Purification of secreted leukotoxin (LtxA) from Actinobacillus actinomycetemcomitans

The RTX (repeats in toxin) family of toxins is important in the pathogenesis of many Gram-negative bacteria. The oral and systemic human pathogen Actinobacillus actinomycetemcomitans produces a member of this family known as leukotoxin (LtxA). Previously, we found that LtxA is secreted into culture supernatants of A. actinomycetemcomitans and that this protein is abundant and relatively pure. Here, we report a large-scale method for the isolation and purification of LtxA from culture supernatants of A. actinomycetemcomitans strain JP2. The purification scheme involves ammonium sulfate precipitation of culture supernatants, dialysis, and ultrafiltration to concentrate LtxA to approximately 10mg/ml. We found that LtxA remained soluble in buffer that contained at least 250mM NaCl. Purified LtxA was >98% pure and the final preparations were active against HL-60 cells. The entire purification protocol can be completed within 2 days. The ability to readily obtain a large amount of purified leukotoxin should accelerate investigations into the structure and biology of this important virulence factor.     

tfoX (sxy)-dependent transformation of Aggregatibacter (Actinobacillus) actinomycetemcomitans

tfoX (sxy) is a regulatory gene needed to turn on competence genes. Aggregatibacter (Actinobacillus) actinomycetemcomitans has a tfoX gene that is important for transformation. We cloned this gene on an IncQ plasmid downstream of the inducible tac promoter. When this plasmid was resident in cells of A. actinomycetemcomitans and tfoX was induced, the cells became competent for transformation. Several strains of A. actinomycetemcomitans, including different serotypes, as well as rough (adherent) and isogenic smooth (nonadherent) forms were tested. Only our two serotype f strains failed to be transformed. With the other strains, we could easily get transformants with extrachromosomal plasmid DNA when closed circular, replicative plasmid carrying an uptake signal sequence (USS) was used. When a replicative plasmid carrying a USS and cloned DNA from the chromosome of A. actinomycetemcomitans was linearized by digestion with a restriction endonuclease or when genomic DNA was used directly, the outcome was allelic exchange. To facilitate allelic exchange, we constructed a suicide plasmid (pMB78) that does not replicate in A. actinomycetemcomitans and carries a region with two inverted copies of a USS. This vector gave allelic exchange in the presence of cloned and induced tfoX easily and without digestion. Using transposon insertions in cloned katA DNA, we found that as little as 78 bp of homology at one of the ends was sufficient for that end to participate in allelic exchange. The cloning and induction of tfoX makes it possible to transform nearly any strain of A. actinomycetemcomitans, and allelic exchange has proven to be important for site-directed mutagenesis.     

Aggregatibacter actinomycetemcomitans leukotoxin cytotoxicity occurs through bilayer destabilization

The Gram-negative bacterium, Aggregatibacter actinomycetemcomitans, is a common inhabitant of the human upper aerodigestive tract. The organism produces an RTX (Repeats in ToXin) toxin (LtxA) that kills human white blood cells. LtxA is believed to be a membrane-damaging toxin, but details of the cell surface interaction for this and several other RTX toxins have yet to be elucidated. Initial morphological studies suggested that LtxA was bending the target cell membrane. Because the ability of a membrane to bend is a function of its lipid composition, we assessed the proficiency of LtxA to release of a fluorescent dye from a panel of liposomes composed of various lipids. Liposomes composed of lipids that form nonlamellar phases were susceptible to LtxA-induced damage while liposomes composed of lipids that do not form non-bilayer structures were not. Differential scanning calorimetry demonstrated that the toxin decreased the temperature at which the lipid transitions from a bilayer to a nonlamellar phase, while (31) P nuclear magnetic resonance studies showed that the LtxA-induced transition from a bilayer to an inverted hexagonal phase occurs through the formation of an isotropic intermediate phase. These results indicate that LtxA cytotoxicity occurs through a process of membrane destabilization.     

