Gram-negative Bacterial Membrane Research Articles

Role of the Cyclization in De Novo Design of Antimicrobial Peptide Mimics

Synthetic compounds mimicking the structure of natural antimicrobial peptides (AMPs) have a great promise as potential anti-infectious agents due to their stability towards enzymatic degradation, high antibiotic efficiency, and broad adjustability of physicochemical properties. Recently we have demonstrated that antimicrobial activity of AMP synthetic analogs depends on their conformational rigidity. Cyclization is one of the strategies to restrain the flexibility of antimicrobial agents. Herein we present results of a study aimed to establish how the cyclization affects the ability of cyclic N- substituted glycine oligomers (peptoids) to disrupt selectively bacterial, but not mammalian cell, membrane mimics. Lipid monolayers at the air/liquid interface composed of LPS or DPPG were used to model the outer leaflets of Gram-negative and Gram-positive bacterial membranes, respectively, while the DPPC/Cholesterol 6/4 mixed film was used to mimic the mammalian plasma membrane. Interactions of cyclic and linear peptoids with model lipid membranes were investigated using constant-pressure insertion assays, epifluorescence microscopy (EFM), and synchrotron X-ray reflectivity (XR) and grazing incidence X-ray diffraction (GIXD). Insertion assays show that both cyclic and non-cyclic peptoids readily incorporate into the bacterial, but not mammalian, membrane mimics. Moreover their insertion into the bacterial membrane mimics was accompanied by rapid deterioration of the structural order in the lipid acyl chains. Electron density profiles across the film, derived from XR data, demonstrate that both peptoids penetrate into the hydrophobic core of DPPG more efficiently than that of LPS, which might be due to a difference in packing of the hydrophobic core. Nevertheless, our data indicate that, despite these similarities, the mechanisms of action of cyclic and linear peptoids on bacterial membranes are different.

Highly dynamic biodegradable micelles capable of lysing Gram-positive and Gram-negative bacterial membrane

The development of biodegradable antimicrobial polymers adds to the toolbox of attractive antimicrobial agents against antibiotic-resistant microbes. To this end, the potential of polycarbonate polymers as such materials were explored. A series of random polycarbonate polymers consisting of monomers MTC-OEt and MTC-CH2CH3Cl were designed and synthesized using metal-free organocatalytic ring-opening polymerization. Random polycarbonate polymers self-assembled in solution but appeared highly dynamic; such behaviors are desirable as ready disassembly of polymers at the microbial membrane facilitates membrane disruption. Their activities against clinically relevant Gram-positive (Staphylococcus aureus) and Gram-negative bacteria (E.coli and Pseudomonas aeruginosa) revealed that the hydrophobic-hydrophilic composition balance in polymers are important to render antimicrobial potency. Scanning electron microscopy (SEM) studies indicated microbial cell surface damage after treatment with polymers, and confocal microscopy studies also showed entry of FITC-dextran dye in Escherichia coli as a result of membrane disruption. On the other hand, the polymers exhibited minimal toxicity against red blood cells in hemolysis tests. Therefore, these random polycarbonate polymers are promising antimicrobial agents against both Gram-positive and Gram-negative bacteria for various biomedical applications.

Human Defensin 5 Disulfide Array Mutants: Disulfide Bond Deletion Attenuates Antibacterial Activity against Staphylococcus aureus

