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1.  Gangliosides Block Aggregatibacter Actinomycetemcomitans Leukotoxin (LtxA)-Mediated Hemolysis  
Toxins  2010;2(12):2824-2836.
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.
doi:10.3390/toxins2122824
PMCID: PMC3153184  PMID: 22069577
erythrocytes; toxin; periodontal disease; endocarditis; RTX toxin
2.  Human CD18 Is the Functional Receptor for Aggregatibacter actinomycetemcomitans Leukotoxin▿  
Infection and Immunity  2007;75(10):4851-4856.
Aggregatibacter (Actinobacillus) actinomycetemcomitans is the causative organism of localized aggressive periodontitis, a rapidly progressing degenerative disease of the gingival and periodontal ligaments, and is also implicated in causing subacute infective endocarditis in humans. The bacterium produces a variety of virulence factors, including an exotoxic leukotoxin (LtxA) that is a member of the repeats-in-toxin (RTX) family of bacterial cytolysins. LtxA exhibits a unique specificity to macrophages and polymorphonuclear cells of humans and other primates. Human lymphocyte function-associated antigen 1 (LFA-1) has been implicated as the putative receptor for LtxA. Human LFA-1 comprises the CD11a and CD18 subunits. It is not clear, however, which of its subunits serves as the functional receptor that confers species-specific susceptibility to LtxA. Here we demonstrate that the human CD18 is the receptor for LtxA based on experiments performed with chimeric β2-integrins recombinantly expressed in a cell line that is resistant to LtxA effects. In addition, we show that the cysteine-rich tandem repeats encompassing integrin-epidermal growth factor-like domains 2, 3, and 4 of the extracellular region of human CD18 are critical for conferring susceptibility to LtxA-induced biological effects.
doi:10.1128/IAI.00314-07
PMCID: PMC2044523  PMID: 17635865
3.  Regulation of Aggregatibacter (Actinobacillus) actinomycetemcomitans Leukotoxin Secretion by Iron▿  
Journal of Bacteriology  2006;188(24):8658-8661.
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.
doi:10.1128/JB.01253-06
PMCID: PMC1698250  PMID: 17041062
4.  Sialic Acid Residues Are Essential for Cell Lysis Mediated by Leukotoxin from Aggregatibacter actinomycetemcomitans 
Infection and Immunity  2014;82(6):2219-2228.
Leukotoxin (LtxA) from Aggregatibacter actinomycetemcomitans is known to target and lyse β2-integrin-expressing cells such as polymorphonuclear leukocytes and macrophages. LtxA is an important virulence factor that facilitates chronic inflammation and is strongly associated with a fast-progressing form of periodontitis caused by the JP2 clone of the bacterium. Here, we show that sialic acid residues are important for LtxA-induced cell lysis, regardless of whether the cell express β2-integrin or not. Clearly, removal of sialic acid groups significantly reduces a β2-integrin-specific LtxA-induced lysis. Moreover, sialic acid presented on alternative proteins, such as, for instance, on erythrocytes that do not express β2-integrin, also makes the cells more sensitive to LtxA. The data also illustrate the importance of the negative charge in order for the sialic acid to associate LtxA with the membrane. Removal of sialic acid is in itself sufficient to significantly reduce the negative charge on the erythrocytes. Moreover, we found that on human erythrocytes there is a positive association between the sensitivity to LtxA and the amount of negative charge caused by sialic acid. Interestingly, these features are not shared by all RTX toxins, since α-hemolysin from Escherichia coli induced cell lysis of both β2-integrin-expressing and nonexpressing cells and this lysis is independent of the presence of sialic acid residues. In conclusion, LtxA not only is cytotoxic to β2-integrin-expressing cells but can potentially initiate cell lysis in all cells that present a sufficient density of sialic acid groups on their plasma membrane.
doi:10.1128/IAI.01647-14
PMCID: PMC4019149  PMID: 24643533
5.  Screen for Leukotoxin Mutants in Aggregatibacter actinomycetemcomitans: Genes of the Phosphotransferase System Are Required for Leukotoxin Biosynthesis▿  
Infection and Immunity  2008;76(8):3561-3568.
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.
doi:10.1128/IAI.01687-07
PMCID: PMC2493242  PMID: 18541661
6.  TdeA, a TolC-like protein required for toxin and drug export in Aggregatibacter (Actinobacillus) actinomycetemcomitans 
Gene  2006;388(1-2):83-92.