Identification and expression of the Actinobacillus actinomycetemcomitans leukotoxin gene

The leukotoxin produced by the oral bacterium Actinobacillus actinomycetemcomitans has been implicated in the pathogenesis of juvenile periodontitis. In order to elucidate the structure of the leukotoxin, molecular cloning of the leukotoxin gene was carried out. A DNA library of A. actinomycetemcomitans, strain JP2, was constructed by partial digestion of genomic DNA with Sau3AI and ligation of 0.5 to 5.0 kilobase pair fragments into the Bam HI site of the plasmid vector pENN-vrf. After transformation into E. coli RR1 (lambda cI857), the clones were screened for the production of A. actinomycetemcomitans leukotoxin with polyclonal antibody. Six immunoreactive clones were identified. The clones expressed proteins which ranged from 21-80 kilodaltons, and the clone designated pII-2, producing the largest protein was selected for further study. Antibodies eluted from immobilized pII-2 protein also recognized the native A. actinomycetemcomitans leukotoxin molecule indicating that both molecules shared at least one epitope. DNA sequence analysis demonstrated that there are regions of significant amino acid sequence homology between the cloned A. actinomycetemcomitans leukotoxin and two other cytolysins, Escherichia coli alpha-hemolysin and Pasteurella haemolytica leukotoxin, suggesting that a family of cytolysins may exist which share a common mechanism of killing but vary in their target cell specificity.     

Cellular and molecular response of human macrophages exposed to Aggregatibacter actinomycetemcomitans leukotoxin

Aggregatibacter (Actinobacillus) actinomycetemcomitans is a facultative anaerobic gram-negative bacterium associated with severe forms of periodontitis. A leukotoxin, which belongs to the repeats-in-toxin family, is believed to be one of its virulence factors and to have an important role in the bacterium''s pathogenicity. This toxin selectively kills human leukocytes by inducing apoptosis and lysis. Here, we report that leukotoxin-induced cell death of macrophages proceeded through a process that differs from the classical characteristics of apoptosis and necrosis. A. actinomycetemcomitans leukotoxin-induced several cellular and molecular mechanisms in human macrophages that led to a specific and excessive pro-inflammatory response with particular secretion of both interleukin (IL)-1β and IL-18. In addition, this pro-inflammatory cell death was inhibited by oxidized ATP, which indicates involvement of the purinergic receptor P2X₇ in this process. This novel virulence mechanism of the leukotoxin may have an important role in the pathogenic potential of this bacterium and can be a target for future therapeutic agents.     

Differential interaction of Aggregatibacter (Actinobacillus) actinomycetemcomitans LsrB and RbsB proteins with autoinducer 2

Our previous studies showed that the Aggregatibacter actinomycetemcomitans RbsB protein interacts with cognate and heterologous autoinducer 2 (AI-2) signals and suggested that the rbsDABCK operon encodes a transporter that may internalize AI-2 (D. James et al., Infect. Immun. 74:4021-4029, 2006.). However, A. actinomycetemcomitans also possesses genes related to the lsr operon of Salmonella enterica serovar Typhimurium which function to import AI-2. Here, we show that A. actinomycetemcomitans LsrB protein competitively inhibits the interaction of the Vibrio harveyi AI-2 receptor (LuxP) with AI-2 from either A. actinomycetemcomitans or V. harveyi. Interestingly, LsrB was a more potent inhibitor of LuxP interaction with AI-2 from V. harveyi whereas RbsB competed more effectively with LuxP for A. actinomycetemcomitans AI-2. Inactivation of lsrB in wild-type A. actinomycetemcomitans or in an isogenic RbsB-deficient strain reduced the rate by which intact bacteria depleted A. actinomycetemcomitans AI-2 from solution. Consistent with the results from the LuxP competition experiments, the LsrB-deficient strain depleted AI-2 to a lesser extent than the RbsB-deficient organism. Inactivation of both lsrB and rbsB virtually eliminated the ability of the organism to remove AI-2 from the extracellular environment. These results suggest that A. actinomycetemcomitans possesses two proteins that differentially interact with AI-2 and may function to inactivate or facilitate internalization of AI-2.     

