Specificity and levels of oral and systemic antibodies to Actinobacillus actinomycetemcomitans

Abstract Salivary and gingival crevicular fluid antibodies and systemic antibodies were analysed for levels and specificity against Actinobacillus actinomycetemcomitans components. The major reactivity of salivary and serum IgA1 and IgA2 antibodies to the periodontal pathogen A. actinomycetemcomitans was against bands between 14 and 83 kD for IgA1 and bands between 14 and 68 kD for IgA2 in Western blot. In addition to specific binding, there was also a hitherto unrecognized Fc-mediated binding of IgG antibodies to an A. actinomycetemcomitans component around 50 kD. Serum IgG antibodies to A. actinomycetemcomitans leukotoxin displayed the highest median value and only 1 individual showed salivary IgM antibodies in ELISA. Elevated levels of gingival crevicular fluid lgA2 antibodies indicated a local production of IgA from periodontal tissues. Using synthetic peptides, several distinct epitopes on the leukotoxin were recognized by both salivary and serum IgA antibodies.     

Aggregatibacter actinomycetemcomitans,a potent immunoregulator of the periodontal host defense system and alveolar bone homeostasis

Aggregatibacter actinomycetemcomitans is a perio‐pathogenic bacteria that has long been associated with localized aggressive periodontitis. The mechanisms of its pathogenicity have been studied in humans and preclinical experimental models. Although different serotypes of A. actinomycetemcomitans have differential virulence factor expression, A. actinomycetemcomitans cytolethal distending toxin (CDT), leukotoxin, and lipopolysaccharide (LPS) have been most extensively studied in the context of modulating the host immune response. Following colonization and attachment in the oral cavity, A. actinomycetemcomitans employs CDT, leukotoxin, and LPS to evade host innate defense mechanisms and drive a pathophysiologic inflammatory response. This supra‐physiologic immune response state perturbs normal periodontal tissue remodeling/turnover and ultimately has catabolic effects on periodontal tissue homeostasis. In this review, we have divided the host response into two systems: non‐hematopoietic and hematopoietic. Non‐hematopoietic barriers include epithelium and fibroblasts that initiate the innate immune host response. The hematopoietic system contains lymphoid and myeloid‐derived cell lineages that are responsible for expanding the immune response and driving the pathophysiologic inflammatory state in the local periodontal microenvironment. Effector systems and signaling transduction pathways activated and utilized in response to A. actinomycetemcomitans will be discussed to further delineate immune cell mechanisms during A. actinomycetemcomitans infection. Finally, we will discuss the osteo‐immunomodulatory effects induced by A. actinomycetemcomitans and dissect the catabolic disruption of balanced osteoclast–osteoblast‐mediated bone remodeling, which subsequently leads to net alveolar bone loss.     

Membrane association and destabilization by Aggregatibacter actinomycetemcomitans leukotoxin requires changes in secondary structures

Aggregatibacter actinomycetemcomitans is a common inhabitant of the upper aerodigestive tract of humans and non‐human primates and is associated with disseminated infections, including lung and brain abscesses, pediatric infective endocarditis, and localized aggressive periodontitis. Aggregatibacter actinomycetemcomitans secretes a repeats‐in‐toxin protein, leukotoxin, which exclusively kills lymphocyte function‐associated antigen‐1‐bearing cells. The toxin's pathological mechanism is not fully understood; however, experimental evidence indicates that it involves the association with and subsequent destabilization of the target cell's plasma membrane. We have long hypothesized that leukotoxin secondary structure is strongly correlated with membrane association and destabilization. In this study, we tested this hypothesis by analysing lipid‐induced changes in leukotoxin conformation. Upon incubation of leukotoxin with lipids that favor leukotoxin‐membrane association, we observed an increase in leukotoxin α‐helical content that was not observed with lipids that favor membrane destabilization. The change in leukotoxin conformation after incubation with these lipids suggests that membrane binding and membrane destabilization have distinct secondary structural requirements, suggesting that they are independent events. These studies provide insight into the mechanism of cell damage that leads to disease progression by A. actinomycetemcomitans.     

