Interaction between Yersinia pestis Ail Outer Membrane Protein and the C-Terminal Domain of Human Vitronectin.

Yersinia pestis, the causative agent of plague, is capable of evading the human immune system response by recruiting the plasma circulating vitronectin proteins, which act as a shield and avoid its lysis. Vitronectin recruitment is mediated by its interaction with the bacterial transmembrane protein Ail, protruding from the Y. pestis outer membrane. By using all-atom long-scale molecular dynamic simulations of Ail embedded in a realistic model of the bacterial membrane, we have shown that vitronectin forms a stable complex, mediated by interactions between the disordered moieties of the two proteins. The main amino acids driving the complexation have also been evidenced, thus favoring the possible rational design of specific peptides which, by inhibiting vitronectin recruitment, could act as original antibacterial agents.

Application of the surface engineered recombinant Escherichia coli to the industrial battery waste solution for lithium recovery.

Escherichia coli were engineered to selectively adsorb and recover lithium from the environment by employing a bacterial cell surface display strategy. Lithium binding peptide (LBP1) was integrated into the Escherichia coli membrane protein OmpC. The effect of environmental conditions on the adsorption of lithium by a recombinant strain was evaluated, and lithium particles on the cellular surface were analyzed by FE-SEM and XRD. To elevate the lithium adsorption, dimeric, trimeric, and tetrameric repeats of the LBP1 peptide were constructed and displayed on the surface of E. coli. The constructed recombinant E. coli displaying the LBP1 trimer was applied to real industrial lithium battery wastewater to recover lithium.

Members of the paralogous gene family 12 from the Lyme disease agent Borrelia burgdorferi are non-specific DNA-binding proteins.

Lyme disease is the most prevalent vector-borne infectious disease in Europe and the USA. Borrelia burgdorferi, as the causative agent of Lyme disease, is transmitted to the mammalian host during the tick blood meal. To adapt to the different encountered environments, Borrelia has adjusted the expression pattern of various, mostly outer surface proteins. The function of most B. burgdorferi outer surface proteins remains unknown. We determined the crystal structure of a previously uncharacterized B. burgdorferi outer surface protein BBK01, known to belong to the paralogous gene family 12 (PFam12) as one of its five members. PFam12 members are shown to be upregulated as the tick starts its blood meal. Structural analysis of BBK01 revealed similarity to the coiled coil domain of structural maintenance of chromosomes (SMC) protein family members, while functional studies indicated that all PFam12 members are non-specific DNA-binding proteins. The residues involved in DNA binding were identified and probed by site-directed mutagenesis. The combination of SMC-like proteins being attached to the outer membrane and exposed to the environment or located in the periplasm, as observed in the case of PFam12 members, and displaying the ability to bind DNA, represents a unique feature previously not observed in bacteria.

A comprehensive computational study to explore promising natural bioactive compounds targeting glycosyltransferase MurG in Escherichia coli for potential drug development.

Peptidoglycan is a carbohydrate with a cross-linked structure that protects the cytoplasmic membrane of bacterial cells from damage. The mechanism of peptidoglycan biosynthesis involves the main synthesizing enzyme glycosyltransferase MurG, which is known as a potential target for antibiotic therapy. Many MurG inhibitors have been recognized as MurG targets, but high toxicity and drug-resistant Escherichia coli strains remain the most important problems for further development. In addition, the discovery of selective MurG inhibitors has been limited to the synthesis of peptidoglycan-mimicking compounds. The present study employed drug discovery, such as virtual screening using molecular docking, drug likeness ADMET proprieties predictions, and molecular dynamics (MD) simulation, to identify potential natural products (NPs) for Escherichia coli. We conducted a screening of 30,926 NPs from the NPASS database. Subsequently, 20 of these compounds successfully passed the potency, pharmacokinetic, ADMET screening assays, and their validation was further confirmed through molecular docking. The best three hits and the standard were chosen for further MD simulations up to 400 ns and energy calculations to investigate the stability of the NPs-MurG complexes. The analyses of MD simulations and total binding energies suggested the higher stability of NPC272174. The potential compounds can be further explored in vivo and in vitro for promising novel antibacterial drug discovery.

