Size Exclusion Chromatography to Analyze Bacterial Outer Membrane Vesicle Heterogeneity

The cell wall of Gram-negative bacteria consists of an inner (cytoplasmic) and outer membrane (OM), separated by a thin peptidoglycan layer. Throughout ...

Lehigh University

The cholesterol recognition/interaction amino acid consensus (CRAC) motif is a primary structure pattern used to identify regions that may be responsible for preferential cholesterol binding in many proteins. The leukotoxin LtxA, which is produced by a pathogenic bacterium, contains two CRAC seqences, only one of which is responsible for cholesterol binding, and the binding is required for cytotoxicity. The factors, in addition to the CRAC definition, that may be responsible for cholesterol-binding functionality and atomistic interactions between the CRAC region and cholesterol are as yet unknown. This study uses molecular dynamics simulations to identify structural characteristics and specific interactions of the two LtxA CRAC peptides with both pure phospholipid and binary cholesterol-phospholipid bilayers. We have identified changes in the secondary structure of these peptides that occur upon cholesterol binding, which are not seen when it is associated with a cholesterol-devoid membrane, and which show salient coupling of structural disorder and function. Additionally, the central tyrosine residue of the CRAC motif was found to play a significant role in cholesterol binding, though residues outside of the CRAC motif also influence membrane interactions and functionality of the CRAC region. 

Disorder in cholesterol-binding functionality of CRAC peptides: a molecular dynamics study.

The leukotoxin (LtxA) produced by Aggregatibacter actinomycetemcomitans kills host immune cells, allowing the bacterium to establish an ecological niche in the upper aerodigestive tract of its human host. The interaction of LtxA with human immune cells is both complex and multifaceted, involving membrane lipids as well as cell-surface proteins. In the initial encounter with the host cell, LtxA associates with lymphocyte function-associated antigen-1, a cell surface adhesion glycoprotein. However, we have also demonstrated that the toxin associates strongly with the plasma membrane lipids, specifically cholesterol. This association with cholesterol is regulated by a cholesterol recognition amino acid consensus (CRAC) motif, with a sequence of (334) LEEYSKR(340), in the N-terminal region of the toxin. Here, we have demonstrated that removal of cholesterol from the plasma membrane or mutation of the LtxA CRAC motif inhibits the activity of the toxin in THP-1 cells. To inhibit LtxA activity, we designed a short peptide corresponding to the CRAC(336) motif of LtxA (CRAC(336WT)). This peptide binds to cholesterol and thereby inhibits the toxicity of LtxA in THP-1 cells. Previously, we showed that this peptide inhibits LtxA toxicity against Jn.9 (Jurkat) cells, indicating that peptides derived from the cholesterol-binding site of LtxA may have a potential clinical applicability in controlling infections of repeats-in-toxin-producing organisms.

Inhibition of LtxA toxicity by blocking cholesterol binding with peptides.

The repeats-in-toxin family of toxins includes proteins produced by Gram negative bacteria such as Escherichia coli (α-hemolysin), Bordetella pertussis (adenylate cyclase toxin), and Aggregatibacter actinomycetemcomitans (LtxA), which contribute to the pathogenesis of these organisms by killing host cells. In the case of LtxA produced by A. actinomycetemcomitans, white blood cells are targeted, allowing the bacteria to avoid clearance by the host immune system. In its association with target cells, LtxA binds to a receptor, lymphocyte function-associated antigen-1, as well as membrane lipids and cholesterol, before being internalized via a lysosomal-mediated pathway. The motivation for this project comes from our discovery that DRAQ5™, a membrane-permeable nuclear stain, prevents the internalization of LtxA in a Jurkat T cell line. We hypothesized that DRAQ5™, in crossing the plasma membrane, alters the properties of the membrane to inhibit LtxA internalization. To investigate how DRAQ5™ interacts with the lipid membrane to prevent LtxA internalization, we used studied DRAQ5™-mediated membrane changes in model membranes using a variety of techniques, including differential scanning calorimetry and fluorescence spectroscopy. Our results suggest that DRAQ5™ inhibits the activity of LtxA by decreasing the fluidity of the cellular lipid membrane, which decreases LtxA binding. These results present an interesting possible anti-virulence strategy; by altering bacterial toxin activity by modifying membrane fluidity, it may be possible to inhibit the pathogenicity of A. actinomycetemcomitans.

