MolPhase, an advanced prediction algorithm for protein phase separation.

We introduce MolPhase, an advanced algorithm for predicting protein phase separation (PS) behavior that improves accuracy and reliability by utilizing diverse physicochemical features and extensive experimental datasets. MolPhase applies a user-friendly interface to compare distinct biophysical features side-by-side along protein sequences. By additional comparison with structural predictions, MolPhase enables efficient predictions of new phase-separating proteins and guides hypothesis generation and experimental design. Key contributing factors underlying MolPhase include electrostatic pi-interactions, disorder, and prion-like domains. As an example, MolPhase finds that phytobacterial type III effectors (T3Es) are highly prone to homotypic PS, which was experimentally validated in vitro biochemically and in vivo in plants, mimicking their injection and accumulation in the host during microbial infection. The physicochemical characteristics of T3Es dictate their patterns of association for multivalent interactions, influencing the material properties of phase-separating droplets based on the surrounding microenvironment in vivo or in vitro. Robust integration of MolPhase's effective prediction and experimental validation exhibit the potential to evaluate and explore how biomolecule PS functions in biological systems.

The sorting platform in the type III secretion pathway: From assembly to function.

The type III secretion system (T3SS) is a specialized nanomachine that enables bacteria to secrete proteins in a specific order and directly deliver a specific set of them, collectively known as effectors, into eukaryotic organisms. The core structure of the T3SS is a syringe-like apparatus composed of multiple building blocks, including both membrane-associated and soluble proteins. The cytosolic components organize together in a chamber-like structure known as the sorting platform (SP), responsible for recruiting, sorting, and initiating the substrates destined to engage this secretion pathway. In this article, we provide an overview of recent findings on the SP's structure and function, with a particular focus on its assembly pathway. Furthermore, we discuss the molecular mechanisms behind the recruitment and hierarchical sorting of substrates by this cytosolic complex. Overall, the T3SS is a highly specialized and complex system that requires precise coordination to function properly. A deeper understanding of how the SP orchestrates T3S could enhance our comprehension of this complex nanomachine, which is central to the host-pathogen interface, and could aid in the development of novel strategies to fight bacterial infections.

Exploring the 'N-terminal arm' & 'Convex surface' Binding Interfaces of the T3SS Chaperone-Translocator Complexes from P. Aeruginosa.

One infection method widely used by many gram-negative bacteria involves a protein nanomachine called the Type Three Secretion System (T3SS). The T3SS enables the transportation of bacterial "toxins" via a proteinaceous channel that directly links the cytosol of the bacteria and host cell. The channel from the bacteria is completed by a translocon pore formed by two proteins named the major and minor translocators. Prior to pore formation, the translocator proteins are bound to a small chaperone within the bacterial cytoplasm. This interaction is crucial to effective secretion. Here we investigated the specificity of the binding interfaces of the translocator-chaperone complexes from Pseudomonas aeruginosa via the selection of peptide and protein libraries based on its chaperone PcrH. Five libraries encompassing PcrH's N-terminal and central α-helices were panned, using ribosome display, against both the major (PopB) and minor (PopD) translocator. Both translocators were shown to significantly enrich a similar pattern of WT and non-WT sequences from the libraries. This highlighted key similarities/differences between the interactions of the major and minor translocators with their chaperone. Moreover, as the enriched non-WT sequences were specific to each translocator, it would suggest that PcrH can be adapted to bind each translocator individually. The ability to evolve such proteins indicates that these molecules may provide promising anti-bacterial candidates.

Exploring structural features and potential lipid interactions of Pseudomonas aeruginosa type three secretion effector PemB by spectroscopic and calorimetric experiments.

Type Three Secretion System (T3SS) is a sophisticated nano-scale weapon utilized by several gram negative bacteria under stringent spatio-temporal regulation to manipulate and evade host immune systems in order to cause infection. To the best of our knowledge, this present study is the first report where we embark upon characterizing inherent features of native type three secretion effector protein PemB through biophysical techniques. Herein, first, we demonstrate binding affinity of PemB for phosphoinositides through isothermal calorimetric titrations. Second, we shed light on its strong homo-oligomerization propensity in aqueous solution through multiple biophysical methods. Third, we also employ several spectroscopic techniques to delineate its disordered and helical conformation. Lastly, we perform a phylogenetic analysis of this new effector to elucidate evolutionary relationship with other organisms. Taken together, our results shall surely contribute to our existing knowledge of Pseudomonas aeruginosa secretome.

