Structural mechanism of bacteriophage lambda tail's interaction with the bacterial receptor.

Bacteriophage infection, a pivotal process in microbiology, initiates with the phage's tail recognizing and binding to the bacterial cell surface, which then mediates the injection of viral DNA. Although comprehensive studies on the interaction between bacteriophage lambda and its outer membrane receptor, LamB, have provided rich information about the system's biochemical properties, the precise molecular mechanism remains undetermined. This study revealed the high-resolution cryo-electron microscopy (cryo-EM) structures of the bacteriophage lambda tail complexed with its irreversible Shigella sonnei 3070 LamB receptor and the closed central tail fiber. These structures reveal the complex processes that trigger infection and demonstrate a substantial conformational change in the phage lambda tail tip upon LamB binding. Providing detailed structures of bacteriophage lambda infection initiation, this study contributes to the expanding knowledge of lambda-bacterial interaction, which holds significance in the fields of microbiology and therapeutic development.

YdbH and YnbE form an intermembrane bridge to maintain lipid homeostasis in the outer membrane of Escherichia coli.

The outer membrane (OM) of didermic gram-negative bacteria is essential for growth, maintenance of cellular integrity, and innate resistance to many antimicrobials. Its asymmetric lipid distribution, with phospholipids in the inner leaflet and lipopolysaccharides (LPS) in the outer leaflet, is required for these functions. Lpt proteins form a transenvelope bridge that transports newly synthesized LPS from the inner membrane (IM) to OM, but how the bulk of phospholipids are transported between these membranes is poorly understood. Recently, three members of the AsmA-like protein family, TamB, YhdP, and YdbH, were shown to be functionally redundant and were proposed to transport phospholipids between IM and OM in Escherichia coli. These proteins belong to the repeating ß-groove superfamily, which includes eukaryotic lipid-transfer proteins that mediate phospholipid transport between organelles at contact sites. Here, we show that the IM-anchored YdbH protein interacts with the OM lipoprotein YnbE to form a functional protein bridge between the IM and OM in E. coli. Based on AlphaFold-Multimer predictions, genetic data, and in vivo site-directed cross-linking, we propose that YnbE interacts with YdbH through ß-strand augmentation to extend the continuous hydrophobic ß-groove of YdbH that is thought to shield acyl chains of phospholipids as they travel through the aqueous intermembrane periplasmic compartment. Our data also suggest that the periplasmic protein YdbL prevents extensive amyloid-like multimerization of YnbE in cells. We, therefore, propose that YdbL has a chaperone-like function that prevents uncontrolled runaway multimerization of YnbE to ensure the proper formation of the YdbH-YnbE intermembrane bridge.

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.

Bridging DNA contacts allow Dps from E. coli to condense DNA.

The DNA-binding protein from starved cells (Dps) plays a crucial role in maintaining bacterial cell viability during periods of stress. Dps is a nucleoid-associated protein that interacts with DNA to create biomolecular condensates in live bacteria. Purified Dps protein can also rapidly form large complexes when combined with DNA in vitro. However, the mechanism that allows these complexes to nucleate on DNA remains unclear. Here, we examine how DNA topology influences the formation of Dps-DNA complexes. We find that DNA supercoils offer the most preferred template for the nucleation of condensed Dps structures. More generally, bridging contacts between different regions of DNA can facilitate the nucleation of condensed Dps structures. In contrast, Dps shows little affinity for stretched linear DNA before it is relaxed. Once DNA is condensed, Dps forms a stable complex that can form inter-strand contacts with nearby DNA, even without free Dps present in solution. Taken together, our results establish the important role played by bridging contacts between DNA strands in nucleating and stabilizing Dps complexes.

Neisseria gonorrhoeae scavenges host sialic acid for Siglec-mediated, complement-independent suppression of neutrophil activation.

Gonorrhea, caused by the bacterium Neisseria gonorrhoeae (Gc), is characterized by neutrophilic influx to infection sites. Gc has developed mechanisms to resist killing by neutrophils that include modifications to its surface lipooligosaccharide (LOS). One such LOS modification is sialylation: Gc sialylates its terminal LOS sugars with cytidine-5'-monophosphate-N-acetylneuraminic acid, which is scavenged from the host using LOS sialyltransferase (Lst) since Gc cannot make its sialic acid. Sialylation enables sensitive strains of Gc to resist complement-mediated killing in a serum-dependent manner. However, little is known about the contribution of sialylation to complement-independent, direct Gc-neutrophil interactions. In the absence of complement, we found sialylated Gc expressing opacity-associated (Opa) proteins decreased the oxidative burst and granule exocytosis from primary human neutrophils. In addition, sialylated Opa+ Gc survived better than vehicle treated or Δlst Gc when challenged with neutrophils. However, Gc sialylation did not significantly affect Opa-dependent association with or internalization of Gc by neutrophils. Previous studies have implicated sialic acid-binding immunoglobulin-type lectins (Siglecs) in modulating neutrophil interactions with sialylated Gc. Blocking neutrophil Siglecs with antibodies that bind to their extracellular domains eliminated the ability of sialylated Opa+ Gc to suppress the oxidative burst and resist neutrophil killing. These findings highlight a new role for sialylation in Gc evasion of human innate immunity, with implications for the development of vaccines and therapeutics for gonorrhea. IMPORTANCE: Neisseria gonorrhoeae, the bacterium that causes gonorrhea, is an urgent global health concern due to increasing infection rates, widespread antibiotic resistance, and its ability to thwart protective immune responses. The mechanisms by which Gc subverts protective immune responses remain poorly characterized. One way N. gonorrhoeae evades human immunity is by adding sialic acid that is scavenged from the host onto its lipooligosaccharide, using the sialyltransferase Lst. Here, we found that sialylation enhances N. gonorrhoeae survival from neutrophil assault and inhibits neutrophil activation, independently of the complement system. Our results implicate bacterial binding of sialic acid-binding lectins (Siglecs) on the neutrophil surface, which dampens neutrophil antimicrobial responses. This work identifies a new role for sialylation in protecting N. gonorrhoeae from cellular innate immunity, which can be targeted to enhance the human immune response in gonorrhea.