Inhibition of Bacterial Toxin Activity by the Nuclear Stain, DRAQ5™.

Recognition of and binding to cholesterol on the host cell membrane is an initial step in the mechanism of numerous pathogens, including viruses, bacteria, and bacterial toxins; however, a viable method of inhibiting this interaction has not yet been uncovered. Here, we describe the mechanism by which a cholesterol recognition amino acid consensus peptide interacts with cholesterol and inhibits the activity of a cholesterol-binding bacterial leukotoxin (LtxA). Using a series of biophysical techniques, we have shown that the peptide recognizes the hydroxyl group of cholesterol with nanomolar affinity and does not disrupt membrane packing, suggesting that it sits primarily near the membrane surface. As a result, LtxA is unable to bind to cholesterol or subsequently become internalized in host cells. Additionally, because cholesterol is not being removed from the cell membrane, the peptide-treated target cells remain viable over extended periods of time. We have demonstrated the use of this peptide in the inhibition of toxin activity for an antivirulence approach to the treatment of bacterial disease, and we anticipate that this approach might have broad utility in the inhibition of viral and bacterial pathogenesis.

Use of a Cholesterol Recognition Amino Acid Consensus Peptide To Inhibit Binding of a Bacterial Toxin to Cholesterol.

The primary virulence factor of Vibrio cholerae, cholera toxin (CT), initiates a pathway in epithelial cells that leads to the severe diarrhoea characteristic of cholera. Secreted CT binds to GM1 on the surface of host cells to facilitate internalisation. Many bacterial toxins, including CT, have been shown to be additionally delivered via outer membrane vesicles (OMVs). A fraction of the closely related heat labile toxin produced by enterotoxigenic Escherichia coli has been demonstrated to reside on the surface of OMVs, where it binds GM1 to facilitate OMV internalisation by host cells. In this work, we investigated whether OMV-associated CT is likewise trafficked to host cells in a GM1-dependent mechanism. We demonstrated that a majority of CT is secreted in its OMV-associated form and is located exclusively inside the vesicle. Therefore, the toxin is unable to bind GM1 on the host cell surface, and the OMVs are trafficked to the host cells in a GM1-independent mechanism. These findings point to a secondary, noncompeting mechanism for secretion and delivery of CT, beyond its well-studied secretion via a Type II secretion system and underscore the importance of focusing future studies on understanding this GM1-independent delivery mechanism to fully understand Vibrio cholerae pathogenesis.

Association of Vibrio cholerae 569B outer membrane vesicles with host cells occurs in a GM1-independent manner.

The Gram-negative bacterium Aggregatibacter actinomycetemcomitans, commonly associated with localized aggressive periodontitis (LAP), secretes an RTX (repeats-in-toxin) protein leukotoxin (LtxA) that targets human white blood cells, an interaction that is driven by its recognition of the lymphocyte function-associated antigen-1 (LFA-1) integrin. In this study, we report on the inhibition of LtxA-LFA-1 binding as an antivirulence strategy to inhibit LtxA-mediated cytotoxicity. Specifically, we designed and synthesized peptides corresponding to the reported LtxA binding domain on LFA-1 and characterized their capability to inhibit LtxA binding to LFA-1 and subsequent cytotoxic activity in human immune cells. We found that several of these peptides, corresponding to sequential β-strands in the LtxA-binding domain of LFA-1, inhibit LtxA activity, demonstrating the effectiveness of this approach. Further investigations into the mechanism by which these peptides inhibit LtxA binding to LFA-1 reveal a correlation between toxin-peptide affinity and LtxA-mediated cytotoxicity, leading to a diminished association between LtxA and LFA-1 on the cell membrane. Our results demonstrate the possibility of using target-based peptides to inhibit LtxA activity, and we expect that a similar approach could be used to hinder the activity of other RTX toxins.