Evolutionary Conservation, Variability, and Adaptation of Type III Secretion Systems.

Type III secretion (T3S) systems are complex bacterial structures used by many pathogens to inject proteins directly into the cytosol of the host cell. These secretion machines evolved from the bacterial flagella and they have been grouped into families by phylogenetic analysis. The T3S system is composed of more than 20 proteins grouped into five complexes: the cytosolic platform, the export apparatus, the basal body, the needle, and the translocon complex. While the proteins located inside the bacterium are conserved, those exposed to the external media present high variability among families. This suggests that the T3S systems have adapted to interact with different cells or tissues in the host, and/or have been subjected to the evolutionary pressure of the host immune defenses. Such adaptation led to changes in the sequence of the T3S needle tip and translocon suggesting differences in the mechanism of assembly and structure of this complex.

Inherent Minor Conformer of Bordetella Effector BteA Directs Chaperone-Mediated Unfolding.

The pathogen Bordetella pertussis uses a type-3 secretion system (T3SS) to inject its cytotoxic effector BteA into the host cell via a designated needle structure. Prior to injection BteA is bound to its cognate chaperone BtcA presumed to assist in effector unfolding en route to needle passage. We utilized NMR and EPR spectroscopy to uncover the molecular mechanism of BtcA-mediated unfolding of BteA. BtcA induces a global structural change in the effector, which adopts a more extended and partially unfolded conformation. EPR distance measurements further show that the structured helical-bundle form of free BteA exists in conformational equilibrium with a lowly populated minor species. The nature of this equilibrium was probed using NMR relaxation dispersion experiments. At 283 K structural effects are most pronounced for a contiguous surface spanning the A- and B-helices of BteA, extending at 303 K to a second surface including the D- and E-helices. Residues perturbed in the minor conformation coincide with those exhibiting a BtcA-induced increase in flexibility, identifying this conformation as the BtcA-bound form of the effector. Our findings hint at a conformational-selectivity mechanism for the chaperone interaction with the effector, a paradigm that may be common to effector-chaperones secretion complexes in this family of pathogens.

Cryo-EM of the injectisome and type III secretion systems.

Double-membrane-spanning protein complexes, such as the T3SS, had long presented an intractable challenge for structural biology. As a consequence, until a few years ago, our molecular understanding of this fascinating complex was limited to composite models, consisting of structures of isolated domains, positioned within the overall complex. Most of the membrane-embedded components remained completely uncharacterized. In recent years, the emergence of cryo-electron microscopy (cryo-EM) as a method for determining protein structures to high resolution, has be transformative to our capacity to understand the architecture of this complex, and its mechanism of substrate transport. In this review, we summarize the recent structures of the various T3SS components, determined by cryo-EM, and highlight the regions of the complex that remain to be characterized. We also discuss the recent structural insights into the mechanism of effector transport through the T3SS. Finally, we highlight some of the challenges that remain to be tackled.

Interactions and substrate selectivity within the SctRST complex of the type III secretion system of enteropathogenic Escherichia coli.

Many bacterial pathogens employ a protein complex, termed the type III secretion system (T3SS), to inject bacterial effectors into host cells. These effectors manipulate various cellular processes to promote bacterial growth and survival. The T3SS complex adopts a nano-syringe shape that is assembled across the bacterial membranes, with an extracellular needle extending toward the host cell membrane. The assembly of the T3SS is initiated by the association of three proteins, known as SctR, SctS, and SctT, which create an entry portal to the translocation channel within the bacterial inner membrane. Using the T3SS of enteropathogenic Escherichia coli, we investigated, by mutational and functional analyses, the role of two structural construction sites formed within the SctRST complex and revealed that they are mutation-resistant components that are likely to act as seals preventing leakage of ions and metabolites rather than as substrate gates. In addition, we identified two residues in the SctS protein, Pro23, and Lys54, that are critical for the proper activity of the T3SS. We propose that Pro23 is critical for the physical orientation of the SctS transmembrane domains that create the tip of the SctRST complex and for their positioning with regard to other T3SS substructures. Surprisingly, we found that SctS Lys54, which was previously suggested to mediate the SctS self-oligomerization, is critical for T3SS activity due to its essential role in SctS-SctT hetero-interactions.