Analysis of genotypic variation in genes associated with virulence in Aggregatibacter actinomycetemcomitans clinical isolates

Pinheiro ET, Kawamoto D, Ota‐Tsuzuki C, Almeida LRS, Nunes ACR, Longo PL, Wikstrom M, Mayer MPA. Analysis of genotypic variation in genes associated with virulence in Aggregatibacter actinomycetemcomitans clinical isolates. J Periodont Res 2011; 46: 310–317. © 2011 John Wiley & Sons A/S Background and Objective: Although certain serotypes of Aggregatibacter actinomycetemcomitans are associated more with aggressive periodontitis than are other serotypes, the correlation between distinct lineages and virulence traits in this species is poorly understood. This study aimed to evaluate the polymorphism of genes encoding putative virulence factors of clinical isolates, and to correlate these findings with A. actinomycetemcomitans serotypes, genotypes and periodontal status of the hosts. Material and Methods: Twenty‐six clinical isolates from diverse geographic populations with different periodontal conditions were evaluated. Genotyping was performed using pulse‐field gel electrophoresis. Polymorphisms in the genes encoding leukotoxin, Aae, ApaH and determinants for serotype‐specific O polysaccharide were investigated. Results: The isolates were classified into serotypes a–f, and exhibited three apaH genotypes, five aae alleles and 25 macrorestriction profiles. Two serotype b isolates (7.7%), obtained from Brazilian patients with aggressive periodontitis, were associated with the highly leukotoxic genotype; these isolates showed identical fingerprint patterns and aae and apaH genotypes. Serotype c, obtained from various periodontal conditions, was the most prevalent among Brazilian isolates, and isolates were distributed in two aae alleles, but formed a genetically distinct group based on apaH analysis. Cluster analysis showed a close relationship between fingerprinting genotypes and serotypes/apaH genotypes, but not with aae genotypes. Conclusion: Apart from the deletion in the ltx promoter region, no disease‐associated markers were identified. Non‐JP2‐like strains recovered from individuals with periodontal disease exhibited considerable genetic variation regarding aae/apaH genotypes, serotypes and XhoI DNA fingerprints.     

The in vivo T helper type 17 and regulatory T cell immune responses to Aggregatibacter actinomycetemcomitans

The periodontal pathogen Aggregatibacter actinomycetemcomitans is known to elicit a systemic immune response in the infected host, and occasionally causes non‐oral infections. Detailed information on its immunopathological responses and the involvement of bacterial virulence factors remains to be elucidated. The aim of this study was to assess the systemic immune response to A. actinomycetemcomitans oral infection. We used an animal model that simulates systemic dissemination of the bacteria by injecting live wild‐type (WT) D7S‐1 and a double knockout mutant of leukotoxin and cytolethal distending toxin (ΔltxΔcdt) A. actinomycetemcomitans strains in rat oral mucosa. Draining lymph nodes were examined for regulatory T (Treg) and T helper type 17 (Th17) cell subsets and their associated mediators. An increase in the proportion of Th17 cells and a decrease in Treg cells over the experimental period of 3 weeks were similarly observed for rats challenged with WT and ΔltxΔcdt. Significant upregulation and downregulation of proinflammatory cytokines in the Th17 gene pathway was noted, as well as several qualitative differences between WT and ΔltxΔcdt. Furthermore, we observed differential fold regulation in key genes associated with a proinflammatory response in ΔltxΔcdt‐inoculated rats relative to D7S‐1 group. This suggests that although the knockout of these two virulence factors (ΔltxΔcdt) may suppress certain proinflammatory genes, it causes similar over‐expression of other genes compared with D7S‐1, indicating a common factor that still remains in the pathogenicity of A. actinomycetemcomitans.     