Actinobacillus actinomycetemcomitans leukotoxin requires lipid microdomains for target cell cytotoxicity

Actinobacillus actinomycetemcomitans produces a leukotoxin (Ltx) that kills leukocyte function-associated antigen-1 (LFA-1)-bearing cells from man, the Great Apes and Old World monkeys. The unique specificity of Ltx for the beta2 integrin, LFA-1, suggests it is capable of providing insight into the pathogenic mechanisms of Ltx and other RTX toxins. Using the Jurkat T cell line and an LFA-1-deficient Jurkat mutant (Jbeta2.7) as models, we found the initial effect of Ltx is to elevate cytosolic Ca2+ [Ca2+]c, an event that is independent of the Ltx/LFA-1 interaction. [Ca2+]c increases initiate a series of events that involve the activation of calpain, talin cleavage, mobilization to, and subsequent clustering of, LFA-1 in cholesterol and sphingolipid-rich regions of the plasma membrane known as lipid rafts. The association of Ltx and LFA-1 within lipid rafts is essential for cell lysis. Jbeta2.7 cells fail to accumulate Ltx in their raft fractions and are not killed, while cholesterol depletion experiments demonstrate the necessity of raft integrity for Ltx function. We propose that toxin-induced Ca2+ fluxes mobilize LFA-1 to lipid rafts where it associates with Ltx. These findings suggest that Ltx utilizes the raft to stimulate an integrin signalling pathway that leads to apoptosis of target cells.     

Aggregatibacter actinomycetemcomitans leukotoxin causes activation of lymphocyte function‐associated antigen 1

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 α_L/β₂)‐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 α_L domain and a yellow fluorescent protein‐tagged β₂ 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 α_L and β₂ were determined using surface plasmon resonance. LtxA has a strong affinity for the cytosolic domains of both the α_L and β₂ subunits (K_d = 15 and 4.2 nM, respectively) and a significantly lower affinity for the cytoplasmic domains of other integrin α_M, α_X, and β₃ subunits (K_d = 400, 180, and 230 nM, respectively), used as controls. Peptide fragments of α_L and β₂ show that LtxA binds membrane‐proximal domain of α_L and intermediate domain of β₂.     

Conformational studies of Actinobacillus actinomycetemcomitans leukotoxin: partial denaturation enhances toxicity

A 114 kDa, water-soluble, cytotoxin secreted by the Gram-negative bacterium Actinobacillus actinomycetemcomitans (Aa) is similar in sequence to Escherichia coli alpha-hemolysin, but is non-hemolytic, killing leukocytes of select species, including humans. In this work, we investigated aspects of the water-soluble conformation of Aa toxin which relate to its biological, pore-forming activity. The toxin has five native tryptophans and fluorescence spectra were monitored in aqueous solutions in the presence of varying denaturants. Significant changes in the fluorescence spectra, without significant wavelength shifts, were induced by small additions of denaturants and changes in the temperature or pH. The fluorescence changes suggested that small perturbations in the aqueous environment resulted in structural changes in the toxin related not to a large unfolding but to more subtle conformational changes. Analytical ultracentrifugation showed the toxin to be a globular monomer in dilute aqueous solution. Circular dichroism spectroscopy showed about 25% alpha-helical structure which is largely maintained up to a temperature (65 degrees C) known to deactivate toxin activity. Changes in the cytotoxic properties of the toxin were monitored with flow cytometric analysis following preincubation of the toxin under mild conditions similar to those used in the fluorescence studies. These experiments showed that the pretreated toxin exhibited enhanced cell-killing potency on toxin-sensitive cells. The correlation of cytotoxicity with the changes in Trp fluorescence is consistent with the idea that partial unfolding of Aa toxin is an early, obligate step in toxin-induced cell kill.     

The regulatory effect of fermentable sugar levels on the production of leukotoxin by Actinobacillus actinomycetemcomitans

The relationship between sugar availability and RTX (repeats in toxin) cytotoxin (leukotoxin) production in the periodontopathic bacterium, Actinobacillus actinomycetemcomitans, was investigated using a chemostat. A. actinomycetemcomitans 301-b produced significant amounts of leukotoxin in anaerobic fructose-limited chemostat cultures at a dilution rate of 0.15 h⁻¹ and at pH 7.0. When the growth limitation was relieved by pulsing the cultures with 50 or 150 mM fructose (final concentrations), leukotoxin production immediately stopped and the amount of cellular leukotoxin decreased until the culture was returned to fructose-limited conditions. Leukotoxin synthesis was also repressed in the chemostat cultures by pulsing with glucose but not with the non-fermentable sugar analog, α-methyl-d-glucoside. Leukotoxin production was also repressed by fructose in chemostat cultures of ATCC 33384, which is generally recognized as a non-leukotoxin-producing or minimally leukotoxic strain.     

