ABSTRACT
Bacterial membrane vesicles (BMVs) are small, spherical structures released by Gram-positive and Gram-negative bacteria that play essential roles in intercellular communication, nutrient acquisition, and antibiotic resistance. BMVs typically range from 40 to 400 nm in diameter and contain a single membrane derived from the bacterial membrane, comprising proteins, lipids, nucleic acids, and other biomolecules. Notably, the molecules located on the surface of BMVs facilitate interactions with neighboring cells, including the transfer of functional genes, coordination of bacterial growth through quorum sensing, and delivery of toxins during infections. In addition, BMVs exhibit heterogeneity in their surface composition, which influences their interactions with host and bacterial cells. It is therefore essential to understand not just the composition of BMVs, but the localization of the molecules of interest, particularly those on the surface. In this chapter, we describe several methods that can be used to quantify and characterize the protein and nucleic acid composition, particularly on the surface of BMVs. We describe quantitative immunoblot and ELISA protocols that enable quantification of the concentration of a particular protein of interest. We also describe an enzymatic digestion protocol to determine whether the protein of interest is located on the surface or within the lumen of the BMV, as well as a nucleic acid staining procedure that enables quantification of dsDNA specifically located on the surface of the BMVs. Together, these tools provide a detailed analysis of the protein and nucleic acid composition of BMVs that can be further combined with various separation techniques to study variations within different populations.
Subject(s)
Cell Membrane , Cell Membrane/metabolism , Bacterial Proteins/metabolism , Enzyme-Linked Immunosorbent Assay/methods , Extracellular Vesicles/metabolism , Extracellular Vesicles/chemistry , Bacteria/metabolism , Bacteria/geneticsABSTRACT
The development of novel antibacterial agents that are effective against Gram-negative bacteria is limited primarily by transport issues. This class of bacteria maintains a complex cell envelope consisting of two membrane bilayers, preventing the passage of most antibiotics. These drugs must therefore pass through protein channels called porins; however, many antibiotics are too large to pass through porins, and a common mechanism of acquired resistance is down-regulation of porins. To overcome this transport limitation, we have proposed the use of outer membrane vesicles (OMVs), released by Gram-negative bacteria, which deliver cargo to other bacterial cells in a porin-independent manner. In this work, we systematically studied the ability to load fluoroquinolones into purified Escherichia coli OMVs using in vivo and in vitro passive loading methods, and active loading methods such as electroporation and sonication. We observed limited loading of all of the antibiotics using passive loading techniques; sonication and electroporation significantly increased the loading, with electroporation at low voltages (200 and 400V) resulting in the greatest encapsulation efficiencies. We also demonstrated that imipenem, a carbapenem antibiotic, can be readily loaded into OMVs, and its administration via OMVs increases the effectiveness of the drug against E. coli. Our results demonstrate that small molecule antibiotics can be readily incorporated into OMVs to create novel delivery vehicles to improve antibiotic activity.
ABSTRACT
Gram-negative bacteria produce outer membrane vesicles (OMVs) that play a critical role in cell-cell communication and virulence. OMVs have emerged as promising therapeutic agents for various biological applications such as vaccines and targeted drug delivery. However, the full potential of OMVs is currently constrained by inherent heterogeneities, such as size and cargo differences, and traditional ensemble assays are limited in their ability to reveal OMV heterogeneity. To overcome this issue, we devised an innovative approach enabling the identification of various characteristics of individual OMVs. This method, employing fluorescence microscopy, facilitates the detection of variations in size and surface markers. To demonstrate our method, we utilize the oral bacterium Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) which produces OMVs with a bimodal size distribution. As part of its virulence, A. actinomycetemcomitans secretes leukotoxin (LtxA) in two forms: soluble and surface associated with the OMVs. We observed a correlation between the size and toxin presence where larger OMVs were much more likely to possess LtxA compared to the smaller OMVs. In addition, we noted that, among the smallest OMVs (<100 nm diameter), the fractions that are toxin positive range from 0 to 30%, while the largest OMVs (>200 nm diameter) are between 70 and 100% toxin positive.
