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Journal of Molecular Biology Nov 2023In protein evolution, diversification is generally driven by genetic duplication. The hallmarks of this mechanism are visible in the repeating topology of various...
In protein evolution, diversification is generally driven by genetic duplication. The hallmarks of this mechanism are visible in the repeating topology of various proteins. In outer membrane β-barrels, duplication is visible with β-hairpins as the repeating unit of the barrel. In contrast to the overall use of duplication in diversification, a computational study hypothesized evolutionary mechanisms other than hairpin duplications leading to increases in the number of strands in outer membrane β-barrels. Specifically, the topology of some 16- and 18-stranded β-barrels appear to have evolved through a loop to β-hairpin transition. Here we test this novel evolutionary mechanism by creating a chimeric protein from an 18-stranded β-barrel and an evolutionarily related 16-stranded β-barrel. The chimeric combination of the two was created by replacing loop L3 of the 16-stranded barrel with the sequentially matched transmembrane β-hairpin region of the 18-stranded barrel. We find the resulting chimeric protein is stable and has characteristics of increased strand number. This study provides the first experimental evidence supporting the evolution through a loop to β-hairpin transition.
Topics: Bacterial Outer Membrane Proteins; Directed Molecular Evolution; Porins; Protein Domains; Recombinant Fusion Proteins; Protein Folding; Protein Conformation, beta-Strand
PubMed: 37769963
DOI: 10.1016/j.jmb.2023.168292 -
Journal of Cell Science Oct 2023The malaria-causing parasite, Plasmodium falciparum completely remodels its host red blood cell (RBC) through the export of several hundred parasite proteins, including...
The malaria-causing parasite, Plasmodium falciparum completely remodels its host red blood cell (RBC) through the export of several hundred parasite proteins, including transmembrane proteins, across multiple membranes to the RBC. However, the process by which these exported membrane proteins are extracted from the parasite plasma membrane for export remains unknown. To address this question, we fused the exported membrane protein, skeleton binding protein 1 (SBP1), with TurboID, a rapid, efficient and promiscuous biotin ligase (SBP1TbID). Using time-resolved proximity biotinylation and label-free quantitative proteomics, we identified two groups of SBP1TbID interactors - early interactors (pre-export) and late interactors (post-export). Notably, two promising membrane-associated proteins were identified as pre-export interactors, one of which possesses a predicted translocon domain, that could facilitate the export of membrane proteins. Further investigation using conditional mutants of these candidate proteins showed that these proteins were essential for asexual growth and localize to the host-parasite interface during early stages of the intraerythrocytic cycle. These data suggest that they might play a role in ushering membrane proteins from the parasite plasma membrane for export to the host RBC.
Topics: Animals; Humans; Biotinylation; Erythrocytes; Malaria; Plasmodium falciparum; Porins; Protein Transport; Protozoan Proteins
PubMed: 37772444
DOI: 10.1242/jcs.260506 -
International Journal of Molecular... Feb 2023Pore-forming proteins (PFPs) play a central role in many biological processes related to infection, immunity, cancer, and neurodegeneration. A common feature of PFPs is... (Review)
Review
Pore-forming proteins (PFPs) play a central role in many biological processes related to infection, immunity, cancer, and neurodegeneration. A common feature of PFPs is their ability to form pores that disrupt the membrane permeability barrier and ion homeostasis and generally induce cell death. Some PFPs are part of the genetically encoded machinery of eukaryotic cells that are activated against infection by pathogens or in physiological programs to carry out regulated cell death. PFPs organize into supramolecular transmembrane complexes that perforate membranes through a multistep process involving membrane insertion, protein oligomerization, and finally pore formation. However, the exact mechanism of pore formation varies from PFP to PFP, resulting in different pore structures with different functionalities. Here, we review recent insights into the molecular mechanisms by which PFPs permeabilize membranes and recent methodological advances in their characterization in artificial and cellular membranes. In particular, we focus on single-molecule imaging techniques as powerful tools to unravel the molecular mechanistic details of pore assembly that are often obscured by ensemble measurements, and to determine pore structure and functionality. Uncovering the mechanistic elements of pore formation is critical for understanding the physiological role of PFPs and developing therapeutic approaches.