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.
doi:10.1016/j.gene.2006.10.004
PMCID: PMC1831674  PMID: 17116373
leukotoxin; periodontitis; endocarditis; outer membrane protein; antibiotics
7.  Aggregatibacter actinomycetemcomitans leukotoxin is post-translationally modified by addition of either saturated or hydroxylated fatty acyl chains 
Molecular Oral Microbiology  2011;26(4):262-276.
SUMMARY
Aggregatibacter actinomycetemcomitans, a common inhabitant of the human upper aerodigestive tract, produces a repeat in toxin (RTX), leukotoxin (LtxA). The LtxA is transcribed as a 114-kDa inactive protoxin with activation being achieved by attachment of short chain fatty acyl groups to internal lysine residues. Methyl esters of LtxA that were isolated from A. actinomycetemcomitans strains JP2 and HK1651 and subjected to gas chromatography/mass spectrometry contained palmitoyl (C16:0, 27–29%) and palmitolyl (C16:1 cis Δ9, 43–44%) fatty acyl groups with smaller quantities of myristic (C14:0, 14%) and stearic (C18:0, 12–14%) fatty acids. Liquid chromatography/mass spectrometry of tryptic peptides from acylated and unacylated recombinant LtxA confirmed that Lys562 and Lys687 are the sites of acyl group attachment. During analysis of recombinant LtxA peptides, we observed peptide spectra that were not observed as part of the RTX acylation schemes of either Escherichia coli α-hemolysin or Bordetella pertussis cyclolysin. Mass calculations of these spectra suggested that LtxA was also modified by the addition of monohydroxylated forms of C14 and C16 acyl groups. Multiple reaction monitoring mass spectrometry identified hydroxymyristic and hydroxypalmitic acids in wild-type LtxA methyl esters. Single or tandem replacement of Lys562 and Lys687 with Arg blocks acylation, resulting in a >75% decrease in cytotoxicity when compared with wild-type toxin, suggesting that these posttranslational modifications are playing a critical role in LtxA-mediated target cell cytotoxicity.
doi:10.1111/j.2041-1014.2011.00617.x
PMCID: PMC3404814  PMID: 21729247
Aggregatibacter actinomycetemcomitans; RTX toxins; leukotoxin; acylation; post-translational protein modification; gas chromatography/mass spectrometry; reverse-phase liquid chromatography/tandem mass spectrometry
8.  Anti-leukemia activity of a bacterial toxin with natural specificity for LFA-1 on white blood cells 
Leukemia research  2009;34(6):777-785.
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.
doi:10.1016/j.leukres.2009.08.022
PMCID: PMC2859097  PMID: 19747730
Acute myeloid leukemia; lymphoma; immunotoxin; targeted therapy
9.  Mlc Is a Transcriptional Activator with a Key Role in Integrating Cyclic AMP Receptor Protein and Integration Host Factor Regulation of Leukotoxin RNA Synthesis in Aggregatibacter actinomycetemcomitans 
Journal of Bacteriology  2013;195(10):2284-2297.
Aggregatibacter actinomycetemcomitans, a periodontal pathogen, synthesizes leukotoxin (LtxA), a protein that helps the bacterium evade the host immune response. Transcription of the ltxA operon is induced during anaerobic growth. The cyclic AMP (cAMP) receptor protein (CRP) indirectly increases ltxA expression, but the intermediary regulator is unknown. Integration host factor (IHF) binds to and represses the leukotoxin promoter, but neither CRP nor IHF is responsible for the anaerobic induction of ltxA RNA synthesis. Thus, we have undertaken studies to identify other regulators of leukotoxin transcription and to demonstrate how these proteins work together to modulate leukotoxin synthesis. First, analyses of ltxA RNA expression from defined leukotoxin promoter mutations in the chromosome identify positions −69 to −35 as the key control region and indicate that an activator protein modulates leukotoxin transcription. We show that Mlc, which is a repressor in Escherichia coli, functions as a direct transcriptional activator in A. actinomycetemcomitans; an mlc deletion mutant reduces leukotoxin RNA synthesis, and recombinant Mlc protein binds specifically at the −68 to −40 region of the leukotoxin promoter. Furthermore, we show that CRP activates ltxA expression indirectly by increasing the levels of Mlc. Analyses of Δmlc, Δihf, and Δihf Δmlc strains demonstrate that Mlc can increase RNA polymerase (RNAP) activity directly and that IHF represses ltxA RNA synthesis mainly by blocking Mlc binding. Finally, a Δihf Δmlc mutant still induces ltxA during anaerobic growth, indicating that there are additional factors involved in leukotoxin transcriptional regulation. A model for the coordinated regulation of leukotoxin transcription is presented.