Receptor-Based Peptides for Inhibition of Leukotoxin Activity.

A leukotoxin (LtxA) that is produced by Aggregatibacter actinomycetemcomitans (Aa) is an important virulence determinant in an aggressive form of periodontitis in adolescents. Understanding the function of this protein at the molecular level is critical to elucidating its role in the disease process. To accomplish genetic analysis of the protein structure and relating these observations to toxin function, we have developed an E. coli expression system for the generation and rapid purification of LtxA. Cloning the structural toxin gene, ltxA, from Aa strain JP2 under control of T7 promoter-1 of pCDFDuet-1 vector resulted in expression of a 114 KDa protein which could be easily purified by the presence of a carboxy-terminal engineered double hexahistidine (double-His) tag and was immunologically reactive with an anti-LtxA monoclonal antibody, but was not cytotoxic. Cloning a second gene, ltxC, an acyltransferase gene, into the vector under control of T7 promoter-2, resulted in expression of the biologically active LtxA. The toxin was extracted from E. coli inclusion bodies, purified by immobilized metal affinity chromatography, and refolded by dialysis. When compared by circular dichroism (CD) spectroscopy analysis, acylated recombinant LtxA has a secondary structure consistent with wt LtxA, while variations in α-helical structure of nonacylated LtxA were observed. No modifications in α-helix were found upon the toxin's binding with liposome-incorporated cholesterol. Our results suggest that pure, biologically active recombinant LtxA can be isolated by a one-step affinity chromatography from E. coli. The toxic and structural properties of the recombinant LtxA are similar to its wt counterpart.

Generation of a recombinant Aggregatibacter actinomycetemcomitans RTX toxin in Escherichia coli.

The Gram-negative bacterium,  has been associated with localized aggressive periodontitis (LAP). In particular, highly leukotoxic strains of  have been more closely associated with this disease, suggesting that LtxA is a key virulence factor for . LtxA is secreted across both the inner and outer membranes via the Type I secretion system, but has also been found to be enriched within outer membrane vesicles (OMVs), derived from the bacterial outer membrane. We have characterized the association of LtxA with OMVs produced by the highly leukotoxic strain, JP2, and investigated the interaction of these OMVs with host cells to understand how LtxA is delivered to host cells in this OMV-associated form. Our results demonstrated that a significant fraction of the secreted LtxA exists in an OMV-associated form. Furthermore, we have discovered that in this OMV-associated form, the toxin is trafficked to host cells by a cholesterol- and receptor-independent mechanism in contrast to the mechanism by which free LtxA is delivered. Because OMV-associated toxin is trafficked to host cells in an entirely different manner than free toxin, this study highlights the importance of studying both free and OMV-associated forms of LtxA to understand  virulence.

 Leukotoxin Is Delivered to Host Cells in an LFA-1-Indepdendent Manner When Associated with Outer Membrane Vesicles.

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.

Catechin-mediated restructuring of a bacterial toxin inhibits activity.

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.

Cytotoxic activity of Kingella kingae RtxA toxin depends on post-translational acylation of lysine residues and cholesterol binding.

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.

Inhibition of bacterial toxin recognition of membrane components as an anti-virulence strategy.

Cholera toxin (CT), the major virulence factor of , 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.

Cholera Toxin Encapsulated within Several  O1 Serotype Inaba Outer Membrane Vesicles Lacks a Functional B-Subunit.

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.

Epigallocatechin gallate inhibits leukotoxin release by Aggregatibacter actinomycetemcomitans by promoting association with the bacterial membrane.

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.

Angela C. Brown

Department of Chemical and Biomolecular Engineering

Lehigh University

Size Exclusion Chromatography to Analyze Bacterial Outer Membrane Vesicle Heterogeneity

Angela C. Brown

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Chemical and Biomolecular Engineering

Lehigh University

Size Exclusion Chromatography to Analyze Bacterial Outer Membrane Vesicle Heterogeneity

Department of Chemical and Biomolecular Engineering