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.
Acute myeloid leukemia; lymphoma; immunotoxin; targeted therapy
Aggregatibacter actinomycetemcomitansW is an oral bacterium that causes localized aggressive periodontitis (LAP) and extra-oral infections such as sub-acute infective endocarditis. As part of its array of virulence factors, A. actinomycetemcomitans produces leukotoxin (LtxA), a member of the RTX family of toxins. LtxA kills human leukocytes and we have recently shown that the toxin is required for β -hemolysis by A. actinomycetemcomitans on solid medium. In other RTX toxin-producing bacteria, an outer membrane channel-forming protein, TolC, is required for toxin secretion and drug export. We have identified an ORF in A. actinomycetemcomitans that encodes a putative protein having predicted structural properties similar to TolC. Inactivation of this ORF resulted in a mutant that was no longer β -hemolytic and did not secrete LtxA. This mutant was significantly more sensitive to antimicrobial agents compared to the wild type strain and was unable to export the antimicrobial agent berberine. Thus, this ORF was named tdeA for “toxin and drug export”. Examination of the DNA sequence surrounding tdeA revealed two upstream ORFs that encode proteins similar to the drug efflux proteins, MacA and MacB. Inactivation of macB in A. actinomycetemcomitans did not alter the drug sensitivity profile or the hemolytic activity of the mutant. The genes macA, macB and tdeA are organized as an operon and are constitutively expressed as a single transcript. These results show that A. actinomycetemcomitans indeed requires a TolC-like protein for LtxA secretion and that this protein, TdeA, also functions as part of a drug efflux system.
leukotoxin; periodontitis; endocarditis; outer membrane protein; antibiotics
Aggregatibacter actinomycetemcomitans is a human pathogen that produces leukotoxin (LtxA) as a major virulence factor. In this study the effect of LtxA on microvascular endothelial cell viability and phenotype was studied. High doses of single LtxA treatment (500 ng/ml to 5 μg/ml) significantly and irreversibly decreased cell proliferation and induced apoptosis, as assessed by tetrazolium salt and annexin V assay, respectively. Apoptosis was partially inhibited by the pan-caspase inhibitor, z-VAD-fmk. LtxA caused a cell cycle arrest in the G2/M phase after 72 h. Between 500 ng/ml and 5 μg/ml, after long- or short-term stimulation LtxA increased the expression of ICAM-1 and VCAM-1, as well as the percentages of endothelial cells expressing these adhesion molecules. Thus, A. actinomycetemcomitans LtxA has substantial pro-inflammatory effects on human brain endothelial cells by upregulation of ICAM-1 and VCAM-1. Furthermore, LtxA in higher concentration was found to decrease proliferation and induces apoptosis in microvascular endothelial cells.
•LtxA has anti-proliferative effects on endothelial cells.•LtxA induces a G2/M phase cell cycle arrest in endothelial cells.•LtxA induces apoptosis in endothelial cells.•LtxA increased expression of ICAM-1 and VCAM-1 in endothelial cells.
Endothelium; Leukotoxin; Aggregatibacter actinomycetemcomitans; Apoptosis; Activation
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.
Cell-free extracts prepared from Kingella kingae colony biofilms were found to inhibit biofilm formation by Aggregatibacter actinomycetemcomitans, Klebsiella pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Candida albicans, and K. kingae. The extracts evidently inhibited biofilm formation by modifying the physicochemical properties of the cell surface, the biofilm matrix, and the substrate. Chemical and biochemical analyses indicated that the biofilm inhibition activity in the K. kingae extract was due to polysaccharide. Structural analyses showed that the extract contained two major polysaccharides. One was a linear polysaccharide with the structure →6)-α-d-GlcNAcp-(1→5)-β-d-OclAp-(2→, which was identical to a capsular polysaccharide produced by Actinobacillus pleuropneumoniae serotype 5. The second was a novel linear polysaccharide, designated PAM galactan, with the structure →3)-β-d-Galf-(1→6)-β-d-Galf-(1→. Purified PAM galactan exhibited broad-spectrum biofilm inhibition activity. A cluster of three K. kingae genes encoding UDP-galactopyranose mutase (ugm) and two putative galactofuranosyl transferases was sufficient for the synthesis of PAM galactan in Escherichia coli. PAM galactan is one of a growing number of bacterial polysaccharides that exhibit antibiofilm activity. The biological roles and potential technological applications of these molecules remain unknown.