ABSTRACT
Antibiotic resistance has become an urgent threat to health care in recent years. The use of drug delivery systems provides advantages over conventional administration of antibiotics and can slow the development of antibiotic resistance. In the current study, we developed a toxin-triggered liposomal antibiotic delivery system, in which the drug release is enabled by the leukotoxin (LtxA) produced by the Gram-negative pathogen, Aggregatibacter actinomycetemcomitans. LtxA has previously been shown to mediate membrane disruption by promoting a lipid phase change in nonlamellar lipids, such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-methyl (N-methyl-DOPE). In addition, LtxA has been observed to bind strongly and nearly irreversibly to membranes containing large amounts of cholesterol. Here, we designed a liposomal delivery system composed of N-methyl-DOPE and cholesterol to take advantage of these interactions. Specifically, we hypothesized that liposomes composed of N-methyl-DOPE and cholesterol, encapsulating antibiotics, would be sensitive to LtxA, enabling controlled antibiotic release. We observed that liposomes composed of N-methyl-DOPE were sensitive to the presence of low concentrations of LtxA, and cholesterol increased the extent and kinetics of content release. The liposomes were stable under various storage conditions for at least 7 days. Finally, we showed that antibiotic release occurs selectively in the presence of an LtxA-producing strain of A. actinomycetemcomitans but not in the presence of a non-LtxA-expressing strain. Together, these results demonstrate that the designed liposomal vehicle enables toxin-triggered delivery of antibiotics to LtxA-producing strains of A. actinomycetemcomitans.
Subject(s)
Aggregatibacter actinomycetemcomitans , Anti-Bacterial Agents , Liposomes , Liposomes/chemistry , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry , Aggregatibacter actinomycetemcomitans/drug effects , Delayed-Action Preparations/chemistry , Delayed-Action Preparations/pharmacology , Drug Liberation , Cholesterol/chemistry , Cholesterol/metabolism , Microbial Sensitivity Tests , Exotoxins/metabolism , Exotoxins/chemistry , Phosphatidylethanolamines/chemistry , Drug Delivery SystemsABSTRACT
Aggregatibacter actinomycetemcomitans is a Gram-negative bacterium associated with localized aggressive periodontitis as well as some systemic diseases. The strains of A. actinomycetemcomitans most closely associated with disease produce more of a secreted leukotoxin (LtxA) than isolates from healthy carriers, suggesting a key role for this toxin in disease progression. LtxA is released into the bacterial cytosol in a free form as well as in association with the surface of outer membrane vesicles (OMVs). We previously observed that the highly leukotoxic A. actinomycetemcomitans strain JP2 produces two populations of OMVs: a highly abundant population of small (<100 nm) OMVs and a less abundant population of large (>300 nm) OMVs. Here, we have developed a protocol to isolate the OMVs produced during each specific phase of growth and used this to demonstrate that small OMVs are produced throughout growth and lack LtxA, while large OMVs are produced only during the exponential phase and are enriched with LtxA. Our results indicate that surface-associated DNA drives the selective sorting of LtxA into large OMVs. This study provides valuable insights into the observed heterogeneity of A. actinomycetemcomitans vesicles and emphasizes the importance of understanding these variations in the context of bacterial pathogenesis.
Subject(s)
Aggregatibacter actinomycetemcomitans , Toxins, Biological , Cytosol , Biological Transport , Cell MovementABSTRACT
Antibiotic resistance has become an urgent threat to health care in recent years. The use of drug delivery systems provides advantages over conventional administration of antibiotics and can slow the development of antibiotic resistance. In the current study, we developed a toxin-triggered liposomal antibiotic delivery system, in which the drug release is enabled by the leukotoxin (LtxA) produced by the Gram-negative pathogen, Aggregatibacter actinomycetemcomitans. LtxA has previously been shown to mediate membrane disruption by promoting a lipid phase change in nonlamellar lipids, such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-methyl (N-methyl-DOPE). In addition, LtxA has been observed to bind strongly and nearly irreversibly to membranes containing large amounts of cholesterol. Here, we designed a liposomal delivery system composed of N-methyl-DOPE and cholesterol to take advantage of these interactions. Specifically, we hypothesized that liposomes composed of N-methyl-DOPE and cholesterol, encapsulating antibiotics, would be sensitive to LtxA, enabling controlled antibiotic release. We observed that liposomes composed of N-methyl-DOPE were sensitive to the presence of low concentrations of LtxA, and cholesterol increased the extent and kinetics of content release. The liposomes were stable under various storage conditions for at least 7 days. Finally, we showed that antibiotic release occurs selectively in the presence of an LtxA-producing strain of A. actinomycetemcomitans but not in the presence of a non-LtxA-expressing strain. Together, these results demonstrate that the designed liposomal vehicle enables toxin-triggered delivery of antibiotics to LtxA-producing strains of A. actinomycetemcomitans.
