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Type III protein secretion is widespread in Gram-negative pathogens. It comprises the injectisome with a surface-exposed needle and an inner membrane translocase. The translocase contains the SctRSTU export channel enveloped by the export gate subunit SctV that binds chaperone/exported clients and forms a putative ante-chamber. We probed the assembly, function, structure and dynamics of SctV from enteropathogenic E. coli (EPEC). In both EPEC and E. coli lab strains, SctV forms peripheral oligomeric clusters that are detergent-extracted as homo-nonamers. Membrane-embedded SctV 9 is necessary and sufficient to act as a receptor for different chaperone/exported protein pairs with distinct C-domain binding sites that are essential for secretion. Negative staining electron microscopy revealed that peptidisc-reconstituted His-SctV 9 forms a tripartite particle of ∼22 nm with a N-terminal domain connected by a short linker to a C-domain ring structure with a ∼5 nm-wide inner opening. The isolated C-domain ring was resolved with cryo-EM at 3.1 Å and structurally compared to other SctV homologues. Its four sub-domains undergo a three-stage "pinching" motion. Hydrogen-deuterium exchange mass spectrometry revealed this to involve dynamic and rigid hinges and a hyper-flexible sub-domain that flips out of the ring periphery and binds chaperones on and between adjacent protomers. These motions are coincident with local conformational changes at the pore surface and ring entry mouth that may also be modulated by the ATPase inner stalk. We propose that the intrinsic dynamics of the SctV protomer are modulated by chaperones and the ATPase and could affect allosterically the other subunits of the nonameric ring during secretion., Competing Interests: Competing interests The authors declare they have no competing financial interests or other conflicts of interest, (Crown Copyright © 2021. Published by Elsevier Ltd. All rights reserved.)

The increasing problem of bacterial resistance to antibiotics underscores the urgent need for new antibacterials. Protein export pathways are attractive potential targets. The Sec pathway is essential for bacterial viability and includes components that are absent from eukaryotes. Here, we used a new high-throughput in vivo screen based on the secretion and activity of alkaline phosphatase (PhoA), a Sec-dependent secreted enzyme that becomes active in the periplasm. The assay was optimized for a luminescence-based substrate and was used to screen a ~240K small molecule compound library. After hit confirmation and analoging, 14 HTS secretion inhibitors (HSI), belonging to eight structural classes, were identified with IC 50 < 60 µM. The inhibitors were evaluated as antibacterials against 19 Gram-negative and Gram-positive bacterial species (including those from the WHO's top pathogens list). Seven of them-HSI#6, 9; HSI#1, 5, 10; and HSI#12, 14-representing three structural families, were bacteriocidal. HSI#6 was the most potent hit against 13 species of both Gram-negative and Gram-positive bacteria with IC 50 of 0.4 to 8.7 μM. HSI#1, 5, 9 and 10 inhibited the viability of Gram-positive bacteria with IC 50 ~6.9-77.8 μM. HSI#9, 12, and 14 inhibited the viability of E. coli strains with IC 50 < 65 μM. Moreover, HSI#1, 5 and 10 inhibited the viability of an E. coli strain missing TolC to improve permeability with IC 50 4 to 14 μM, indicating their inability to penetrate the outer membrane. The antimicrobial activity was not related to the inhibition of the SecA component of the translocase in vitro, and hence, HSI molecules may target new unknown components that directly or indirectly affect protein secretion. The results provided proof of the principle that the new broad HTS approach can yield attractive nanomolar inhibitors that have potential as new starting compounds for optimization to derive potential antibiotics.

The type 3 secretion system is essential for pathogenesis of several human and animal Gram-negative bacterial pathogens. The T3SS comprises a transmembrane injectisome, providing a conduit from the bacterial cytoplasm to the host cell cytoplasm for the direct delivery of effectors (including toxins). Functional studies of T3SS commonly monitor the extracellular secretion of proteins by SDS-PAGE and western blot analysis, which are slow and semi-quantitative in nature. Here, we describe an enzymatic reporter-based quantitative and rapid in vivo assay for T3SS secretion studies in enteropathogenic E. coli (EPEC). The assay monitors the secretion of the fusion protein SctA-PhoA through the injectisome based on a colorimetric assay that quantifies the activity of alkaline phosphatase. We validated the usage of this reporter system by following the secretion in the absence of various injectisome components, including domains of the gatekeeper essential for T3SS function. This platform can now be used for the isolation of mutations, functional analysis and anti-virulence compound screening.