Human α-defensin 5 (HD5, HD5(ox) to specify the oxidized and disulfide linked form) is a 32-residue cysteine-rich host-defense peptide, expressed and released by small intestinal Paneth cells, that exhibits antibacterial activity against a number of Gram-negative and -positive bacterial strains. To ascertain the contributions of its disulfide array to structure, antimicrobial activity, and proteolytic stability, a series of HD5 double mutant peptides where pairs of cysteine residues corresponding to native disulfide linkages (Cys(3)-Cys(31), Cys(5)-Cys(20), Cys(10)-Cys(30)) were mutated to Ser or Ala residues, overexpressed in E. coli, purified, and characterized. A hexa mutant peptide, HD5[Ser(hexa)], where all six native Cys residues are replaced by Ser residues, was also evaluated. Removal of a single native S-S linkage influences oxidative folding and regioisomerization, antibacterial activity, Gram-negative bacterial membrane permeabilization, and proteolytic stability. Whereas the majority of the HD5 mutant peptides show low micromolar activity against Gram-negative E. coli ATCC 25922 in colony counting assays, the wild-type disulfide array is essential for low micromolar activity against Gram-positive S. aureus ATCC 25923. Removal of a single disulfide bond attenuates the activity observed for HD5(ox) against this Gram-positive bacterial strain. This observation supports the notion that the HD5(ox) mechanism of antibacterial action differs for Gram-negative and Gram-positive species [Wei et al. (2009) J. Biol. Chem. 284, 29180-29192] and that the native disulfide array is a requirement for its activity against S. aureus.

Lipopolysaccharide, Mediator of Sepsis Enigma: Recognition and Signaling

The outer leaflet of Gram-negative bacterial membrane contains a great amount of lipopolysaccharides, also known as endotoxins, which play a central role in the pathogenesis of sepsis and ultimately septic shock. Lipopolysaccharide (LPS) is potent inducer of acute sepsis or chronic inflammation. Sepsis can strike anyone, but is most likely to develop from infection associated with events such as pneumonia, trauma, surgery, and burns, or serious illnesses such as cancer and AIDS. In fact, people whose deaths are ascribed to complications of cancer, AIDS, or pneumonia, often actually die as a direct result of sepsis. Sepsis involves a complex interaction between bacterial toxins and the host immune system. LPS stimulates Toll-like receptor (TLR)-4 which leads to the formation and release of range of proinflammatory mediators which are essential for the potent immune response. The massive host response to this single bacterial pattern recognition molecule is sufficient to generate diffuse endothelial injury, tissue hypoperfusion, disseminated intravascular coagulation and refractory shock. LPS recognition involves LPS binding protein (LBP), CD14 ending up in TLR4/MD-2/LPS complex. The complex leads to activation of TLR4 and subsequent signaling cascade via two pathways i. e., myeloid differentiation protein 88 (MyD88)-dependent and TRIF-dependent. Here is a brief review of TLR4 signaling and LPS recognition biology with its impact if any on downstream pathways.

Interaction of Recombinant Surfactant Protein D with Lipopolysaccharide: Conformation and Orientation of Bound Protein by IRRAS and Simulations

Effective innate host defense requires early recognition of pathogens. Surfactant protein D (SP-D), shown to play a role in host defense, binds to the lipopolysaccharide (LPS) component of Gram-negative bacterial membranes. Binding takes place via the carbohydrate recognition domain (CRD) of SP-D. Recombinant trimeric neck+CRDs (NCRD) have proven valuable in biophysical studies of specific interactions. Although X-ray crystallography has provided atomic level information on NCRD binding to carbohydrates and other ligands, molecular level information about interactions between SP-D and biological ligands under physiologically relevant conditions is lacking. Infrared reflection-absorption spectroscopy (IRRAS) provides molecular structure information from films at the air/water interface where protein adsorption to LPS monolayers serves as a model for protein-lipid interaction. In the current studies, we examine the adsorption of NCRDs to Rd 1 LPS monolayers using surface pressure measurements and IRRAS. Measurements of surface pressure, Amide I band intensities, and LPS acyl chain conformational ordering, along with the introduction of EDTA, permit discrimination of Ca (2+)-mediated binding from nonspecific protein adsorption. The findings support the concept of specific binding between the CRD and heptoses in the core region of LPS. In addition, a novel simulation method that accurately predicts the IR Amide I contour from X-ray coordinates of NCRD SP-D is applied and coupled to quantitative IRRAS equations providing information on protein orientation. Marked differences in orientation are found when the NCRD binds to LPS compared to nonspecific adsorption. The geometry suggests that all three CRDs are simultaneously bound to LPS under conditions that support the Ca (2+)-mediated interaction.