ABSTRACT
Gram-negative bacteria produce outer membrane vesicles (OMVs) that play a critical role in cell-cell communication and virulence. Despite being isolated from a single population of bacteria, OMVs can exhibit heterogeneous size and toxin content, which can be obscured by assays that measure ensemble properties. To address this issue, we utilize fluorescence imaging of individual OMVs to reveal size-dependent toxin sorting. Our results showed that the oral bacterium Aggregatibacter actinomycetemcomitans (A.a.) produces OMVs with a bimodal size distribution, where larger OMVs were much more likely to possess leukotoxin (LtxA). Among the smallest OMVs (< 100 nm diameter), the fraction that are toxin positive ranges from 0-30%, while the largest OMVs (> 200 nm diameter) are between 70-100% toxin positive. Our single OMV imaging method provides a non-invasive way to observe OMV surface heterogeneity at the nanoscale level and determine size-based heterogeneities without the need for OMV fraction separation.
ABSTRACT
The development of high quality, non-toxic (i.e., heavy-metal-free), and functional quantum dots (QDs) via 'green' and scalable synthesis routes is critical for realizing truly sustainable QD-based solutions to diverse technological challenges. Herein, we demonstrate the low-temperature all-aqueous-phase synthesis of silver indium sulfide/zinc (AIS/Zn) QDs with a process initiated by the biomineralization of highly crystalline indium sulfide nanocrystals, and followed by the sequential staging of Ag+ cation exchange and Zn2+ addition directly within the biomineralization media without any intermediate product purification. Therein, we exploit solution phase cation concentration, the duration of incubation in the presence of In2S3 precursor nanocrystals, and the subsequent addition of Zn2+ as facile handles under biomineralization conditions for controlling QD composition, tuning optical properties, and improving the photoluminescence quantum yield of the AIS/Zn product. We demonstrate how engineering biomineralization for the synthesis of intrinsically hydrophilic and thus readily functionalizable AIS/Zn QDs with a quantum yield of 18% offers a 'green' and non-toxic materials platform for targeted bioimaging in sensitive cellular systems. Ultimately, the decoupling of synthetic steps helps unravel the complexities of ion exchange-based synthesis within the biomineralization platform, enabling its adaptation for the sustainable synthesis of 'green', compositionally diverse QDs.
Subject(s)
Quantum Dots , Biomineralization , Cations , Indium/chemistry , Quantum Dots/chemistry , Sulfides/chemistry , Temperature , Water/chemistry , Zinc/chemistryABSTRACT
Bacterial outer membrane vesicles (OMVs) are nanometer-scale, spherical vehicles released by Gram-negative bacteria into their surroundings throughout growth. These OMVs have been demonstrated to play key roles in pathogenesis by delivering certain biomolecules to host cells, including toxins and other virulence factors. In addition, this biomolecular delivery function enables OMVs to facilitate intra-bacterial communication processes, such as quorum sensing and horizontal gene transfer. The unique ability of OMVs to deliver large biomolecules across the complex Gram-negative cell envelope has inspired the use of OMVs as antibiotic delivery vehicles to overcome transport limitations. In this review, we describe the advantages, applications, and biotechnological challenges of using OMVs as antibiotic delivery vehicles, studying both natural and engineered antibiotic applications of OMVs. We argue that OMVs hold great promise as antibiotic delivery vehicles, an urgently needed application to combat the growing threat of antibiotic resistance.