Cellular proteomes are distributed in multiple compartments: on DNA, ribosomes, on and inside membranes, or they become secreted. Structural properties that allow polypeptides to occupy subcellular niches, particularly to after crossing membranes, remain unclear. We compared intrinsic and extrinsic features in cytoplasmic and secreted polypeptides of the Escherichia coli K-12 proteome. Structural features between the cytoplasmome and secretome are sharply distinct, such that a signal peptide-agnostic machine learning tool distinguishes cytoplasmic from secreted proteins with 95.5% success. Cytoplasmic polypeptides are enriched in aliphatic, aromatic, charged and hydrophobic residues, unique folds and higher early folding propensities. Secretory polypeptides are enriched in polar/small amino acids, β folds, have higher backbone dynamics, higher disorder and contact order and are more often intrinsically disordered. These non-random distributions and experimental evidence imply that evolutionary pressure selected enhanced secretome flexibility, slow folding and looser structures, placing the secretome in a distinct protein class. These adaptations protect the secretome from premature folding during its cytoplasmic transit, optimize its lipid bilayer crossing and allowed it to acquire cell envelope specific chemistries. The latter may favor promiscuous multi-ligand binding, sensing of stress and cell envelope structure changes. In conclusion, enhanced flexibility, slow folding, looser structures and unique folds differentiate the secretome from the cytoplasmome. These findings have wide implications on the structural diversity and evolution of modern proteomes and the protein folding problem.

More than a third of all bacterial polypeptides, comprising the 'exportome', are transported to extracytoplasmic locations. Most of the exportome is targeted and inserts into ('membranome') or crosses ('secretome') the plasma membrane. The membranome and secretome use distinct targeting signals and factors, and driving forces, but both use the ubiquitous and essential Sec translocase and its SecYEG protein-conducting channel. Membranome export is co-translational and uses highly hydrophobic N-terminal signal anchor sequences recognized by the signal recognition particle on the ribosome, that also targets C-tail anchor sequences. Translating ribosomes drive movement of these polypeptides through the lateral gate of SecY into the inner membrane. On the other hand, secretome export is post-translational and carries two types of targeting signals: cleavable N-terminal signal peptides and multiple short hydrophobic targeting signals in their mature domains. Secretome proteins remain translocation competent due to occupying loosely folded to completely non-folded states during targeting. This is accomplished mainly by the intrinsic properties of mature domains and assisted by signal peptides and/or chaperones. Secretome proteins bind to the dimeric SecA subunit of the translocase. SecA converts from a dimeric preprotein receptor to a monomeric ATPase motor and drives vectorial crossing of chains through SecY aided by the proton motive force. Signal peptides are removed by signal peptidases and translocated chains fold or follow subsequent trafficking.

Investigations of cellular responses involved in injury and repair processes have generated valuable information contributing to the advancement of wound healing and treatments. Intra- and extracellular regulators of healing mechanisms, such as cytokines, signaling proteins, and growth factors, have been described to possess significant roles in facilitating optimal recovery. This study explored a collection of 30 spatiotemporal responses comprised of cytokines (IL-1α, IL-1ß, IL-2, IL-6, TNF-α, MIP-1α), intracellular proteins (Akt, c-Jun, CREB, ERK1/2, JNK, MEK1, p38, p53, p90RSK), phosphorylated proteins (p-Akt, p-c-Jun, p-CREB, p-ERK1/2, p-GSK-3α/ß, p-HSP27, p-IκBα, p-JNK, p-MEK1, p-p38, p-p70S6K, p-p90RSK, p-STAT2, p-STAT3), and a protease (Caspase-3), measured in skeletal muscle tissue following a traumatic injury (rodent Gustilo IIIB fracture). To optimize the analysis of context-specific data sets, a network centrality parameter approach was used to assess the impact of each response in relation to all other measured responses. This approach identified proteins that were substantially amplified and potentially central in the wound healing network by evaluation of their corresponding centrality parameter, radiality. Network analysis allowed us to distinguish the progression of healing that occurred at certain time points and regions of injury. Notably, new tissue formation was proposed to occur by 168 h post-injury in severely injured tissue, while tissue 1-cm away from the site of injury that experienced relatively minor injury appeared to exhibit signs of new tissue formation as early as 24 h post-injury. In particular, hallmarks of inflammation, cytokines IL-1ß, IL-6, and IL-2, appear to have a pronounced impact at earlier time points (0-24 h post-injury), while intracellular proteins involved in cell proliferation, differentiation, or proteolysis (c-Jun, CREB, JNK, p38, p-c-Jun; p-MEK1, p-p38, p-STAT3) are more significant at later times (24-168 h). Overall, this study demonstrates the feasibility of a network analysis approach to extract significant information and also offers a spatiotemporal visualization of the intra- and extracellular signaling responses that regulate healing mechanisms., (Published by Elsevier Ltd.)