A quinol peroxidase inhibitor prevents secretion of a leukotoxin from Aggregatibacter actinomycetemcomitans

Takashima E, Yamada H, Yajima A, Shiomi K, ōmura S, Kiyoshi K. A quinol peroxidase inhibitor prevents secretion of a leukotoxin from Aggregatibacter actinomycetemcomitans. J Periodont Res 2009; doi: 10.1111/j.1600‐0765.2009.01211.x. © 2009 John Wiley & Sons A/S Background and Objective: Quinol peroxidase (QPO) catalyzes peroxidase activity using quinol in the respiratory chain as a substrate. Quinol peroxidase is essential for the secretion of leukotoxin (LtxA), which destroys leukocytes and erythrocytes in humans and is one of the major virulence factors of Aggregatibacter actinomycetemcomitans, which is associated with localized aggressive periodontitis. In the present study, we aimed to find a highly potent QPO inhibitor to attenuate the virulence of A. actinomycetemcomitans. Material and Methods: For screening of QPO inhibitors, QPO activity was measured kinetically by SpectraMax Plus with 96‐well UV plates. Three hundred compounds in the Kitasato Institute for Life Sciences Chemical Library were screened. Secretion of LtxA in the culture supernatant was examined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Cytotoxicity against human promyelocytic leukemia cell line (HL‐60) cells from the culture supernatant was measured by Trypan Blue exclusion test. Results: The present study characterized ascofuranone as a highly potent inhibitor of QPO (K_i = 9.557 ± 0.865 nm ). Ascofuranone inhibited secretion of LtxA by A. actinomycetemcomitans in a dose‐dependent manner, making A. actinomycetemcomitans less pathogenic to HL‐60 cells. Conclusion: Quinol peroxidase inhibitors are promising candidates as alternative drugs for the treatment and prevention of the onset of localized aggressive periodontitis. Using ascofuranone as a seed compound, further study of QPO inhibitors could provide novel chemotherapeutic strategies for controlling localized aggressive periodontitis.     

Systemic Aggregatibacter actinomycetemcomitans Leukotoxin‐Neutralizing Antibodies in Periodontitis

Background: Leukotoxin (Ltx) expressed by Aggregatibacter actinomycetemcomitans is a powerful exotoxin, which can cause imbalance in host response. Immunoreactivity to Ltx is a marker for presence of leukotoxic A. actinomycetemcomitans, a presence that may modify the disease pattern of colonized individuals. The aim of the present study is to examine presence of systemic immunoreactivity to A. actionmycetemcomitans Ltx with respect to clinical and inflammatory findings in individuals with or without periodontitis (N = 88). Methods: Periodontal status was examined in patients with severe periodontitis (n = 49) and healthy controls (n = 39), and patients received periodontal treatment. Systemic biomarkers associated with inflammation and infections as well as clinical parameters were analyzed at baseline and 3 and 6 months after treatment. Results: Presence of immunoreactivity against Ltx was associated with impaired remission of disease after periodontal treatment. This immunoreactivity was also significantly associated with increased systemic levels of A. actinomycetemcomitans‐specific immunoglobulins and increasing age. Conclusion: Presence and levels of systemic immunoreactivity against A. actinomycetemcomitans Ltx are associated with decreased remission after otherwise successful periodontal treatment.     

Microbiological and immunological characteristics of young Moroccan patients with aggressive periodontitis with and without detectable Aggregatibacter actinomycetemcomitans JP2 infection

Cross‐sectional and longitudinal studies identify the JP2 clone of Aggregatibacter actinomycetemcomitans as an aetiological agent of aggressive periodontitis (AgP) in adolescents of northwest African descent. To gain information on why a significant part of Moroccan adolescents show clinical signs of periodontal disease in the absence of this pathogen we performed comprehensive mapping of the subgingival microbiota of eight young Moroccans, four of whom were diagnosed with clinical signs of AgP. The analysis was carried out by sequencing and phylogenetic analysis of a total of 2717 cloned polymerase chain reaction amplicons of the phylogenetically informative 16S ribosomal RNA gene. The analyses revealed a total of 173 bacterial taxa of which 39% were previously undetected. The JP2 clone constituted a minor proportion of the complex subgingival microbiota in patients with active disease. Rather than identifying alternative aetiologies to AgP, the recorded infection history of the subjects combined with remarkably high concentrations of antibodies against the A. actinomycetemcomitans leukotoxin suggest that disease activity was terminated in some patients with AgP as a result of elimination of the JP2 clone. This study provides information on the microbial context of the JP2 clone activity in a JP2‐susceptible population and suggests that such individuals may develop immunity to AgP.     

