Gram-negative Bacterial Outer Membrane Research Articles

A Novel Role for the Bactericidal/Permeability Increasing Protein in Interactions of Gram-Negative Bacterial Outer Membrane Blebs with Dendritic Cells

The bactericidal/permeability-increasing protein (BPI) is thought to play an important role in killing and clearance of Gram-negative bacteria and the neutralization of endotoxin. A possible role for BPI in clearance of cell-free endotoxin has also been suggested based on studies with purified endotoxin aggregates and blood monocytes. Because the interaction of BPI with cell-free endotoxin, during infection, occurs mainly in tissue and most likely in the form of shed bacterial outer membrane vesicles ("blebs"), we examined the effect of BPI on interactions of metabolically labeled ([(14)C]-acetate) blebs purified from Neisseria meningitidis serogroup B with either human monocyte-derived macrophages or monocyte-derived dendritic cells (MDDC). BPI produced a dose-dependent increase (up to 3-fold) in delivery of (14)C-labeled blebs to MDDC, but not to monocyte-derived macrophages in the presence or absence of serum. Both, fluorescently labeled blebs and BPI were internalized by MDDC under these conditions. The closely related LPS-binding protein, in contrast to BPI, did not increase association of the blebs with MDDC. BPI-enhanced delivery of the blebs to MDDC did not increase cell activation but permitted CD14-dependent signaling by the blebs as measured by changes in MDDC morphology, surface expression of CD80, CD83, CD86, and MHC class II and secretion of IL-8, RANTES, and IP-10. These findings suggest a novel role of BPI in the interaction of bacterial outer membrane vesicles with dendritic cells that may help link innate immune recognition of endotoxin to Ag delivery and presentation.

A group of Escherichia coli and Salmonella enterica O antigens sharing a common backbone structure

The O-antigen moiety of the LPS is one of the most variable cell surface components of the Gram-negative bacterial outer membrane. Variation is due to the presence of different sugars and sugar linkages. Here, it is reported that a group of Escherichia coli O serogroups (O17, O44, O73, O77 and O106), and the Salmonella enterica serogroup O : 6,14 (H), share a common four-sugar backbone O-subunit structure, and possess almost identical O-antigen gene clusters. Whereas the E. coli O77 antigen does not have any substitutions, the other O antigens in this group differ by the addition of one or two glucose side branches at various positions of the backbone. The O-antigen gene clusters for all members of the group encode only the proteins required for biosynthesis of the common four-sugar backbone. The identification of three genes within a putative prophage in the E. coli O44 genome is also reported; these genes are presumably involved in the glucosylation of the basic tetrasaccharide unit. This was confirmed by deletion of one of the genes, which encodes a putative glucosyltransferase. Structural analysis of the O antigen produced by the mutant strain demonstrated the absence of glucosylation. An O-antigen structure shared by five E. coli and one S. enterica serogroups, all of which have a long evolutionary history, suggests that the common backbone may be important for the survival of E. coli strains in the environment, or for their pathogenicity.

Bound To Shock: Protection from Lethal Endotoxemic Shock by a Novel, Nontoxic, Alkylpolyamine Lipopolysaccharide Sequestrant

Lipopolysaccharide (LPS), or endotoxin, a structural component of gram-negative bacterial outer membranes, plays a key role in the pathogenesis of septic shock, a syndrome of severe systemic inflammation which leads to multiple-system organ failure. Despite advances in antimicrobial chemotherapy, sepsis continues to be the commonest cause of death in the critically ill patient. This is attributable to the lack of therapeutic options that aim at limiting the exposure to the toxin and the prevention of subsequent downstream inflammatory processes. Polymyxin B (PMB), a peptide antibiotic, is a prototype small molecule that binds and neutralizes LPS toxicity. However, the antibiotic is too toxic for systemic use as an LPS sequestrant. Based on a nuclear magnetic resonance-derived model of polymyxin B-LPS complex, we had earlier identified the pharmacophore necessary for optimal recognition and neutralization of the toxin. Iterative cycles of pharmacophore-based ligand design and evaluation have yielded a synthetically easily accessible N(1),mono-alkyl-mono-homologated spermine derivative, DS-96. We have found that DS-96 binds LPS and neutralizes its toxicity with a potency indistinguishable from that of PMB in a wide range of in vitro assays, affords complete protection in a murine model of LPS-induced lethality, and is apparently nontoxic in vertebrate animal models.