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.

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.

Exposure to imipenem at sub-minimum inhibitory concentration leads to altered expression of major outer membrane proteins in Acinetobacter baumannii.

AIMS: Acinetobacter baumannii is a nosocomial pathogen known to be multidrug-resistant (MDR), especially to drugs of the carbapenem class. Several factors contribute to resistance, including efflux pumps, ß-lactamases, alteration of target sites, and permeability defects. In addition, outer membrane proteins (OMPs), like porins are involved in the passage of antibiotics, and their alteration could lead to resistance development. This study aimed to explore the possible involvement of porins and OMPs in developing carbapenem resistance due to differential expression. METHODS AND RESULTS: The antibiotic-susceptible and MDR isolates of A. baumannii were first studied for differences in their transcriptional levels of OMP expression and OMP profiles. The antibiotic-susceptible isolates were further treated with imipenem, and it was found that the omp genes were differentially expressed. Six of the nine genes studied were upregulated at 1 h of exposure to imipenem. Their expression gradually decreased with time, further confirmed by their OMP profile and two-dimensional gel electrophoresis. CONCLUSIONS: This study could identify OMPs that were differentially expressed on exposure to imipenem. Hence, this study provides insights into the role of specific OMPs in antibiotic resistance in A. baumannii.

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.

Proteome analysis of outer membrane vesicles from Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease.

A specific strain of Vibrio parahaemolyticus (VpAHPND) causes acute hepatopancreatic necrosis disease (AHPND), leading to significant losses in shrimp aquaculture. Outer membrane vesicles (OMVs) are naturally secreted by Gram-negative bacteria, and their significant roles in host-pathogen interactions and pathogenicity have been recognized. In the present study, OMVs were isolated from VpAHPND by differential-ultracentrifugation and used for proteomics analysis. In the Nano-HPLC-MS/MS analysis, totally 645 proteins were determined, including virulence factors, immunogenic proteins, outer membrane protein, bacterial secretory proteins, ribosomal proteins, protease, and iron regulation proteins. Furthermore, GO and KEGG annotations indicated that proteins identified in VpAHPND-OMVs are involved in metabolism, regulation of multiple biological processes, genetic information processes, immunity and more. Meanwhile, toxin proteins PirAvp and PirBvp, associated with VpAHPND pathogenicity, were also identified in the proteome of VpAHPND-OMVs. Our objective is to identify the protein composition of OMVs released by VpAHPND, analyzing the potential for cytotoxicity and immunomodulatory activity of these granule hosts. This study is crucial for understanding the roles played by bacterial-derived vesicles in the disease process, given that these vesicles carry relevant activities inherent to the bacteria that produce them.

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.

Bacteriophage LHE83 targeting OmpA as a receptor exhibited synergism with spectinomycin against Escherichia coli.

Understanding the characteristics of bacteriophages is crucial for the optimization of phage therapy. In this study, the biological and genomic characteristics of coliphage LHE83 were determined and its synergistic effects with different types of antibiotics against E. coli E82 were investigated. Phage LHE83 displayed a contractile tail morphology and had a titer of 3.02 × 109 pfu/mL at an optimal MOI of 0.01. Meanwhile, phage LHE83 exhibited good physical and chemical factors tolerance. The 1-step growth analysis revealed a latent period of approx. 10 min with a burst size of 87 pfu/infected cell. Phage LHE83 belongs to the genus Dhakavirus. Its genome consists of 170,464 bp with a 40% GC content, and a total of 268 Open Reading Frames (ORF) were predicted with no detected virulent or resistant genes. ORF 213 was predicted to encode the receptor binding protein (RBP) and confirmed by the antibody-blocking assay. Furthermore, a phage-resistant strain E. coli E82R was generated by co-culturing phage LHE83 with E. coli E82. Genomic analysis revealed that OmpA served as the receptor for phage LHE83, which was further confirmed by phage adsorption assay using E. coli BL21ΔOmpA, E. coli BL21ΔOmpA: OmpA and E. coli BL21:OmpA strains. Additionally, a synergistic effect was observed between phage LHE83 and spectinomycin against the drug-resistant strain E. coli E82. These results provide a theoretical basis for understanding the interactions between phages, antibiotics, and host bacteria, which can assist in the clinical application of phages and antibiotics against drug-resistant bacteria.

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.