Topology and Contribution to the Pore Channel Lining of Plasma Membrane-Embedded Shigella flexneri Type 3 Secretion Translocase IpaB.

Shigella spp. are human bacterial pathogens that cause bacillary dysentery. Virulence depends on a type 3 secretion system (T3SS), a highly conserved structure present in multiple important human and plant pathogens. Upon host cell contact, the T3SS translocon is delivered to the host membrane, facilitates bacterial docking to the membrane, and enables delivery of effector proteins into the host cytosol. The Shigella translocon is composed of two proteins, IpaB and IpaC, which together form this multimeric structure within host plasma membranes. Upon interaction of IpaC with host intermediate filaments, the translocon undergoes a conformational change that allows for bacterial docking onto the translocon and, together with host actin polymerization, enables subsequent effector translocation through the translocon pore. To generate additional insights into the translocon, we mapped the topology of IpaB in plasma membrane-embedded pores using cysteine substitution mutagenesis coupled with site-directed labeling and proximity-enabled cross-linking by membrane-permeant sulfhydryl reactants. We demonstrate that IpaB function is dependent on posttranslational modification by a plasmid-encoded acyl carrier protein. We show that the first transmembrane domain of IpaB lines the interior of the translocon pore channel such that the IpaB portion of the channel forms a funnel-like shape leading into the host cytosol. In addition, we identify regions of IpaB within its cytosolic domain that protrude into and are closely associated with the pore channel. Taken together, these results provide a framework for how IpaB is arranged within translocons natively delivered by Shigella during infection. IMPORTANCE Type 3 secretion systems are nanomachines employed by many bacteria, including Shigella, which deliver into human cells bacterial virulence proteins that alter cellular function in ways that promote infection. Delivery of Shigella virulence proteins occurs through a pore formed in human cell membranes by the IpaB and IpaC proteins. Here, we define how IpaB contributes to the formation of pores natively delivered into human cell membranes by Shigella flexneri. We show that a specific domain of IpaB (transmembrane domain 1) lines much of the pore channel and that portions of IpaB that lie in the inside of the human cell loop back into and/or are closely associated with the pore channel. These findings provide new insights into the organization and function of the pore in serving as the conduit for delivery of virulence proteins into human cells during Shigella infection.

Genetic Analysis of the Salmonella FliE Protein That Forms the Base of the Flagellar Axial Structure.

The FliE component of the bacterial flagellum is the first protein secreted through the flagellar type III secretion system (fT3SS) that is capable of self-assembly into the growing bacterial organelle. The FliE protein plays dual roles in the assembly of the Salmonella flagellum as the final component of the flagellar type III secretion system (fT3SS) and as an adaptor protein that anchors the rod (drive shaft) of the flagellar motor to the membrane-imbedded MS-ring structure. This work has identified the interactions between FliE and other proteins at the inner membrane base of the flagellar machine. The fliE sequence coding for the 104-amino-acid protein was subject to saturating mutagenesis. Single-amino-acid substitutions were generated in fliE, resulting in motility phenotypes. From these mutants, intergenic suppressor mutations were generated, isolated, and characterized. Single-amino-acid mutations defective in FliE function were localized to the N- and C-terminal helices of the protein. Motile suppressors of amino acid mutations in fliE were found in rod protein genes flgB and flgC, the MS ring gene, fliF, and one of the core T3SS genes, fliR. These results support the hypothesis that FliE acts as a linker protein consisting of an N-terminal α-helix that is involved in the interaction with the MS ring with a rotational symmetry and a C-terminal coiled coil that interacts with FliF, FliR, FlgB, and FlgC, and these interactions open the exit gate of the protein export channel of the fT3SS. IMPORTANCE The bacterial flagellum represents one of biology's most complex molecular machines. Its rotary motor spins at speeds of more than 2,000 cycles per second, and its type 3 secretion (T3S) system secretes proteins at rates of tens of thousands of amino acids per second. Within the complex flagellar motility machine resides a unique protein, FliE, which serves as an adaptor to connect a planar, inner membrane-embedded ring structure, the MS-ring, the core T3S secretion complex at the cytoplasmic base, and a rigid, axial structure that spans the periplasmic space, penetrates the outer membrane, and extends 10 to 20 microns from the cell surface. This work combines genetic mutant suppressor analysis with the structural data for the core T3S system, the MS-ring, and the axial drive shaft (rod) that transverses the periplasm to provide insight into the essential adaptor role of FliE in flagellum assembly and function.