The cellular adhesion molecule LFA-1 and its ICAM-1 ligand play an important role in promoting HIV-1 infectivity and transmission. These molecules are present on the envelope of HIV-1 virions and are integral components of the HIV virological synapse. However, cellular activation is required to convert LFA-1 to the active conformation that has high affinity binding for ICAM-1. This study evaluates whether such activation can be induced by HIV itself. The data show that HIV-1 gp120 was sufficient to trigger LFA-1 activation in fully quiescent naïve CD4 T cells in a CD4-dependent manner, and these CD4 T cells became more susceptible to killing by LtxA, a bacterial leukotoxin that preferentially targets leukocytes expressing high levels of the active LFA-1. Moreover, virus p24-expressing CD4 T cells in the peripheral blood of HIV-infected subjects were found to have higher levels of surface LFA-1, and LtxA treatment led to significant reduction of the viral DNA burden. These results demonstrate for the first time the ability of HIV to directly induce LFA-1 activation on CD4 T cells. Although LFA-1 activation may enhance HIV infectivity and transmission, it also renders the cells more susceptible to an LFA-1-targeting bacterial toxin, which may be harnessed as a novel therapeutic strategy to deplete virus reservoir in HIV-infected individuals.
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.
erythrocytes; toxin; periodontal disease; endocarditis; RTX toxin
The structure and composition of the oocyst wall are primary factors determining the survival and hydrologic transport of Cryptosporidium parvum oocysts outside the host. Microscopic and biochemical analyses of whole oocysts and purified oocyst walls were undertaken to better understand the inactivation kinetics and hydrologic transport of oocysts in terrestrial and aquatic environments. Results of microscopy showed an outer electron-dense layer, a translucent middle layer, two inner electron-dense layers, and a suture structure embedded in the inner electron-dense layers. Freeze-substitution showed an expanded glycocalyx layer external to the outer bilayer, and Alcian Blue staining confirmed its presence on some but not all oocysts. Biochemical analyses of purified oocyst walls revealed carbohydrate components, medium- and long-chain fatty acids, and aliphatic hydrocarbons. Purified walls contained 7.5% total protein (by the Lowry assay), with five major bands in SDS-PAGE gels. Staining of purified oocyst walls with magnesium anilinonaphthalene-8-sulfonic acid indicated the presence of hydrophobic proteins. These structural and biochemical analyses support a model of the oocyst wall that is variably impermeable and resistant to many environmental pressures. The strength and flexibility of oocyst walls appear to depend on an inner layer of glycoprotein. The temperature-dependent permeability of oocyst walls may be associated with waxy hydrocarbons in the electron-translucent layer. The complex chemistry of these layers may explain the known acid-fast staining properties of oocysts, as well as some of the survival characteristics of oocysts in terrestrial and aquatic environments. The outer glycocalyx surface layer provides immunogenicity and attachment possibilities, and its ephemeral nature may explain the variable surface properties noted in oocyst hydrologic transport studies.