Consequences of the expression of lipopolysaccharide-modifying enzymes for the efficacy and reactogenicity of whole-cell pertussis vaccines

Lipopolysaccharide is one of the major constituents of the Gram-negative bacterial outer membrane and is, due to its endotoxic activity, responsible for the relatively high reactogenicity of whole-cell vaccines. In addition, lipopolysaccharide has strong immune stimulating properties, which makes it, potentially, an interesting vaccine component. In a previous study, we have shown that expression of two lipopolysaccharide-modifying enzymes, i.e., PagP and PagL, modulates the endotoxic activity of the Gram-negative bacterium Bordetella pertussis, the causative agent of whooping cough. To assess the consequences of PagP and PagL expression on the efficacy and reactogenicity of whole-cell pertussis vaccines, we have immunised mice and challenged them intranasally with wild-type B. pertussis. Vaccine efficacy, B. pertussis-specific antibody responses, and cytokine profiles were evaluated. The results show that expression of PagL, but not of PagP, significantly increases vaccine efficacy without altering vaccine reactogenicity. Therefore, PagL-expressing B. pertussis strains may form a basis for the development of a new and safer whole-cell pertussis vaccine, as higher vaccine efficacies may allow a reduced vaccine dosage. These data show, for the first time, that lipopolysaccharide composition is an important determinant for the efficacy of whole-cell pertussis vaccines.

Lipopolysaccharide in bacterial chronic infection: Insights from Helicobacter pylori lipopolysaccharide and lipid A

Lipopolysaccharides are generally considered toxic components of the Gram-negative bacterial outer membrane with potent immunomodulating and immunostimulating properties, but their contribution to adaptation of a given bacterial species to its microbial niche is, however, predominantly overlooked. Helicobacter pylori, as a cause of long-term infection in the gastroduodenal tract, has been proposed as a model for investigating and understanding the dynamics of bacterial persistence and parasitism in chronic infections. This review examines the structure and properties of H. pylori lipopolysaccharide and its lipid A moiety, and the insights that have been gained into their contribution to chronic infection and pathogenesis, including evasion and dampening of innate immune responses.

Structure and reactivity of LpxD, the N -acyltransferase of lipid A biosynthesis

The external layer of the Gram-negative bacterial outer membrane is primarily composed of a protective, selectively permeable LPS. The biosynthesis of LPS relies on UDP-3-O-acyl-glucosamine N-acyltransferase (LpxD), which transfers 3-hydroxy-arachidic acid from acyl carrier protein to the 2' amine of UDP-3-O-myristoyl glucosamine in Chlamydia trachomatis. Our crystallographic study reveals that LpxD is a homotrimer, each subunit of which is constructed from a novel combination of an N-terminal uridine-binding domain, a core lipid-binding domain, and a C-terminal helical extension. Highly conserved residues dominate nucleotide binding. Phe-43 and Tyr-49 form pi-stacking interactions with uracil, and Asn-46 and His-284 form hydrogen bonds with the phosphate groups. These interactions place the glucosamine moiety at the catalytic center formed by two adjacent subunits. Here His-247 and His-284 contribute to a mechanism involving nucleophilic attack by the amine of one substrate on the carbonyl carbon of an acyl carrier protein thioester conjugate. Serendipitously, our study reveals a fatty acid (FA) binding groove near the catalytic center. MS elucidated the presence of a FA mixture binding to LpxD, with palmitic acid the most prevalent. The placement of UDP-N-acetylglucosamine and the FA provides details of N-acyltransferase ligand interactions and allows for a description of structure and reactivity at an early stage of LPS assembly.