Oxygen-Induced Conformational Changes in the PAS-Heme Domain of the Pseudomonas aeruginosa Aer2 Receptor.

The Aer2 receptor from Pseudomonas aeruginosa has an O2-binding PAS-heme domain that stabilizes O2 via a Trp residue in the distal heme pocket. Trp rotates ∼90° to bond with the ligand and initiate signaling. Although the isolated PAS domain is monomeric, both in solution and in a cyanide-bound crystal structure, an unliganded structure forms a dimer. An overlay of the two structures suggests possible signaling motions but also predicts implausible clashes at the dimer interface when the ligand is bound. Moreover, in a full-length Aer2 dimer, PAS is sandwiched between multiple N- and C-terminal HAMP domains, which would feasibly restrict PAS motions. To explore the PAS dimer interface and signal-induced motions in full-length Aer2, we introduced Cys substitutions and used thiol-reactive probes to examine in vivo accessibility and residue proximities under both aerobic and anaerobic conditions. In vivo, PAS dimers were retained in full-length Aer2 in the presence and absence of O2, and the dimer interface was consistent with the isolated PAS dimer structure. O2-mediated changes were also consistent with structural predictions in which the PAS N-terminal caps move apart and the C-terminal DxT region moves closer together. The DxT motif links PAS to the C-terminal HAMP domains and was critical for PAS-HAMP signaling. Removing the N-terminal HAMP domains altered the distal PAS dimer interface and prevented signaling, even after signal-on lesions were introduced into PAS. The N-terminal HAMP domains thus facilitate the O2-dependent shift of PAS to the signal-on conformation, clarifying their role upstream of the PAS-sensing domain.

Conserved Salt Bridges Facilitate Assembly of the Helical Core Export Apparatus of a Salmonella enterica Type III Secretion System.

Virulence-associated type III secretion systems (T3SS) are utilized by Gram negative bacterial pathogens for injection of effector proteins into eukaryotic host cells. The transmembrane export apparatus at the core of T3SS is composed of a unique helical complex of the hydrophobic proteins SctR, SctS, SctT, and SctU. These components comprise a number of highly conserved charged residues within their hydrophobic domains. The structure of the closed state of the core complex SctR5S4T1 revealed that several of these residues form inter- and intramolecular salt bridges, some of which have to be broken for pore opening. Mutagenesis of individual residues was shown to compromise assembly or secretion of both, the virulence-associated and the related flagellar T3SS. However, the exact role of these conserved charged residues in the assembly and function of T3SS remains elusive. Here we performed an in-depth mutagenesis analysis of these residues in the T3SS of Salmonella Typhimurium, coupled to blue native PAGE, in vivo photocrosslinking and luciferase-based secretion assays. Our data show that these conserved salt bridges are not critical for assembly of the respective protein but rather facilitate the incorporation of the following subunit into the assembling complex. Our data also indicate that these conserved charged residues are critical for type III-dependent secretion and reveal a functional link between SctSE44 and SctTR204 and the cytoplasmic domain of SctU in gating the T3SS injectisome. Overall, our analysis provides an unprecedented insight into the delicate requirements for the assembly and function of the machinery at the core of T3SS.

Substrate-engaged type III secretion system structures reveal gating mechanism for unfolded protein translocation.