Subject(s)
Anti-Bacterial Agents/administration & dosage , Bacterial Outer Membrane/metabolism , Drug Carriers , Extracellular Vesicles/metabolism , Gram-Negative Aerobic Bacteria/metabolism , Animals , Anti-Bacterial Agents/metabolism , Drug Compounding , Extracellular Vesicles/genetics , Gram-Negative Aerobic Bacteria/genetics , HumansABSTRACT
Analytical characterization of small biological particles, such as extracellular vesicles (EVs), is complicated by their extreme heterogeneity in size, lipid, membrane protein, and cargo composition. Analysis of individual particles is essential for illuminating particle property distributions that are obscured by ensemble measurements. To enable high-throughput analysis of individual particles, liftoff nanocontact printing (LNCP) is used to define hexagonal antibody and toxin arrays that have a 425 nm dot size, on average, and 700 nm periodicity. The LNCP process is rapid, simple, and does not require access to specialized nanofabrication tools. These densely packed, highly ordered arrays are used to capture liposomes and bacterial outer membrane vesicles on the basis of their surface biomarkers, with a maximum of one particle per array dot, resulting in densely packed arrays of particles. Despite the high particle density, the underlying antibody or toxin array ensured that neighboring individual particles are optically resolvable. Provided target particle biomarkers and suitable capture molecules are identified, this approach can be used to generate high density arrays of a wide variety of small biological particles, including other types of EVs like exosomes.
Subject(s)
Exosomes , Extracellular Vesicles , Bacterial Outer Membrane , Lipids , LiposomesABSTRACT
Tea is the second most commonly consumed beverage worldwide. Along with its aromatic and delicate flavors that make it an enjoyable beverage, studies report numerous health advantages in tea consumption, including applications in antimicrobial therapy. The antimicrobial properties of tea are related to catechin and its derivatives, which are natural flavonoids that are abundant in tea. Increasing evidence from in vitro studies demonstrated antimicrobial effects of catechins on both gram-positive and gram-negative bacteria, and proposed direct and indirect therapeutic mechanisms. Additionally, catechins were reported to be effective anti-virulence agents. Furthermore, a number of studies presented evidence that catechins display synergistic effects with certain antibiotics, thus potentiating the activity of antibiotics in resistant bacteria. Despite their numerous beneficial properties, catechins face many challenges in their development as therapeutic agents, including poor absorption, low bioavailability, and rapid degradation. The introduction of nanobiotechnology provides target-based and stable delivery, which enhances catechin bioavailability and optimizes drug efficacy. As further research continues to focus on overcoming the unresolved challenges, catechins are likely to see additional promising applications in our continual fight against bacterial infections.
ABSTRACT
The cell wall of Gram-negative bacteria consists of an inner (cytoplasmic) and outer membrane (OM), separated by a thin peptidoglycan layer. Throughout growth, the outer membrane can bleb to form spherical outer membrane vesicles (OMVs). These OMVs are involved in numerous cellular functions including cargo delivery to host cells and communication with bacterial cells. Recently, the therapeutic potential of OMVs has begun to be explored, including their use as vaccines and drug delivery vehicles. Although OMVs are derived from the OM, it has long been appreciated that the lipid and protein cargo of the OMV differs, often significantly, from that of the OM. More recently, evidence that bacteria can release multiple types of OMVs has been discovered, and evidence exists that size can impact the mechanism of their uptake by host cells. However, studies in this area are limited by difficulties in efficiently separating the heterogeneously sized OMVs. Density gradient centrifugation (DGC) has traditionally been used for this purpose; however, this technique is time-consuming and difficult to scale-up. Size exclusion chromatography (SEC), on the other hand, is less cumbersome and lends itself to the necessary future scale-up for therapeutic use of OMVs. Here, we describe a SEC approach that enables reproducible separation of heterogeneously sized vesicles, using as a test case, OMVs produced by Aggregatibacter actinomycetemcomitans, which range in diameter from less than 150 nm to greater than 350 nm. We demonstrate separation of "large" (350 nm) OMVs and "small" (<150 nm) OMVs, verified by dynamic light scattering (DLS). We recommend SEC-based techniques over DGC-based techniques for separation of heterogeneously sized vesicles due to its ease of use, reproducibility (including user-to-user), and possibility for scale-up.
