrate for Tse8. This suggests Tse8 might have evolved to recognize a unique physiological target, possibly associated with its secreted effector function during interspecies or host-pathogen interactions. Alternatively, Tse8 may require additional cofactors or post-translational modifications, or function in a multiprotein complex in vivo for full activity.

The interaction of Tse8 with its immunity protein, Tsi8, likely blocks its catalytic cleft, preventing autotoxicity within the producing cell. Further studies elucidating the Tse8–Tsi8 complex, as well as in vivo identification of its physiological substrate, will be necessary to fully clarify the mechanisms underlying effector delivery and toxicity in the Type VI secretion repertoire of Pseudomonas aeruginosa.

Conclusion

The high-resolution crystal structures of Tse8 provide the first atomic insights into this newly identified Type VI secretion system effector. The structural features highlight Tse8’s capacity to act as an amidase, in line with its placement within the AS superfamily. The identification of a functional Lys84-Ser162-Ser186 triad and the binding of the serine inhibitor PMSF to Ser186 confirm the active character of this residue.

The structural groundwork presented here opens opportunities for both the biochemical dissection of Tse8′s role in bacterial competition and the broader understanding of T6SS effector mechanisms. Future investigations will focus on elucidating the exact nature of Tse8’s target substrate(s), the mode of its delivery via the secretion system, and its interplay with immunity proteins in vivo. This knowledge will advance our comprehension of bacterial warfare and could eventually inform the development of strategies against pathogenic strains of P. aeruginosa.