Molecular mimicry of Aggregatibacter actinomycetemcomitans with β2 glycoprotein I

Introduction: β2‐Glycoprotein I (β2GPI) is important in the suppression of coagulation, and antibodies against TLRVYK peptides on the β2GPI molecule are related to thrombosis. According to the Swiss‐Prot database, Aggregatibacter actinomycetemcomitans leukotoxin c has sequences (SIRVYK) that are homologous to the TLRVYK peptides. The aim of this study was to investigate the effects of A. actinomycetemcomitans infection on the antibody response against SIRVYK peptides in patients with periodontitis. Methods: Serum immunoglobulin G (IgG) antibody and IgG subclass antibody titers against SIRVYK or TLRVYK peptides were measured by enzyme‐linked immunosorbent assay in 46 patients with aggressive periodontitis (eight with localized disease, 38 with generalized disease), 28 patients with chronic periodontitis, and 20 periodontally healthy subjects. The presence of A. actinomycetemcomitans in plaque and saliva samples was determined using polymerase chain reaction. Results: The level of anti‐SIRVYK antibodies was significantly higher in patients who were A. actinomycetemcomitans‐positive than in A. actinomycetemcomitans‐negative patients (P < 0.05) in the chronic periodontitis group. A similar trend was found in the antibody response to TLRVYK peptide; however, no statistically significant difference was seen between A. actinomycetemcomitans‐positive and ‐negative patients. The A. actinomycetemcomitans‐positive patients displayed significantly higher levels of anti‐SIRVYK IgG2 and IgG3 antibodies than A. actinomycetemcomitans‐negative patients (P < 0.05 and P < 0.05, respectively). The level of IgG2 was highest among the four IgG subclasses and it predominantly increased in patients who were A. actinomycetemcomitans‐positive. Anti‐TLRVYK antibody levels were significantly correlated with anti‐SIRVYK IgG antibody levels. Conclusion: The results suggest that A. actinomycetemcomitans infection may elicit anti‐SIRVYK IgG antibodies and modify the anti‐TLRVYK antibody response in patients with periodontitis by molecular mimicry with β2GPI.     

Prevalence of Aggregatibacter actinomycetemcomitans in Sudanese patients with aggressive periodontitis: a case-control study

Elamin A, Albandar JM, Poulsen K, Ali RW, Bakken V. Prevalence ofAggregatibacter actinomycetemcomitansin Sudanese patients with aggressive periodontitis: a case–control study. J Periodont Res 2011; 46: 285–291.©2011 John Wiley & Sons A/S Background and Objective: Aggregatibacter actinomycetemcomitans is considered a possible etiological agent for aggressive periodontitis. The aim of this study was to determine the prevalence of the JP2 clone and non‐JP2 genotypes of A. actinomycetemcomitans in the subgingival plaque of patients with aggressive periodontitis and controls among Sudanese high‐school students. Material and Methods: In a previous study we examined a large representative sample of students attending high schools in Khartoum, Sudan. In this population, 17 patients with aggressive periodontitis and 17 controls (14–19 years of age) consented to participate in the present study. The subjects underwent a clinical periodontal examination, and subgingival dental plaque samples were collected using paper points. The presence of the A. actinomycetemcomitans JP2 clone and non‐JP2 genotypes were assessed using loop‐mediated isothermal amplification (LAMP) and the PCR. Results: The JP2 clone of A. actinomycetemcomitans was not detected in the subgingival plaque of either the cases or the controls. Non‐JP2 types of A. actinomycetemcomitans were detected in the subgingival plaque of 12 (70.6%) patients with aggressive periodontitis and from only one (5.9%) control subject, showing a significantly higher frequency of detection in cases than in controls (p = 0.0001). The odds ratio for the detection of A. actinomycetemcomitans in the subgingival plaque of the patients with aggressive periodontitis was 38.4 (95% confidence interval: 4.0–373.0; p = 0.002). The PCR and LAMP methods showed identical results pertaining to the identification of non‐JP2 types of A. actinomycetemcomitans. Conclusions: The JP2 clone of A. actinomycetemcomitans was not detected in the subgingival plaque of high school subjects in Sudan. The detection of non‐JP2 types of A. actinomycetemcomitans may be a useful marker of increased risk for development of aggressive periodontitis in young subjects.     

