Delivery Determinants of an Type VI Secretion System Bifunctional Peptidoglycan Hydrolase

Overview

Journal mBio

Publisher American Society for Microbiology

Specialty Microbiology

Date 2025 Jan 2

PMID 39745415

Authors

Valeriya Bezkorovayna

Brooke K Hayes

Francesca N Gillett

Amy Wright

David I Roper

Marina Harper

Sheena McGowan

John D Boyce

Affiliations

Soon will be listed here.

Abstract

is a Gram-negative opportunistic pathogen and is a common cause of nosocomial infections. The increasing development of antibiotic resistance in this organism is a global health concern. The clinical isolate AB307-0294 produces a type VI secretion system (T6SS) that delivers three antibacterial effector proteins that give this strain a competitive advantage against other bacteria in polymicrobial environments. Each effector, Tse15, Tde16, and Tae17, is delivered a non-covalent interaction with a specific T6SS VgrG protein (VgrG15, VgrG16, and VgrG17, respectively). Here we define the regions of interaction between Tae17 and its cognate delivery protein VgrG17 and identify that amino acids G1069 and W1075 in VgrG17 are essential for Tae17 delivery the T6SS, the first time such specific delivery determinants of T6SS cargo effectors have been defined. Furthermore, we determine that the Tae17 effector is a multidomain, bifunctional, peptidoglycan-degrading enzyme that has both amidase activity, which targets the sugar-peptide bonds, and lytic transglycosylase activity, which targets the peptidoglycan sugar backbone. Moreover, we show that the Tae17 transglycosylase activity is more important than amidase activity for the killing of . This study provides molecular insight into how the T6SS allows strains to gain dominance in polymicrobial communities and thus improve their chances of survival and transmission.IMPORTANCEWe have shown that the T6SS effector Tae17 is a modular, bifunctional, peptidoglycan-degrading enzyme that has both lytic transglycosylase and amidase activities. Both activities contribute to the ability to degrade peptidoglycan, but the transglycosylase activity was more important for the killing of . We have defined the specific regions of Tae17 and its cognate delivery protein VgrG17 that are necessary for the non-covalent interactions and, for the first time, identified specific amino acids essential for T6SS cargo effector delivery. This work contributes to our molecular understanding of bacterial competition strategies in polymicrobial environments and may provide a window to design new therapeutic approaches for combating infection by .

References

Bondage D, Lin J, Ma L, Kuo C, Lai E . VgrG C terminus confers the type VI effector transport specificity and is required for binding with PAAR and adaptor-effector complex. Proc Natl Acad Sci U S A. 2016; 113(27):E3931-40. PMC: 4941472. DOI: 10.1073/pnas.1600428113. View

Bateman A, Rawlings N . The CHAP domain: a large family of amidases including GSP amidase and peptidoglycan hydrolases. Trends Biochem Sci. 2003; 28(5):234-7. DOI: 10.1016/S0968-0004(03)00061-6. View

Gurnani Serrano C, Winkle M, Martorana A, Biboy J, More N, Moynihan P . ActS activates peptidoglycan amidases during outer membrane stress in Escherichia coli. Mol Microbiol. 2021; 116(1):329-342. PMC: 8360153. DOI: 10.1111/mmi.14712. View

Gulliver E, Sy B, Wong J, Lucas D, Powell D, Harper M . The Role and Targets of the RNA-Binding Protein ProQ in the Gram-Negative Bacterial Pathogen Pasteurella multocida. J Bacteriol. 2022; 204(4):e0059221. PMC: 9017301. DOI: 10.1128/jb.00592-21. View

Dong C, Zhang H, Gao Z, Wang W, She Z, Liu G . Structural insights into the inhibition of type VI effector Tae3 by its immunity protein Tai3. Biochem J. 2013; 454(1):59-68. DOI: 10.1042/BJ20130193. View