Proteomics of Protein Secretion by Aggregatibacter actinomycetemcomitans

The extracellular proteome (secretome) of periodontitis-associated bacteria may constitute a major link between periodontitis and systemic diseases. To obtain an overview of the virulence potential of Aggregatibacter actinomycetemcomitans, an oral and systemic human pathogen implicated in aggressive periodontitis, we used a combined LC-MS/MS and bioinformatics approach to characterize the secretome and protein secretion pathways of the rough-colony serotype a strain D7S. LC-MS/MS revealed 179 proteins secreted during biofilm growth. Further to confirming the release of established virulence factors (e.g. cytolethal distending toxin [CDT], and leukotoxin [LtxA]), we identified additional putative virulence determinants in the secretome. These included DegQ, fHbp, LppC, Macrophage infectivity protein (MIP), NlpB, Pcp, PotD, TolB, and TolC. This finding indicates that the number of extracellular virulence-related proteins is much larger than previously demonstrated, which was also supported by in silico analysis of the strain D7S genome. Moreover, our LC-MS/MS and in silico data revealed that at least Type I, II, and V secretion are actively used to excrete proteins directly into the extracellular space, or via two-step pathways involving the Sec/Tat systems for transport across the inner membrane, and outer membrane factors, secretins and auto-transporters, respectively for delivery across the outer membrane. Taken together, our results provide a molecular basis for further elucidating the role of A. actinomycetemcomitans in periodontal and systemic diseases.     

Actinobacillus (Haemophilus) actinomycetemcomitans in periodontal disease

Actinobacillus (Haemophilus) actinomycetemcomitans is a facultatively anaerobic, gram-negative coccobacillus which is a possible etiological agent in juvenile periodontitis (JP). In this study, bacterial flora, especially the occurrence of A. actinomycetemcomitans, in the periodontal pockets of one juvenile with gingivitis (G), one JP patients, five rapidly progressive periodontitis (RP) patients and one adult periodontitis(AP) patient, and one adult with healthy periodontium was investigated using a blood agar medium and a selective medium for A. actinomycetemcomitans. Eight hundred and sixty-five bacteria were isolated from the periodontal pockets, examined for their gram-stain, cell morphologies, relations to O2 and CO2 and catalase reaction, and divided into 21 groups on the basis of these characters. Among the isolates, 604 isolates were further characterized biochemically and identified. A. actinomycetemcomitans was found as 0.2% of the flora of a site in the JP patient, as 9% of the flora of a site in the G patient, and as 19% and 1%, respectively, of the flora of a site in the two RP patients. However, the organism was not detected in another lesion site of the JP patient. In our JP and RP patients, Fusobacterium, Wolinella, Streptococcus, and obligately anaerobic, gram-positive cocci were frequently found at high levels. The bacterial flora of the G and AP patients were more heterogeneous and included Bacteroides at relatively high proportions. These results indicate that A. actinomycetemcomitans is not always associated with JP but occurred in some patients with RP and G.     

Neutrophil-derived resistin release induced by Aggregatibacter actinomycetemcomitans

Resistin is an adipokine that induces insulin resistance in mice. In humans, resistin is not produced in adipocytes, but in various leukocytes instead, and it acts as a proinflammatory molecule. The present investigation demonstrated high levels of resistin in culture supernatants of neutrophils that are stimulated by a highly leukotoxic strain of Aggregatibacter actinomycetemcomitans. In contrast, the level of resistin was remarkably low when neutrophils were exposed to two other strains that produce minimal levels of leukotoxin and a further isogenic mutant strain incapable of producing leukotoxin. Pretreatment of neutrophils with a monoclonal antibody to CD18, β chain of lymphocyte function-associated molecule 1 (LFA-1), or an Src family tyrosine kinase inhibitor before incubation with the highly leukotoxic strain inhibited the release of resistin. These results show that A. actinomycetemcomitans-expressed leukotoxin induces extracellular release of human neutrophil-derived resistin by interacting with LFA-1 on the surface of neutrophils and, consequently, activating Src family tyrosine kinases.     