Frequency of 530‐bp deletion in Actinobacillus actinomycetemcomitans leukotoxin promoter region

Actinobacillus actinomycetemcomitans strains showing a 530‐bp deletion in the promoter region of the leukotoxin gene operon elaborate high amounts of leukotoxin that may play a role in the pathogenesis of periodontal disease. This study used polymerase chain reaction detection to determine the occurrence of the 530‐bp deletion in 94 A. actinomycetemcomitans strains from individuals of various ethnic backgrounds. Eleven blacks and one Hispanic subject but no Caucasian or Asian subjects showed the 530‐bp deletion in the leukotoxin promoter region, suggesting that the deletion is mainly a characteristic of individuals of African descent. A. actinomycetemcomitans strains exhibiting a deletion in the leukotoxin promotor region occurred both in individuals having severe periodontitis and in adolescents revealing no evidence of destructive periodontal disease.     

Human salivary cystatin SA exhibits antimicrobial effect against Aggregatibacter actinomycetemcomitans

Ganeshnarayan K, Velliyagounder K, Furgang D, Fine DH. Human salivary cystatin SA exhibits antimicrobial effect against Aggregatibacter actinomycetemcomitans. J Periodont Res 2012; 47: 661–673. © 2012 John Wiley & Sons A/S Background and Objective: Healthy subjects who do not have Aggregatibacter actinomycetemcomitans in their oral cavity may possess factors in saliva that might demonstrate antibacterial activity against the bacterium. The aim of this study was to identify and purify proteins from saliva of healthy subjects that might demonstrate antibacterial activity against A. actinomycetemcomitans and test the same against the bacteria. Material and Methods: Saliva from 10 healthy volunteers was tested individually for its anti‐A. actinomycetemcomitans activity. Among the 10 subjects, eight demonstrated anti‐A. actinomycetemcomitans activity. Saliva was collected from one healthy volunteer who demonstrated the highest antimicrobial activity against A. actinomycetemcomitans. After clarifying the saliva, it was subjected to an affinity chromatography column with A. actinomycetemcomitans. The proteins bound to A. actinomycetemcomitans were eluted from the column and identified using mass spectrometry (MALDI‐TOF/TOF MS). Among other proteins that bound to A. actinomycetemcomitans, which included lactoferrin, immunoglobulin A and kallikrein, cystatin SA was observed in significantly higher concentrations, and this was purified from the eluate. The purified cystatin SA was tested at different concentrations for its ability to kill A. actinomycetemcomitans in a 2 h cell killing assay. The bacteria were also treated with a proteinase inhibitor, leupeptin, to clarify whether the antimicrobial effect of cystatin SA was related to its protease inhibitory function. Cystatin SA was also tested for its ability to prevent binding of A. actinomycetemcomitans to buccal epithelial cells (BECs) in an A. actinomycetemcomitans–BEC binding assay. Results: Cystatin SA (0.1 mg/mL) demonstrated a statistically significant antimicrobial activity against A. actinomycetemcomitans. The effect of cystatin SA decreased with lower concentrations, with 0.01 mg/mL showing no effect. The addition of monoclonal cystatin SA antibodies to the purified sample completely negated the antimicrobial effect. Treatment of A. actinomycetemcomitans with leupeptin resulted in no antimicrobial effect, suggesting that the antimicrobial activity of cystatin SA is independent of its protease inhibitory function. A. actinomycetemcomitans pretreated with cystatin SA showed reduced binding to BECs, suggesting a potential role for cystatin SA in decreasing the colonization of A. actinomycetemcomitans. Conclusion: The present study shows that cystatin SA demonstrates antimicrobial activity against the periodontopathogen A. actinomycetemcomitans, and future studies determining the mechanism of action are necessary. The study also shows the ability of cystatin SA to reduce significantly the binding of A. actinomycetemcomitans to BECs.     