Synthetic Antimicrobial Oligomers Induce a Composition-Dependent Topological Transition in Membranes

Antimicrobial peptides (AMPs) are cationic amphiphiles that comprise a key component of innate immunity. Synthetic analogues of AMPs, such as the family of phenylene ethynylene antimicrobial oligomers (AMOs), recently demonstrated broad-spectrum antimicrobial activity, but the underlying molecular mechanism is unknown. Homologues in this family can be inactive, specifically active against bacteria, or nonspecifically active against bacteria and eukaryotic cells. Using synchrotron small-angle X-ray scattering (SAXS), we show that observed antibacterial activity correlates with an AMO-induced topological transition of small unilamellar vesicles into an inverted hexagonal phase, in which hexagonal arrays of 3.4-nm water channels defined by lipid tubes are formed. Polarized and fluorescence microscopy show that AMO-treated giant unilamellar vesicles remain intact, instead of reconstructing into a bulk 3D phase, but are selectively permeable to encapsulated macromolecules that are smaller than 3.4 nm. Moreover, AMOs with different activity profiles require different minimum threshold concentrations of phosphoethanolamine (PE) lipids to reconstruct the membrane. Using ternary membrane vesicles composed of DOPG:DOPE:DOPC with a charge density fixed at typical bacterial values, we find that the inactive AMO cannot generate the inverted hexagonal phase even when DOPE completely replaces DOPC. The specifically active AMO requires a threshold ratio of DOPE:DOPC = 4:1, and the nonspecifically active AMO requires a drastically lower threshold ratio of DOPE:DOPC = 1.5:1. Since most gram-negative bacterial membranes have more PE lipids than do eukaryotic membranes, our results imply that there is a relationship between negative-curvature lipids such as PE and antimicrobial hydrophobicity that contributes to selective antimicrobial activity.

Molecular Mechanisms that Govern the Specificity of Sushi Peptides for Gram-Negative Bacterial Membrane Lipids

Factor C-derived Sushi peptides (S1 and S3) have been shown to bind lipopolysaccharide (LPS) and inhibit the growth of Gram-negative bacteria but do not affect mammalian cells. On the premise that the composition of membrane phospholipids differs between the microbial and human cells, we studied the modes of interaction between S1 and S3 and the bacterial membrane phospholipids, POPG, in comparison to that with the mammalian cell membrane phospholipids, POPC and POPE. S1 exhibits specificity against POPG, suggesting its preference for bacterial anionic phospholipids, regardless of whether the phospholipids form vesicles in a solution or a monolayer on a solid surface. The specificity of the Sushi peptides for POPG is a consequence of the electrostatic and hydrophobic forces. The unsaturated nature of POPG confers fluidity to the lipid layer, and being in the proximity of LPS in the microenvironmental milieu, POPG probably enhances the insertion of the peptide-LPS complex into the bacterial inner membrane. Furthermore, during its interaction with POPG, the S1 peptide underwent a transition from random to alpha-helical coil, while S3 became a mixture of beta-sheet and alpha-helical structures. This differential structural change in the peptides could be responsible for their different modes of disruption of POPG vesicles. Conceivably, the selectivity for POPG spares the mammalian membranes from undesirable effects of antimicrobial peptides, which could be helpful in designing and developing a new generation of antibiotics and in offering some clues about the specific function of Factor C, a LPS biosensor.

