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Virulence Dec 2023subspecies ( ) is increasingly recognized as an emerging bacterial pathogen, particularly in cystic fibrosis (CF) patients and CF centres' respiratory outbreaks. We...
BACKGROUND
subspecies ( ) is increasingly recognized as an emerging bacterial pathogen, particularly in cystic fibrosis (CF) patients and CF centres' respiratory outbreaks. We characterized genomic and phenotypic changes in 15 serial isolates from two CF patients (1S and 2B) with chronic pulmonary M. massiliense infection leading to death, as well as four isolates from a CF centre outbreak in which patient 2B was the index case.
RESULTS
Comparative genomic analysis revealed the mutations affecting growth rate, metabolism, transport, lipids (loss of glycopeptidolipids), antibiotic susceptibility (macrolides and aminoglycosides resistance), and virulence factors. Mutations in 23S rRNA, 4, porin locus and R genes occurred in isolates from both CF patients. Interestingly, we identified two different spontaneous mutation events at the mycobacterial porin locus: a fusion of two tandem porin paralogs in patient 1S and a partial deletion of the first porin paralog in patient 2B. These genomic changes correlated with reduced porin protein expression, diminished C-glucose uptake, slower bacterial growth rates, and enhanced TNF-α induction in mycobacteria-infected THP-1 human cells. Porin gene complementation of porin mutants partly restored C-glucose uptake, growth rate and TNF-α levels to those of intact porin strains.
CONCLUSIONS
We hypothesize that specific mutations accumulated and maintained over time in , including mutations shared among transmissible strains, collectively lead to more virulent, host adapted lineages in CF patients and other susceptible hosts.
Topics: Humans; Anti-Bacterial Agents; Cystic Fibrosis; Genomics; Glucose; Lung; Mutation; Mycobacterium; Mycobacterium abscessus; Tumor Necrosis Factor-alpha; Porins
PubMed: 37221835
DOI: 10.1080/21505594.2023.2215602 -
Nature Oct 2021Dimeric IgA secreted across mucous membranes in response to nonpathogenic taxa of the microbiota accounts for most antibody production in mammals. Diverse binding...
Dimeric IgA secreted across mucous membranes in response to nonpathogenic taxa of the microbiota accounts for most antibody production in mammals. Diverse binding specificities can be detected within the polyclonal mucosal IgA antibody response, but limited monoclonal hybridomas have been studied to relate antigen specificity or polyreactive binding to functional effects on microbial physiology in vivo. Here we use recombinant dimeric monoclonal IgAs (mIgAs) to finely map the intestinal plasma cell response to microbial colonization with a single microorganism in mice. We identify a range of antigen-specific mIgA molecules targeting defined surface and nonsurface membrane antigens. Secretion of individual dimeric mIgAs targeting different antigens in vivo showed distinct alterations in the function and metabolism of intestinal bacteria, largely through specific binding. Even in cases in which the same microbial antigen is targeted, microbial metabolic alterations differed depending on IgA epitope specificity. By contrast, bacterial surface coating generally reduced motility and limited bile acid toxicity. The overall intestinal IgA response to a single microbe therefore contains parallel components with distinct effects on microbial carbon-source uptake, bacteriophage susceptibility, motility and membrane integrity.
Topics: Animals; Antibodies, Monoclonal; Antigens, Bacterial; Escherichia coli; Germ-Free Life; Immunoglobulin A, Secretory; Intestines; Mice; Mice, Inbred C57BL; Microbiota; Plasma Cells; Porins
PubMed: 34646015
DOI: 10.1038/s41586-021-03973-7 -
Advances in Experimental Medicine and... 2023Aquaporins (AQPs) are a family of membrane water channels that basically function as regulators of intracellular and intercellular water flow. To date, 13 AQPs,...
Aquaporins (AQPs) are a family of membrane water channels that basically function as regulators of intracellular and intercellular water flow. To date, 13 AQPs, distributed widely in specific cell types in various organs and tissues, have been characterized in humans. A pair of NPA boxes forming a pore is highly conserved among all aquaporins and is also key residues for the classification of AQP superfamily into four groups according to primary sequences. AQPs may also be classified based on their transport properties. So far, chromosome localization and gene structure of 13 human AQPs have been identified, which is definitely helpful for studying phenotypes and potential targets in naturally occurring and synthetic mutations in human or cells.