Subject(s)
Bacterial Outer Membrane Proteins/metabolism , Chromatography, Gel/methods , Genetic Heterogeneity , Reproducibility of ResultsABSTRACT
OBJECTIVES: We and others have previously shown that epigallocatechin gallate (EGCg) inhibits the activity of an important virulence factor, leukotoxin (LtxA), produced by the oral bacterium Aggregatibacter actinomycetemcomitans, suggesting the potential use of this molecule as an anti-virulence strategy to treat periodontal infections. Here, we sought to better understand the effects of EGCg on toxin secretion and A. actinomycetemcomitans pathogenicity in a co-culture model. METHODS: We used a quantitative immunoblot assay to determine the concentrations of LtxA in the bacterial supernatant and on the bacterial cell surface. Using a co-culture model, consisting of A. actinomycetemcomitans and THP-1 cells, we studied the impact of EGCg-mediated changes in LtxA secretion on the toxicity of A. actinomycetemcomitans. KEY FINDINGS: EGCg increased production of LtxA and changed the localization of secreted LtxA from the supernatant to the surface of the bacterial cells. In the co-culture model, a single low dose of EGCg did not protect host THP-1 cells from A. actinomycetemcomitans-mediated cytotoxicity, but a multiple dosing strategy had improved effects. CONCLUSIONS: Together, these results demonstrate that EGCg has important, but complicated, effects on toxin secretion and activity; new dosing strategies and comprehensive model systems may be required to properly develop these anti-virulence activities.
Subject(s)
Aggregatibacter actinomycetemcomitans , Catechin/analogs & derivatives , Exotoxins , Periodontitis , Aggregatibacter actinomycetemcomitans/drug effects , Aggregatibacter actinomycetemcomitans/metabolism , Aggregatibacter actinomycetemcomitans/pathogenicity , Anti-Bacterial Agents/pharmacology , Bacterial Toxins/antagonists & inhibitors , Bacterial Toxins/metabolism , Catechin/pharmacology , Coculture Techniques/methods , Dose-Response Relationship, Drug , Exotoxins/antagonists & inhibitors , Exotoxins/metabolism , Humans , Periodontitis/drug therapy , Periodontitis/microbiology , Virulence/drug effectsABSTRACT
Aggregatibacter actinomycetemcomitans is a Gram-negative bacterium associated with localized aggressive periodontitis, as well as other systemic diseases. This organism produces a number of virulence factors, all of which provide some advantage to the bacterium. Several studies have demonstrated that clinical isolates from diseased patients, particularly those of African descent, frequently belong to specific clones of A. actinomycetemcomitans that produce significantly higher amounts of a protein exotoxin belonging to the repeats-in-toxin (RTX) family, leukotoxin (LtxA), whereas isolates from healthy patients harbor minimally leukotoxic strains. This finding suggests that LtxA might play a key role in A. actinomycetemcomitans pathogenicity. Because of this correlation, much work over the past 30 years has been focused on understanding the mechanisms by which LtxA interacts with and kills host cells. In this article, we review those findings, highlight the remaining open questions, and demonstrate how knowledge of these mechanisms, particularly the toxin's interactions with lymphocyte function-associated antigen-1 (LFA-1) and cholesterol, enables the design of targeted anti-LtxA strategies to prevent/treat disease.
Subject(s)
Aggregatibacter actinomycetemcomitans , Exotoxins , Humans , Lymphocyte Function-Associated Antigen-1 , Virulence FactorsABSTRACT
The oral pathogen, Aggregatibacter actinomycetemcomitans, produces a number of virulence factors, including a leukotoxin (LtxA), which specifically kills human white blood cells, to provide a colonization advantage to the bacterium. Strains of A. actinomycetemcomitans that produce more LtxA have been more closely linked to disease, indicating that this toxin plays a key role in pathogenesis of the bacterium. Disruption of the activity of LtxA thus represents a promising approach to reducing the pathogenicity of the bacterium. Catechins are polyphenolic molecules derived from plants, which have shown potent antibacterial and antitoxin activities. We have previously shown that galloylated catechins are able to prevent LtxA delivery to host cells by altering the toxin's secondary structure and preventing binding to cholesterol on the host cell membrane. Here, we have investigated how one particular galloylated catechin, epigallocatechin gallate (EGCg), affects A. actinomycetemcomitans growth and toxin secretion. Our results demonstrate that EGCg, at micromolar concentrations, inhibits A. actinomycetemcomitans growth, as has been reported for other bacterial species. At subinhibitory concentrations, EGCg promotes LtxA production, but the toxicity of the bacterial supernatant against human immune cells is reduced. The results of our biophysical studies indicate that this seemingly contradictory result is caused by an EGCg-mediated enhancement of LtxA affinity for the bacterial cell surface. Together, these results demonstrate the potential of EGCg in the treatment of virulent A. actinomycetemcomitans infections.