A novel gene required for natural competence in Aggregatibacter actinomycetemcomitans

Tanaka A, Fujise O, Chen C, Miura M, Hamachi T, Maeda K. A novel gene required for natural competence inAggregatibacter actinomycetemcomitans. J Periodont Res 2012; 47: 129–134. © 2011 John Wiley & Sons A/S Background and Objective: Natural competence is the ability of bacteria to take up extracellular DNA and incorporate it into their genomes. Some strains of Aggregatibacter actinomycetemcomitans, a critical periodontal pathogen, are naturally competent for transformation. However, information on natural competence genes is limited for this species. The aim of this study was to confirm the involvement of a novel gene identified near the fimbriae gene cluster in natural competence. Material and Methods: The functions of putative open reading frames (ORFs), designated AA00863–AA00865, in the Oralgen project database for A. actinomycetemcomitans strain HK1651, have not been determined. Using naturally transformable A. actinomycetemcomitans strains D7S‐1 and ATCC29523, we created deletion mutants of homologous genes of these ORFs. Natural competence in the study strains was determined using an agar‐based transformation frequency assay. Results: Mutation of the AA00865 homolog, which we named urpA in A. actinomycetemcomitans strain D7S‐1, resulted in the loss of natural competence, whereas mutations of the AA00864 and AA00863 homologs, located downstream of urpA gene, did not. Similar results were also observed in the mutants of A. actinomycetemcomitans ATCC29523. Complementation of the deleted sequence in the urpA mutant restored natural competence. Conclusion: The urpA gene is a novel gene required for natural competence in A. actinomycetemcomitans and does not exhibit significant homology with any natural competence genes previously identified in other bacterial species.     

Specific infections as the etiology of destructive periodontal disease: a systematic review

Destructive periodontal disease has been primarily defined and investigated as an infectious disease. The aim of this study was to systematically search for cohort studies where microbiological diagnoses were performed before the onset of destructive periodontal disease and where statistically significant associations were identified. A search was executed in PubMed. The results showed that three studies published after 2005 supported the infection hypothesis for one putative periodontal pathogen: Aggregatibacter actinomycetemcomitans. These three studies were conducted in predominantly non‐Caucasian pediatric populations living in geographic areas with an elevated child‐mortality rate. These studies did not obtain physical or laboratory markers of health, making it possible that A. actinomycetemcomitans was not a cause but a marker for poor environmental or systemic health. No cohort studies were identified supporting the infection hypothesis in adults, Caucasians or in a population residing in areas with child‐mortality rates reflective of healthy population goals. While the possibility cannot be excluded that A. actinomycetemcomitans has an etiological role in certain specific pediatric populations, there are no cohort studies supporting an infectious etiology of destructive periodontal disease in adults.     

Interaction of oral bacteria with gingival epithelial cell multilayers

Primary gingival epithelial cells were cultured in multilayers as a model for the study of interactions with oral bacteria associated with health and periodontal disease. Multilayers maintained at an air–liquid interface in low‐calcium medium displayed differentiation and cytokeratin properties characteristic of junctional epithelium. Multilayers were infected with fluorescently labeled Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum or Streptococcus gordonii, and bacterial association was determined by confocal microscopy and quantitative image analysis. Porphyromonas gingivalis invaded intracellularly and spread from cell to cell; A. actinomycetemcomitans and F. nucleatum remained extracellular and showed intercellular movement through the multilayer; whereas S. gordonii remained extracellular and predominantly associated with the superficial cell layer. None of the bacterial species disrupted barrier function as measured by transepithelial electrical resistance. P. gingivalis did not elicit secretion of proinflammatory cytokines. However, A. actinomycetemcomitans and S. gordonii induced interleukin‐1β (IL‐1β), tumor necrosis factor‐α (TNF‐α), IL‐6 and IL‐8 secretion; and F. nucleatum stimulated production of IL‐1β and TNF‐α. Aggregatibacter actinomycetemcomitans, F. nucleatum and S. gordonii, but not P. gingivalis, increased levels of apoptosis after 24 h infection. The results indicate that the organisms with pathogenic potential were able to traverse the epithelium, whereas the commensal bacteria did not. In addition, distinct host responses characterized the interaction between the junctional epithelium and oral bacteria.     