Topics: Humans; Aquaporins; Mutation; Phenotype
PubMed: 36717483
DOI: 10.1007/978-981-19-7415-1_1 -
Journal of Bioenergetics and... Dec 2022Salinibacter ruber is an extremophilic bacterium able to grow in high-salts environments, such as saltern crystallizer ponds. This halophilic bacterium is red-pigmented...
Salinibacter ruber is an extremophilic bacterium able to grow in high-salts environments, such as saltern crystallizer ponds. This halophilic bacterium is red-pigmented due to the production of several carotenoids and their derivatives. Two of these pigment molecules, salinixanthin and retinal, are reported to be essential cofactors of the xanthorhodopsin, a light-driven proton pump unique to this bacterium. Here, we isolate and characterize an outer membrane porin-like protein that retains salinixanthin. The characterization by mass spectrometry identified an unknown protein whose structure, predicted by AlphaFold, consists of a 8 strands beta-barrel transmembrane organization typical of porins. The protein is found to be part of a functional network clearly involved in the outer membrane trafficking. Cryo-EM micrographs showed the shape and dimensions of a particle comparable with the ones of the predicted structure. Functional implications, with respect to the high representativity of this protein in the outer membrane fraction, are discussed considering its possible role in primary functions such as the nutrients uptake and the homeostatic balance. Finally, also a possible involvement in balancing the charge perturbation associated with the xanthorhodopsin and ATP synthase activities is considered.
Topics: Porins; Bacteroidetes; Carotenoids
PubMed: 36229623
DOI: 10.1007/s10863-022-09950-7 -
Advances in Protein Chemistry and... 2022The outer membrane of a gram-negative bacteria encapsulates the plasma membrane thereby protecting it from the harsh external environment. This membrane acts as a... (Review)
Review
The outer membrane of a gram-negative bacteria encapsulates the plasma membrane thereby protecting it from the harsh external environment. This membrane acts as a sieving barrier due to the presence of special membrane-spanning proteins called "porins." These porins are β-barrel channel proteins that allow the passive transport of hydrophilic molecules and are impermeable to large and charged molecules. Many porins form trimers in the outer membrane. They are abundantly present on the bacterial surface and therefore play various significant roles in the host-bacteria interactions. These include the roles of porins in the adhesion and virulence mechanisms necessary for the pathogenesis, along with providing resistance to the bacteria against the antimicrobial substances. They also act as the receptors for phage and complement proteins and are involved in modulating the host cellular responses. In addition, the potential use of porins as adjuvants, vaccine candidates, therapeutic targets, and biomarkers is now being exploited. In this review, we focus briefly on the structure of the porins along with their important functions and roles in the host-bacteria interactions.
Topics: Adjuvants, Vaccine; Anti-Bacterial Agents; Cell Membrane; Gram-Negative Bacteria; Porins
PubMed: 35034723
DOI: 10.1016/bs.apcsb.2021.09.004 -
Advances in Experimental Medicine and... 2023Aquaporins (AQPs) allow water molecules and other small, neutral solutes to quickly pass through membrane. The protein structures of AQPs solved by crystallographic...
Aquaporins (AQPs) allow water molecules and other small, neutral solutes to quickly pass through membrane. The protein structures of AQPs solved by crystallographic methods or cryo-electron microscopy technology show that AQP monomer consists of six membrane-spanning alpha-helices that form the central water-transporting pore. AQP monomers assemble to form tetramers, forming the functional units in the membrane, to transport water or other small molecules. The biological functions of AQPs are regulated by posttranslational modifications, e.g., phosphorylation, ubiquitination, glycosylation, subcellular distribution, degradation and protein interactions. Modifications of AQP combined with structural properties contribute to a better functional mechanism of AQPs. Insight into the molecular mechanisms responsible for AQP modifications as well as gating and transport properties proved to be fundamental to the development of new therapeutic targets or reliable diagnostic and prognostic biomarkers.
Topics: Cryoelectron Microscopy; Aquaporins; Protein Processing, Post-Translational; Biological Transport; Water
PubMed: 36717484
DOI: 10.1007/978-981-19-7415-1_2 -
Sovremennye Tekhnologii V Meditsine 2021was to develop a new software tool for identifying gene mutations that determine the porin-mediated resistance to antibiotics in gram-negative bacteria and to...
UNLABELLED
was to develop a new software tool for identifying gene mutations that determine the porin-mediated resistance to antibiotics in gram-negative bacteria and to demonstrate the functionality of this program by detecting porin-mediated resistance to carbapenems in clinical isolates of .
