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Microbiology and Molecular Biology... Jun 2023Clinical management of Staphylococcus aureus infections presents a challenge due to the high incidence, considerable virulence, and emergence of drug resistance... (Review)
Review
Clinical management of Staphylococcus aureus infections presents a challenge due to the high incidence, considerable virulence, and emergence of drug resistance mechanisms. The treatment of drug-resistant strains, such as methicillin-resistant S. aureus (MRSA), is further complicated by the development of tolerance and persistence to antimicrobial agents in clinical use. To address these challenges, membrane disruptors, that are not generally considered during drug discovery for agents against S. aureus, should be explored. The cell membrane protects S. aureus from external stresses and antimicrobial agents, but membrane-targeting antimicrobial agents are probably less likely to promote bacterial resistance. Nontypical linear cationic antimicrobial peptides (AMPs), highly modified AMPs such as daptomycin (lipopeptide), bacitracin (cyclic peptide), and gramicidin S (cyclic peptide), are currently in clinical use. Recent studies have demonstrated that AMPs and small molecules can penetrate the cell membrane of S. aureus, inhibit phospholipid biosynthesis, or block the passage of solutes between the periplasm and the exterior of the cell. In addition to their primary mechanism of action (MOA) that targets the bacterial membrane, AMPs and small molecules may also impact bacteria through secondary mechanisms such as targeting the biofilm, and downregulating virulence genes of S. aureus. In this review, we discuss the current state of research into cell membrane-targeting AMPs and small molecules and their potential mechanisms of action against drug-resistant physiological forms of S. aureus, including persister cells and biofilms.
Topics: Humans; Staphylococcus aureus; Anti-Bacterial Agents; Methicillin-Resistant Staphylococcus aureus; Antimicrobial Peptides; Anti-Infective Agents; Peptides, Cyclic; Cell Membrane; Biofilms; Staphylococcal Infections
PubMed: 37129495
DOI: 10.1128/mmbr.00037-22 -
Methods in Molecular Biology (Clifton,... 2024Protein function is generally dependent on its subcellular localization. In gram-negative bacteria such as Escherichia coli, a protein can be targeted to five different...
Protein function is generally dependent on its subcellular localization. In gram-negative bacteria such as Escherichia coli, a protein can be targeted to five different compartments: the cytoplasm, the inner membrane, the periplasm, the outer membrane, and the extracellular medium. Different approaches can be used to determine the protein localization within cell such as in silico identification of protein signal sequences and motifs, electron microscopy and immunogold labeling, optical fluorescence microscopy, and biochemical technics. In this chapter, we describe a simple and efficient method to isolate the different compartments of Escherichia coli by a fractionation method and to determine the presence of the protein of interest. For inner membrane proteins, we propose a method to discriminate between integral and peripheral membrane proteins.
Topics: Cell Fractionation; Chemical Fractionation; Cytoplasm; Escherichia coli; Membrane Proteins
PubMed: 37930520
DOI: 10.1007/978-1-0716-3445-5_3 -
Nature Aug 2023To replicate inside macrophages and cause tuberculosis, Mycobacterium tuberculosis must scavenge a variety of nutrients from the host. The mammalian cell entry (MCE)...
To replicate inside macrophages and cause tuberculosis, Mycobacterium tuberculosis must scavenge a variety of nutrients from the host. The mammalian cell entry (MCE) proteins are important virulence factors in M. tuberculosis, where they are encoded by large gene clusters and have been implicated in the transport of fatty acids and cholesterol across the impermeable mycobacterial cell envelope. Very little is known about how cargos are transported across this barrier, and it remains unclear how the approximately ten proteins encoded by a mycobacterial mce gene cluster assemble to transport cargo across the cell envelope. Here we report the cryo-electron microscopy (cryo-EM) structure of the endogenous Mce1 lipid-import machine of Mycobacterium smegmatis-a non-pathogenic relative of M. tuberculosis. The structure reveals how the proteins of the Mce1 system assemble to form an elongated ABC transporter complex that is long enough to span the cell envelope. The Mce1 complex is dominated by a curved, needle-like domain that appears to be unrelated to previously described protein structures, and creates a protected hydrophobic pathway for lipid transport across the periplasm. Our structural data revealed the presence of a subunit of the Mce1 complex, which we identified using a combination of cryo-EM and AlphaFold2, and name LucB. Our data lead to a structural model for Mce1-mediated lipid import across the mycobacterial cell envelope.
Topics: Animals; Bacterial Proteins; Cryoelectron Microscopy; Lipids; Membrane Transport Proteins; Mycobacterium tuberculosis; Tuberculosis; Virus Internalization; Virulence Factors; ATP-Binding Cassette Transporters; Periplasm; Protein Domains; Hydrophobic and Hydrophilic Interactions; Multiprotein Complexes
PubMed: 37495693
DOI: 10.1038/s41586-023-06366-0 -
Nature Nov 2023Gram-negative bacteria are surrounded by two membranes. A special feature of the outer membrane is its asymmetry. It contains lipopolysaccharide (LPS) in the outer...