Aggregatibacter actinomycetemcomitans leukotoxin: From mechanism to targeted anti‐toxin therapeutics

Aggregatibacter actinomycetemcomitans is a Gram‐negative bacterium associated with localized aggressive periodontitis, as well as other systemic diseases. This organism produces a number of virulence factors, all of which provide some advantage to the bacterium. Several studies have demonstrated that clinical isolates from diseased patients, particularly those of African descent, frequently belong to specific clones of A. actinomycetemcomitans that produce significantly higher amounts of a protein exotoxin belonging to the repeats‐in‐toxin (RTX) family, leukotoxin (LtxA), whereas isolates from healthy patients harbor minimally leukotoxic strains. This finding suggests that LtxA might play a key role in A. actinomycetemcomitans pathogenicity. Because of this correlation, much work over the past 30 years has been focused on understanding the mechanisms by which LtxA interacts with and kills host cells. In this article, we review those findings, highlight the remaining open questions, and demonstrate how knowledge of these mechanisms, particularly the toxin's interactions with lymphocyte function‐associated antigen‐1 (LFA‐1) and cholesterol, enables the design of targeted anti‐LtxA strategies to prevent/treat disease.     

Sustained mitogen-activated protein kinase activation with Aggregatibacter actinomycetemcomitans causes inflammatory bone loss

Aggregatibacter actinomycetemcomitans is a gram‐negative facultative capnophile involved in pathogenesis of aggressive forms of periodontal disease. In the present study, we interrogated the ability of A. actinomycetemcomitans to stimulate innate immune signaling and cytokine production and established that A. actinomycetemcomitans causes bone loss in a novel rat calvarial model. In vitro studies indicated that A. actinomycetemcomitans stimulated considerable production of soluble cytokines, tumor necrosis factor‐α, interleukin‐6 and interleukin‐10 in both primary bone marrow‐derived macrophages and NR8383 macrophages. Immunoblot analysis indicated that A. actinomycetemcomitans exhibits sustained activation of all major mitogen‐activated protein kinase (MAPK) pathways, as well as the negative regulator of MAPK signaling, MAPK phosphatase‐1 (MKP‐1), for at least 8 h. In a rat calvarial model of inflammatory bone loss, high and low doses of formalin‐fixed A. actinomycetemcomitans were microinjected into the supraperiosteal calvarial space for 1–2 weeks. Histological staining and micro‐computed tomography of rat calvariae revealed a significant increase of inflammatory and fibroblast infiltrate and increased bone resorption as measured by total lacunar pit formation. From these data, we provide new evidence that fixed whole cell A. actinomycetemcomitans stimulation elicits a pro‐inflammatory host response through sustained MAPK signaling, leading to enhanced bone resorption within the rat calvarial bone.     

Host–microbial co-evolution in periodontitis associated with Aggregatibacter actinomycetemcomitans infection

Periodontitis is caused by bacterial biofilms composed of more than 800 bacterial species. Not all biofilm bacteria are equally pathogenic; Aggregatibacter actinomycetemcomitans is recognized as one of the most virulent periodontopathic bacteria. A. actinomycetemcomitans is also associated with systemic diseases, including cardiovascular disease and brain abscesses. Molecular mimicry between A. actinomycetemcomitans and human β2-glycoprotein I may be associated with systemic diseases such as threatened preterm labor (TPL), preterm birth (PB), and Buerger's disease. Patients with certain genetic neutrophil dysfunction disorders (e.g., Kostmann and Papillon-Lefevre syndromes) also suffer from severe periodontitis. These patients are frequently infected with A. actinomycetemcomitans, suggesting that an impaired immune system might be relevant in the pathogenesis of A. actinomycetemcomitans-associated periodontitis. Virulence of A. actinomycetemcomitans differs amongst clones. JP2 is a highly pathogenic A. actinomycetemcomitans clone that has a deletion in the leukotoxin promoter, resulting in the production of large amounts of leukotoxin. Dissemination of the JP2 clone is skewed to certain regions and ethnic groups. JP2 has not been reported in East Asia, e.g., Japan, Korea, and China. However, an A. actinomycetemcomitans clone with an insertion sequence in the leukotoxin promoter was reported as a highly pathogenic clone in Japanese periodontitis patients. This evidence suggests that host–microbial co-evolution might exist in A. actinomycetemcomitans-associated periodontitis, and elucidation of the co-evolution of A. actinomycetemcomitans and its host might enable molecular diagnosis and treatment of A. actinomycetemcomitans-associated periodontitis.