Topics: Single Molecule Imaging; Cell Membrane; Porins
PubMed: 36901959
DOI: 10.3390/ijms24054528 -
Emerging Microbes & Infections Dec 2024Carbapenem-resistant Enterobacterales (CRE), specifically those resistant to only ertapenem among carbapenems (ETP-mono-resistant), are increasingly reported, while the... (Randomized Controlled Trial)
Randomized Controlled Trial
Carbapenem-resistant Enterobacterales (CRE), specifically those resistant to only ertapenem among carbapenems (ETP-mono-resistant), are increasingly reported, while the optimal therapy options remain uncertain. To investigate the prevalence and characteristics of ETP-mono-resistant CRE, CRE strains were systematically collected from 102 hospitals across China between 2018 and 2021. A 1:1 randomized matching study was conducted with ETP-mono-resistant strains to meropenem- and/or imipenem-resistant (MEM/IPM-resistant) strains. Antimicrobial susceptibility testing, whole-genome sequencing, carbapenem-hydrolysing activity and the expression of carbapenemase genes were determined. In total, 18.8% of CRE strains were ETP-mono-resistant, with relatively low ertapenem MIC values. ETP-mono-resistant strains exhibited enhanced susceptibility to β-lactams, β-lactam/β-lactamase inhibitor combinations, levofloxacin, fosfomycin, amikacin and polymyxin than MEM/IPM-resistant strains ( < 0.05). Phylogenetic analysis revealed high genetic diversity among ETP-mono-resistant strains. Extended-spectrum β-lactamases (ESBLs) and/or AmpC, as well as porin mutations, were identified as potential major mechanisms mediating ETP-mono-resistance, while the presence of carbapenemases was found to be the key factor distinguishing the carbapenem-resistant phenotypes between the two groups ( < 0.001). Compared with the MEM/IPM-resistant group, limited carbapenemase-producing CRE (CP-CRE) strains in the ETP-mono-resistant group showed a significantly lower prevalence of ESBLs and porin mutations, along with reduced expression of carbapenemase. Remarkably, spot assays combined with modified carbapenem inactivation method indicated that ETP-mono-resistant CP-CRE isolates grew at meropenem concentrations eightfold above their corresponding MIC values, accompanied by rapidly enhanced carbapenem-hydrolysing ability. These findings illustrate that ETP-mono-resistant CRE strains are relatively prevalent and that caution should be exercised when using meropenem alone for treatment. The detection of carbapenemase should be prioritized.
Topics: Anti-Bacterial Agents; Bacterial Proteins; beta-Lactamases; Carbapenems; Ertapenem; Meropenem; Microbial Sensitivity Tests; Phylogeny; Porins; Prevalence; China
PubMed: 38517707
DOI: 10.1080/22221751.2024.2332658 -
Journal of the American Chemical Society Feb 2019The porinACj is an α-helical porin that spans the mycolic acid outer membrane of Gram-positive mycolate, Corynebacterium jeikeium. Here, we report that a...
The porinACj is an α-helical porin that spans the mycolic acid outer membrane of Gram-positive mycolate, Corynebacterium jeikeium. Here, we report that a 40-amino acid, synthetic peptide, pPorA corresponding to porin PorACj, inserts into the lipid bilayers and forms well-defined pores. By electrical recordings, we measured the single-channel properties that revealed the autonomous assembly of large conductance ion-selective synthetic pores. Further, we characterized the functional properties by blocking the peptide pores by cyclodextrins of different charge and symmetry. We deduced the subunit stoichiometry and putative structure of the pore by site-specific chemical modification in single-channel electrical recordings and gel electrophoresis. On the basis of these findings, we suggest that this is a large functional uniform transmembrane pore built entirely from short synthetic α-helical peptides. Accordingly, we propose a model demonstrating structural assembly of large α-helix-based peptide pores for understanding the action of antimicrobial peptides and for the design of pores with applications in biotechnology.
Topics: Amino Acid Sequence; Corynebacterium; Cyclodextrins; Cysteine; Lipid Bilayers; Models, Molecular; Peptides; Porins; Protein Binding; Protein Conformation, alpha-Helical; Protein Structure, Quaternary
PubMed: 30702873
DOI: 10.1021/jacs.8b09973 -
Cell Reports Jun 2023To kill bacteria, bacteriophages (phages) must first bind to a receptor, triggering the release of the phage DNA into the bacterial cell. Many bacteria secrete...
