Jul 2026· Protein Science· Vol 35· 0 citations· 87 references
Medicine
TL;DR
Using atomic‐force‐microscopy‐based force spectroscopy, it is shown that effector NleC is mechanically labile and mechanically compliant, supporting the notion that mechanical lability is an evolved, structurally encoded feature underlying effector secretion.
Abstract
Many Gram‐negative pathogens critically depend on the Type III secretion system (T3SS) to inject effector proteins into host cells for colonization. Because the channel of the T3SS is narrow (~2 nm), effectors must be unfolded for secretion. However, the T3SS cannot unfold mechanically robust substrates (GFP, ubiquitin, and dihydrofolate reductase), severely impairing their secretion. Consistent with this, effectors are exceptionally mechanically labile, unfolding at low forces. Thus, secretion competency is correlated with mechanical properties. Effector sequences have significantly diverged from non‐effectors, suggesting that secretion exerts evolutionary pressure selecting mechanical lability. Here, using atomic‐force‐microscopy‐based force spectroscopy, we show that effector NleC is mechanically labile (Funfold = 13.5 pN at 100 nm/s) and mechanically compliant, as characterized by a large distance to the transition state (Δx‡ = 2.7 nm). In contrast, the non‐effector homolog protealysin is mechanically stable (Funfold = 50.7 pN at 100 nm/s) and brittle (Δx‡ = 0.7 nm), comparable to proteins known to impair secretion (Funfold >80 pN; Δx‡ <0.4 nm). Denaturant‐induced unfolding assays demonstrate that effectors exhibit rates typical of their fold, further reinforcing mechanical properties rather than fast unfolding kinetics (k0) predicts secretion. Steered molecular dynamic simulations revealed NleC unfolding initiates at the N‐terminus, consistent with current secretion models, whereas protealysin unfolding initiates at the C‐terminus. Notably, the NleC N‐terminus is primarily α‐helical while non‐effector homologs contain β‐sheets, which may account for the distinct unfolding pathway. Together, these results support the notion that mechanical lability is an evolved, structurally encoded feature underlying effector secretion.
Key determinants of secretion specificity and endopilus stability are identified, revealing how minor sequence variations in conserved nanomachines drive functional adaptation to diverse environments.
Abstract The type III secretion system (T3SS) is a virulence mechanism commonly used by Gram-negative bacterial pathogens to deliver virulence proteins, known as effectors, into infected cells. The T3SS secretes a range of different substrates: first the needle subunits, then the translocon pore components and finally a pathogen-specific range of effector proteins. Each of these classes of substrates interacts with a corresponding class of bacterial chaperones, which are required for their efficient secretion. The requirement for these chaperones has been attributed to multiple functions, including preventing premature substrate activity, maintaining substrate stability in the bacterial cytoplasm and mediating substrate targeting and secretion hierarchy. Here, we bring together what is known about the function of T3SS chaperones in a range of different bacterial pathogens. Through analysis of the conservation of chaperone sequence and structure, we discuss how these proteins interact with and support the secretion of diverse substrates. Finally, we evaluate the extent to which chaperones are universally required for effector secretion.
Kyra Roepke, Alexia J Galsworthy, Adam Agbamu et al.· Microbiology· 0 citations
Abstract Pseudomonas aeruginosa is a major nosocomial pathogen in which the type VI secretion system (T6SS) contributes to interbacterial competition and virulence. While most strains encode three T6SSs, additional T6SS clusters have been identified in clinical isolates through comparative genomics, but their functions and effector biology remain undefined. Here, we identify TseMt as a major antibacterial effector associated with an H4‐T6SS in a clinical P. aeruginosa isolate LYSZa7. TseMt is a periplasmically active toxin whose activity is neutralized by a cognate immunity protein, TsiMt. Biochemical assays show that TseMt binds membranes and forms ion‐conducting pores, establishing it as a pore‐forming effector. A 3.0 Å cryo‐electron microscopy structure reveals a distinct three‐domain architecture comprising an N‐terminal MIX‐like domain, a central α‐helical scaffold, and a C‐terminal toxin domain. Genetic analysis and structural modeling indicate that TseMt is delivered through a dedicated PAAR−VgrG−chaperone pathway. Together, these findings define the structural basis, functional mechanism, and delivery pathway of the H4‐T6SS effector TseMt from a clinical P. aeruginosa isolate and reveal its role in mediating bacterial competition.