Two virulence factors produced by the periodontopathogen Aggregatibacter actinomycetemcomitans are leukotoxin, a secreted lipoprotein that kills human polymorphonuclear leukocytes and macrophages, and poly-N-acetylglucosamine (PGA), a surface polysaccharide that mediates intercellular adhesion, biofilm formation and detergent resistance. In this study we examined the roles of leukotoxin and PGA in protecting A. actinomycetemcomitans cells from killing by the human macrophage cell line THP-1. Monolayers of THP-1 cells were infected with single-cell suspensions of a wild-type A. actinomycetemcomitans strain, or of isogenic leukotoxin or PGA mutant strains. After 48 h, viable bacteria were enumerated by dilution plating, macrophage morphology was evaluated microscopically, and macrophage viability was measured by a Trypan blue dye exclusion assay. The number of A. actinomycetemcomitans CFUs increased approximately 2-fold in wells infected with the wild-type strain, but decreased by approximately 70–90% in wells infected with the leukotoxin and PGA mutant strains. Infection with the wild-type or leukotoxin mutant strain caused a significant decrease in THP-1 cell viability, whereas infection with the PGA mutant strain did not result in any detectable changes in THP-1 viability. Pre-treatment of wild-type A. actinomycetemcomitans cells with the PGA-hydrolyzing enzyme dispersin B rendered them sensitive to killing by THP-1 cells. We concluded that both leukotoxin and PGA are necessary for evasion of macrophage killing by A. actinomycetemcomitans.
Aggregatibacter actinomycetemcomitans; biofilm; dispersin B; HL-60; J774-1; leukotoxin; macrophage; poly-N-acetylglucosamine; THP-1
Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans is a pathogen that causes localized aggressive periodontitis and extraoral infections including infective endocarditis. Recently, we reported that A. actinomycetemcomitans is beta-hemolytic on certain growth media due to the production of leukotoxin (LtxA). Based on this observation and our ability to generate random transposon insertions in A. actinomycetemcomitans, we developed and carried out a rapid screen for LtxA mutants. Using PCR, we mapped several of the mutations to genes that are known or predicted to be required for LtxA production, including ltxA, ltxB, ltxD, and tdeA. In addition, we identified an insertion in a gene previously not recognized to be involved in LtxA biosynthesis, ptsH. ptsH encodes the protein HPr, a phosphocarrier protein that is part of the sugar phosphotransferase system. HPr results in the phosphorylation of other proteins and ultimately in the activation of adenylate cyclase and cyclic AMP (cAMP) production. The ptsH mutant showed only partial hemolysis on blood agar and did not produce LtxA. The phenotype was complemented by supplying wild-type ptsH in trans, and real-time PCR analysis showed that the ptsH mutant produced approximately 10-fold less ltxA mRNA than the wild-type strain. The levels of cAMP in the ptsH mutant were significantly lower than in the wild-type strain, and LtxA production could be restored by adding exogenous cAMP to the culture.
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.
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.
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 toxin's ability to destroy red blood cells.
Cells of Actinobacillus actinomycetemcomitans, a gram-negative pathogen responsible for an aggressive form of juvenile periodontitis, form tenaciously adherent biofilms on solid surfaces. The bacteria produce long fibrils of bundled pili, which are required for adherence. Mutations in flp-1, which encodes the major subunit of the pili, or any of seven downstream tad genes (tadABCDEFG) cause defects in fibril production, autoaggregation, and tenacious adherence. We proposed that the tad genes specify part of a novel secretion system for the assembly and transport of Flp pili. The predicted amino acid sequence of TadA (426 amino acids, 47,140 Da) contains motifs for nucleotide binding and hydrolysis common among secretion NTP hydrolase (NTPase) proteins. In addition, the tadA gene is the first representative of a distinct subfamily of potential type IV secretion NTPase genes. Here we report studies on the function of TadA. The tadA gene was altered to express a modified version of TadA that has the 11-residue epitope (T7-TAG) fused to its C terminus. The TadA-T7 protein was indistinguishable from the wild type in its ability to complement the fibril and adherence defects of A. actinomycetemcomitans tadA mutants. Although TadA is not predicted to have a transmembrane domain, the protein was localized to the inner membrane and cytoplasmic fractions of A. actinomycetemcomitans cells, indicating a possible peripheral association with the inner membrane. TadA-T7 was purified and found to hydrolyze ATP in vitro. The ATPase activity is stimulated by Triton X-100, with maximal stimulation at the critical micellar concentration. TadA-T7 forms multimers that are stable during sodium dodecyl sulfate-polyacrylamide gel electrophoresis in nonreducing conditions, and electron microscopy revealed that TadA-T7 can form structures closely resembling the hexameric rings of other type IV secretion NTPases. Site-directed mutagenesis was used to substitute Ala and Gln residues for the conserved Lys residue of the Walker A box for nucleotide binding. Both mutants were found to be defective in their ability to complement tadA mutants. We suggest that the ATPase activity of TadA is required to energize the assembly or secretion of Flp pili for tight adherence of A. actinomycetemcomitans.
