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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 -
Trends in Microbiology Jun 2022The cell envelope is essential for survival and adaptation of bacteria. Bacterial membrane proteins include the major porins that mediate the influx of nutrients and... (Review)
Review
The cell envelope is essential for survival and adaptation of bacteria. Bacterial membrane proteins include the major porins that mediate the influx of nutrients and several classes of antimicrobial drugs. Consequently, membrane remodelling is closely linked to antimicrobial resistance (AMR). Knowledge of bacterial membrane protein biogenesis and turnover underpins our understanding of bacterial membrane remodelling and the consequences that this process have in the evolution of AMR phenotypes. At the population level, the evolution of phenotypes is a reversible process, and we can use these insights to deploy evolutionary principles to resensitize bacteria to existing antimicrobial drugs. In our opinion, fundamental knowledge is opening a new way of thinking towards sustainable solutions to the mounting crisis in AMR. Here we discuss what is known about outer-membrane remodelling in bacteria and how the process could be targeted as a means to restore sensitivity to antimicrobial drugs. Bacteriophages are highlighted as a powerful means to exert this control over membrane remodelling but they require careful selection so as to reverse, and not exacerbate, AMR phenotypes.
Topics: Anti-Bacterial Agents; Bacteria; Bacterial Proteins; Drug Resistance, Bacterial; Drug Resistance, Multiple, Bacterial; Porins
PubMed: 34872824
DOI: 10.1016/j.tim.2021.11.002 -
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 -
Frontiers in Cellular and Infection... 2022P66, a bifunctional integral outer membrane protein, is necessary for to establish initial infection and to disseminate in mice. The integrin binding function of P66...
P66, a bifunctional integral outer membrane protein, is necessary for to establish initial infection and to disseminate in mice. The integrin binding function of P66 facilitates extravasation and dissemination, but the role of its porin function during murine infection has not been investigated. A limitation to studying P66 porin function during mammalian infection has been the lack of structural information for P66. In this study, we experimentally characterized specific domains of P66 with regard to structure and function. First, we aligned the amino acid sequences of P66 from Lyme disease-causing and relapsing fever-causing to identify conserved and unique domains between these disease-causing clades. Then, we examined whether specific domains of P66 are exposed on the surface of the bacteria by introducing c-Myc epitope tags into each domain of interest. The c-Myc epitope tag inserted C-terminally to E33 (highly conserved domain), to T187 (integrin binding region domain and a non-conserved domain), and to E334 (non-conserved domain) were all detected on the surface of . The c-Myc epitope tag inserted C-terminally to E33 and D303 in conserved domains disrupted P66 oligomerization and porin function. In a murine model of infection, the E33 and D303 mutants exhibited decreased infectivity and dissemination. Taken together, these results suggest the importance of these conserved domains, and potentially P66 porin function, .
Topics: Amino Acids; Animals; Bacterial Proteins; Borrelia; Borrelia burgdorferi; Borrelia burgdorferi Group; Epitopes; Integrins; Lyme Disease; Mammals; Mice; Porins
PubMed: 36211976
DOI: 10.3389/fcimb.2022.991689 -
Microbiology Spectrum Apr 2022Helicobacter pylori is a human pathogen competent for natural transformation. Intrinsic and acquired antibiotic resistance contribute to the survival and multiplication...
HopE and HopD Porin-Mediated Drug Influx Contributes to Intrinsic Antimicrobial Susceptibility and Inhibits Streptomycin Resistance Acquisition by Natural Transformation in Helicobacter pylori.
Helicobacter pylori is a human pathogen competent for natural transformation. Intrinsic and acquired antibiotic resistance contribute to the survival and multiplication of H. pylori under antibiotics. While drug-resistance dissemination by natural transformation (NT)-mediated horizontal gene transfer remains poorly understood in H. pylori. The purpose of the study was to investigate the role of H. pylori porins (HopA, HopB, HopC, HopD, and HopE) in the intrinsic antibiotic resistance and to preliminarily reveal the potential effect of HopE and HopD porins in streptomycin resistance acquisition after NT in the presence of antibiotics. Using traditional antibiotic susceptibility tests and growth curve analysis, we found the MIC values of metronidazole, clarithromycin, levofloxacin, tetracycline, rifampin, and streptomycin in mutants lacking HopE and/or HopD were significantly elevated compare to those in wild-type strain. The quantitative analysis of the tetramethyl rhodamine isothiocyanate (TRITC)-labeled streptomycin accumulation at the single-cell level showed reduced streptomycin intracellular fluorescence in Δ and Δ mutant cells. Furthermore, in the presence of translation-inhibiting antibiotic streptomycin, the resistance acquisition frequency was decreased in the wild-type strain, which could be reversed by mutants lacking HopE and HopD that restored relatively high resistance acquisition frequencies. By transforming a pUC19-mut- linear plasmid carrying a streptomycin conferring mutation, we observed that the impaired ability of rpsLmut synthesis in the wild-type strain was restored in the Δ and Δ mutant transformants. Our study revealed that in the presence of streptomycin, resistance acquisition at least partially relied on the deletion of the and genes, because their loss reduced streptomycin concentration in the cell and thus restored the expression of the resistance-conferring gene, which was inhibited by streptomycin in wild-type strain. The loss of HopE and HopD influx activity may also preserve resistance acquisition by transformation in the presence of antibiotics with other modes of action. Helicobacter pylori is constitutively competent for natural transformation (NT) and possesses an efficient system for homologous recombination, which could be utilized to study the NT-mediated horizontal gene transfer induced antibiotic resistance acquisition. Bacterial porins have drawn renewed attention because of their crucial role in antibiotic susceptibility. From the perspective of porin-mediated influx in H. pylori, our study preliminarily revealed the important role of HopE and HopD porins not only in preserving the intrinsic susceptibility to specific antibiotic but also in evading acquired antibiotic resistance by NT in the presence of translation-inhibiting antimicrobial. Therefore, the loss of HopE or HopD porin in H. pylori genomes, combined with the large number of secreted or cell-free genetic elements carrying mutations conferring antibiotic resistance, may raise the possibility that this mechanism plays a potential role in the propagation of antibiotic resistance within H. pylori communities.