Aggregatibacter actinomycetemcomitans leukotoxin requires beta-sheets 1 and 2 of the human CD11a beta-propeller for cytotoxicity

Aggregatibacter actinomycetemcomitans leukotoxin (Ltx) is a repeats-in-toxin (RTX) cytolysin that kills human leukocyte function-associated antigen-1 (LFA-1; alpha(L)/beta(2))-bearing cells. In order to determine whether the alpha(L) portion of the heterodimer is involved in Ltx recognition, we transfected human, mouse and bovine alpha(L) cDNAs into J-beta(2).7, an alpha(L)-deficient cell line, and looked for restoration of Ltx susceptibility. Cells expressing either bovine or human alpha(L) in conjunction with human beta(2) were efficiently killed by Ltx, an indication that bovine alpha(L) could substitute for its human counterpart in critical regions used by Ltx for attachment to LFA-1. On the other hand, cells expressing murine alpha(L) and human beta(2) were not susceptible to the lethal effects of Ltx indicating that the toxin recognition sites are not present in the corresponding mouse sequence. To further identify the region(s) of alpha(L) recognized by Ltx, we constructed and evaluated a panel of chimeric human/murine alpha(L) genes in J-beta(2).7 cells. Analysis of the alpha(L) mutant panel showed that the presence of human N-terminal 128 amino acids on a mouse CD11a background, a region that includes beta-sheets 1 and 2 of the beta-propeller of the human alpha(L) chain, was sufficient for Ltx cytolysis.     

Interaction of Actinobacillus actinomycetemcomitans outer membrane vesicles with HL60 cells does not require leukotoxin

Outer membrane derived vesicles (MVs) secreted by Actinobacillus actinomycetemcomitans JP2 contain a membranolytic leukotoxin and are toxic to human HL60 cells. To determine how MVs interact with human target cells, HL60 cells were incubated with vesicles, reacted with anti-vesicle antibodies and a FITC-labelled reporter, and visualized by confocal scanning laser microscopy. Target cells rapidly became reactive with anti-vesicle antibodies upon exposure to vesicles. Confocal microscopy showed that labelling occurred primarily in the cytoplasmic membrane and that very little internal fluorescence was observed. The cytoplasmic membrane of HL60 cells was also strongly labelled after exposure to MVs that contained the fluorescent phospholipid, SP-DiOC18. In contrast, incubation of cells with free SP-DiOC18 resulted primarily in the labelling of internal structures of HL60 cells. These results suggest that A. actinomycetemcomitans MVs associate with, or are incorporated into the cytoplasmic membrane of HL60 cells. The leukotoxin is a membranolytic cytotoxin and cells exposed to MVs were lysed by vesicle-associated toxin in a time and dose-dependent manner. However, cells became reactive with anti-vesicle antibodies when MVs were added in the presence of inhibitors of leukotoxin-mediated lysis or when sublytic doses of MVs were analysed. In addition, MVs produced by an isogenic leukotoxin-deficient strain of A. actinomycetemcomitans JP2 were non-toxic but rapidly interacted with HL60 cells. These results suggest that A. actinomycetemcomitans MVs can deliver leukotoxin to HL60 cells but that the association of vesicles with the cytoplasmic membrane occurs independently of the leukotoxin polypeptide.     