Type III secretion (T3S), a protein export pathway common to Gram-negative pathogens, comprises a trans-envelope syringe, the injectisome, with a cytoplasm-facing translocase channel. Exported substrates are chaperone-delivered to the translocase, EscV in enteropathogenic Escherichia coli, and cross it in strict hierarchical manner, for example, first "translocators", then "effectors". We dissected T3S substrate targeting and hierarchical switching by reconstituting them in vitro using inverted inner membrane vesicles. EscV recruits and conformationally activates the tightly membrane-associated pseudo-effector SepL and its chaperone SepD. This renders SepL a high-affinity receptor for translocator/chaperone pairs, recognizing specific chaperone signals. In a second, SepD-coupled step, translocators docked on SepL become secreted. During translocator secretion, SepL/SepD suppress effector/chaperone binding to EscV and prevent premature effector secretion. Disengagement of the SepL/SepD switch directs EscV to dedicated effector export. These findings advance molecular understanding of T3S and reveal a novel mechanism for hierarchical trafficking regulation in protein secretion channels., (© 2017 The Authors.)

Molecular mechanisms of wound healing have been extensively characterized, providing a better understanding of the processes involved in wound repair and offering advances in treatment methods. Both spatial and temporal investigations of injury biomarkers have helped to pinpoint significant time points and locations during the recovery process, which may be vital in managing the injury and making the appropriate diagnosis. This study addresses spatial and temporal differences of phosphoproteins found in skeletal muscle tissue following a traumatic femur fracture, which were further compared to co-localized cytokine responses. In particular, several proteins (Akt, ERK, c-Jun, CREB, JNK, MEK1, and p38) and post-translational phosphorylations (p-Akt, p-c-Jun, p-CREB, p-ERK1/2, p-MEK1, p-p38, p-GSK3α/β, p-HSP27, p-p70S6K, and p-STAT3) associated with inflammation, new tissue formation, and remodeling were found to exhibit significant spatial and temporal differences in response to the traumatic injury. Quadratic discriminant analysis of all measured responses, including cytokine concentrations from previously published findings, was used to classify temporal and spatial observations at high predictive rates, further confirming that distinct spatiotemporal distributions for total protein, phosphorylation signaling, and cytokine (IL-1α, IL-1ß, IL2, IL6, TNF-α, and MIP-1α) responses exist. Finally, phosphoprotein measurements were found to be significantly correlated to cytokine concentrations, suggesting coordinated intracellular and extracellular activity during crucial periods of repair. This study represents a first attempt to monitor and assess integrated changes in extracellular and intracellular signaling in response to a traumatic injury in muscle tissues, which may provide a framework for future research to improve both our understanding of wounds and their treatment options., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The Type III secretion system (T3SS) is a protein export pathway that is widespread in Gram-negative bacteria and delivers effector proteins directly into eukaryotic cells. At its core lie the injectisome (a sophisticated transmembrane secretion apparatus) and a complex network of specialized chaperones that target secretory proteins to the antechamber of the injectisome. The assembly of the system, and the subsequent secretion of proteins through it, undergo fine-tuned, hierarchical regulation. Here, we present the current understanding of the injectisome assembly process, secretion hierarchy, and the role of chaperones. We discuss these events in light of available structural and biochemical dissection and propose future directions essential to revealing mechanistic insight into this fascinating nanomachine., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Temporal changes in cytokine concentrations following traumatic injuries have been extensively studied. Less is known regarding spatial differences in cytokine concentrations following traumatic injury. The primary aim of this study was to determine the spatial relationship between cytokines and the zone of injury (ZOI). Muscle and vessel tissues obtained from rats subjected to an open femoral fracture were analyzed to determine if spatial cytokine gradients exist that could potentially be used as biomarkers of the ZOI. Samples were collected at 4 time points following fracture from 3 distinct locations: at the fracture site, 1-cm away from the fracture, and from