Bioinformatic Analyses of Gram-Negative Bacterial OstA Outer Membrane Assembly Homologues

Bioinformatic Analyses of Gram-Negative Bacterial OstA Outer Membrane Assembly Homologues

Role of Free Radicals in Sepsis: Antioxidant Therapy

Severe sepsis leading to shock is the principal cause of death in intensive care units. It is a systemic inflammatory response caused by excessive secretion of pro-inflammatory mediators, such as tumor necrosis factor-alpha (TNFalpha) and reactive oxygen species (ROS), mainly induced by endotoxin (a major component of the Gram-negative bacterial outer membrane). Immune cells use ROS in order to support their functions and need adequate levels of antioxidant defenses to avoid harmful effects of an excessive ROS production. In addition, nitric oxide (NO) is thought to play a key role in the pathogenesis of sepsis and in the development of multiple organ failure. This article discusses the toxic effects of endotoxin, paying particular attention to cardiovascular damage. It continues by analysing the mechanism by which endotoxin is recognized by specific cells of the immune system, and the pathway leading to nuclear factor-kappaB (NF-kappaB) activation and pro-inflammatory gene transcription. In relation to this process, this review focuses on the involvement of reactive oxygen and nitrogen species. Finally, the protective role of antioxidants against homeostatic disturbances such as those caused by endotoxin toxicity, their potential clinical use and the effects on the redox state of the immune cells is discussed.

Microextraction of bacterial lipid A: easy and rapid method for mass spectrometric characterization

Endotoxins (lipopolysaccharides) are the main components of Gram-negative bacterial outer membranes. A quick and simple way to isolate their lipid region (lipid A) directly from whole bacterial cells was devised. This method using hot ammonium-isobutyrate solvent was applied to small quantities of cells and proved to be indispensable when a rapid characterization of lipid A structure by mass spectrometry was required. Biological activities of endotoxins are directly related to the lipid A structures, which vary greatly with cell growth conditions. This method is suitable for rough- and smooth-type bacteria and very efficient for screening variations in lipid A structures. Data are acquired in a few hours and avoid the use of phenol in extraction.

Dissemination of Lipid A Deacylases (PagL) among Gram-negative Bacteria: IDENTIFICATION OF ACTIVE-SITE HISTIDINE AND SERINE RESIDUES

Lipopolysaccharide (LPS) is one of the main constituents of the Gram-negative bacterial outer membrane. It usually consists of a highly variable O-antigen, a less variable core oligosaccharide, and a highly conserved lipid moiety, designated lipid A. Several bacteria are capable of modifying their lipid A architecture in response to external stimuli. The outer membrane-localized lipid A 3-O-deacylase, encoded by the pagL gene of Salmonella enterica serovar Typhimurium, removes the fatty acyl chain from the 3 position of lipid A. Although a similar activity was reported in some other Gram-negative bacteria, the corresponding genes could not be identified. Here, we describe the presence of pagL homologs in a variety of Gram-negative bacteria. Although the overall sequence similarity is rather low, a conserved domain could be distinguished in the C-terminal region. The activity of the Pseudomonas aeruginosa and Bordetella bronchiseptica pagL homologs was confirmed upon expression in Escherichia coli, which resulted in the removal of an R-3-hydroxymyristoyl group from lipid A. Upon deacylation by PagL, E. coli lipid A underwent another modification, which was the result of the activity of the endogenous palmitoyl transferase PagP. Furthermore, we identified a conserved histidine-serine couple as active site residues, suggesting a catalytic mechanism similar to serine hydrolases. The biological function of PagL remains unclear. However, because PagL homologs were found in both pathogenic and nonpathogenic species, PagL-mediated deacylation of lipid A probably does not have a dedicated role in pathogenicity.