To kill bacteria, bacteriophages (phages) must first bind to a receptor, triggering the release of the phage DNA into the bacterial cell. Many bacteria secrete polysaccharides that had been thought to shield bacterial cells from phage attack. We use a comprehensive genetic screen to distinguish that the capsule is not a shield but is instead a primary receptor enabling phage predation. Screening of a transposon library to select phage-resistant Klebsiella shows that the first receptor-binding event docks to saccharide epitopes in the capsule. We discover a second step of receptor binding, dictated by specific epitopes in an outer membrane protein. This additional and necessary event precedes phage DNA release to establish a productive infection. That such discrete epitopes dictate two essential binding events for phages has profound implications for understanding the evolution of phage resistance and what dictates host range, two issues critically important to translating knowledge of phage biology into phage therapies.
Topics: Klebsiella pneumoniae; Bacteriophages; Porins; Polysaccharides
PubMed: 37224021
DOI: 10.1016/j.celrep.2023.112551 -
Microbial Pathogenesis Jan 2022A. baumannii is a multi-drug resistant pathogen with a relatively high mortality rate. To date, no vaccine has been approved against this bacterium. DcaP is a high...
A. baumannii is a multi-drug resistant pathogen with a relatively high mortality rate. To date, no vaccine has been approved against this bacterium. DcaP is a high abundance porin during infection that its structure has been recently determined, but no information about its immunogenic properties has been reported yet. So, in this study DcaP properties were analyzed and its vaccine potential was evaluated. The results showed this porin is an extremely conserved antigen with no allergenicity and toxicity that bears no resemblance to human proteins. Six potential immunogen areas in the DcaP sequence were detected based on in-silico B and T-cell epitope mapping and other approaches. A multiple-epitope potential vaccine was designed based on the predicted linear epitopes and amplified by overlap extension PCR technique. In-vivo results indicated that active and passive immunization of mice with the DcaP protein or its designed subunit vaccine raises the antibody titers and decreases the mortality rate of the immunized mice infected with A. baumannii. Based on the results, DcaP and its indicated immunogen regions can be considered as a peptide or subunit vaccine. The immunogen regions could also be applied in multivalent subunit vaccine candidates against A. baumannii and other bacteria.
Topics: Acinetobacter baumannii; Animals; Bacterial Vaccines; Epitopes, T-Lymphocyte; Mice; Porins; Vaccines, Subunit
PubMed: 34864145
DOI: 10.1016/j.micpath.2021.105346 -
Applied and Environmental Microbiology Apr 2022Bacterial porin-encoding genes are often found under positive selection. Local recombination has also been identified in a few of them to facilitate bacterial rapid...
Bacterial porin-encoding genes are often found under positive selection. Local recombination has also been identified in a few of them to facilitate bacterial rapid adaptation, although it remains unknown whether it is a common evolutionary mechanism for the porins or outer membrane proteins in Gram-negative bacteria. In this study, we investigated the beta-barrel (β-barrel) porin-encoding genes in Escherichia coli that were reported under positive Darwinian selection. Besides that was found with ingenic local recombination previously, we identified four other genes, i.e., , , , and , all showing the similar mosaic evolution patterns. Comparative analysis of the protein sequences disclosed a list of highly variable regions in each family, which are mostly located in the convex of extracellular loops and coinciding with the binding sites of bacteriophages. For each of the porin families, mosaic recombination leads to unique combinations of the variable regions with different sequence patterns, generating diverse protein groups. Structural modeling indicated a conserved global topology among the different porins, with the extracellular surface varying a lot due to individual or combinatorial variable regions. The conservation of global tertiary structure would ensure the channel activity, while the wide diversity of variable regions may represent selection to avoid the invasion of phages, antibiotics or immune surveillance factors. Our study identified multiple bacterial porin genes with mosaic evolution. We hypothesize that this could be generalized strategy for outer membrane proteins to both maintain normal life processes and evade the attack of unfavored factors rapidly. Microevolution studies can disclose more elaborate evolutionary mechanisms of genes, appearing especially important for genes with multifaceted function such as those encoding outer membrane proteins. However, in most cases, the gene is considered as a whole unit, and the evolutionary patterns are disclosed. Here, we report that multiple bacterial porin proteins follow mosaic evolution, with local ingenic recombination combined with spontaneous mutations based on positive Darwinian selection, and conservation for most structural regions. This could represent a common mechanism for bacterial outer membrane proteins. The variable regions within each porin family showed large coincidence with the binding sites of bacteriophages, antibiotics, and immune factors and therefore would represent effective targets for the development of new antibacterial agents or vaccines.
