Nataliya V. Balashova

Leukotoxin Confers Beta-Hemolytic Activity to Actinobacillus actinomycetemcomitans

ABSTRACT Actinobacillus actinomycetemcomitans is the etiologic agent of localized aggressive periodontitis, a rapidly progressing oral disease that occurs in adolescents. A. actinomycetemcomitans can also cause systemic disease, including infective endocarditis. In early work on A. actinomycetemcomitans workers concluded that this bacterium is not beta-hemolytic. More recent reports have suggested that A. actinomycetemcomitans does have the potential to be beta-hemolytic. While growing A. actinomycetemcomitans on several types of growth media, we noticed a beta-hemolytic reaction on media from one manufacturer. Beta-hemolysis occurred on Columbia agar from Accumedia with either sheep or horse blood, but not on similar media from other manufacturers. A surprising result was that mutants of A. actinomycetemcomitans defective for production of leukotoxin, a toxin that is reportedly highly specific for only human and primate white blood cells, are not beta-hemolytic. Purified leukotoxin was able to lyse sheep and human erythrocytes in vitro. This work showed that in contrast to the accepted view, A. actinomycetemcomitans leukotoxin can indeed destroy erythrocytes and that the production of this toxin results in beta-hemolytic colonies on solid medium. In light of these results, the diagnostic criteria for clinical identification of A. actinomycetemcomitans and potentially related bacteria should be reevaluated. Furthermore, in studies on A. actinomycetemcomitans leukotoxin workers should now consider this toxins ability to destroy red blood cells.

Anti-leukemia activity of a bacterial toxin with natural specificity for LFA-1 on white blood cells

The oral bacterium, Aggregatibacter actinomycetemcomitans, produces a leukotoxin (LtxA) that is specific for white blood cells (WBCs) from humans and Old World primates by interacting with lymphocyte function antigen-1 (LFA-1) on susceptible cells. To determine if LtxA could be used as a therapeutic agent for the treatment of WBC diseases, we tested the in vitro and in vivo anti-leukemia activity of the toxin. LtxA kills human malignant WBC lines and primary leukemia cells from acute myeloid leukemia patients, but healthy peripheral blood mononuclear cells (PBMCs) are relatively resistant to LtxA-mediated cytotoxicity. Levels of LFA-1 on cell lines correlated with killing by LtxA and the toxin preferentially killed cells expressing the activated form of LFA-1. In a SCID mouse model for human leukemia, LtxA had potent therapeutic value resulting in long-term survival in LtxA-treated mice. Intravenous infusion of LtxA into a rhesus macaque resulted in a drop in WBC counts at early times post-infusion; however, red blood cells, platelets, hemoglobin and blood chemistry values remained unaffected. Thus, LtxA may be an effective and safe novel therapeutic agent for the treatment of hematologic malignancies.

Aggregatibacter actinomycetemcomitans LtxC is required for leukotoxin activity and initial interaction between toxin and host cells.

Aggregatibacter actinomycetemcomitans is a human pathogen that produces the RTX toxin (repeats in toxin), leukotoxin (LtxA). Based on other RTX toxin systems, the product of ltxC, the first gene of the ltx operon, is predicted to be involved in fatty acid modification of LtxA. To determine the function of ltxC in A. actinomycetemcomitans, we generated an ltxC mutation in the highly leukotoxic strain JP2N using random mutagenesis. The toxin from the ltxC mutant (LtxA(ltxC)) was expressed and secreted into the cell culture supernatant but could not lyse human leukocytes or erythrocytes. Mass spectrometric analysis of LtxA(ltxC) and LtxA from strain JP2N (LtxA(wt)) revealed two peptides that differed and this data suggests that two internal lysine residues of LtxA from the wild-type strain are modified. In blocking experiments, pre-treatment of cells with LtxA(ltxC) was unable to prevent LtxA(wt) from killing cells. Furthermore, in contrast to LtxA(wt), LtxA(ltxC) did not cause an increase in intracellular calcium levels in human leukocytes. Taken together, our data show that ltxC is required for full activity and modification of LtxA in A. actinomycetemcomitans and that modification is important for initial binding of toxin to host cells, as defined by an increase in intracellular calcium levels.

