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 IC50 < 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 IC50 of 0.4 to 8.7 μM. HSI#1, 5, 9 and 10 inhibited the viability of Gram-positive bacteria with IC50 ~6.9–77.8 μM. HSI#9, 12, and 14 inhibited the viability of E. coli strains with IC50 < 65 μM. Moreover, HSI#1, 5 and 10 inhibited the viability of an E. coli strain missing TolC to improve permeability with IC50 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 favour 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.
Abstract 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 pore surface and ring entry mouth local conformational changes that are also modulated by the ATPase inner stalk. We propose a model 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.
This brief review was inspired by discussions relating to the IIIrd. International C1 Workshop (this volume) and the realization that certain functional properties of the C1q molecule are limited exclusively to the A-chain. The collagen-like region of the A-chain contains a major binding site for non-immunoglobulin substances, which include C-reactive protein, serum amyloid P, LPS and DNA. This binding site is immediately adjacent to, and partially overlapping with, an arthritis-modulating epitope common to the C1q A-chain and various types of collagen, including cartilage type II collagen. At the N-terminal end of the C1q A-chain is a leader peptide sequence that anchors the intact C1q molecule firmly in the membrane of macrophages, the C1q molecule can thus be classified as a type II membrane protein, functioning as an additional receptor for molecules known to react with C1q in fluid phase such as the Fc region of IgG, LPS and polyanionic molecules (e.g. chondroitin sulphate, heparin, dextran sulphate etc.). The various domains within the A-chain, and their respective functions (or potential functions), are presented and discussed in the context of the intact C1 molecule and with regard to any wider functional relevance.
The Fc-recognizing, collagen-like C1q molecule, a subcomponent of the first component of complement, C1, is present on the cell surface of guinea pig peritoneal macrophages and human peritoneal and monocyte-derived macrophages. On closer examination, C1q appears to be a membrane protein (membrane C1q) of macrophages since it is i) anchored into the membrane throughout the biosynthetic pathway, ii) tightly and irreversibly bound to the cell surface and iii) only liberated if the intact membrane structure is disrupted by a detergent or repeated freeze/thawing. Additionally, the amino acid sequence of the A chain of human C1q displays properties that are characteristic for integral type II membrane proteins. The membrane C1q of guinea pig macrophages has a "lighter" B chain than serum C1q. Under physiological conditions in culture guinea pig macrophages release membrane C1q thereby converting it into the serum form. Moreover, membrane C1q appears to be involved in various cellular events such as binding of Fc, polyanions, lipid A and gram-negative bacteria to macrophages.
Abstract The increasing problem of bacterial resistance to antibiotics underscores the urgent need for new antibacterials. The Sec preprotein export pathway is an attractive potential alternative target. It 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, fourteen HTS secretion inhibitors (HSI), belonging to 8 structural classes, were identified (IC 50 <60 μM). The inhibitors were also evaluated as antibacterials against 19 Gram − and Gram + bacterial species (including those from the WHO top pathogens list). Seven of them, HSI#6, 9; HSI#1, 5, 10 and HSI#12, 14 representing three structural families were microbicidals. HSI#6 was the most potent (IC 50 of 0.4-8.7 μM), against 13 species of both Gram − and Gram + bacteria. HSI#1, 5, 9 and 10 inhibited viability of Gram + bacteria with IC 50 ~6.9-77.8 μM. HSI#9, 12 and 14 inhibited viability of E. coli strains with IC 50 <65 μM. Moreover, HSI#1, 5 and 10 inhibited viability of an E. coli strain missing TolC to improve permeability with IC 50 4-14 μM, indicating their inability to penetrate the outer membrane. In vitro assays revealed that antimicrobial activity was not related to inhibition of the SecA component of the translocase and hence HSI molecules may target new unknown components that affect secretion. The results provide proof of principle for our approach, and new starting compounds for optimization.
