Structural roles and independent secretion of TseP domains

A, Secretion analysis of TseP, TsePN, and TsePC in the SSU triple effector deletion mutant (Δ3eff). A schematic of the TseP N-terminus (TsePN, 1-603 aa) and C-terminus (TsePC, 604-845 aa) is depicted at the top. Hcp serves as a positive control for T6SS secretion. Hcp, RpoB, and 3V5-tagged TseP proteins were detected using specific antibodies. B, Time-lapse imaging of VipA-sfGFP signals in the Δ3eff mutant complemented with different TseP variants. Each sample was captured every 10 s for 5 min and temporally color-coded. Color scale used to temporally color code the VipA-sfGFP signals is shown at the bottom. A 30-× 30-μm representative field of cells is shown. Scale bars, 5 μm. C, Statistical analysis of T6SS sheath assemblies in the Δ3eff mutant complemented with different TseP variants. Error bars indicate the mean ± standard deviation of three biological replicates, and statistical significance was calculated using a two-tailed Student’s t-test. ns, not significant; **, P<0.01. D, Secretion analysis of TseP, TsePN, and TsePC in SSU wild type, ΔvasK, ΔtseP, and Δ3eff mutants. For B and D, TseP, TsePN, and TsePC were tagged with a 3V5 C-terminal tag and expressed on pBAD vectors. RpoB serves as an equal loading and autolysis control. E, Pull-down analysis of VgrG2 with TseP, TsePN, and TsePC. His-tagged VgrG2 and 3V5-tagged TseP, TsePN, or TsePC were used. His-tagged sfGFP and 3V5-tagged MBP were used as controls.

Functional analysis of TseP reveals an amidase activity

A, In vitro amidase activity of the TseP, TsePN, TsePC, and the lysozyme inactivated mutants TsePE663A and TsePC-E663A. Cell-wall digestion products after incubation with the TseP or its mutants were analyzed by the UPLC/MS. B, MS analysis of cell-wall digestion products (NAM-NAC, tetrapeptides, and tripeptides) following treatment with TseP. C, Protein sequence alignment of the TseP amidase domain with other amidase homologs (top), and the structural superimposition of the TseP amidase domain and 3SLU (bottom). Cartoon representations of TseP and 3SLU are shown in green and cyan, respectively. The key residues involved in Zn2+ binding are shown in a stick model, and the zinc ion is indicated by the green sphere. D, In vitro amidase activity of TsePN and its amidase site mutated variants. E, In vitro amidase activity of TseP under cationic conditions of 2 mM EDTA, Zn2+, Ca2+, or a combination of both cations. TseP represents protein purified without EDTA treatment while TsePEDTA refers to protein purified in presence of EDTA. F, PG digestion analysis of TsePN with or without TsiP. The immunity protein TsiP was incubated with TsePN on ice for 12 h before being mixed with PG. Products in D, E, and F were analyzed by UPLC-QTOF MASS. G, Pull-down analysis of TsiP with TseP, TsePN, and TsePC. His-tagged TsiP and 3V5-tagged TseP, TsePN, or TsePC were used. His-tagged sfGFP and 3V5-tagged MBP were used as controls.

Amidase activity of TseP is not essential for T6SS assembly or lysozyme function

A, Secretion analysis of Hcp in the Δ3eff mutant complemented with different TseP variants. RpoB serves as an equal loading and autolysis control. Hcp, RpoB, and 3V5-tagged TseP proteins were detected using specific antibodies. B, Time-lapse imaging of VipA-sfGFP signals in the Δ3eff mutant complemented with TseP or its amidase-inactive mutant TsePH339A. Each sample was captured every 10 s for 5 min and temporally color-coded. Color scale used to temporally color code the VipA-sfGFP signals is shown at the right. A 30 × 30 μm representative field of cells is shown. Scale bars, 5 μm. C, Competition analysis of the Δ3eff mutant complemented with different TseP variants. Competition assays were repeated once. D, Enzymatic activity of the TseP, TsePC and amidase-inactive mutant TsePH339A. The error bars indicate the mean ± standard deviation of three biological replicates.