Regulation of Aggregatibacter (Actinobacillus) actinomycetemcomitans Leukotoxin Secretion by Iron

The gram-negative oral and systemic pathogen Aggregatibacter (Actinobacillus) actinomycetemcomitans produces a leukotoxin (LtxA) that is a member of the RTX (repeats in toxin) family of secreted bacterial toxins. We have recently shown that LtxA has the ability to lyse erythrocytes, which results in a beta-hemolytic phenotype on Columbia blood agar. To determine if LtxA is regulated by iron, we examined beta-hemolysis under iron-rich and iron-limiting conditions. Beta-hemolysis was suppressed in the presence of FeCl3. In contrast, strong beta-hemolysis occurred in the presence of the iron chelator deferoxamine. We found that secretion of LtxA was completely inhibited by free iron, but expression of ltxA was not regulated by iron. Free chromium, cobalt, and magnesium did not affect LtxA secretion. Other LtxA-associated genes were not regulated by iron. Thus, iron appears to play an important role in the regulation of LtxA secretion in A. actinomycetemcomitans in a manner independent of gene regulation.

Aggregatibacter actinomycetemcomitans Leukotoxin Utilizes a Cholesterol Recognition/Amino Acid Consensus Site for Membrane Association

Background: A repeats-in-toxin (RTX) leukotoxin and its integrin receptor aggregate in cholesterol-rich lipid rafts. Results: The affinity of the toxin to cholesterol is driven by a cholesterol recognition/amino acid consensus (CRAC) motif. Conclusion: Leukotoxin cytotoxicity is regulated by the CRAC motif. Significance: Other RTX toxins contain this CRAC motif, suggesting a role for cholesterol recognition in RTX cytolysis. Aggregatibacter actinomycetemcomitans produces a repeats-in-toxin (RTX) leukotoxin (LtxA) that selectively kills human immune cells. Binding of LtxA to its β2 integrin receptor (lymphocyte function-associated antigen-1 (LFA-1)) results in the clustering of the toxin·receptor complex in lipid rafts. Clustering occurs only in the presence of LFA-1 and cholesterol, and LtxA is unable to kill cells lacking either LFA-1 or cholesterol. Here, the interaction of LtxA with cholesterol was measured using surface plasmon resonance and differential scanning calorimetry. The binding of LtxA to phospholipid bilayers increased by 4 orders of magnitude in the presence of 40% cholesterol relative to the absence of cholesterol. The affinity was specific to cholesterol and required an intact secondary structure. LtxA contains two cholesterol recognition/amino acid consensus (CRAC) sites; CRAC336 (333LEEYSKR339) is highly conserved among RTX toxins, whereas CRAC503 (501VDYLK505) is unique to LtxA. A peptide corresponding to CRAC336 inhibited the ability of LtxA to kill Jurkat (Jn.9) cells. Although peptides corresponding to both CRAC336 and CRAC503 bind cholesterol, only CRAC336 competitively inhibited LtxA binding to this sterol. A panel of full-length LtxA CRAC mutants demonstrated that an intact CRAC336 site was essential for LtxA cytotoxicity. The conservation of CRAC336 among RTX toxins suggests that this mechanism may be conserved among RTX toxins.

Interaction between leukotoxin and Cu,Zn superoxide dismutase in Aggregatibacter actinomycetemcomitans.