Actinobacillus actinomycetemcomitans, the etiologic agent for localized juvenile periodontitis and certain other human infections, such as endocarditis, expresses a leukotoxin that acts on polymorphonuclear leukocytes and macrophages. Leukotoxin is a member of the highly conserved repeat toxin (RTX) family of bacterial toxins expressed by a variety of pathogenic bacteria. While the RTX toxins of other bacterial species are secreted, the leukotoxin of A. actinomycetemcomitans is thought to remain associated with the bacterial cell. We have examined leukotoxin production and localization in rough (adherent) and smooth (nonadherent) strains of A. actinomycetemcomitans. We found that leukotoxin expressed by the rough, adherent, clinical isolate CU1000N is indeed cell associated, as expected. However, we were surprised to find that smooth, nonadherent strains of A. actinomycetemcomitans, including Y4, JP2 (a strain expressing a high level of toxin), and CU1060N (an isogenic smooth variant of CU1000N), secrete an abundance of leukotoxin into the culture supernatants during early stages of growth. After longer times of incubation, leukotoxin disappears from the supernatants, and its loss is accompanied by the appearance of a number of low-molecular-weight polypeptides. The secreted leukotoxin is active, as evidenced by its ability to kill HL-60 cells in vitro. We found that the growth phase and initial pH of the growth medium significantly affect the abundance of secreted leukotoxin, and we have developed a rapid (<2 h) method to partially purify large amounts of leukotoxin. Remarkably, mutations in the tad genes, which are required for tight nonspecific adherence of A. actinomycetemcomitans to surfaces, cause leukotoxin to be released from the bacterial cell. These studies show that A. actinomycetemcomitans has the potential to secrete abundant leukotoxin. It is therefore appropriate to consider a possible role for leukotoxin secretion in the pathogenesis of A. actinomycetemcomitans.
The gram-negative coccobacillus, Actinobacillus actinomycetemcomitans, is the putative agent for localized juvenile periodontitis, a particularly destructive form of periodontal disease in adolescents. This bacterium has also been isolated from a variety of other infections, notably endocarditis. Fresh clinical isolates of A. actinomycetemcomitans form tenacious biofilms, a property likely to be critical for colonization of teeth and other surfaces. Here we report the identification of a locus of seven genes required for nonspecific adherence of A. actinomycetemcomitans to surfaces. The recently developed transposon IS903φkan was used to isolate mutants of the rough clinical isolate CU1000 that are defective in tight adherence to surfaces (Tad−). Unlike wild-type cells, Tad− mutant cells adhere poorly to surfaces, fail to form large autoaggregates, and lack long, bundled fibrils. Nucleotide sequencing and genetic complementation analysis revealed a 6.7-kb region of the genome with seven adjacent genes (tadABCDEFG) required for tight adherence. The predicted TadA polypeptide is similar to VirB11, an ATPase involved in macromolecular transport. The predicted amino acid sequences of the other Tad polypeptides indicate membrane localization but no obvious functions. We suggest that the tad genes are involved in secretion of factors required for tight adherence of A. actinomycetemcomitans. Remarkably, complete and highly conserved tad gene clusters are present in the genomes of the bubonic plague bacillus Yersinia pestis and the human and animal pathogen Pasteurella multocida. Partial tad loci also occur in strikingly diverse Bacteria and Archaea. Our results show that the tad genes are required for tight adherence of A. actinomycetemcomitans to surfaces and are therefore likely to be essential for colonization and pathogenesis. The occurrence of similar genes in a wide array of microorganisms indicates that they have important functions. We propose that tad-like genes have a significant role in microbial colonization.