Lipopolysaccharide microarrays for the detection of antibodies

Lipopolysaccharide (LPS) is the major component of Gram-negative bacterial outer membrane. LPS are immunogenic and show species/strain specificity. The demonstration of anti-LPS antibodies in clinical samples is of diagnostic value in certain Gram-negative bacterial infections. In the present study we explored the possibility of immobilizing LPS isolated from different bacteria in a microarray format for the detection of anti-LPS antibodies. LPS was successfully immobilized on nitrocellulose-coated glass slides, preserving the accessibility of epitopes for antibody binding. Specificity of the LPS arrays was established using four different monoclonal antibodies specific for Escherichia coli O111, E. coli O157, Francisella tularensis and Salmonella typhimurium O-antigens and a panel of LPS preparations. The detection limit of antibodies was found to be 10 ng/ml, which is about a 100-fold greater sensitivity compared to conventional immunofluorescence assays. Furthermore, using LPS arrays, tularemia positive canine serum samples could be differentiated from negative samples based on the presence of significantly higher levels of anti- F. tularensis LPS antibodies in positive samples. LPS arrays will facilitate simultaneous screening of samples against multiple antigens and are expected to find applications in diagnostics and seroepidemiology.

Polymyxin B: An ode to an old antidote for endotoxic shock

Endotoxic shock, a syndrome characterized by deranged hemodynamics, coagulation abnormalities, and multiple system organ failure is caused by the release into the circulation of lipopolysaccharide (LPS), the structurally diverse component of Gram-negative bacterial outer membranes, and is responsible for 60% mortality in humans. Polymyxin B (PMB), a cyclic, cationic peptide antibiotic, neutralizes endotoxin but induces severe side effects in the process. The potent endotoxin neutralizing ability of PMB, however, offers possibilities for designing non-toxic therapeutic agents for combating endotoxicosis. Amongst the numerous approaches for combating endotoxic shock, peptide mediated neutralization of LPS seems to be the most attractive one. The precise mode of binding of PMB to LPS and the structural features involved therein have been elucidated only recently using a variety of biophysical approaches. These suggest that efficient neutralization of endotoxin by PMB is not achieved by mere binding to LPS but requires its sequestration from the membrane. Incorporation of this feature into the design of endotoxin neutralizing peptides should lead to the development of effective antidotes for endotoxic shock.

Lipid composition and lipopolysaccharide binding capacity of lipoproteins in plasma and lymph of patients with systemic inflammatory response syndrome and multiple organ failure.

Lipopolysaccharide (LPS), the major glycolipid component of Gram-negative bacterial outer membranes, is a potent endotoxin responsible for many of the directly or indirectly induced symptoms of infection. Lipoproteins (in particular, high-density lipoproteins) sequester LPS, thereby acting as a humoral detoxification mechanism. Differences in the lipoprotein composition in human plasma and lymph of a control patient group (n = 5) without systemic inflammatory response syndrome (non-SIRS/MOF) and patients with SIRS and multiple organ failure (MOF, n = 9) were studied. The LPS binding capacity of the lipoproteins in SIRS/MOF and non-SIRS/MOF patients was investigated by rechallenge of the plasma and lymph with fluorescently labeled LPS ex vivo. The lipoprotein composition was analyzed using immunochemical techniques and high-performance gel permeation chromatography. In the non-SIRS/MOF patient group, plasma and lymph levels of apolipoprotein A-I (600 and 450 mg/L, respectively), apolipoprotein B (440 and 280 mg/L, respectively), total cholesterol (2.88 and 1.05 mM, respectively), and total triglycerides (0.67 and 0.97 mM, respectively) were observed. In the SIRS/MOF group, a decrease of apolipoprotein A-I (-55% in plasma and lymph), a decrease of apolipoprotein B (-43% in plasma and -38% in lymph), and a decrease of total cholesterol levels (-54% in plasma and -37% in lymph) were demonstrated. However, the triglyceride levels in the SIRS/MOF group showed a 30% increase in plasma and a 47% decrease in lymph compared with the non-SIRS/MOF patients. In SIRS/MOF patients, a 2.8-fold increase in plasma and a 1.8-fold increase in lymph of the LPS low-density lipoprotein/high-density lipoprotein ratio was observed, indicating that the relative LPS binding capacity of the lipoproteins in the SIRS/MOF patient group showed a trend to be shifted mainly toward low-density lipoproteins. Furthermore, in plasma and lymph of four SIRS/MOF patients, a novel cholesterol-containing high-density lipoprotein-like particle was found that barely had LPS binding capacity (<5%). In the SIRS/MOF patients, the changes in lipoprotein composition in lymph are a reflection of those in plasma, except for the triglyceride levels. In comparison with the non-SIRS/MOF patients, the SIRS/MOF patients show a shifted LPS binding capacity of high-density lipoproteins toward low-density lipoproteins in plasma and in lymph. Moreover, in plasma and lymph, novel cholesterol-containing particles, resembling high-density lipoprotein, were identified in the SIRS/MOF patient group.