The TAM, a Translocation and Assembly Module for protein assembly and potential conduit for phospholipid transfer.

The assembly of ß-barrel proteins into the bacterial outer membrane is an essential process enabling the colonization of new environmental niches. The TAM was discovered as a module of the ß-barrel protein assembly machinery; it is a heterodimeric complex composed of an outer membrane protein (TamA) bound to an inner membrane protein (TamB). The TAM spans the periplasm, providing a scaffold through the peptidoglycan layer and catalyzing the translocation and assembly of ß-barrel proteins into the outer membrane. Recently, studies on another membrane protein (YhdP) have suggested that TamB might play a role in phospholipid transport to the outer membrane. Here we review and re-evaluate the literature covering the experimental studies on the TAM over the past decade, to reconcile what appear to be conflicting claims on the function of the TAM.

The inner membrane protein YhiM links copper and CpxAR envelope stress responses in uropathogenic E. coli.

Urinary tract infection (UTI) is a ubiquitous infectious condition, and uropathogenic Escherichia coli (UPEC) is the predominant causative agent of UTI. Copper (Cu) is implicated in innate immunity, including against UPEC. Cu is a trace element utilized as a co-factor, but excess Cu is toxic due to mismetalation of non-cognate proteins. E. coli precisely regulates Cu homeostasis via efflux systems. However, Cu import mechanisms into the bacterial cell are not clear. We hypothesized that Cu import defective mutants would exhibit increased resistance to Cu. This hypothesis was tested in a forward genetic screen with transposon (Tn5) insertion mutants in UPEC strain CFT073, and we identified 32 unique Cu-resistant mutants. Transposon and defined mutants lacking yhiM, which encodes a hypothetical inner membrane protein, were more resistant to Cu than parental strain. Loss of YhiM led to decreased cellular Cu content and increased expression of copA, encoding a Cu efflux pump. The CpxAR envelope stress response system was activated in the ΔyhiM mutant as indicated by increased expression of cpxP. Transcription of yhiM was regulated by CueR and CpxR, and the CpxAR system was essential for increased Cu resistance in the ΔyhiM mutant. Importantly, activation of CpxAR system in the ΔyhiM mutant was independent of NlpE, a known activator of this system. YhiM was required for optimal fitness of UPEC in a mouse model of UTI. Our findings demonstrate that YhiM is a critical mediator of Cu homeostasis and links bacterial adaptation to Cu stress with the CpxAR-dependent envelope stress response in UPEC.IMPORTANCEUPEC is a common bacterial infection. Bacterial pathogens are exposed to host-derived Cu during infection, including UTI. Here, we describe detection of genes involved in Cu homeostasis in UPEC. A UPEC mutant lacking YhiM, a membrane protein, exhibited dramatic increase in resistance to Cu. Our study demonstrates YhiM as a nexus between Cu stress and the CpxAR-dependent envelope stress response system. Importantly, our findings establish NlpE-independent activation of CpxAR system during Cu stress in UPEC. Collectively, YhiM emerges as a critical mediator of Cu homeostasis in UPEC and highlights the interlinked nature of bacterial adaptation to survival during Cu and envelope stress.

The Name Is Barrel, ß-Barrel.

ß-barrels are a class of membrane proteins made up of a cylindrical, anti-parallel ß-sheet with a hydrophobic exterior and a hydrophilic interior. The majority of proteins found in the outer membranes (OMs) of Gram-negative bacteria, mitochondria, and chloroplasts are ß-barrel outer membrane proteins (OMPs). ß-barrel OMPs have a diverse repertoire of functions, including nutrient transport, secretion, bacterial virulence, and enzymatic activity. Here, we discuss the broad functional classes of ß-barrel OMPs, how they are folded into the membrane, and the future of ß-barrel OMP research and its applications.