Gram-negative bacteria are surrounded by two membranes. A special feature of the outer membrane is its asymmetry. It contains lipopolysaccharide (LPS) in the outer leaflet and phospholipids in the inner leaflet. The proper assembly of LPS in the outer membrane is required for cell viability and provides Gram-negative bacteria intrinsic resistance to many classes of antibiotics. LPS biosynthesis is completed in the inner membrane, so the LPS must be extracted, moved across the aqueous periplasm that separates the two membranes and translocated through the outer membrane where it assembles on the cell surface. LPS transport and assembly requires seven conserved and essential LPS transport components (LptA-G). This system has been proposed to form a continuous protein bridge that provides a path for LPS to reach the cell surface, but this model has not been validated in living cells. Here, using single-molecule tracking, we show that Lpt protein dynamics are consistent with the bridge model. Half of the inner membrane Lpt proteins exist in a bridge state, and bridges persist for 5-10 s, showing that their organization is highly dynamic. LPS facilitates Lpt bridge formation, suggesting a mechanism by which the production of LPS can be directly coupled to its transport. Finally, the bridge decay kinetics suggest that there may be two different types of bridges, whose stability differs according to the presence (long-lived) or absence (short-lived) of LPS. Together, our data support a model in which LPS is both a substrate and a structural component of dynamic Lpt bridges that promote outer membrane assembly.
Topics: Bacterial Outer Membrane; Bacterial Outer Membrane Proteins; Biological Transport; Carrier Proteins; Escherichia coli; Escherichia coli Proteins; Gram-Negative Bacteria; Lipopolysaccharides; Membrane Proteins
PubMed: 37938784
DOI: 10.1038/s41586-023-06709-x -
Nature Microbiology Aug 2023Akkermansia muciniphila, a mucophilic member of the gut microbiota, protects its host against metabolic disorders. Because it is genetically intractable, the mechanisms...
Akkermansia muciniphila, a mucophilic member of the gut microbiota, protects its host against metabolic disorders. Because it is genetically intractable, the mechanisms underlying mucin metabolism, gut colonization and its impact on host physiology are not well understood. Here we developed and applied transposon mutagenesis to identify genes important for intestinal colonization and for the use of mucin. An analysis of transposon mutants indicated that de novo biosynthesis of amino acids was required for A. muciniphila growth on mucin medium and that many glycoside hydrolases are redundant. We observed that mucin degradation products accumulate in internal compartments within bacteria in a process that requires genes encoding pili and a periplasmic protein complex, which we term mucin utilization locus (MUL) genes. We determined that MUL genes were required for intestinal colonization in mice but only when competing with other microbes. In germ-free mice, MUL genes were required for A. muciniphila to repress genes important for cholesterol biosynthesis in the colon. Our genetic system for A. muciniphila provides an important tool with which to uncover molecular links between the metabolism of mucins, regulation of lipid homeostasis and potential probiotic activities.
Topics: Animals; Mice; Mucins; Sterols; Verrucomicrobia; Intestines; Specific Pathogen-Free Organisms; DNA Transposable Elements; Mutagenesis; Host Microbial Interactions; Intracellular Space; Bacterial Proteins; Transcription, Genetic
PubMed: 37337046
DOI: 10.1038/s41564-023-01407-w -
Drug Resistance Updates : Reviews and... Jan 2024Antibacterial drug resistance of gram-negative bacteria (GNB) results in high morbidity and mortality of GNB infection, seriously threaten human health globally.... (Review)
Review
Antibacterial drug resistance of gram-negative bacteria (GNB) results in high morbidity and mortality of GNB infection, seriously threaten human health globally. Developing new antibiotics has become the critical need for dealing with drug-resistant bacterial infections. Cefiderocol is an iron carrier cephalosporin that achieves drug accumulation through a unique "Trojan horse" strategy into the bacterial periplasm. It shows high antibacterial activity against multidrug-resistant (MDR) Enterobacteriaceae and MDR non-fermentative bacteria. The application of cefiderocol offers new hope for treating clinical drug-resistant bacterial infections. However, limited clinical data and uncertainties about its resistance mechanisms constrain the choice of its therapeutic use. This review aimed to summarize the clinical applications, drug resistance mechanisms, and co-administration of cefiderocol.
Topics: Humans; Cefiderocol; Siderophores; Anti-Bacterial Agents; Cephalosporins; Gram-Negative Bacterial Infections; Gram-Negative Bacteria; Drug Resistance, Multiple, Bacterial; Microbial Sensitivity Tests
PubMed: 38134561
DOI: 10.1016/j.drup.2023.101034 -
Science Advances Jul 2023Semiconductor-based biointerfaces are typically established either on the surface of the plasma membrane or within the cytoplasm. In Gram-negative bacteria, the...