Topics: Anti-Bacterial Agents; Anti-Infective Agents; Drug Resistance, Bacterial; Helicobacter Infections; Helicobacter pylori; Humans; Microbial Sensitivity Tests; Porins; Streptomycin
PubMed: 35234510
DOI: 10.1128/spectrum.01987-21 -
IUBMB Life 2001Trafficking of metabolites across the outer mitochondrial membrane is believed to be mediated primarily by the pore-forming voltage-dependent anion channel, VDAC (also... (Review)
Review
Trafficking of metabolites across the outer mitochondrial membrane is believed to be mediated primarily by the pore-forming voltage-dependent anion channel, VDAC (also known as mitochondrial porin). An expanding body of in vitro studies have strongly suggest that the pore formed by VDAC can be regulated in a number of ways that implicate it as a site for the regulation of mitochondrial function, yet technical limitations have prevented the extension these studies to a relevant cellular context. The goal of this brief review is to summarize recent data that examine the role of VDAC and its regulation in the context not of the isolated protein or organelles but in cells, focusing on the application of genetic strategies in a number of experimental systems.
Topics: Animals; Humans; Intracellular Membranes; Ion Channels; Mitochondria; Porins; Voltage-Dependent Anion Channels
PubMed: 11798022
DOI: 10.1080/15216540152845902 -
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 -
Enzyme and Microbial Technology Jan 2022TprK is a key virulence factor of Treponema pallidum subsp. pallidum (T. pallidum) due to its ability to undergo intra-strain antigenic variation through gene...
TprK is a key virulence factor of Treponema pallidum subsp. pallidum (T. pallidum) due to its ability to undergo intra-strain antigenic variation through gene conversion. This mechanism can generate millions of tprK gene and protein variants to allow immune evasion and pathogen persistence during infection. In silico structural modeling supports that TprK is an outer membrane β-barrel with porin function and with several surface-exposed loops, seven of which corresponding to the variable regions. No definitive structural of functional data, however, exist for this protein aside from its role in immune evasion. Studies to elucidate TprK biological function as a porin, are hindered by the evidence that TprK is not abundant on T. pallidum outer membrane, and by the fragility of T. pallidum envelope. To gain insight onto TprK structure and possible function as a porin, we used an Escherichia coli - based expression system that yielded highly pure full-length TprK without any intermediate denaturation step, and proceeded to reconstitute it in detergents and lipid nanodiscs. Visualization of TprK in nanodiscs using negative staining electron microscopy supported that TprK is a monomeric porin in an artificial lipid environment mimicking T. pallidum membrane. Our work provided evidence that TprK is a possible porin transporter of T. pallidum, a biological function compatible with its structural models. These results bring us closer to a comprehensive understanding of the function of this important virulence factor in syphilis pathogenesis and T. pallidum biology.
Topics: Bacterial Outer Membrane Proteins; Escherichia coli; Lipids; Porins; Treponema
PubMed: 34670182
DOI: 10.1016/j.enzmictec.2021.109897 -
Journal of Bacteriology May 2008Ehrlichia chaffeensis, an obligatory intracellular gram-negative bacterium, must take up various nutrients and metabolic compounds because it lacks many genes involved...
Ehrlichia chaffeensis, an obligatory intracellular gram-negative bacterium, must take up various nutrients and metabolic compounds because it lacks many genes involved in metabolism. Nutrient uptake by a gram-negative bacterium occurs primarily through pores or channels in the bacterial outer membrane. Here we demonstrate that isolated E. chaffeensis outer membranes have porin activities, as determined by a proteoliposome swelling assay. The activity was partially blocked by an antibody that recognizes the two most abundant outer membrane proteins, P28/OMP-19 and OMP-1F/OMP-18. Both proteins were predicted to have structural features characteristic of porins, including 12 transmembrane segments comprised of amphipathic and antiparallel beta-strands. The sodium dodecyl sulfate stability of the two proteins was consistent with a beta-barrel structure. Isolated native P28 and OMP-1F exhibited porin activities, with pore sizes similar to and larger than, respectively, that of OprF, which is the porin with the largest pore size known to date. E. chaffeensis experiences temperature changes during transmission by ticks. During the intracellular development of E. chaffeensis, both P28 and OMP-1F were expressed mostly in the mid-exponential growth phase at 37 degrees C and the late-exponential growth phase at 28 degrees C. The porin activity of proteoliposomes reconstituted with proteins from the outer membrane fractions derived from bacteria in the mid- and late-exponential growth phases at 28 degrees C and 37 degrees C correlated with the expression levels of P28 and OMP-1F. These results imply that P28 and OMP-1F function as porins with large pore sizes, suggesting that the differential expression of these two proteins might regulate nutrient uptake during intracellular E. chaffeensis development at both temperatures.
Topics: Antibodies, Monoclonal; Antigens, Bacterial; Bacterial Outer Membrane Proteins; Ehrlichia chaffeensis; Gene Expression; Porins; Protein Structure, Secondary; Proteolipids
PubMed: 18359808
DOI: 10.1128/JB.02017-07 -
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