Structural Correlates of Antibacterial and Membrane-Permeabilizing Activities in Acylpolyamines

A homologous series of mono- and bis-acyl polyamines with varying acyl chain lengths originally synthesized for the purpose of sequestering lipopolysaccharide were evaluated for antimicrobial activity to test the hypothesis that these bis-cationic amphipathic compounds may also bind to and permeabilize intact gram-negative bacterial membranes. Some compounds were found to possess significant antimicrobial activity, mediated via permeabilization of bacterial membranes. Structure-activity relationship studies revealed a strong dependence of the acyl chain length on antimicrobial potency and permeabilization activity. Homologated spermine, bis-acylated with C8 or C9 chains, was found to profoundly sensitize Escherichia coli to hydrophobic antibiotics such as rifampin. Nonspecific cytotoxicity is a potential drawback of these membranophilic compounds. However, the surface activity of these cationic amphipaths is strongly attenuated under physiological conditions via binding to serum albumin. Significant antibacterial activity is still retained in the presence of physiological concentrations of human serum albumin, suggesting that these compounds may serve as leads in the development of novel adjuncts to conventional antimicrobial chemotherapy.

Novel endotoxin-sequestering compounds with terephthalaldehyde-bis-guanylhydrazone scaffolds

We have shown that lipopolyamines bind to the lipid A moiety of lipopolysaccharide, a constituent of Gram-negative bacterial membranes, and neutralize its toxicity in animal models of endotoxic shock. In an effort to identify non-polyamine scaffolds with similar endotoxin-recognizing features, we had observed an unusually high frequency of hits containing guanylhydrazone scaffolds in high-throughput screens. We now describe the syntheses and preliminary structure–activity relationships in a homologous series of bis-guanylhydrazone compounds decorated with hydrophobic functionalities. These first-generation compounds bind and neutralize lipopolysaccharide with a potency comparable to that of polymyxin B, a peptide antibiotic known to sequester LPS.

Limiting the Damage

Toll-like receptors (TLR), found in cells of the innate immune system, recognize conserved microbial patterns to mediate proinflammatory responses and thereby eliminate pathogens. When out of control, however, these responses can be dangerous to the host; indeed, activation of TLR4 by lipopolysaccharide (LPS, found in Gram-negative bacterial membranes) can lead to life-threatening endotoxic shock. Tissue damage can lead to the release of adenosine triphosphate (ATP); thus, Kaufmann et al . investigated the possible role of extracellular ATP as a "damage signal" that modulates TLR signaling. The authors used reverse transcription polymerase chain reaction (RT-PCR) to show that human peripheral blood monocytes expressed various P2Y and P2X purinergic receptors. Exposure to ATP increased monocyte adenosine 3′,5′-monophosphate (cAMP) content, elicited a biphasic intracellular calcium signal, stimulated pseudopodial retraction, and promoted nondirectional cell migration. LPS stimulated the production of tumor necrosis factor α (TNFα); ATPγS inhibited LPS-stimulated secretion of TNFα and monocyte chemoattractant protein-1 (MCP-1), whereas it enhanced LPS-stimulated secretion of the anti-inflammatory interleukin-10 (IL-10). Pharmacological analysis indicated that this likely involved stimulation of the cAMP signaling pathway, mediated through P2Y 11 . Similarly, ATPγS inhibited the production of TNFα and MCP-1, and enhanced that of IL-10, elicited by stimulating TLR2 or the TLR2/6 complex with the appropriate ligands. Thus, the authors suggest that ATP released from injured cells acts through P2Y 11 and the cAMP pathway to limit the proinflammatory response to TLR activation. A. Kaufmann, B. Musset, S. H. Limberg, V. Renigunta, R. Sus, A. H. Dalpke, K. M. Heeg, B. Robaye, P. J. Hanley, "Host tissue damage" signal ATP promotes non-directional migration and negatively regulates Toll-like receptor signaling in human monocytes. J. Biol. Chem. 280 , 32459-32467 (2005). [Abstract] [Full Text]

Origins of Cell Selectivity of Cationic Steroid Antibiotics

A key factor in the potential clinical utility of membrane-active antibiotics is their cell selectivity (i.e., prokaryote over eukaryote). Cationic steroid antibiotics were developed to mimic the lipid A binding character of polymyxin B and are shown to bind lipid A derivatives with affinity greater than that of polymyxin B. The outer membranes of Gram-negative bacteria are comprised primarily of lipid A, and a fluorophore-appended cationic steroid antibiotic displays very high selectivity for Gram-negative bacterial membranes over Gram-positive bacteria and eukaryotic cell membranes. This cell selectivity of cationic steroid antibiotics may be due, in part, to the affinity of these compounds for lipid A.