Fighting the stranger—antioxidant protection against endotoxin toxicity

Septic shock is a serious problem in critically ill and surgical patients throughout the world. It is a systemic inflammatory response caused by excessive secretion of proinflammatory mediators, such as tumor necrosis factor-α, mainly induced by endotoxin, a major component of the Gram-negative bacterial outer membrane. Experimental evidence suggests that reactive oxygen species (ROS) may be important mediators of cellular injury during endotoxemia, either as a result of macromolecular damage or by interfering with extracellular and intracellular regulatory processes. In addition, nitric oxide is thought to play a key role in the pathogenesis of sepsis. This review begins with a brief overview of the toxic effects of endotoxin at organism level, paying particular attention to cardiovascular damage. It continues by analysing the mechanism by which endotoxin is recognized by specific cells of the immune system, which then respond to bacterial infection and the pathway leading to nuclear factor-κB activation and proinflammatory gene transcription. With regard to this process, the review focuses on the involvement of reactive oxygen and nitrogen species. Lastly, the protective role of antioxidants against endotoxin toxicity and their potential clinical use is discussed.

Distribution and kinetics of lipoprotein-bound endotoxin.

Lipopolysaccharide (LPS), the major glycolipid component of gram-negative bacterial outer membranes, is a potent endotoxin responsible for pathophysiological symptoms characteristic of infection. The observation that the majority of LPS is found in association with plasma lipoproteins has prompted the suggestion that sequestering of LPS by lipid particles may form an integral part of a humoral detoxification mechanism. Previous studies on the biological properties of isolated lipoproteins used differential ultracentrifugation to separate the major subclasses. To preserve the integrity of the lipoproteins, we have analyzed the LPS distribution, specificity, binding capacity, and kinetics of binding to lipoproteins in human whole blood or plasma by using high-performance gel permeation chromatography and fluorescent LPS of three different chemotypes. The average distribution of O111:B4, J5, or Re595 LPS in whole blood from 10 human volunteers was 60% (+/-8%) high-density lipoprotein (HDL), 25% (+/-7%) low-density lipoprotein, and 12% (+/-5%) very low density lipoprotein. The saturation capacity of lipoproteins for all three LPS chemotypes was in excess of 200 microg/ml. Kinetic analysis however, revealed a strict chemotype dependence. The binding of Re595 or J5 LPS was essentially complete within 10 min, and subsequent redistribution among the lipoprotein subclasses occurred to attain similar distributions as O111:B4 LPS at 40 min. We conclude that under simulated physiological conditions, the binding of LPS to lipoproteins is highly specific, HDL has the highest binding capacity for LPS, the saturation capacity of lipoproteins for endotoxin far exceeds the LPS concentrations measured in clinical situations, and the kinetics of LPS association with lipoproteins display chemotype-dependent differences.