Subject(s)
Bacterial Outer Membrane , Aggregatibacter actinomycetemcomitans , Animals , Bacteria , Catechin/analogs & derivatives , Catechin/pharmacology , Exotoxins , Humans , MiceABSTRACT
Cholera toxin (CT), the major virulence factor of Vibrio cholerae, is an AB5 toxin secreted through the type II secretion system (T2SS). Upon secretion, the toxin initiates endocytosis through the interaction of the B pentamer with the GM1 ganglioside receptor on small intestinal cells. In addition to the release of CT in the free form, the bacteria secrete CT in association with outer membrane vesicles (OMVs). Previously, we demonstrated that strain 569B releases OMVs that encapsulate CT and which interact with host cells in a GM1-independent mechanism. Here, we have demonstrated that OMV-encapsulated CT, while biologically active, does not exist in an AB5 form; rather, the OMVs encapsulate two enzymatic A-subunit (CTA) polypeptides. We further investigated the assembly and secretion of the periplasmic CT and found that a major fraction of periplasmic CTA does not participate in the CT assembly process and instead is continuously encapsulated within the OMVs. Additionally, we found that the encapsulation of CTA fragments in OMVs is conserved among several Inaba O1 strains. We further found that under conditions in which the amount of extracellularly secreted CT increases, the concentration of OMV-encapsulated likewise CTA increases. These results point to a secondary mechanism for the secretion of biologically active CT that does not depend on the CTB-GM1 interaction for endocytosis.
Subject(s)
Bacterial Outer Membrane/metabolism , Cholera Toxin/metabolism , Extracellular Vesicles/metabolism , Vibrio cholerae O1/metabolism , Cell Line , G(M1) Ganglioside/metabolism , Humans , Osmolar Concentration , Protein Subunits/metabolism , Serogroup , Sodium Chloride/pharmacology , Vibrio cholerae O1/drug effectsABSTRACT
Over recent years, the development of new antibiotics has not kept pace with the rate at which bacteria develop resistance to these drugs. For this reason, many research groups have begun to design and study alternative therapeutics, including molecules to specifically inhibit the virulence of pathogenic bacteria. Because many of these pathogenic bacteria release protein toxins, which cause or exacerbate disease, inhibition of the activity of bacterial toxins is a promising anti-virulence strategy. In this review, we describe several approaches to inhibit the initial interactions of bacterial toxins with host cell membrane components. The mechanisms by which toxins interact with the host cell membrane components have been well-studied over the years, leading to the identification of therapeutic targets, which have been exploited in the work described here. We review efforts to inhibit binding to protein receptors and essential membrane lipid components, complex assembly, and pore formation. Although none of these molecules have yet been demonstrated in clinical trials, the in vitro and in vivo results presented here demonstrate their promise as novel alternatives and/or complements to traditional antibiotics.
ABSTRACT
BACKGROUND: Catechins, polyphenols derived from tea leaves, have been shown to have antibacterial properties, through direct killing of bacteria as well as through inhibition of bacterial toxin activity. In particular, certain catechins have been shown to have bactericidal effects on the oral bacterium, Aggregatibacter actinomycetemcomitans, as well as the ability to inhibit a key virulence factor of this organism, leukotoxin (LtxA). The mechanism of catechin-mediated inhibition of LtxA has not been shown. METHODS: In this work, we studied the ability of six catechins to inhibit LtxA-mediated cytotoxicity in human white blood cells, using Trypan blue staining, and investigated the mechanism of action using a combination of techniques, including fluorescence and circular dichroism spectroscopy, confocal microscopy, and surface plasmon resonance. RESULTS: We found that all the catechins except (-)-catechin inhibited the activity of this protein, with the galloylated catechins having the strongest effect. Pre-incubation of the toxin with the catechins increased the inhibitory action, indicating that the catechins act on the protein, rather than the cell. The secondary structure of LtxA was dramatically altered in the presence of catechin, which resulted in an inhibition of toxin binding to cholesterol, an important initial step in the cytotoxic mechanism of the toxin. CONCLUSIONS: These results demonstrate that the catechins inhibit LtxA activity by altering its structure to prevent interaction with specific molecules present on the host cell surface. GENERAL SIGNIFICANCE: Galloylated catechins modify protein toxin structure, inhibiting the toxin from binding to the requisite molecules on the host cell surface.