The Measurement and Health Impact of Endotoxin Contamination in Organic Dusts from Multiple Sources: Focus on the Cotton Industry

Endotoxin is derived from Gram-negative bacterial membranes, and its inflammatory effects following inhalation are well characterized. The significance of this fact becomes apparent when the wide-ranging environments containing high levels of this microbial product are considered. Endotoxin is present in numerous industrial environments, especially where organic fibers are processed. Microbial contamination of these fibers mainly occurs at the agricultural stage. Materials such as flax and hemp are affected in this way, but the most important product in this context is cotton, from which chronic dust inhalation causes the disease byssinosis. Despite the fact that endotoxin constitutes a significant threat to public health, there are currently no occupational exposure limits for this toxicant. This communication describes the toxicology of endotoxin, and its role in inhalation-induced disease, focusing on measurement of airborne endotoxin in the occupational and domestic environments using the Limulus amebocyte lysate (LAL) enzyme assay. Following the success of the LAL assay for measuring endotoxin in dusts, our laboratory has examined its application to aqueous washes from cotton fibers. Reproducibility of the results was high, and data are presented displaying levels of endotoxin contamination in fibers from different cotton producing countries. Hence, worldwide comparison of industrial endotoxin concentrations can be readily made using this test. It would be highly desirable if the performance of the LAL assay facilitated introduction of industrial endotoxin safety limits, and in spite of minor surmountable shortcomings, the test is accurate, reliable, and well field-tested, so its continued widespread use may achieve this goal.

Groups IV, V, and X Phospholipases A2s in Human Neutrophils: ROLE IN EICOSANOID PRODUCTION AND GRAM-NEGATIVE BACTERIAL PHOSPHOLIPID HYDROLYSIS

The bacterial tripeptide formyl-Met-Leu-Phe (fMLP) induces the secretion of enzyme(s) with phospholipase A(2) (PLA(2)) activity from human neutrophils. We show that circulating human neutrophils express groups V and X sPLA(2) (GV and GX sPLA(2)) mRNA and contain GV and GX sPLA(2) proteins, whereas GIB, GIIA, GIID, GIIE, GIIF, GIII, and GXII sPLA(2)s are undetectable. GV sPLA(2) is a component of both azurophilic and specific granules, whereas GX sPLA(2) is confined to azurophilic granules. Exposure to fMLP or opsonized zymosan results in the release of GV but not GX sPLA(2) and most, if not all, of the PLA(2) activity in the extracellular fluid of fMLP-stimulated neutrophils is due to GV sPLA(2). GV sPLA(2) does not contribute to fMLP-stimulated leukotriene B(4) production but may support the anti-bacterial properties of the neutrophil, because 10-100 ng per ml concentrations of this enzyme lead to Gram-negative bacterial membrane phospholipid hydrolysis in the presence of human serum. By use of a recently described and specific inhibitor of cytosolic PLA(2)-alpha (group IV PLA(2)alpha), we show that this enzyme produces virtually all of the arachidonic acid used for the biosynthesis of leukotriene B(4) in fMLP- and opsonized zymosan-stimulated neutrophils, the major eicosanoid produced by these pro-inflammatory cells.

Antibacterial agents based on the cyclic D,L-alpha-peptide architecture.

The rapid emergence of bacterial infections that are resistant to many drugs underscores the need for new therapeutic agents. Here we report that six- and eight-residue cyclic d,l-alpha-peptides act preferentially on Gram-positive and/or Gram-negative bacterial membranes compared to mammalian cells, increase membrane permeability, collapse transmembrane ion potentials, and cause rapid cell death. The effectiveness of this class of materials as selective antibacterial agents is highlighted by the high efficacy observed against lethal methicillin-resistant Staphylococcus aureus infections in mice. Cyclic d,l-alpha-peptides are proteolytically stable, easy to synthesize, and can be derived from a potentially vast membrane-active sequence space. The unique abiotic structure of the cyclic peptides and their quick bactericidal action may also contribute to limit temporal acquirement of drug resistant bacteria. The low molecular weight d,l-alpha-peptides offer an attractive complement to the current arsenal of naturally derived antibiotics, and hold considerable potential in combating a variety of existing and emerging infectious diseases.