In Vitro Reconstruction of Bacterial ß-Barrel Membrane Protein Assembly Using E. coli Microsomal (Mid-Density) Membrane.

The in vitro reconstruction assay enables us to evaluate in detail the insertion and proper protein folding (together termed assembly) of ß-barrel membrane proteins. Here, we introduce an in vitro reconstitution experiments using isolated membrane fractions from Escherichia coli (E. coli). Membrane fractions isolated from E. coli cells and disrupted by sonication, which we have termed E. coli microsomal (mid-density) membrane (EMM), are ideal for biochemical experiments, as they can be harvested by high-speed centrifugation and do not require ultra-centrifugation. EMM pretreated with detergent can assemble externally supplemented ß-barrel membrane proteins via intact ß-barrel assembly machinery (BAM) complex retained in EMM. This method not only allows assembly analysis with inexpensive equipment but it also can be applied to drug screening using assembly as an indicator with high reproducibility. In this chapter, we introduce our method of evaluating assembled ß-barrel membrane proteins by demonstrating four representative ß-barrel membrane proteins: E. coli major porins OmpA and OmpF; enterohemorrhagic E. coli (EHEC) autotransporter EspP, and Haemophilus influenzae (H. influenzae) adhesin Hia.

Examining Protein Translocation by ß-Barrel Membrane Proteins Using Reconstituted Proteoliposomes.

ß-barrel membrane proteins populate the outer membrane of Gram-negative bacteria, mitochondria, and chloroplasts, playing significant roles in multiple key cellular pathways. Characterizing the functions of these membrane proteins in vivo is often challenging due to the complex protein network in the periplasm of Gram-negative bacteria (or intermembrane space in mitochondria and chloroplasts) and the presence of other outer membrane proteins. In vitro reconstitution into lipid-bilayer-like environments such as nanodiscs or proteoliposomes provides an excellent method for examining the specific function and mechanism of these membrane proteins in an isolated system. Here, we describe the methodologies employed to investigate Slam, a 14-stranded ß-barrel membrane protein also known as the type XI secretion system that is responsible for translocating proteins across the outer membrane of many bacterial species.

In Vivo Disulfide-Bond Crosslinking to Study ß-Barrel Membrane Protein Interactions, Dynamicity, and Folding Intermediates.

Membrane-embedded ß-barrels are the major building blocks of the Gram-negative outer membrane and are involved in antibiotic resistance, virulence, and the maintenance of bacterial cell physiology. The increased frequency of multidrug resistant Gram-negative infections warrants the sharing of accessible methods for the study of ß-barrels. One such method is "in vivo disulfide-bond crosslinking" which is a highly informative and cost-effective approach to study the structure, topology, dynamicity, and function of ß-barrels in situ. The approach can also be used to identify and finely map both stable or transient interactions between ß-barrels and other interacting proteins. In this chapter, I describe the conceptual basis of in vivo disulfide-bond crosslinking and the potential pitfalls in experimental design. I also provide a general protocol for high-efficiency in vivo disulfide-bond crosslinking and modified protocols as examples for how the method can be adapted to different scenarios.

Recombinant Expression and Overproduction of Transmembrane ß-Barrel Proteins.

Transmembrane ß-barrel proteins reside in the outer membrane of Gram-negative bacteria and are thus in direct contact with the environment. Because of that, they are involved in many key processes stretching from cellular survival to virulence. Hence, they are an attractive target for the development of novel antimicrobials, in addition to being of fundamental biological interest. To study this class of proteins, they are often required to be expressed in Escherichia coli. Recombinant expression of ß-barrel proteins can be achieved using two fundamentally different strategies. The first alternative uses a complete coding sequence that includes a signal peptide for targeting the protein to its native cellular location, the bacterial outer membrane. The second alternative omits the signal peptide in the gene, leading to mislocalization and aggregation of the protein in the bacterial cytoplasm. These aggregates, called inclusion bodies, can be solubilized and the protein can be folded into its native form in vitro. In this chapter, we present example protocols for both strategies and discuss their advantages and disadvantages.