Subject(s)
Aggregatibacter actinomycetemcomitans/chemistry , Bacterial Toxins/chemistry , Catechin/chemistry , Cholesterol/chemistry , Exotoxins/chemistry , Leukocytes/microbiology , Cell Membrane/metabolism , Cell Survival , Circular Dichroism , Humans , Leukocytes/metabolism , Membrane Fluidity , Microscopy, Confocal , Periodontitis/therapy , Protein Structure, Secondary , Surface Plasmon Resonance , THP-1 CellsABSTRACT
Repeats-in-toxin leukotoxin (LtxA) produced by the oral bacterium Aggregatibacter actinomycetemcomitans kills human leukocytes in a lymphocyte function-associated antigen 1 (LFA-1, integrin αL /ß2 )-dependent manner, although the mechanism for this interaction has not been identified. The LtxA internalisation by LFA-1-expressing cells was explored with florescence resonance energy transfer (FRET) microscopy using a cell line that expresses LFA-1 with a cyan fluorescent protein-tagged cytosolic αL domain and a yellow fluorescent protein-tagged ß2 domain. Phorbol 12-myristate 13-acetate activation of LFA-1 caused transient cytosolic domain separation. However, addition of LtxA resulted in an increase in FRET, indicating that LtxA brings the cytosolic domains closer together, compared with the inactive state. Unlike activation, this effect was not transient, lasting more than 30 min. Equilibrium constants of LtxA binding to the cytoplasmic domains of both αL and ß2 were determined using surface plasmon resonance. LtxA has a strong affinity for the cytosolic domains of both the αL and ß2 subunits (Kd = 15 and 4.2 nM, respectively) and a significantly lower affinity for the cytoplasmic domains of other integrin αM , αX , and ß3 subunits (Kd = 400, 180, and 230 nM, respectively), used as controls. Peptide fragments of αL and ß2 show that LtxA binds membrane-proximal domain of αL and intermediate domain of ß2 .
Subject(s)
Aggregatibacter actinomycetemcomitans/immunology , Exotoxins/metabolism , Host-Pathogen Interactions , Immunosuppressive Agents/metabolism , Lymphocyte Function-Associated Antigen-1/metabolism , Fluorescence Resonance Energy Transfer , Humans , Jurkat Cells , Microscopy, Fluorescence , Protein BindingABSTRACT
Kingella kingae is a member of the commensal oropharyngeal flora of young children. Improvements in detection methods have led to the recognition of K. kingae as an emerging pathogen that frequently causes osteoarticular infections in children and a severe form of infective endocarditis in children and adults. Kingella kingae secretes a membrane-damaging RTX (Repeat in ToXin) toxin, RtxA, which is implicated in the development of clinical infections. However, the mechanism by which RtxA recognizes and kills host cells is largely unexplored. To facilitate structure-function studies of RtxA, we have developed a procedure for the overproduction and purification of milligram amounts of biologically active recombinant RtxA. Mass spectrometry analysis revealed the activation of RtxA by post-translational fatty acyl modification on the lysine residues 558 and/or 689 by the fatty-acyltransferase RtxC. Acylated RtxA was toxic to various human cells in a calcium-dependent manner and possessed pore-forming activity in planar lipid bilayers. Using various biochemical and biophysical approaches, we demonstrated that cholesterol facilitates the interaction of RtxA with artificial and cell membranes. The results of analyses using RtxA mutant variants suggested that the interaction between the toxin and cholesterol occurs via two cholesterol recognition/interaction amino acid consensus motifs located in the C-terminal portion of the pore-forming domain of the toxin. Based on our observations, we conclude that the cytotoxic activity of RtxA depends on post-translational acylation of the K558 and/or K689 residues and on the toxin binding to cholesterol in the membrane.