doi:10.1128/JB.02144-12
PMCID: PMC3650521  PMID: 23475968
10.  Aggregatibacter actinomycetemcomitans leukotoxin cytotoxicity occurs through bilayer destabilization 
Cellular microbiology  2012;14(6):869-881.
Summary
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 31P 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.
doi:10.1111/j.1462-5822.2012.01762.x
PMCID: PMC3412409  PMID: 22309134
11.  Pathogenicity of the highly leukotoxic JP2 clone of Aggregatibacter actinomycetemcomitans and its geographic dissemination and role in aggressive periodontitis 
Journal of Oral Microbiology  2014;6:10.3402/jom.v6.23980.
For decades, Aggregatibacter actinomycetemcomitans has been associated with aggressive forms of periodontitis in adolescents. In the middle of the 1990s, a specific JP2 clone of A. actinomycetemcomitans, belonging to the cluster of serotype b strains of A. actinomycetemcomitans and having a number of other characteristics, was found to be strongly associated with aggressive forms of periodontitis, particularly in North Africa. Although several longitudinal studies still point to the bacterial species, A. actinomycetemcomitans as a risk factor of aggressive periodontitis, it is now also widely accepted that the highly leukotoxic JP2 clone of A. actinomycetemcomitans is implicated in rapidly progressing forms of aggressive periodontitis. The JP2 clone strains are highly prevalent in human populations living in Northern and Western parts of Africa. These strains are also prevalent in geographically widespread populations that have originated from the Northwest Africa. Only sporadic signs of a dissemination of the JP2 clone strains to non-African populations have been found despite Africans living geographically widespread for hundreds of years. It remains an unanswered question if a particular host tropism exists as a possible explanation for the frequent colonization of the Northwest African population with the JP2 clone. Two exotoxins of A. actinomycetemcomitans are known, leukotoxin (LtxA) and cytolethal distending toxin (Cdt). LtxA is able to kill human immune cells, and Cdt can block cell cycle progression in eukaryotic cells and thus induce cell cycle arrest. Whereas the leukotoxin production is enhanced in JP2 clone strains thus increasing the virulence potential of A. actinomycetemcomitans, it has not been possible so far to demonstrate such a role for Cdt. Lines of evidence have led to the understanding of the highly leukotoxic JP2 clone of A. actinomycetemcomitans as an aetiological factor of aggressive periodontitis. Patients, who are colonized with the JP2 clone, are likely to share this clone with several family members because the clone is transmitted through close contacts. This is a challenge to the clinicians. The patients need intense monitoring of their periodontal status as the risk for developing severely progressing periodontal lesions are relatively high. Furthermore, timely periodontal treatment, in some cases including periodontal surgery supplemented by the use of antibiotics, is warranted. Preferably, periodontal attachment loss should be prevented by early detection of the JP2 clone of A. actinomycetemcomitans by microbial diagnostic testing and/or by preventive means.
doi:10.3402/jom.v6.23980
PMCID: PMC4139931  PMID: 25206940
Virulence factors; spreading; geographical dissemination; leukotoxin; cytolethal distending toxin; host response
12.  Effects of Aggregatibacter actinomycetemcomitans leukotoxin on endothelial cells☆ 
Microbial Pathogenesis  2013;61-62(100):43-50.
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.
Highlights
•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.
doi:10.1016/j.micpath.2013.05.001
PMCID: PMC3885975  PMID: 23665198
Endothelium; Leukotoxin; Aggregatibacter actinomycetemcomitans; Apoptosis; Activation
13.  Aggregatibacter actinomycetemcomitans Leukotoxin 
Journal of Dental Research  2010;89(6):561-570.
