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1.
Biochemistry ; 51(16): 3373-82, 2012 Apr 24.
Article in English | MEDLINE | ID: mdl-22482748

ABSTRACT

Changes in the cholesterol content of cell membranes affect many physiological and pathological events, including the formation of arterial plaques, the entry of virus into cells, and receptor organization. Measuring the trafficking and distribution of cholesterol is essential to understanding how cells regulate sterol levels in membranes. Perfringolysin O (PFO) is a cytolysin secreted by Clostridium perfringens that requires cholesterol in the target membrane for binding. The specificity of PFO for high levels of cholesterol makes the toxin an attractive tool for studying the distribution and trafficking of cholesterol in cells. However, the use of the native toxin is limited given that binding is triggered only above a determined cholesterol concentration. To this end, we have identified mutations in PFO that altered the threshold for how much cholesterol is required to trigger binding. The cholesterol threshold among different PFO derivatives varied up to 10 mol % sterol, and these variations were not dependent on the lipid composition of the membrane. We characterized the binding of these PFO derivatives on murine macrophage-like cells whose cholesterol content was reduced or augmented. Our findings revealed that engineered PFO derivatives differentially associated with these cells in response to changes in cholesterol levels in the plasma membrane.


Subject(s)
Bacterial Toxins/chemistry , Cholesterol/metabolism , Hemolysin Proteins/chemistry , Animals , Bacterial Toxins/metabolism , Binding Sites , Cells, Cultured , Clostridium perfringens , Cyclodextrins/chemistry , Cyclodextrins/metabolism , Hemolysin Proteins/metabolism , Liposomes/metabolism , Macrophages/metabolism , Mice
2.
Biochemistry ; 49(44): 9498-507, 2010 Nov 09.
Article in English | MEDLINE | ID: mdl-20886855

ABSTRACT

Clostridium perfringens causes gas gangrene and gastrointestinal disease in humans. These pathologies are mediated by potent extracellular protein toxins, particularly α-toxin and perfringolysin O (PFO). While α-toxin hydrolyzes phosphatidylcholine and sphingomyelin, PFO forms large transmembrane pores on cholesterol-containing membranes. It has been suggested that the ability of PFO to perforate the membrane of target cells is dictated by how much free cholesterol molecules are present. Given that C. perfringens α-toxin cleaves the phosphocholine headgroup of phosphatidylcholine, we reasoned that α-toxin may increase the number of free cholesterol molecules in the membrane. Our present studies reveal that α-toxin action on membrane bilayers facilitates the PFO−cholesterol interaction as evidenced by a reduction in the amount of cholesterol required in the membrane for PFO binding and pore formation. These studies suggest a mechanism for the concerted action of α-toxin and PFO during C. perfringens pathogenesis.


Subject(s)
Bacterial Proteins/metabolism , Bacterial Toxins/metabolism , Calcium-Binding Proteins/metabolism , Clostridium perfringens/physiology , Gas Gangrene/microbiology , Hemolysin Proteins/metabolism , Phospholipids/metabolism , Type C Phospholipases/metabolism , Cholesterol/metabolism , Clostridium perfringens/metabolism , Host-Pathogen Interactions , Humans , Hydrolysis , Liposomes/metabolism , Models, Molecular , Protein Binding
3.
Subcell Biochem ; 51: 551-77, 2010.
Article in English | MEDLINE | ID: mdl-20213558