Topics: Animals; Anti-Bacterial Agents; Bacterial Outer Membrane Proteins; Bacterial Proteins; Escherichia coli; Humans; Porins; Sheep
PubMed: 35285711
DOI: 10.1128/aem.00060-22 -
Microbiology (Reading, England) Mar 2018Bacteria have evolved several strategies to survive a myriad of harmful conditions in the environment and in hosts. In Gram-negative bacteria, responses to nutrient... (Review)
Review
Bacteria have evolved several strategies to survive a myriad of harmful conditions in the environment and in hosts. In Gram-negative bacteria, responses to nutrient limitation, oxidative or nitrosative stress, envelope stress, exposure to antimicrobials and other growth-limiting stresses have been linked to the development of antimicrobial resistance. This results from the activation of protective changes to cell physiology (decreased outer membrane permeability), resistance transporters (drug efflux pumps), resistant lifestyles (biofilms, persistence) and/or resistance mutations (target mutations, production of antibiotic modification/degradation enzymes). In targeting and interfering with essential physiological mechanisms, antimicrobials themselves are considered as stresses to which protective responses have also evolved. In this review, we focus on envelope stress responses that affect the expression of outer membrane porins and their impact on antimicrobial resistance. We also discuss evidences that indicate the role of antimicrobials as signaling molecules in activating envelope stress responses.
Topics: Anti-Bacterial Agents; Bacterial Outer Membrane Proteins; Cell Membrane Permeability; Drug Resistance, Multiple, Bacterial; Enterobacteriaceae; Porins; Stress, Physiological; Transcription Factors
PubMed: 29458656
DOI: 10.1099/mic.0.000613 -
Microbial Drug Resistance (Larchmont,... Sep 2021The aim of this study was to identify antimicrobial resistance genes, virulence factor genes, and porin loss or mutations exhibited by the multidrug-resistant strain....
The aim of this study was to identify antimicrobial resistance genes, virulence factor genes, and porin loss or mutations exhibited by the multidrug-resistant strain. Whole-genome sequencing was done via the Illumina NovaSeq 6000 platform. Strain identification and antibiotic susceptibility testing of strains were performed by the Vitek 2 automated system. Multilocus sequence typing analysis was carried out using seven conserved housekeeping genes. The strain was resistant to penicillins, cephalosporins, carbapenems, aminoglycosides, fluoroquinolones, fosfomycin, and trimethoprim/sulfamethoxazole. The isolate was found to carry KPC-3, CTX-M-27, SHV-11, SHV-67, and TEM-1 β-lactamases. The clonal subtype of the isolate was ST147, and it possessed 64 and 38 alleles. Fifteen different point mutations (N49S, L59V, R146H, V178P, G189T, F198Y, V202L, F207Y, A217S, T222L, D223G, H235N, A280V, N304E, and S346N) were detected in the OmpK36 porin. A frame shift was observed in OmpK35 and two different point mutations (I70M and I128M) were found in the OmpK37 porin, in addition to seven mutations observed on the AcrR. This study demonstrated for the first time that the ST147 clone produced CTX-M-27 as well as KPC-3. In addition, new mutations were detected in the outer membrane proteins. These mutations together with the production of extended-spectrum β-lactamase and carbapenemase were found to contribute to the resistance of the ST147 clone to carbapenem and other antibiotics.
Topics: Anti-Bacterial Agents; Bacterial Proteins; Drug Resistance, Multiple, Bacterial; Genes, Bacterial; Humans; Klebsiella pneumoniae; Microbial Sensitivity Tests; Multilocus Sequence Typing; Point Mutation; Porins; Turkey; beta-Lactamases
PubMed: 33794115
DOI: 10.1089/mdr.2020.0274