Many bacterial pathogens rely on virulent type III secretion systems (T3SSs) or injectisomes to translocate effector proteins in order to establish infection. The central component of the injectisome is the needle complex which assembles a continuous conduit crossing the bacterial envelope and the host cell membrane to mediate effector protein translocation. However, the molecular principles underlying type III secretion remain elusive. Here, we report a structure of an active Salmonella enterica serovar Typhimurium needle complex engaged with the effector protein SptP in two functional states, revealing the complete 800Å-long secretion conduit and unraveling the critical role of the export apparatus (EA) subcomplex in type III secretion. Unfolded substrates enter the EA through a hydrophilic constriction formed by SpaQ proteins, which enables side chain-independent substrate transport. Above, a methionine gasket formed by SpaP proteins functions as a gate that dilates to accommodate substrates while preventing leaky pore formation. Following gate penetration, a moveable SpaR loop first folds up to then support substrate transport. Together, these findings establish the molecular basis for substrate translocation through T3SSs and improve our understanding of bacterial pathogenicity and motility.

Structure of the cytoplasmic domain of SctV (SsaV) from the Salmonella SPI-2 injectisome and implications for a pH sensing mechanism.

Bacterial type III secretion systems assemble the axial structures of both injectisomes and flagella. Injectisome type III secretion systems subsequently secrete effector proteins through their hollow needle into a host, requiring co-ordination. In the Salmonella enterica serovar Typhimurium SPI-2 injectisome, this switch is triggered by sensing the neutral pH of the host cytoplasm. Central to specificity switching is a nonameric SctV protein with an N-terminal transmembrane domain and a toroidal C-terminal cytoplasmic domain. A 'gatekeeper' complex interacts with the SctV cytoplasmic domain in a pH dependent manner, facilitating translocon secretion while repressing effector secretion through a poorly understood mechanism. To better understand the role of SctV in SPI-2 translocon-effector specificity switching, we purified full-length SctV and determined its toroidal cytoplasmic region's structure using cryo-EM. Structural comparisons and molecular dynamics simulations revealed that the cytoplasmic torus is stabilized by its core subdomain 3, about which subdomains 2 and 4 hinge, varying the flexible outside cleft implicated in gatekeeper and substrate binding. In light of patterns of surface conservation, deprotonation, and structural motion, the location of previously identified critical residues suggest that gatekeeper binds a cleft buried between neighboring subdomain 4s. Simulations suggest that a local pH change from 5 to 7.2 stabilizes the subdomain 3 hinge and narrows the central aperture of the nonameric torus. Our results are consistent with a model of local pH sensing at SctV, where pH-dependent dynamics of SctV cytoplasmic domain affect binding of gatekeeper complex.

Type 3 secretion system of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen, mainly affecting severe patients, such as those in intensive care units (ICUs). High levels of antibiotic resistance and a long battery of virulence factors characterise this pathogen. Among virulence factors, the T3SS (Type 3 Secretion Systems) are especially relevant, being one of the most important virulence factors in P. aeruginosa. T3SS are a complex "molecular syringe" able to inject different effectors in host cells, subverting cell machinery influencing immune responses, and increasing bacterial survival rates. While T3SS have been largely studied and the molecular structure and main effector functions have been established, a series of questions and further points remain to be clarified or established. The key role of T3SS in P. aeruginosa virulence has resulted in the search for T3SS-targeting molecules able to impair their functions and subsequently improve patient outcomes. This review aims to summarise the most relevant features of the P. aeruginosa T3SS.

Structure of the Yersinia injectisome in intracellular host cell phagosomes revealed by cryo FIB electron tomography.

Many pathogenic bacteria use the type III secretion system (T3SS), or injectisome, to secrete toxins into host cells. These protruding systems are primary targets for drug and vaccine development. Upon contact between injectisomes and host membranes, toxin secretion is triggered. How this works structurally and functionally is yet unknown. Using cryo-focused ion beam milling and cryo-electron tomography, we visualized injectisomes of Yersinia enterocolitica inside the phagosomes of infected human myeloid cells in a close-to-native state. We observed that a minimum needle length is required for injectisomes to contact the host membrane and bending of host membranes by some injectisomes that contact the host. Through subtomogram averaging, the structure of the entire injectisome was determined, which revealed structural differences in the cytosolic sorting platform compared to other bacteria. These findings contribute to understanding how injectisomes secrete toxins into host cells and provides the indispensable native context. The application of these cryo-electron microscopy techniques paves the way for the study of the 3D structure of infection-relevant protein complexes in host-pathogen interactions.