Lipopolysaccharide-binding protein and phospholipid transfer protein release lipopolysaccharides from gram-negative bacterial membranes.

Although animals mobilize their innate defenses against gram-negative bacteria when they sense the lipid A moiety of bacterial lipopolysaccharide (LPS), excessive responses to this conserved bacterial molecule can be harmful. Of the known ways for decreasing the stimulatory potency of LPS in blood, the binding and neutralization of LPS by plasma lipoproteins is most prominent. The mechanisms by which host lipoproteins take up the native LPS that is found in bacterial membranes are poorly understood, however, since almost all studies of host-LPS interactions have used purified LPS aggregates. Using native Salmonella enterica serovar Typhimurium outer membrane fragments (blebs) that contained (3)H-labeled lipopolysaccharide (LPS) and (35)S-labeled protein, we found that two human plasma proteins, LPS-binding protein (LBP) and phospholipid transfer protein (PLTP), can extract [(3)H]LPS from bacterial membranes and transfer it to human high-density lipoproteins (HDL). Soluble CD14 (sCD14) did not release LPS from blebs yet could facilitate LBP-mediated LPS transfer to HDL. LBP, but not PLTP, also promoted the activation of human monocytes by bleb-derived LPS. Whereas depleting or neutralizing LBP significantly reduced LPS transfer from blebs to lipoproteins in normal human serum, neutralizing serum PLTP had no demonstrable effect. Of the known lipid transfer proteins, LBP is thus most able to transfer LPS from bacterial membranes to the lipoproteins in normal human serum.

Pyrithione biocides as inhibitors of bacterial ATP synthesis.

Sodium pyrithione and zinc pyrithione (NaPT and ZnPT, respectively) are widely used as cosmetic preservatives and general antimicrobial agents. They have been shown to be active against fungal cell walls, associated membranes and bacterial transport processes. Investigations were undertaken into the effect of these antimicrobial agents on substrate catabolism and intracellular ATP levels using an oxygen electrode and luciferin-laciferase technology, respectively. Results indicate that, while both compounds are poor inhibitors of substrate catabolism, sub-inhibitory concentrations of biocide greatly reduces intracellular ATP levels in both Escherichia coli NCIMB 10000 and Pseudomonas aeruginosa NCIMB 10548. This is thought to be due to the action of NaPT and ZnPT on the Gram-negative bacterial membrane.

Polymyxin-B Stimulates Tumor Necrosis Factor-α Production by Human Peripheral Blood Mononuclear Cells