Aggregatibacter actinomycetemcomitans is a Gram-negative bacterium that colonizes the human oral cavity and is the causative agent for localized aggressive periodontitis (LAP), an aggressive form of periodontal disease that occurs in adolescents. A. actinomycetemcomitans secretes a protein toxin, leukotoxin (LtxA), which helps the bacterium evade the host immune response during infection. LtxA is a membrane-active toxin that specifically targets white blood cells (WBCs). In this review, we discuss recent developments in this field, including the identification and characterization of genes and proteins involved in secretion, regulation of LtxA, biosynthesis, newly described activities of LtxA, and how LtxA may be used as a therapy for the treatment of diseases.
doi:10.1177/0022034510363682
PMCID: PMC3144085  PMID: 20200418
leukemia; periodontitis; RTX toxin; LAP
14.  Lactobacillus salivarius and L. gasseri down-regulate Aggregatibacter actinomycetemcomitans exotoxins expression 
Annals of Microbiology  2013;64(2):611-617.
Beneficial microbes, such as lactobacilli establish a symbiosis with the host and confer health-associated effects, by limiting the growth of indigenous pathogens and challenging microbes introduced by altered foods. Nevertheless, there is scarce information on the effects of beneficial microbes on the virulence properties of bacterial species associated with oral diseases, such as periodontitis. Aggregatibacter actinomycetemcomitans is a Gram-negative species highly implicated in the etiology of localized aggressive periodontitis. The objective of this study was to investigate the effect of lactobacilli on the expression of the two major virulence factors of A. actinomycetemcomitans. Lactobacillus salivarius and L. gasseri were selected as beneficial species. The gene expressions of leukotoxin (LtxA) and cytolethal distending toxin (CdtB) by A. actinomycetemcomitans were analyzed in response to challenge by lactobacilli cell-free supernatants. Neither lactobacilli affected the growth, but strongly attenuated the expressions of both CdtB and LtxA in the two A. actinomycetemcomitans strains tested. This reduction of the expression of these two exotoxins was time-dependent. These fundamental findings may indicate that lactobacilli can reduce the virulence of putative opportunistic oral pathogens, and may provide insights to future therapeutic approaches for the respective diseases.
Electronic supplementary material
The online version of this article (doi:10.1007/s13213-013-0694-x) contains supplementary material, which is available to authorized users.
doi:10.1007/s13213-013-0694-x
PMCID: PMC4028514  PMID: 24860281
Probiotics; Oral cavity; Cytolethal distending toxin; Leukotoxin; Periodontal disease
15.  Distribution of biotypes and leukotoxic activity of Aggregatibacter actinomycetemcomitans isolated from Brazilian patients with chronic periodontitis 
Brazilian Journal of Microbiology  2008;39(4):658-663.
Aggregatibacter actinomycetemcomitans is an important etiologic agent of the periodontitis and is associated with extra-oral infections. In this study, the detection of the ltxA gene as well as the ltx promoter region from leukotoxic A. actinomycetemcomitans isolated from 50 Brazilian patients with periodontitis and 50 healthy subjects was performed. The leukotoxic activity on HL-60 cells was also evaluated. Leukotoxic activity was determined using a trypan blue exclusion method. The 530 bp deletion in the promoter region was evaluated by PCR using a PRO primer pair. A. actinomycetemcomitans was detected by culture and directly from crude subgingival biofilm by PCR using specific primers. By culture, A. actinomycetemcomitans was detected in nine (18%) of the periodontal patients and one (2%) healthy subject. However, by PCR, this organism was detected in 44% of the periodontal patients and in 16% of the healthy subjects. It was verified a great discrepancy between PCR detection of the ltx operon promoter directly from crude subgingival biofilm and from bacterial DNA. Only one periodontal sample harbored highly leukotoxic A. actinomycetemcomitans. Moreover, biotype II was the most prevalent and no correlation between biotypes and leukotoxic activity was observed. The diversity of leukotoxin expression by A. actinomycetemcomitans suggests a role of this toxin in the pathogenesis of periodontal disease and other infectious diseases.
doi:10.1590/S1517-838220080004000011
PMCID: PMC3768452  PMID: 24031284
Aggregatibacter actinomycetemcomitans; biotype; leukotoxin; periodontitis
16.  Regulation of Actinobacillus actinomycetemcomitans leukotoxin expression: analysis of the promoter regions of leukotoxic and minimally leukotoxic strains. 
Infection and Immunity  1994;62(2):501-508.