TseP homologs showed the same in vitro PG-hydrolysis activity with the TseP

A, Maximum-likelihood phylogeny of TseP homologs. Phylogeny was constructed using the IQ-tree web server with bootstrap 1000 times. Proteins tested in this study are highlighted in red. B, Amidase activity analysis of TseP homologs. C, In vitro PG-hydrolysis activity of the TseP homologs. Products in B and C were analyzed through UPLC-QTOF MASS. D, GC contents of the tseP gene cluster and AHA_1849 gene cluster. E, Summary of TsePN and TsePC homologs output by Foldseek Search server. F, Structure alignments of TseP and homologs WL1483_2262 and DSB67_24810.

Crystal structure of TsePC

A, Enzymatic activity of TseP, TsePC, and lysozyme in hydrolyzing purified E. coli PG. The error bars indicate the mean standard deviation of three biological replicates. B, The overall structure of the TsePC. The small lobe and large lobe are shown in cyan and green, respectively, with the connecting loop depicted in red. C, Electrostatic potential maps of TsePC with the Y720, R723, and K796 shown as a stick model. The electrostatic surface potentials are colored red for negative charges, blue for positive charges, and white for neutral residues. D, Structural comparison of TsePC and lysozyme (PDB ID: 1LZC). The catalytic sites are shown as a stick model. E, E. coli PG digestion analysis of TsePC and TsePC-E663D mutant. Error bars indicate the mean ± standard deviation of three biological replicates, and statistical significance was calculated using a two-tailed Student’s t-test. ****, P<0.0001.

Data collection and refinement statistics of TsePC crystallization

Modification of surface charge enables TsePC to kill Gram-positive bacteria

A, B. subtilis PG digestion analysis of TseP and TsePC. PG was treated with 0.25 M NaOH for 12 h at 37 °C to remove cross-linked peptides and teichoic acid. The lysis percentage was calculated by detecting the changes of OD600 during 1 h. The hen-egg lysozyme was used as a positive control. B, Electrostatic potential maps of the TsePC and TsePC4+. The active sites and the mutation region are highlighted in red circles. The negatively charged residues D604, E609, D841, and E845 in the mutation region are shown as a stick model and colored in green, and lysines are colored in cyan. C, In vitro PG-hydrolysis activity of the TsePC and TsePC4+. RBB-labelled E. coli PG was used as the substrate and the lysis percentage was detected by dye release. D, B. subtilis PG digestion analysis of TsePC and TsePC4+. Exponential phase B. subtilis cells (OD600∼1.0) were used as substrate. The lysis percentage was calculated by detecting the changes of OD600 during 1 h with the enzyme concentration at 100 nM. E, Secretion analysis of TsePC and TsePC4+ in the ΔtseP mutant. RpoB serves as an equal loading and autolysis control. RpoB and 3V5-tagged TsePC proteins were detected using specific antibodies. F, Statistical analysis of B. subtilis cells in the competition assays. Error bars of statistical analysis in A, C, D, and F indicate the mean ± standard deviation of three biological replicates, and statistical significance was calculated using a two-tailed Student’s t-test. ns, not significant; *, P<0.05; **, P<0.01; ****, P<0.0001.

Model of TseP dual functions and engineering

This schematic illustrates the dual amidase-lysozyme activities of TseP and demonstrates an effective engineering strategy to enhance T6SS and its effector capabilities. The T6SS can independently secrete both the N- and C-terminal domains, with multiple evidence suggesting an evolutionary fusion event. These domains interact directly with the upstream-encoded carrier protein VgrG2 for secretion. TsePN, the N-terminal domain, functions as a Zn2+-dependent amidase, while the C-terminal domain, TsePC, exhibits lysozyme activities. However, in its native form, TsePC does not lyse Gram-positive B. subtilis cells. Through structural-guided design, TsePC was engineered to create TsePC4+ by altering its surface charge. This modification enables TsePC4+ to lyse B. subtilis cells without affecting its T6SS-dependent secretion. Consequently, the T6SS-equipped cell acquires the ability to lyse B. subtilis in a contact-independent manner. Given the diversity of TseP-like effectors and T6SS species, this approach holds significant potential for modulating interspecies competition and combating antimicrobial resistance.