Early changes in cytosolic calcium and membrane potential induced by Actinobacillus actinomycetemcomitans leukotoxin in susceptible and resistant target cells

Actinobacillus actinomycetemcomitans produces a cytolytic peptide leukotoxin which kills susceptible target cells, including human neutrophils, monocytes, lymphocytes, and HL-60 promyelocytic leukemia cells. Cell death occurs as a consequence of colloid osmotic lysis. In the present investigation early leukotoxin-induced changes in membrane permeability were studied by flow cytometry and quantitative spectrofluorimetry in leukotoxin-susceptible and resistant targets. Within 5 s toxin-susceptible cells exhibited concentration-dependent, sustained increases in systolic free Ca2+, and this was rapidly followed by a progressive fall in membrane potential. These early manifestations of membrane injury occurred approximately 10-15 min before cell death, as reflected by flow cytometric analysis of propidium iodide stained cells. The rise in cytosolic Ca2+ was almost entirely due to an influx of extracellular Ca2+. The results of Hill plots for the action of leukotoxin on Ca2+ permeability in human neutrophils or HL-60 cells suggested that two or more toxin molecules participate in the assembly of an ion conducting pore in the plasma membrane. Changes in membrane permeability or cell viability were not observed in response to heat-inactivated toxin. Under appropriate conditions toxin-induced membrane abnormalities were inhibited by leukotoxin-neutralizing mAb or relatively high concentrations (greater than or equal to 2.5 mM) of extracellular Ca2+. Leukotoxin-resistant target cells showed no evidence of membrane injury even when exposed to high concentrations of leukotoxin for prolonged periods of time. These included resistant human K562 erythroleukemia cells and murine SP2 myeloma cells which have previously been shown to adsorb the toxin, suggesting that they possess a protective mechanism(s) which impedes toxin insertion or assembly in the lipid bilayer. These data support the concept that A. actinomycetemcomitans leukotoxin acts as a cell-specific, pore-forming protein which permeabilizes the plasma membrane of susceptible target cells.     

Analysis of the Actinobacillus actinomycetemcomitans leukotoxin gene. Delineation of unique features and comparison to homologous toxins

Actinobacillus actinomycetemcomitans leukotoxin has been implicated as a virulence factor in human infections. To initiate delineation of leukotoxin structure/function relationships, molecular cloning of the leukotoxin gene was carried out. When an A. actinomycetemcomitans genomic DNA library in lambda EMBL3 was screened using a 1.3-kilobase pair restriction fragment containing a portion of the leukotoxin gene, 13 positive recombinants were identified. One recombinant, designated lambda OP8, containing a 16-kilobase pair insert was selected for detailed study. Lysates from lambda OP8, but not control lysates, exhibited leukotoxic activity with target cell specificity identical to the native toxin. Western blots identified the recombinant-produced toxin as a 125-kDa protein doublet identical in mobility to the native toxin. Restriction enzyme and extensive DNA analyses demonstrated that the leukotoxin gene showed strong homology to two other toxins produced by Escherichia coli and Pasteurella haemolytica. As in the other two species, the A. actinomycetemcomitans toxin is contained in a cluster of four genes in which the A gene encodes the toxin and the products of the B, C, and D genes are involved in posttranslational modification of the toxin and its membrane insertion and secretion. The target cell specificity of the A. actinomycetemcomitans toxin differs from the other two toxins and is restricted to human and some non-human primate cells of the monomyelocytic lineage. The A. actinomycetemcomitans leukotoxin is not secreted but remains associated with the bacterial membrane, possibly through a hydrophobic domain at the carboxyl terminus which distinguishes it from the E. coli and P. haemolytica toxins.     

Aggregatibacter actinomycetemcomitans biofilm killing by a targeted ciprofloxacin prodrug

A pH-sensitive ciprofloxacin prodrug was synthesized and targeted against biofilms of the periodontal pathogen Aggregatibacter actinomycetemcomitans (Aa). The dose required to reduce the viability of a mature biofilm of Aa by ～80% was in the range of ng?cm^?2 of colonized area (mean biofilm density 2.33?×?10⁹?cells?cm^?2). A mathematical model was formulated that predicts the temporal change in the concentration of ciprofloxacin in the Aa biofilm as the drug is released and diffuses into the bulk medium. The predictions of the model were consistent with the extent of killing obtained. The results demonstrate the feasibility of the strategy to induce mortality, and together with the mathematical model, provide the basis for design of targeted antimicrobial prodrugs for the topical treatment of oral infections such as periodontitis. The targeted prodrug approach offers the possibility of optimizing the dose of available antimicrobials in order to kill a chosen pathogen while leaving the commensal microbiota relatively undisturbed.