ABSTRACT Aggregatibacter (Actinobacillus) actinomycetemcomitans is a gram-negative oral pathogen that is the etiologic agent of localized aggressive periodontitis and systemic infections. A. actinomycetemcomitans produces leukotoxin (LtxA), which is a member of the RTX (repeats in toxin) family of secreted bacterial toxins and is known to target human leukocytes and erythrocytes. To better understand how LtxA functions as a virulence factor, we sought to detect and study potential A. actinomycetemcomitans proteins that interact with LtxA. We found that Cu,Zn superoxide dismutase (SOD) interacts specifically with LtxA. Cu,Zn SOD was purified from A. actinomycetemcomitans to homogeneity and remained enzymatically active. Purified Cu,Zn SOD allowed us to isolate highly specific anti-Cu,Zn SOD antibody and this antibody was used to further confirm protein interaction. Cu,Zn SOD-deficient mutants displayed decreased survival in the presence of reactive oxygen and nitrogen species and could be complemented with wild-type Cu,Zn SOD in trans. We suggest that A. actinomycetemcomitans Cu,Zn SOD may protect both bacteria and LtxA from reactive species produced by host inflammatory cells during disease. This is the first example of a protein-protein interaction involving a bacterial Cu,Zn SOD.

Genome Sequence of Kingella kingae Septic Arthritis Isolate PYKK081

Kingella kingae is a human oral bacterium that can cause infections of the skeletal system in children. The bacterium is also a cardiovascular pathogen causing infective endocarditis in children and adults. We report herein the draft genome sequence of septic arthritis K. kingae strain PYKK081.

Gangliosides Block Aggregatibacter Actinomycetemcomitans Leukotoxin (LtxA)-Mediated Hemolysis

Aggregatibacter actinomycetemcomitans is an oral pathogen and etiologic agent of localized aggressive periodontitis. The bacterium is also a cardiovascular pathogen causing infective endocarditis. A. actinomycetemcomitans produces leukotoxin (LtxA), an important virulence factor that targets white blood cells (WBCs) and plays a role in immune evasion during disease. The functional receptor for LtxA on WBCs is leukocyte function antigen-1 (LFA-1), a β-2 integrin that is modified with N-linked carbohydrates. Interaction between toxin and receptor leads to cell death. We recently discovered that LtxA can also lyse red blood cells (RBCs) and hemolysis may be important for pathogenesis of A. actinomycetemcomitans. In this study, we further investigated how LtxA might recognize and lyse RBCs. We found that, in contrast to a related toxin, E. coli α-hemolysin, LtxA does not recognize glycophorin on RBCs. However, gangliosides were able to completely block LtxA-mediated hemolysis. Furthermore, LtxA did not show a preference for any individual ganglioside. LtxA also bound to ganglioside-rich C6 rat glioma cells, but did not kill them. Interaction between LtxA and C6 cells could be blocked by gangliosides with no apparent specificity. Gangliosides were only partially effective at preventing LtxA-mediated cytotoxicity of WBCs, and the effect was only observed when a high ratio of ganglioside:LtxA was used over a short incubation period. Based on the results presented here, we suggest that because of the similarity between N-linked sugars on LFA-1 and the structures of gangliosides, LtxA may have acquired the ability to lyse RBCs.

A bioluminescent HL-60 cell line to assay anti-leukaemia therapeutics under physiological conditions.

Screens for compounds and proteins with anti-cancer activity employ viability assays using relevant cancer cell lines. For leukaemia studies, the human leukaemia cell line, HL-60, is often used as a model system. To facilitate the discovery and investigation of anti-leukaemia therapeutics under physiological conditions, we have engineered HL-60 cells that stably express firefly luciferase and produce light that can be detected using an in vivo imaging system (IVIS). Bioluminescent HL-60luc cells could be rapidly detected in whole blood with a sensitivity of approximately 1000 viable cells/200 microl blood. Treatment of HL-60luc cells with the drug chlorambucil revealed that the bioluminescent viability assay is able to detect cell death earlier than the Trypan blue dye exclusion assay. HL-60luc cells administered intraperitoneally (i.p.) or intravenously (i.v.) were visualized in living mice. The rapidity and ease of detecting HL-60luc cells in biological fluid indicates that this cell line could be used in high-throughput screens for the identification of drugs with anti-leukaemia activity under physiological conditions.

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

The Gram-negative bacterium, Aggregatibacter actinomycetemcomitans, has been associated with localized aggressive periodontitis (LAP). In particular, highly leukotoxic strains of A. actinomycetemcomitans have been more closely associated with this disease, suggesting that LtxA is a key virulence factor for A. actinomycetemcomitans. 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 A. actinomycetemcomitans virulence.