Site-Specific Photocrosslinking to Investigate Toxin Delivery Mediated by the Bacterial ß-Barrel Assembly Machine.

Contact-dependent inhibition (CDI) is a mechanism of interbacterial competition in Gram-negative organisms that relies on a specific interaction between a CdiA protein on the surface of one cell and a ß-barrel protein on the surface of a neighboring cell. This interaction triggers the transport of a protein toxin into the neighboring cell where it exerts its lethal activity. Several classes of CdiA proteins that bind to different ß-barrel receptors have been identified, but the molecular mechanism by which they deliver their toxins across the outer membranes of their target cells is poorly understood. Here we describe the use of site-specific photocrosslinking to characterize the interaction between a CdiA protein and its receptor. We describe the method for an E. coli CdiA that utilizes BamA as its receptor. BamA's central role in assembling ß-barrel proteins in the outer membrane makes its role in CDI particularly intriguing; it suggests that these two different protein transport processes might share mechanistic features. Our in vitro photocrosslinking method is useful in elucidating early steps in the CDI mechanism, but it could be adapted to study later steps or to study other CdiA-receptor pairs.

Monitoring the Interaction of the Peptidoglycan with the Bacterial ß-Barrel Assembly Machinery.

Gram-negative bacteria coordinate the biosynthesis of their different cell envelope components. Growth of the outer membrane (OM) requires the essential ß-barrel assembly machine (BAM), which inserts OM proteins (OMPs) into the OM. The underlying peptidoglycan (PG) sacculus grows by the insertion of nascent glycan chains. We have previously identified interactions between BAM and PG in E. coli and showed that these interactions coordinate OM biogenesis with PG growth. BAM responds to the maturation state of the PG, and this mechanism activates preferentially BAM complexes at sites of active PG synthesis. Here we present protocols to purify soluble Bam proteins and full-length BamABCDE, isolate PG and soluble PG fragments, and study BAM-PG interactions with the isolated components. We also describe the protocol to detect interactions between Bam proteins and PG in cells.

Analysis of Transmembrane ß-Barrel Proteins by Native and Semi-native Polyacrylamide Gel Electrophoresis.

Membrane-embedded ß-barrel proteins are important regulators of the outer membrane permeability barrier of Gram-negative bacteria. ß-barrels are highly structured domains formed by a series of antiparallel ß-strands. Each ß-strand is locked in position by hydrogen bonds between its polypeptide backbone and those of the two neighbouring strands in the barrel structure. Some transmembrane ß-barrel proteins form larger homo- or hetero-multimeric complexes that accomplish specific functions. In this chapter, we describe native and semi-native polyacrylamide gel electrophoresis (PAGE) methods to characterize the organization of transmembrane ß-barrel proteins. We illustrate blue native (BN)-PAGE as an analytical method to assess the formation of protein complexes. Furthermore, we describe a heat-modifiability assay via semi-native PAGE as a rapid method to investigate the folding of transmembrane ß-barrels.

Affinity Purification of Membrane ß-Barrel Proteins via Biotin-Tagged Peptidiscs.

ß-barrel membrane proteins play a crucial role in bacterial pathogenesis and antibiotic resistance, making them a prime focus for the development of new antibiotics and therapeutics. However, their inherent hydrophobic nature and limited presence pose challenges for their high-throughput characterization using conventional methods. In this context, we present a simple but efficacious approach using peptidisc, a membrane mimetic, to overcome the low abundance and hydrophobicity of these proteins. Our methodology, illustrated here using Escherichia coli (E. coli) as a model organism, covers the entire process from outer membrane fraction preparation to data analysis. This detailed protocol outlines the purification of a diverse collection of ß-barrel membrane proteins, rendering them water-soluble and readily amenable to mass spectrometry and downstream drug screening strategies.

Characterization of ß-Barrel Outer Membrane Proteins and Their Interactions with Chaperones by Chemical-Crosslinking Mass Spectrometry.