Gram-negative bacterial lipopolysaccharide (LPS) is a well known stimulus for cytokine production, particularly interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha). Polymyxin B (PMX-B) is a cationic polypeptide that binds to LPS, neutralizing its biological effects. PMX-B also disrupts gram-negative bacterial cell membrane phospholipids but is highly toxic to mammalian cells, therefore is of limited use. PMX-B is used as additive to media, as a way to handle LPS contamination. To derive benefit from the ability of PMX-B to neutralize lipid A in vivo while avoiding its systemic toxicity, PMX-B was covalently bound to polystyrene-derivative fibers, creating a hemoperfusion column (PMX-F) for the selective removal of circulating ET. In vitro PMX-F hemoperfusion studies have demonstrated effective ET removal, using either the Limulus amebocyte lysate assay or TNF alpha production by peripheral blood mononuclear cells (PBMC) as an index of ET removal. However, the question whether PMX-B itself could stimulate human PBMC to produce cytokines has not been adequately addressed. We examined the effect of increasing concentrations of PMX-B on cytokine production by PBMC in vitro. PBMC harvested from healthy volunteers were incubated for 24 hours at 37 degrees C with control (tissue culture media RPMI), or 5 microg/ml, 10 microg/ml, 20 microg/ml or 100 microg/ml PMX-B. At the end of 24 hours, PBMC were subjected to three freeze-thaw cycles, and total TNF alpha production (pg/2.5x10(6) PBMC) was measured by radioimmunoassay. Total TNF alpha production by PBMC was 163 +/- 3 pg, 171 +/- 9 pg, 164 +/- 4 pg, 323 +/- 63 pg and 331 +/- 58 pg, in the control, PMX-B 5 microg/ml, 10 microg/ml, 20 microg/ml and 100 microg/ml conditions, respectively. Compared to controls (RPMI), the percentage increase in TNF alpha production by PBMC was 5 +/- 6% (P=0.23), 1 +/- 3% (P=0.45), 99 +/- 40% (P=0.03) and 103 +/- 36% (P=0.02) in the presence of 5 microg/ml, 10 microg/ml, 20 microg/ml and 100 microg/ml of PMX-B, respectively. Furthermore, total TNF alpha production correlated significantly with increasing concentrations of PMX-B (R=0.53, P=0.007). We conclude that the use of PMX-B in in vitro studies as an LPS-neutralizing agent, or in the experimental treatment of endotoxic or septic shock can lead to erroneous interpretations of cytokine production by PBMC, and should be used cautiously in in vitro systems at high concentrations.

A family of extracytoplasmic proteins that allow transport of large molecules across the outer membranes of gram-negative bacteria.

Seventeen fully sequenced and two partially sequenced extracytoplasmic proteins of purple, gram-negative bacteria constitute a homologous family termed the putative membrane fusion protein (MFP) family. Each such protein apparently functions in conjunction with a cytoplasmic membrane transporter of the ATP-binding cassette family, major facilitator superfamily, or heavy metal resistance/nodulation/cell division family to facilitate transport of proteins, peptides, drugs, or carbohydrates across the two membranes of the gram-negative bacterial cell envelope. Evidence suggests that at least some of these transport systems also function in conjunction with a distinct outer membrane protein. We report here that the phylogenies of these proteins correlate with the types of transport systems with which they function as well as with the natures of the substrates transported. Characterization of the MFPs with respect to secondary structure, average hydropathy, and average similarity provides circumstantial evidence as to how they may allow localized fusion of the two gram-negative bacterial cell membranes. The membrane fusion protein of simian virus 5 is shown to exhibit significant sequence similarity to representative bacterial MFPs.

Killing of gram-negative bacteria by complement. Fractionation of cell membranes after complement C5b-9 deposition on to the surface of Salmonella minnesota Re595.

The effect of C5b-9 deposition on the envelope of target Gram-negative bacteria was studied. In order to understand the changes occurring after complement deposition on the bacterial surface, the preparation of Gram-negative bacterial membranes by different methods involving the osmotic lysis of spheroplasts was investigated. Subsequent fractionation of the outer membrane (OM) and cytoplasmic membrane (CM) by sucrose-density-gradient centrifugation showed differences in the membrane profiles obtained. The results indicate that optimum separation of OM and CM components requires effective digestion of DNA in the total membrane preparation before density-gradient fractionation. Salmonella minnesota Re595 carrying the intermediate complement complex C5b-7 (BC1-7) or C5b-8 (BC1-8) were efficiently killed upon incubation with purified C8 + C9 or C9 respectively. Human-alpha-thrombin-cleaved C9 (C9n), which is unable to form tubular poly(C9), was shown to be more effective at killing than native C9. By using an optimized system for the separation of OM and CM, it was found that, subsequent to lethal complement attack, the CM could not be recovered when C9 was used as the terminal complement component, but was recovered with reduced yield when C9n replaced C9. The results show that inability to recover the CM on sucrose density gradients after complement attack may not be a consequence of an essential membrane damage event required for complement-mediated killing of Gram-negative bacteria.