ABSTRACT The type VI secretion system (T6SS) is a nanomachine utilized by Gram-negative bacteria to secrete toxic effectors. The T6SS is employed against both prokaryotic and eukaryotic targets, allowing for competitive advantage and virulence of the attackers in a wide variety of environments. Recent work has revealed the great diversity of T6SS systems. Beyond the canonical, conserved architectural genes of the T6SS, diverse T6SS-associated genes (TAGs) are employed to optimize T6SS activity, compensate for variations in architecture, and dictate T6SS use. Although various TAGs have been discovered in T6SS gene clusters, they display unique activities in modifying their cognate T6SS. In this review, we discuss several of the better-studied TAGs, as well as highlight under-researched TAGs that are likely of great significance. As a means to better understand TAG roles, identify common principles and unifying themes, we present the following six functional categories: Firing Coordinators, Target Recognition Factors, Structural Factors, Transcriptional Regulators, Secretion Regulators, and Unknown TAGs.
C. Latario, Benjamin D. Ross· Journal of Bacteriology· 0 citations
SUMMARY One of the first gram-negative protein secretion systems to be discovered was the type II secretion system (T2SS), in which proteins first cross the inner membrane mainly via the Sec translocon, and then are recognized and carried across the outer membrane by the multiprotein T2SS apparatus. With its erstwhile appellation as the main terminal branch of the general secretory pathway, the T2SS has sometimes been described as (only) secreting a relatively small number of degradative enzymes for the purpose of nutrient acquisition. However, as comprehensively reviewed here, the T2SS is a great deal more than this, e.g., it (i) can secrete and expose on the cell surface over 100 proteins, (ii) is a major mediator of virulence against humans, animals, and plants, secreting diverse substrates that target essentially every step in the pathogenic process, (iii) promotes biofilm formation and environmental reactions such as metal reduction, (iv) has functional interactions with other secretion systems, and (v) has a homolog in mitochondria. Moreover, current assessment of the literature reveals an underappreciated role for the T2SS in both interbacterial antagonism and bacterial competition with fungi, protozoa, and perhaps algae. We also assembled data indicating that the T2SS mediates bacterial resistance to antibiotics. Finally, the T2SS is a fascinatingly complex “machine” whose contributing parts can vary and whose ability to recognize substrates is primarily conformational, involving spatially separated elements that interact with the T2SS apparatus through transient, multivalent interactions rather than a single dedicated binding motif.
N. Cianciotto, Joshua Mayoral, James A Garnett· Microbiology and Molecular B...· 0 citations
The type IX secretion system (T9SS) is a Bacteroidota-specific multiprotein machine that supports a wide range of biological processes, from nutrient acquisition and surface modification to host interaction and gliding motility. T9SS effectors represent a structurally diverse repertoire of enzymes, adhesins, and surface proteins that all possess a C-terminal domain that addresses them to their final destination. Recent structural and mechanistic information has revealed the modular organization of the T9SS and molecular details governing effector selection, transport, processing, and sorting. In motile Bacteroidota, the T9SS has been co-opted, evolved, and specialized for gliding motility. In this review, we summarize current knowledge on T9SS architecture and function, describe the embedded gliding machinery, and highlight conceptual advances and open questions regarding the mechanisms, dynamics, and ecological implications of this unique system.
Yaëlle Aouizerate, Thierry Doan, Eric Cascales· Annual Review of Microbiolog...· 0 citations