Exchangeability of N Termini in the Ligand-gated Porins ofEscherichia coli

The ferric siderophore transporters of the Gram-negative bacterial outer membrane manifest a unique architecture: Their N termini fold into a globular domain that lodges within, and physically obstructs, a transmembrane porin beta-barrel formed by their C termini. We exchanged and deleted the N termini of two such siderophore receptors, FepA and FhuA, which recognize and transport ferric enterobactin and ferrichrome, respectively. The resultant chimeric proteins and empty beta-barrels avidly bound appropriate ligands, including iron complexes, protein toxins, and viruses. Thus, the ability to recognize and discriminate these molecules fully originates in the transmembrane beta-barrel domain. Both the hybrid and the deletion proteins also transported the ferric siderophore that they bound. The FepA constructs showed less transport activity than wild type receptor protein, but the FhuA constructs functioned with turnover numbers that were equivalent to wild type. The mutant proteins displayed the full range of transport functionalities, despite their aberrant or missing N termini, confirming (Braun, M., Killmann, H., and Braun, V. (1999) Mol. Microbiol. 33, 1037-1049) that the globular domain within the pore is dispensable to the siderophore internalization reaction, and when present, acts without specificity during solute uptake. These and other data suggest a transport process in which siderophore receptors undergo multiple conformational states that ultimately expel the N terminus from the channel concomitant with solute internalization.

Outer membrane protein A (OmpA), peptidoglycan-associated lipoprotein (PAL), and murein lipoprotein (MLP) are released in experimental Gram-negative sepsis

We previously showed that Escherichia coli bacteria incubated in normal human serum release complexes that contain three conserved Gram-negative bacterial outer membrane proteins (OMPs) and LPS. We have identified the OMPs as outer membrane protein A (OmpA), peptidoglycan-associated lipoprotein (PAL), and murein lipoprotein (MLP). These OMPs are conserved among enteric Gram-negative bacteria and are bound by IgG in antisera raised to heat-killed rough bacteria such as E. coli J5 (J5 IgG). The present experiments were performed to further analyze the release of these OMPs in a rat wound infection model of sepsis. Plasma was collected from thermally injured rats with E. coli O18 sepsis and filtered. LPS was affinity-purified from plasma filtrates using monoclonal antibody specific for the O-polysaccharide side chain of E. coli O18 LPS. Plasma filtrates were also incubated with J5 IgG conjugated to magnetic beads. Affinity-purified samples were analyzed for the OMPs by immunoblotting. OmpA, PAL, and MLP were released into septic rat blood in complexes with LPS. PAL was consistently present in samples affinity-purified using J5 IgG. The results indicate that OmpA, PAL, and MLP are released and circulate in experimental Gram-negative sepsis and suggest that a proportion of released OMPs are tightly associated with LPS.

Prevention and treatment of multiple organ dysfunction syndrome: lessons learned and future prospects.

Gram-negative bacteria commonly cause serious infections in hospitalized patients, and those that lead to bacteremic episodes and sepsis syndrome are associated with the highest mortality rate. Sepsis syndrome frequently progresses to multisystem organ dysfunction and failure, with as many as 400,000 cases occurring annually. Unfortunately, the associated mortality rate remains about 40%. Lipopolysaccharide (LPS, endotoxin), an integral component of the gram-negative bacterial outer membrane, plays a critical role in the pathophysiology of this lethal disease process. It is capable of interacting with host macrophages, a process that leads to the secretion of an increasingly well-characterized array of macrophage cytokines. Several different classes of compounds that bind directly to LPS and thereby neutralize its effects are being examined. These consist of anti-LPS monoclonal antibodies (mAbs), naturally occurring proteins and their derivatives (e.g., bactericidal/permeability-increasing protein [BPI], Limulus anti-LPS factor [LALF]), and certain antibiotics (polymyxin B, taurolidine). The molecular biology of BPI, LALF, and LPS binding protein (LBP, which augments the host response to LPS) is of considerable interest, as each demonstrates considerable genetic sequence homology. Although two anti-LPS monoclonal antibodies (HA-1A, E5) did not demonstrate efficacy during sepsis syndrome, information obtained from these clinical trials provided investigators with the ability to better understand this disease process. However, a detailed understanding of the biology of endotoxin antagonism is beginning to emerge, and the application of this knowledge in the clinical setting provides hope that it may be possible to reduce the mortality of sepsis syndrome caused by these microorganisms to a statistic well below the current 40%.