the opposite leg. The concentrations of IL-6, IL-1α, IL-1ß, IL-2, GM-CSF, TNF-α, and MIP-1α were quantified in each sample. Temporally and spatially regulated variations in cytokine concentrations were found. IL-6 showed the most promise as a ZOI biomarker with statistically different spatial concentrations that were inversely proportional to the distance from the fracture in both tissues. IL-1ß and IL-2 also showed spatial differences in concentration in both tissues, while GM-CSF, MIP-1α, and TNF-α showed spatial differences in vessel samples. These results demonstrate that spatial cytokine gradients exist following traumatic injury, representing potential biomarkers that may be used to define the ZOI., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The response of tissue to trauma is difficult to define. The zone of injury is an area surrounding a wound that, though traumatized, may not appear nonviable at initial debridement. Because of this, a policy of repeated debridements has been followed to monitor tissues for viability before final tissue coverage. Appreciation of the zone of injury has led to a controversy in the literature about how to define and approach the management of traumatic injuries requiring free-tissue coverage. This review examines the current literature with regard to the definition of the zone of injury, and seeks to establish a consensus statement about the application of free flaps to traumatized wounds. We have investigated the literature supporting the use of free flaps relying on proximally or distally based recipient vessels. Critical appraisal of this literature includes study design, determination of the power of the study, subject classification, inclusion and exclusion criteria, follow-up, and outcomes (free flap success). There has been little attempt in the literature to fully and objectively define the zone of injury. All studies to date have been observational alone. Although it would be impossible to rid a definition of the zone of injury of subjectivity entirely, a more objective, reproducible definition is vitally needed. Without a clear definition of what the zone of injury is, there can only be anecdotal, technique reports of the placement of free flap anastomoses. In this time of rising costs and lower reimbursements, this is one area that could provide vital information to improve care for patients, lessen costs, and further medical knowledge.

Background: The use of free muscle or myocutaneous flaps is well established as a means for reconstructing tissue defects over nearly any part of the body. This free tissue transfer is based on the availability of a robust blood supply in the recipient wound. The reliability of native blood supplies within the zone of injury is suspect even in the best of conditions. There are several causes of flap failure however; one of the most common is vascular compromise. Though refinements in the technique of vascular anastomosis have lessened the risk, it is still significant, especially when dealing with an area of injury at the recipient site. The mechanisms of scar formation and healing that occur within the zone of injury often limit the potential for viable anastomotic targets. This often leads to delay in wound coverage and exposes the patient to all of the risks associated with having an open wound. Free flap failure can be devastating for patients, even leading to the loss of limbs. Therefore, it is vital that all measures possible to ensure the survival of the transferred tissue be employed., Methods: We will examine interpositional vein grafts versus local anastomoses in the context of free tissue transfer for wound coverage in the traumatized and reconstructed patient. We will retrospectively review the case histories of free tissue transfers preformed at West Virginia University Hospital over a 5-year period (2001-2006). We will examine data including the demographics of our patient population, type and locations of the free flaps, length of stay in the hospital, time interval between injury and repair, and the Success and failure rate., Results: The overall success rate was 38 out of 45 or 84%. Success was defined as a healthy flap and preservation of the limb and/or successful wound coverage at time of discharge. Flap success was present in 18 of 23 (78%) of the vein interposition grafts, and flap failure occurred in 5 (22%). In patients with local anastomoses, 20 out of 22 flaps survived (91%) with 2 failures (9%)., Goals: We will demonstrate that the use of interpositional vein grafts for free tissue transfer is a viable option in the wound coverage. We will demonstrate that this is especially 'true in the patient population that exists in rural America.