The leukotoxin of Actinobacillus actinomycetemcomitans has been implicated as a virulence determinant in various human infections and is encoded by a multigene operon consisting of four known genes, designated ltxC, ltxA, ltxB, and ltxD. The ltx operon appears to be present in all A. actinomycetemcomitans strains, but levels of toxin expression vary greatly among strains. Thus, to gain a better understanding of the expression and regulation of the ltx operon, we have analyzed the ltx promoters of a highly toxic (JP2) and a minimally toxic (652) strain of A. actinomycetemcomitans. The nucleotide sequence of the JP2 ltx promoter contains -10 and -35 elements situated 350 bases upstream of ltxC, and primer extension of JP2 RNA confirmed that they are functional in vivo. However, a second primer extension product of 40 bases was present, and analysis of a series of truncated JP2 promoters fused to lacZ suggested that the region immediately upstream of ltxC also promotes transcription in Escherichia coli. These results suggest that two promoters may direct ltx expression in JP2. In addition, a small open reading frame capable of encoding a peptide of 78 amino acids was identified upstream of ltxC. Northern blots showed that this open reading frame is transcribed as part of a 4.2-kb mRNA, a transcript not previously identified as being derived from the ltx operon. In contrast, strain 652 expresses low steady-state levels of ltx mRNA, and its intact ltx promoter was inefficient in transcribing lacZ in E. coli. The nucleotide sequence of the 652 promoter is similar to that of the JP2 promoter but contains a region of 530 bp that is not present in JP2. Of 15 additional strains of A. actinomycetemcomitans that were analyzed, 13 contained promoters resembling the 652 sequence and 2 possessed JP2-like promoters. Both strains possessing the JP2-like promoter expressed 10- to 20-fold-higher levels of leukotoxin than did the strains possessing promoters resembling the 652 promoter. These results suggest that high levels of leukotoxin expression may correlate with the presence of the JP2-like promoter.
Images
PMCID: PMC186135  PMID: 8300209
17.  Actinobacillus actinomycetemcomitans leukotoxin requires lipid microdomains for target cell cytotoxicity 
Cellular Microbiology  2006;8(11):1753-1767.
Summary
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 β2 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 (Jβ2.7) 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. Jβ2.7 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.
doi:10.1111/j.1462-5822.2006.00746.x
PMCID: PMC3404838  PMID: 16827908
18.  A Comparison of Aggregatibacter actinomycetemcomitans (Aa) Virulence Traits in a Rat Model for Periodontal Disease 
PLoS ONE  2013;8(7):e69382.
Our aim was to explore the effects of Cytolethal Distending toxin (Cdt) in a well established rat model of periodontal disease where leukotoxin (LtxA) was thought to have no known effect. In vitro studies, were used to assess CdtB activity using Aa Leukotoxin as a negative control. These studies showed that both CdtB and LtxA (unexpectedly) exerted significant effects on CD4+ T cells. As a result we decided to compare the effects of these two prominent Aa virulence factors on bone loss using our rat model of Aa-induced periodontitis. In this model, Aa strains, mutant in cdtB and ltxA, were compared to their parent non-mutant strains and evaluated for colonization, antibody response to Aa, bone loss and disease. We found that bone loss/disease caused by the ltxA mutant strain, in which cdtB was expressed, was significantly less (p<0.05) than that due to the wild type strain. On the other hand, the disease caused by cdtB mutant strain, in which ltxA was expressed, was not significantly different from the wild type strain. This data indicates that Aa LtxA exerts a greater effect on bone loss than Cdt in this rat model of periodontal disease and supports the utility of this model to dissect specific virulence factors as they relate to immunopathology in studies of Aa-induced disease.
doi:10.1371/journal.pone.0069382
PMCID: PMC3720274  PMID: 23936002
19.  Porcine CD18 mediates Actinobacillus pleuropneumoniae ApxIII species-specific toxicity 
Veterinary Research  2009;40(4):33.
Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, produces Apx toxins that are recognized as major virulence factors. Recently, we showed that ApxIIIA-cytotoxic activity specifically targets Sus scrofa leukocytes. Since both LtxA from Aggregatibacter actinomycetemcomitans (aggressive periodontitis in humans) and LktA from Mannheimia haemolytica (pneumonia in ruminants) share this characteristic, respectively towards human and ruminant leukocytes, and because both use the CD18 subunit to interact with their respective LFA-1, we hypothesized that ApxIIIA was likely to bind porcine CD18 to exercise its deleterious effects on pig leukocytes. A β 2−integrin-deficient ApxIIIA-resistant human erythroleukemic cell line was transfected either with homologous or heterologous CD11a/CD18 heterodimers using a set of plasmids coding for human (ApxIIIA-resistant), bovine (-resistant) and porcine (-susceptible) CD11a and CD18 subunits. Cell preparations that switched from ApxIIIA-resistance to -susceptibility were then sought to identify the LFA-1 subunit involved. The results showed that the ApxIIIA-resistant recipient cell line was rendered susceptible only if the CD18 partner within the LFA-1 heterodimer was that of the pig. It is concluded that porcine CD18 is necessary to mediate A. pleuropneumoniae ApxIIIA toxin-induced leukolysis.
doi:10.1051/vetres/2009016
PMCID: PMC2701182  PMID: 19356397
Actinobacillus pleuropneumoniae; ApxIIIA; porcine; LFA-1; CD18
20.  Lipopolysaccharides mediate leukotoxin secretion in Aggregatibacter actinomycetemcomitans 
Molecular Oral Microbiology  2011;27(2):70-82.
SUMMARY
We previously reported that lipopolysaccharide (LPS) related sugars are associated with the glycosylation of the collagen adhesin EmaA, a virulence determinant of Aggregatibacter actinomycetemcomitans. In this study, the role of LPS in the secretion of other virulence factors was investigated. The secretion of the epithelial adhesin Aae, the immunoglobulin Fc receptor Omp34 and leukotoxin were examined in a mutant strain with inactivated TDP-4-keto-6-deoxy-d-glucose 3,5-epimerase (rmlC), which resulted in altered O-antigen polysaccharides (O-PS) of LPS. The secretion of Aae and Omp34 was not affected. However, the leukotoxin secretion, which is mediated by the TolC-dependent Type I secretion system, was altered in the rmlC mutant. The amount of secreted leukotoxin in the bacterial growth medium was reduced 9-fold, with a concurrent 4-fold increase of the membrane-bound toxin in the mutant compared with the wild type strain. The altered leukotoxin secretion pattern was restored to the wild-type by complementation of the rmlC gene in trans. Examination of the ltxA mRNA levels indicated that the leukotoxin secretion was posttranscriptionally regulated in the modified O-PS containing strain. The mutant strain also showed increased resistance to vancomycin, an antibiotic dependent on TolC for internalization, indicating that TolC was affected. Overexpression of TolC in the rmlC mutant resulted in an increased TolC level in the outer membrane but did not restore the leukotoxin secretion profile to the wild-type phenotype. The data suggest that O-PS mediate leukotoxin secretion in A. actinomycetemcomitans.
doi:10.1111/j.2041-1014.2011.00632.x
PMCID: PMC3296970  PMID: 22394466
Lipopolysaccharides; Leukotoxin; Type I secretion system; TolC
21.  Secretion of RTX Leukotoxin by Actinobacillus actinomycetemcomitans 
Infection and Immunity  2000;68(11):6094-6100.
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.
PMCID: PMC97685  PMID: 11035711
22.  Membrane Morphology and Leukotoxin Secretion Are Associated with a Novel Membrane Protein of Aggregatibacter actinomycetemcomitans▿  
Journal of Bacteriology  2008;190(17):5972-5980.
Gram-negative bacteria display either a flat or an irregular outer membrane. The periodontal pathogen Aggregatibacter (Actinobacillus) actinomycetemcomitans has an irregular outer membrane. We have identified a gene that is associated with the biogenesis of this morphology. The gene is part of a three-gene operon and codes for a 141-kDa protein designated morphogenesis protein C (MorC), which is conserved in several gram-negative bacteria including Haemophilus influenzae and Pasteurella multocida. Insertional inactivation of this gene resulted in the conversion of an irregularly shaped membrane to a flat membrane. Associated with this morphological change were the autoaggregation of the bacteria during planktonic growth and a concomitant increase in the surface hydrophobicity of the bacterium. The absence of MorC also resulted in the loss of the secretion of leukotoxin but not the ltxA transcription. Our findings suggest that MorC is critical for membrane morphology and leukotoxin secretion in A. actinomycetemcomitans.
doi:10.1128/JB.00548-08
PMCID: PMC2519528  PMID: 18621903
23.  Differential transcription of virulence genes in Aggregatibacter actinomycetemcomitans serotypes 
Journal of Oral Microbiology  2013;5:10.3402/jom.v5i0.21473.