Permeability barrier of the Gram-negative bacterial outer membrane with special reference to nisin

The effect of nisin pretreatment on organic acid-induced permeability increase in strains of Escherichia coli, Pseudomonas aeruginosa, P. marginalis, and Salmonella enterica sv. Typhimurium was investigated, using assays based on the uptake of a fluorescent dye 1- N-phenylnaphthylamine (NPN) and on the bacterial susceptibility to detergent-induced bacteriolysis. The outer membrane of bacteria which had been pretreated with nisin was shown to be less stable against 1 mM EDTA, as indicated by their significantly higher NPN uptake levels as compared to untreated bacteria. Upon challenge with a tenfold lower concentration of EDTA (0.1 mM) some nisin-treated strains (Typhimurium, P. marginalis) exhibited, however, NPN uptake levels which were lower than those seen in control bacteria, suggesting that nisin had stabilized their outer membrane. Nisin pretreatment also decreased the NPN uptake induced by citric or lactic acid or both in E. coli, P. marginalis, and Typhimurium, whereas in P. aeruginosa the pretreatment resulted in increased NPN uptake in response to citric and lactic acid. These results suggest that, with the exception of P. aeruginosa, nisin could protect bacteria from the outer membrane-disrupting effect caused by the acids. P. aeruginosa was, however, shown to be protected against bacteriolysis induced by the detergents sodium dodecylsulfate and Triton X-100. With a pair of isogenic mutants of Typhimurium differing in their cell surface charge it was shown that the NPN uptake response to 1 mM EDTA of the abnormally cationic strain was not significantly affected by nisin, whereas in the normal anionic strain nisin strongly strengthened the uptake. Our hypothesis based on these findings is that the normally anionic cell surface of Gram-negative bacteria has a tendency to bind the cationic nisin. The binding of nisin to the surface does not proceed to the cytoplasmic membrane, but in the outer membrane the bound nisin actually stabilizes its structure through electrostatic interactions. With the exception of EDTA, the organic acids at pH 4 did not cause leakage of cell contents from Typhimurium, indicating that these acids do not permeabilize the outer membrane to an extent required for cytoplasmic pore formation by nisin.

Lactic acid permeabilizes gram-negative bacteria by disrupting the outer membrane.

The effect of lactic acid on the outer membrane permeability of Escherichia coli O157:H7, Pseudomonas aeruginosa, and Salmonella enterica serovar Typhimurium was studied utilizing a fluorescent-probe uptake assay and sensitization to bacteriolysis. For control purposes, similar assays were performed with EDTA (a permeabilizer acting by chelation) and with hydrochloric acid, the latter at pH values corresponding to those yielded by lactic acid, and also in the presence of KCN. Already 5 mM (pH 4.0) lactic acid caused prominent permeabilization in each species, the effect in the fluorescence assay being stronger than that of EDTA or HCl. Similar results were obtained in the presence of KCN, except for P. aeruginosa, for which an increase in the effect of HCl was observed in the presence of KCN. The permeabilization by lactic and hydrochloric acid was partly abolished by MgCl(2). Lactic acid sensitized E. coli and serovar Typhimurium to the lytic action of sodium dodecyl sulfate (SDS) more efficiently than did HCl, whereas both acids sensitized P. aeruginosa to SDS and to Triton X-100. P. aeruginosa was effectively sensitized to lysozyme by lactic acid and by HCl. Considerable proportions of lipopolysaccharide were liberated from serovar Typhimurium by these acids; analysis of liberated material by electrophoresis and by fatty acid analysis showed that lactic acid was more active than EDTA or HCl in liberating lipopolysaccharide from the outer membrane. Thus, lactic acid, in addition to its antimicrobial property due to the lowering of the pH, also functions as a permeabilizer of the gram-negative bacterial outer membrane and may act as a potentiator of the effects of other antimicrobial substances.