Chemical crosslinking-mass spectrometry (XL-MS) is an established tool that can be used to study the architecture and dynamics of proteins and protein assemblies. Here the application of XL-MS to study outer membrane proteins (OMPs) and their interactions with periplasmic chaperones is described, to inform on the molecular mechanisms underpinning OMP assembly. XL-MS data are especially powerful when used to complement high-resolution structural data, data from structural prediction or to drive molecular modeling of proteins and protein assemblies. The approach described here could be applied to the study of any protein assembly (including other membrane proteins).

Expression, Purification, and Cryo-EM Structural Analysis of an Outer Membrane Secretin Channel.

Secretin proteins form pores in the outer membranes of Gram-negative bacteria, and as such provide a means of transporting a wide variety of molecules out of or in to the cell. They are important components of several different bacterial secretion systems, surface filament assembly machineries, and virus assembly complexes. Despite accommodating a diverse assortment of molecules, including virulence factors, folded proteins, and whole viruses, the secretin family of proteins is highly conserved, particularly in their membrane-embedded ß-barrel domain. We describe here a protocol for the expression, purification and cryo-EM structural determination of the pIV secretin from the Ff family of filamentous bacteriophages.

Recent Advances in Modeling Membrane ß-Barrel Proteins Using Molecular Dynamics Simulations: From Their Lipid Environments to Their Assemblies.

Spurred by advances in AI-driven modeling and experimental methods, molecular dynamics simulations are now acting as a platform to integrate these different approaches. This combination of methods is especially useful to understand ß-barrel proteins from the molecular level, e.g., identifying specific interactions with lipids or small molecules, up to assemblies comprised of hundreds of proteins and thousands of lipids. In this minireview, we will discuss recent advances, mainly from the last 5 years, in modeling ß-barrel proteins and their assemblies. These approaches require specific kinds of modeling and potentially different model resolutions that we will first describe in Subheading 1. We will then focus on different aspects of ß-barrel protein modeling: how different types of molecules can diffuse through ß-barrel proteins (Subheading 2); how lipids can interact with these proteins (Subheading 3); how ß-barrel proteins can interact with membrane partners (Subheading 4) or periplasmic extensions and partners (Subheading 5) to form large assemblies.

Structural Modeling of T9SS Outer Membrane Proteins and Their Complexes.

The type 9 secretion system (T9SS) is a recently discovered machinery that both transports cargo proteins across the Gram-negative bacterial outer membrane and attaches them to lipopolysaccharides on the extracellular surface. Outer membrane proteins (OMPs) are key components of the T9SS and are involved in both steps. In this chapter, we describe a method for the in silico modeling of T9SS OMPs and their complexes, and model validation. This is useful when the production of recombinant OMPs is difficult, and these protocols can also be applied to OMP complexes outside of the T9SS.

Stress-Based Screening for Compounds That Inhibit ß-Barrel Outer Membrane Protein Assembly in Gram-Negative Bacteria.

Biogenesis of the outer membrane (OM) of Gram-negative bacteria involves two processes essential for growth, that is, the insertion of ß-barrel outer membrane proteins (OMPs) by the Bam complex and the assembly of the LPS-containing outer leaflet of the OM by the LptD/E complex from the Lpt pathway. These processes have only recently gained attention as targets for antimicrobial drugs. Our laboratory has developed a simple screening tool to identify compounds that target processes that disrupt the biogenesis of the cell envelope, among which the activity of the Bam complex. The tool is based on the observation that such a disruption triggers cell envelope stress response systems, such as the σE, Rcs, and Cpx responses. In essence, specific stress-responsive promoters are fused to a gene encoding a bright fluorescent protein to serve as a panel of easy-to-monitor stress reporter plasmids. Using these plasmids, compounds triggering these stress systems and, therefore, putatively disrupting the biogenesis of the cell envelope can be identified by the nature and kinetics of the induced stress responses. We describe here the use of the stress reporter plasmids in high-throughput phenotypic screening using multi-well plates.