Semiconductor-based biointerfaces are typically established either on the surface of the plasma membrane or within the cytoplasm. In Gram-negative bacteria, the periplasmic space, characterized by its confinement and the presence of numerous enzymes and peptidoglycans, offers additional opportunities for biomineralization, allowing for nongenetic modulation interfaces. We demonstrate semiconductor nanocluster precipitation containing single- and multiple-metal elements within the periplasm, as observed through various electron- and x-ray-based imaging techniques. The periplasmic semiconductors are metastable and display defect-dominant fluorescent properties. Unexpectedly, the defect-rich (i.e., the low-grade) semiconductor nanoclusters produced in situ can still increase adenosine triphosphate levels and malate production when coupled with photosensitization. We expand the sustainability levels of the biohybrid system to include reducing heavy metals at the primary level, building living bioreactors at the secondary level, and creating semi-artificial photosynthesis at the tertiary level. The biomineralization-enabled periplasmic biohybrids have the potential to serve as defect-tolerant platforms for diverse sustainable applications.
Topics: Periplasm; Biomineralization; Cell Membrane; Cytoplasm; Photosynthesis
PubMed: 37478187
DOI: 10.1126/sciadv.adg5858 -
Journal of Bacteriology Jun 2023The outer membranes (OM) of Gram-negative bacteria contain a class of proteins (TBDTs) that require energy for the import of nutrients and to serve as receptors for... (Review)
Review
The outer membranes (OM) of Gram-negative bacteria contain a class of proteins (TBDTs) that require energy for the import of nutrients and to serve as receptors for phages and protein toxins. Energy is derived from the proton motif force (pmf) of the cytoplasmic membrane (CM) through the action of three proteins, namely, TonB, ExbB, and ExbD, which are located in the CM and extend into the periplasm. The leaky phenotype of exbB exbD mutants is caused by partial complementation by homologous tolQ tolR. TonB, ExbB, and ExbD are genuine components of an energy transmission system from the CM into the OM. Mutant analyses, cross-linking experiments, and most recently X-ray and cryo-EM determinations were undertaken to arrive at a model that describes the energy transfer from the CM into the OM. These results are discussed in this paper. ExbB forms a pentamer with a pore inside, in which an ExbD dimer resides. This complex harvests the energy of the pmf and transmits it to TonB. TonB interacts with the TBDT at the TonB box, which triggers a conformational change in the TBDT that releases bound nutrients and opens the pore, through which nutrients pass into the periplasm. The structurally altered TBDT also changes the interactions of its periplasmic signaling domain with anti-sigma factors, with the consequence being that the sigma factors initiate transcription.
Topics: Escherichia coli Proteins; Escherichia coli; Membrane Proteins; Cell Membrane; Biological Transport; Bacterial Proteins
PubMed: 37219427
DOI: 10.1128/jb.00035-23 -
Trends in Biochemical Sciences Feb 2024Tripartite ATP-independent periplasmic (TRAP) transporters are nutrient-uptake systems found in bacteria and archaea. These evolutionary divergent transporter systems... (Review)
Review
Tripartite ATP-independent periplasmic (TRAP) transporters are nutrient-uptake systems found in bacteria and archaea. These evolutionary divergent transporter systems couple a substrate-binding protein (SBP) to an elevator-type secondary transporter, which is a first-of-its-kind mechanism of transport. Here, we highlight breakthrough TRAP transporter structures and recent functional data that probe the mechanism of transport. Furthermore, we discuss recent structural and biophysical studies of the ion transporter superfamily (ITS) members and highlight mechanistic principles that are relevant for further exploration of the TRAP transporter system.
Topics: Bacterial Proteins; Membrane Transport Proteins; Carrier Proteins; Bacteria; Biological Transport
PubMed: 38102017
DOI: 10.1016/j.tibs.2023.11.006 -
BioRxiv : the Preprint Server For... Aug 2023Many temperate phages encode prophage-expressed functions that interfere with superinfection of the host bacterium by external phages. phage P22 has four such systems...
Many temperate phages encode prophage-expressed functions that interfere with superinfection of the host bacterium by external phages. phage P22 has four such systems that are expressed from the prophage in a lysogen that are encoded by the (repressor), , , and genes. Here we report that the P22-encoded SieA protein is the only phage protein required for exclusion by the SieA system, and that it is an inner membrane protein that blocks DNA injection by P22 and its relatives, but has no effect on infection by other tailed phage types. The P22 virion injects its DNA through the host cell membranes and periplasm via a conduit assembled from three "ejection proteins" after their release from the virion. Phage P22 mutants were isolated that overcome the SieA block, and they have amino acid changes in the C-terminal regions of the gene and encoded ejection proteins. Three different single amino acid changes in these proteins are required to obtain nearly full resistance to SieA. Hybrid P22 phages that have phage HK620 ejection protein genes are also partially resistant to SieA. There are three sequence types of extant phage-encoded SieA proteins that are less than 30% identical to one another, yet comparison of two of these types found no differences in target specificity. Our data are consistent with a model in which the inner membrane protein SieA interferes with the assembly or function of the periplasmic gp20 and membrane-bound gp16 DNA delivery conduit.
PubMed: 37645741
DOI: 10.1101/2023.08.15.553423