MATERIALS AND METHODS
The proposed algorithm is based on searching for a correspondence between the reference and the studied genes. When the sought nucleotide sequence is found in the analyzed genome, it is compared with the reference one and analyzed. The genomic analysis is then verified by comparing between the amino acid sequences encoded by the reference and studied genes. The genes of the susceptible ATCC 27853 strain were used as the reference nucleotide sequences encoding for porins (OprD, OpdD, and OpdP) involved in the transport of carbapenems into the bacterial cell. The complete genomes of clinical isolates from the PATRIC database 3.6.9 and our own collection were used to test the functionality of the proposed program. The analyzed isolates were phenotypically characterized according to the CLSI standard. The search for carbapenemase genes in the studied genomes of was carried out using the ResFinder 4.1.
RESULTS
The developed program for detecting the genetic determinants of non-plasmid antibiotic resistance made it possible to identify mutations of various types and significance in the porin genes of clinical isolates. These mutations led to modifications of the peptide structure of porin proteins. Single amino acid substitutions prevailed in the OpdD and OpdP porins of carbapenem-susceptible and carbapenem-resistant isolates. In the carbapenem-resistant strains, the gene encoding for OprD porin was found heavily modified, including insertions and/or deletions, which led to premature termination of porin synthesis. In several isolates resistant to meropenem, no mutations were detected in the gene encoding for OprD, which might be associated with alternative mechanisms of resistance to carbapenems.
CONCLUSION
The proposed software product can become an effective tool for deciphering the molecular genetic mechanisms of bacterial chromosomal resistance to antibiotics. Testing the program revealed differences between the occurrences of mutations significant for carbapenem resistance in the , , and genes.
Topics: Drug Resistance, Bacterial; Microbial Sensitivity Tests; Porins; Pseudomonas aeruginosa; Software
PubMed: 35265355
DOI: 10.17691/stm2021.13.6.02 -
Nature Sep 2020Transmembrane channels and pores have key roles in fundamental biological processes and in biotechnological applications such as DNA nanopore sequencing, resulting in...
Transmembrane channels and pores have key roles in fundamental biological processes and in biotechnological applications such as DNA nanopore sequencing, resulting in considerable interest in the design of pore-containing proteins. Synthetic amphiphilic peptides have been found to form ion channels, and there have been recent advances in de novo membrane protein design and in redesigning naturally occurring channel-containing proteins. However, the de novo design of stable, well-defined transmembrane protein pores that are capable of conducting ions selectively or are large enough to enable the passage of small-molecule fluorophores remains an outstanding challenge. Here we report the computational design of protein pores formed by two concentric rings of α-helices that are stable and monodisperse in both their water-soluble and their transmembrane forms. Crystal structures of the water-soluble forms of a 12-helical pore and a 16-helical pore closely match the computational design models. Patch-clamp electrophysiology experiments show that, when expressed in insect cells, the transmembrane form of the 12-helix pore enables the passage of ions across the membrane with high selectivity for potassium over sodium; ion passage is blocked by specific chemical modification at the pore entrance. When incorporated into liposomes using in vitro protein synthesis, the transmembrane form of the 16-helix pore-but not the 12-helix pore-enables the passage of biotinylated Alexa Fluor 488. A cryo-electron microscopy structure of the 16-helix transmembrane pore closely matches the design model. The ability to produce structurally and functionally well-defined transmembrane pores opens the door to the creation of designer channels and pores for a wide variety of applications.
Topics: Cell Line; Computer Simulation; Cryoelectron Microscopy; Crystallography, X-Ray; Electric Conductivity; Escherichia coli; Genes, Synthetic; Hydrazines; Ion Channels; Ion Transport; Liposomes; Models, Molecular; Patch-Clamp Techniques; Porins; Protein Engineering; Protein Structure, Secondary; Solubility; Synthetic Biology; Water
PubMed: 32848250
DOI: 10.1038/s41586-020-2646-5 -
Cells Sep 2021Autophagy is a cellular recycling program which efficiently reduces the cellular burden of ageing. Autophagy is characterised by nucleation of isolation membranes, which...