Cryo-EM structure of the EspA filament from enteropathogenic Escherichia coli: Revealing the mechanism of effector translocation in the T3SS.

The type III secretion system (T3SS) is a virulence mechanism employed by Gram-negative pathogens. The T3SS forms a proteinaceous channel that projects a needle into the extracellular medium where it interacts with the host cell to deliver virulence factors. Enteropathogenic Escherichia coli (EPEC) is unique in adopting a needle extension to the T3SS-a filament formed by EspA-which is absolutely required for efficient colonization of the gut. Here, we describe the cryoelectron microscopy structure of native EspA filaments from EPEC at 3.6-Å resolution. Within the filament, positively charged residues adjacent to a hydrophobic groove line the lumen of the filament in a spiral manner, suggesting a mechanism of substrate translocation mediated via electrostatics. Using structure-guided mutagenesis, in vivo studies corroborate the role of these residues in secretion and translocation function. The high-resolution structure of the EspA filament could aid in structure-guided drug design of antivirulence therapeutics.

Characterization of the Pilotin-Secretin Complex from the Salmonella enterica Type III Secretion System Using Hybrid Structural Methods.

The type III secretion system (T3SS) is a multi-membrane-spanning protein channel used by Gram-negative pathogenic bacteria to secrete effectors directly into the host cell cytoplasm. In the many species reliant on the T3SS for pathogenicity, proper assembly of the outer membrane secretin pore depends on a diverse family of lipoproteins called pilotins. We present structural and biochemical data on the Salmonella enterica pilotin InvH and the S domain of its cognate secretin InvG. Characterization of InvH by X-ray crystallography revealed a dimerized, α-helical pilotin. Size-exclusion-coupled multi-angle light scattering and small-angle X-ray scattering provide supporting evidence for the formation of an InvH homodimer in solution. Structures of the InvH-InvG heterodimeric complex determined by X-ray crystallography and NMR spectroscopy indicate a predominantly hydrophobic interface. Knowledge of the interaction between InvH and InvG brings us closer to understanding the mechanisms by which pilotins assemble the secretin pore.

CryoEM of bacterial secretion systems: A primer for microbiologists.

"CryoEM" has come of age, enabling considerable structural insights into many facets of molecular biology. Here, we present a primer for microbiologists to understand the capabilities and limitations of two complementary cryoEM techniques for studying bacterial secretion systems. The first, single particle analysis, determines the structures of purified protein complexes to resolutions sufficient for molecular modeling, while the second, electron cryotomography and subtomogram averaging, tends to determine more modest resolution structures of protein complexes in intact cells. We illustrate these abilities with examples of insights provided into how secretion systems work by cryoEM, with a focus on type III secretion systems.

The PopN Gate-keeper Complex Acts on the ATPase PscN to Regulate the T3SS Secretion Switch from Early to Middle Substrates in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic bacterium of which the main virulence factor is the Type III Secretion System. The ATPase of this machinery, PscN (SctN), is thought to be localized at the base of the secretion apparatus and to participate in the recognition, chaperone dissociation and unfolding of exported T3SS proteins. In this work, a protein-protein interaction ELISA revealed the interaction of PscN with a wide range of exported T3SS proteins including the needle, translocator, gate-keeper and effector. These interactions were further confirmed by Microscale Thermophoresis that also indicated a preferential interaction of PscN with secreted proteins or protein-chaperone complex rather than with chaperones alone, in line with the release of the chaperones in the bacterial cytoplasm after the dissociation from their exported proteins. Moreover, we suggest a new role of the gate-keeper complex and the ATPase in the regulation of early substrates recognition by the T3SS. This finding sheds a new light on the mechanism of secretion switching from early to middle substrates in P. aeruginosa.