Structural determinants of endopilus assembly, stability, and functional specificity in bacterial type II secretion.

Type III secretion system chaperones: a helping hand for secretion

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.

Intrinsic flexibility of Type VII secretion central pore is required for substrate translocation

Chaperone structure is not a sufficient determinant for the hierarchy of substrate secretion in bacterial type III secretion systems

Functional type III secretion in Gram negative bacteria relies on precise substrate targeting and a strict order of secretion with early, intermediate, and late substrates. Type III secretion chaperones facilitate these processes by maintaining substrates in a partially unfolded, secretion-competent state and serving as order-specific targeting factors. Early needle filament assembling substrates are chaperoned by none or class III chaperones, intermediate translocator-type substrates by class II and late effector-type substrates by class I chaperones. In case of hydrophobic transmembrane effectors, chaperones may also serve to prevent erroneous mistargeting of these substrates to the bacterial inner membrane. Here, we characterized the Salmonella transmembrane effectors SseF and SseG and their chaperone SscB, encoded in the operon sscB-sseF-sseG, in order to gain a deeper understanding of the underlying molecular requirements of targeting of this special class of substrates. We show that the gene linkage of SscB and SseF is critical for these proteins’ stability and SseF secretion. Counterintuitively, SscB revealed to feature a class II chaperone structure with a class I chaperone function. Likewise, SseF and SseG harbour conserved, translocator-like chaperone-binding motifs (PXI/LXXP) but were secreted as late substrates, independent of the gatekeeper protein SsaL. These findings challenge the current chaperone classification and our understanding of the molecular basis of the hierarchy of substrate secretion. They show that chaperone structure is not a sufficient molecular determinant for the correct order of substrate secretion.

Structure and Function of the Type IX Secretion System for Protein Secretion and Cellular Motility.

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.

Export of the pseudopilin XcpT of the Pseudomonas aeruginosa type II secretion system via the SRP/Sec pathway

Evolution of N‐terminal mechanical lability as a determinant for Type III secretion

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.