Autophagy is a cellular recycling program which efficiently reduces the cellular burden of ageing. Autophagy is characterised by nucleation of isolation membranes, which grow in size and further expand to form autophagosomes, engulfing cellular material to be degraded by fusion with lysosomes (vacuole in yeast). Autophagosomal membranes do not bud from a single cell organelle, but are generated de novo. Several lipid sources for autophagosomal membranes have been identified, but the whole process of their generation is complex and not entirely understood. In this study, we investigated how the mitochondrial outer membrane protein porin 1 (Por1), the yeast orthologue of mammalian voltage-dependent anion channel (VDAC), affects autophagy in yeast. We show that deficiency reduces the autophagic capacity and leads to changes in vacuole and lipid homeostasis. We further investigated whether limited phosphatidylethanolamine (PE) availability in ∆ was causative for reduced autophagy by overexpression of the PE-generating phosphatidylserine decarboxylase 1 (Psd1). Altogether, our results show that deficiency is associated with reduced autophagy, which can be circumvented by additional overexpression. This suggests a role for Por1 in Psd1-mediated autophagy regulation.
Topics: Autophagosomes; Autophagy; Carboxy-Lyases; Mitochondria; Mitochondrial Proteins; Phosphatidylethanolamines; Porins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 34572064
DOI: 10.3390/cells10092416 -
Phytomedicine : International Journal... Jan 2024The therapeutic efficacy of liver injuries heavily relies on the liver's remarkable regenerative capacity, necessitating the maintenance of glycose/lipids homeostasis...
BACKGROUND
The therapeutic efficacy of liver injuries heavily relies on the liver's remarkable regenerative capacity, necessitating the maintenance of glycose/lipids homeostasis and oxidative eustasis during the recovery process. Astragali Radix, an herbal tonic widely used in China and many other countries, is believed to have many positive effects, including immune stimulation, nourishing, antioxidant, liver protection, diuresis, anti-diabetes, anti-cancer and expectorant. Astragali Radix is widely integrated into hepatoprotective formulas as it is believed to facilitate liver regeneration. Nevertheless, the precise molecular pharmacological mechanisms underlying this hepatoprotective effect remain elusive.
PURPOSE
To investigate the improving effects of Astragali Radix on liver regeneration and the underlying mechanisms.
METHODS
A mouse model of 70% partial hepatectomy (PHx) was employed to investigate the impact of Radix Astragali decoction (HQD) on liver regeneration. HQD was orally administered for 7 days before the PHx procedure and throughout the experiment. N-acetylcysteine (NAC) was used as a positive control for liver regeneration. Liver regeneration was assessed by evaluating the liver-to-body weight ratio (LW/BW) and the expression of representative cell proliferation marker proteins. Oxidative stress and glucose metabolism were analyzed using biochemical assays, Western blotting, dihydroethidium (DHE) fluorescence, and periodic acid-Schiff (PAS) staining methods. To understand the role of AQP9 as a potential molecular target of HQD in promoting liver regeneration, td-Tomato-tagged AQP9 transgenic mice (AQP9-RFP) were employed to determine the expression pattern of AQP9 protein. AQP9 knockout mice (AQP9) were used to assess the specific targeting of AQP9 in the promotion of liver regeneration by HQD.
RESULTS
HQD significantly upregulated hepatic AQP9 expression, alleviated liver injury and promoted liver regeneration in wild-type (AQP9) mice after 70% PHx. However, the beneficial impact of HQD on liver regeneration was absent in AQP9 gene knockout (AQP9) mice. Moreover, HQD facilitated the uptake of glycerol by hepatocytes, enhanced gluconeogenesis, and concurrently reduced HO content and oxidative stress levels in AQP9 but not AQP9 mouse livers. Additionally, main active substance of Radix Astragali, astragaloside IV (AS-IV) and cycloastragenol (CAG), demonstrated substantial upregulation of AQP9 expression and promoted liver regeneration in AQP9 but not AQP9 mice.
CONCLUSION
This study is the first to demonstrate that Radix Astragali and its main active constituents (AS-IV and CAG) improve liver regeneration by upregulating the expression of AQP9 in hepatocytes to increase gluconeogenesis and reduce oxidative stress. The study revealed novel molecular pharmacological mechanisms of Radix Astragali and provided a promising therapeutic target of liver diseases.
Topics: Mice; Animals; Liver Regeneration; Hydrogen Peroxide; Liver; Drugs, Chinese Herbal; Astragalus Plant; Aquaporins
PubMed: 37918281
DOI: 10.1016/j.phymed.2023.155166