ABSTRACT

The cholesterol-dependent cytolysins (CDCs) are a family of beta-barrel pore-forming toxins secreted by Gram-positive bacteria. These toxins are produced as water-soluble monomeric proteins that after binding to the target cell oligomerize on the membrane surface forming a ring-like pre-pore complex, and finally insert a large beta-barrel into the membrane (about 250 A in diameter). Formation of such a large transmembrane structure requires multiple and coordinated conformational changes. The presence of cholesterol in the target membrane is absolutely required for pore-formation, and therefore it was long thought that cholesterol was the cellular receptor for these toxins. However, not all the CDCs require cholesterol for binding. Intermedilysin, secreted by Streptoccocus intermedius only binds to membranes containing a protein receptor, but forms pores only if the membrane contains sufficient cholesterol. In contrast, perfringolysin O, secreted by Clostridium perfringens, only binds to membranes containing substantial amounts of cholesterol. The mechanisms by which cholesterol regulates the cytolytic activity of the CDCs are not understood at the molecular level. The C-terminus of perfringolysin O is involved in cholesterol recognition, and changes in the conformation of the loops located at the distal tip of this domain affect the toxin-membrane interactions. At the same time, the distribution of cholesterol in the membrane can modulate toxin binding. Recent studies support the concept that there is a dynamic interplay between the cholesterol-binding domain of the CDCs and the excess of cholesterol molecules in the target membrane.


Subject(s)
Cell Membrane/metabolism , Cholesterol/metabolism , Cytotoxins/metabolism , Bacterial Toxins/metabolism , Bacteriocins/metabolism , Cell Membrane/drug effects , Cytotoxins/chemistry , Hemolysin Proteins/metabolism , Liposomes/metabolism , Membrane Lipids/metabolism , Protein Conformation , Sequence Homology, Amino Acid
4.
J Biol Chem ; 280(10): 8784-92, 2005 Mar 11.
Article in English | MEDLINE | ID: mdl-15613476

ABSTRACT

The functions of the mechanosensitive channels from Lactococcus lactis were determined by biochemical, physiological, and electrophysiological methods. Patch-clamp studies showed that the genes yncB and mscL encode MscS and MscL-like channels, respectively, when expressed in Escherichia coli or if the gene products were purified and reconstituted in proteoliposomes. However, unless yncB was expressed in trans, wild type membranes of L. lactis displayed only MscL activity. Membranes prepared from an mscL disruption mutant did not show any mechanosensitive channel activity, irrespective of whether the cells had been grown on low or high osmolarity medium. In osmotic downshift assays, wild type cells survived and retained 20% of the glycine betaine internalized under external high salt conditions. On the other hand, the mscL disruption mutant retained 40% of internalized glycine betaine and was significantly compromised in its survival upon osmotic downshifts. The data strongly suggest that L. lactis uses MscL as the main mechanosensitive solute release system to protect the cells under conditions of osmotic downshift.


Subject(s)
Aquaporins/physiology , Calcium Channels/physiology , Lactococcus lactis/physiology , Amino Acid Sequence , Amino Acid Substitution , Aquaporins/chemistry , Aquaporins/genetics , Base Sequence , Calcium Channels/chemistry , Calcium Channels/genetics , Cell Membrane/physiology , Cystine , DNA Primers , Gene Expression Regulation, Bacterial , Lactococcus lactis/genetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Patch-Clamp Techniques , Sequence Alignment , Sequence Homology, Amino Acid
5.
EMBO J ; 21(20): 5323-30, 2002 Oct 15.
Article in English | MEDLINE | ID: mdl-12374733

ABSTRACT

Three gene products that form independent mechanosensitive channel activities have been identified in Escherichia coli. Two of these, MscL and MscS, play a vital role in allowing the cell to survive acute hypotonic stress. Much less is known of the third protein, MscK (KefA). Here, we characterize the MscK channel activity and compare it with the activity of its structural and functional homologue, MscS. While both show a slight anionic preference, MscK appears to be more sensitive to membrane tension. In addition, MscK, but not MscS activity appears to be regulated by external ionic environment, requiring not only membrane tension but also high concentrations of external K(+), NH(4)(+), Rb(+) or Cs(+) to gate; no activity is observed with Na(+), Li(+) or N-methyl-D-glucamine (NMDG). An MscK gain-of-function mutant gates spontaneously in the presence of K(+) or similar ions, and will gate in the presence of Na(+), Li(+) and NMDG, but only when stimulated by membrane tension. Increased sensitivity and the highly regulated nature of MscK suggest a more specialized physiological role than other bacterial mechanosensitive channels.


Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Ion Channels/metabolism , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Genes, Bacterial , Ion Channel Gating , Ion Channels/drug effects , Ion Channels/genetics , Mechanotransduction, Cellular , Mutation , Phenotype , Potassium/pharmacology , Sodium/pharmacology
6.
J Biol Chem ; 277(31): 27682-8, 2002 Aug 02.
Article in English | MEDLINE | ID: mdl-12015316

ABSTRACT

MscS and MscL are mechanosensitive channels found in bacterial plasma membranes that open large pores in response to membrane tension. These channels function to alleviate excess cell turgor invoked by rapid osmotic downshock. Although much is known of the structure and molecular mechanisms underlying MscL, genes correlating with MscS activity have only recently been identified. Previously, it was shown that eliminating the expression of Escherichia coli yggB removed a major portion of MscS activity. YggB is distinct from MscL by having no obvious structural similarity. Here we have reconstituted purified YggB in proteoliposomes and have successfully detected MscS channel activity, confirming that purified YggB protein encodes MscS activity. Additionally, to define functional regions of the channel protein, we have randomly mutagenized the structural gene and isolated a mutant that evokes a gain-of-function phenotype. Physiological experiments demonstrate that the mutated channel allows leakage of solutes from the cell, suggesting inappropriate channel opening. Interestingly, this mutation is analogous in position and character to mutations yielding a similar phenotype in MscL. Hence, although MscS and MscL mechanosensitive channels are structurally quite distinct, there may be analogies in their gating mechanisms.


Subject(s)
Escherichia coli Proteins/physiology , Escherichia coli/physiology , Ion Channels/physiology , Bacterial Proteins/physiology , Cell Membrane/physiology , DNA Primers , Escherichia coli/growth & development , Escherichia coli Proteins/genetics , Gene Expression Regulation, Bacterial , Hydrogen-Ion Concentration , Ion Channels/genetics , Mutagenesis , Protein Conformation , Recombinant Proteins/metabolism , Sequence Deletion
7.
Mol Microbiol ; 44(4): 889-902, 2002 May.
Article in English | MEDLINE | ID: mdl-12010487

ABSTRACT

Maintenance of cell turgor is a prerequisite for almost any form of life as it provides a mechanical force for the expansion of the cell envelope. As changes in extracellular osmolality will have similar physicochemical effects on cells from all biological kingdoms, the responses to osmotic stress may be alike in all organisms. The primary response of bacteria to osmotic upshifts involves the activation of transporters, to effect the rapid accumulation of osmoprotectants, and sensor kinases, to increase the transport and/or biosynthetic capacity for these solutes. Upon osmotic downshift, the excess of cytoplasmic solutes is released via mechanosensitive channel proteins. A number of breakthroughs in the last one or two years have led to tremendous advances in our understanding of the molecular mechanisms of osmosensing in bacteria. The possible mechanisms of osmosensing, and the actual evidence for a particular mechanism, are presented for well studied, osmoregulated transport systems, sensor kinases and mechanosensitive channel proteins. The emerging picture is that intracellular ionic solutes (or ionic strength) serve as a signal for the activation of the upshift-activated transporters and sensor kinases. For at least one system, there is strong evidence that the signal is transduced to the protein complex via alterations in the protein-lipid interactions rather than direct sensing of ion concentration or ionic strength by the proteins. The osmotic downshift-activated mechanosensitive channels, on the other hand, sense tension in the membrane but other factors such as hydration state of the protein may affect the equilibrium between open and closed states of the proteins.


Subject(s)
Bacterial Proteins/metabolism , Membrane Proteins/metabolism , Water-Electrolyte Balance , Bacterial Proteins/chemistry , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Macromolecular Substances , Membrane Proteins/chemistry , Osmolar Concentration , Osmotic Pressure , Water/metabolism
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