Background
Aggregatibacter actinomycetemcomitans serotypes are clearly associated with periodontitis or health, which suggests distinct strategies for survival within the host.
Objective
We investigated the transcription profile of virulence-associated genes in A. actinomycetemcomitans serotype b (JP2 and SUNY 465) strains associated with disease and serotype a (ATCC 29523) strain associated with health.
Design
Bacteria were co-cultured with immortalized gingival epithelial cells (OBA-9). The adhesion efficiency after 2 hours and the relative transcription of 13 genes were evaluated after 2 and 24 hours of interaction.
Results
All strains were able to adhere to OBA-9, and this contact induced transcription of pgA for polysaccharide biosynthesis in all tested strains. Genes encoding virulence factors as Omp29, Omp100, leukotoxin, and CagE (apoptotic protein) were more transcribed by serotype b strains than by serotype a. ltxA and omp29, encoding the leukotoxin and the highly antigenic Omp29, were induced in serotype b by interaction with epithelial cells. Factors related to colonization (aae, flp, apaH, and pgA) and cdtB were upregulated in serotype a strain after prolonged interaction with OBA-9.
Conclusion
Genes relevant for surface colonization and interaction with the immune system are regulated differently among the strains, which may help explaining their differences in association with disease.
doi:10.3402/jom.v5i0.21473
PMCID: PMC3807012  PMID: 24159369
Aggregatibacter actinomycetemcomitans; gene expression; epithelial cell; infection
24.  Proinflammatory effect in whole blood by free soluble bacterial components released from planktonic and biofilm cells 
BMC Microbiology  2008;8:206.
Background
Aggregatibacter actinomycetemcomitans is an oral bacterium associated with aggressive forms of periodontitis. Increasing evidence points to a link between periodontitis and cardiovascular diseases, however, the underlying mechanisms are poorly understood. This study investigated the pathogenic potential of free-soluble surface material, released from live planktonic and biofilm A. actinomycetemcomitans cells.
Results
By employing an ex vivo insert model (filter pore size 20 nm) we demonstrated that the A. actinomycetemcomitans strain D7S and its derivatives, in both planktonic and in biofilm life-form, released free-soluble surface material independent of outer membrane vesicles. This material clearly enhanced the production of several proinflammatory cytokines (IL-1β, TNF-α, IL-6, IL-8, MIP-1β) in human whole blood, as evidenced by using a cytokine antibody array and dissociation-enhanced-lanthanide-fluorescent-immunoassay. In agreement with this, quantitative real-time PCR indicated a concomitant increase in transcription of each of these cytokine genes. Experiments in which the LPS activity was blocked with polymyxin B showed that the stimulatory effect was only partly LPS-dependent, suggesting the involvement of additional free-soluble factors. Consistent with this, MALDI-TOF-MS and immunoblotting revealed release of GroEL-like protein in free-soluble form. Conversely, the immunomodulatory toxins, cytolethal distending toxin and leukotoxin, and peptidoglycan-associated lipoprotein, appeared to be less important, as evidenced by studying strain D7S cdt/ltx double, and pal single mutants. In addition to A. actinomycetemcomitans a non-oral species, Escherichia coli strain IHE3034, tested in the same ex vivo model also released free-soluble surface material with proinflammatory activity.
Conclusion
A. actinomycetemcomitans, grown in biofilm and planktonic form, releases free-soluble surface material independent of outer membrane vesicles, which induces proinflammatory responses in human whole blood. Our findings therefore suggest that release of surface components from live bacterial cells could constitute a mechanism for systemic stimulation and be of particular importance in chronic localized infections, such as periodontitis.
doi:10.1186/1471-2180-8-206
PMCID: PMC2612679  PMID: 19038023
25.  Proteomics of Protein Secretion by Aggregatibacter actinomycetemcomitans 
PLoS ONE  2012;7(7):e41662.
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.
doi:10.1371/journal.pone.0041662
PMCID: PMC3405016  PMID: 22848560

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