Gunther P, Quentin D, Ahmad S, Sachar K, Gatsogiannis C, Whitney J . Structure of a bacterial Rhs effector exported by the type VI secretion system. PLoS Pathog. 2022; 18(1):e1010182. PMC: 8765631. DOI: 10.1371/journal.ppat.1010182. View

Grant S, Jessee J, Bloom F, Hanahan D . Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants. Proc Natl Acad Sci U S A. 1990; 87(12):4645-9. PMC: 54173. DOI: 10.1073/pnas.87.12.4645. View

Pukatzki S, McAuley S, Miyata S . The type VI secretion system: translocation of effectors and effector-domains. Curr Opin Microbiol. 2009; 12(1):11-7. DOI: 10.1016/j.mib.2008.11.010. View

Kumar V, Mathure S, Lee M, Boorman J, Zeng X, Lin J . Turnover Chemistry and Structural Characterization of the Cj0843c Lytic Transglycosylase of . Biochemistry. 2021; 60(14):1133-1144. PMC: 9067259. DOI: 10.1021/acs.biochem.1c00027. View

10.

Zapun A, Philippe J, Abrahams K, Signor L, Roper D, Breukink E . In vitro reconstitution of peptidoglycan assembly from the Gram-positive pathogen Streptococcus pneumoniae. ACS Chem Biol. 2013; 8(12):2688-96. DOI: 10.1021/cb400575t. View

11.

Smith W, Vettiger A, Winter J, Ryser T, Comstock L, Basler M . The evolution of the type VI secretion system as a disintegration weapon. PLoS Biol. 2020; 18(5):e3000720. PMC: 7274471. DOI: 10.1371/journal.pbio.3000720. View

12.

Bock L . Bacterial biocide resistance: a new scourge of the infectious disease world?. Arch Dis Child. 2019; 104(11):1029-1033. DOI: 10.1136/archdischild-2018-315090. View

13.

Bateman A, Bycroft M . The structure of a LysM domain from E. coli membrane-bound lytic murein transglycosylase D (MltD). J Mol Biol. 2000; 299(4):1113-9. DOI: 10.1006/jmbi.2000.3778. View

14.

Liang X, Pei T, Li H, Zheng H, Luo H, Cui Y . VgrG-dependent effectors and chaperones modulate the assembly of the type VI secretion system. PLoS Pathog. 2021; 17(12):e1010116. PMC: 8668125. DOI: 10.1371/journal.ppat.1010116. View

15.

Russell A, Singh P, Brittnacher M, Bui N, Hood R, Carl M . A widespread bacterial type VI secretion effector superfamily identified using a heuristic approach. Cell Host Microbe. 2012; 11(5):538-49. PMC: 3358704. DOI: 10.1016/j.chom.2012.04.007. View

16.

Karimova G, Pidoux J, Ullmann A, Ladant D . A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proc Natl Acad Sci U S A. 1998; 95(10):5752-6. PMC: 20451. DOI: 10.1073/pnas.95.10.5752. View

17.

Mahmood T, Yang P . Western blot: technique, theory, and trouble shooting. N Am J Med Sci. 2012; 4(9):429-34. PMC: 3456489. DOI: 10.4103/1947-2714.100998. View

18.

Maeda H . A new lysozyme assay based on fluorescence polarization or fluorescence intensity utilizing a fluorescent peptidoglycan substrate. J Biochem. 1980; 88(4):1185-91. DOI: 10.1093/oxfordjournals.jbchem.a133073. View

19.

Chen L, Zou Y, She P, Wu Y . Composition, function, and regulation of T6SS in Pseudomonas aeruginosa. Microbiol Res. 2015; 172:19-25. DOI: 10.1016/j.micres.2015.01.004. View

20.

Heckman K, Pease L . Gene splicing and mutagenesis by PCR-driven overlap extension. Nat Protoc. 2007; 2(4):924-32. DOI: 10.1038/nprot.2007.132. View