Absence of an especially toxic clone among isolates of Actinobacillus actinomycetemcomitans recovered from army recruits

Actinobacillus actinomycetemcomitans is a major periodontal pathogen which is associated with early-onset and adult periodontitis. The organism has been shown to be widely distributed among dentate, healthy individuals as well. It has been demonstrated that A. actinomycetemcomitans shows great genetic diversity. An especially virulent clone of the organism (JP2-like) with a specific 530-base pair (bp) deletion in the promoter region of the leukotoxin gene has been isolated from localized juvenile periodontitis patients and related subjects of African and African-American origin. The aim of the present study was to examine the presence of this specific clone employing specific oligonucleotide primers in a polymerase chain reaction among 51 isolates of A. actinomycetemcomitans recovered from 201, 18- to 25-year-old recruits with no or minor periodontal disease. In addition, clinical isolates from 37 periodontitis patients were analyzed as well as reference strains ATCC 29524, NCTC 9710, Y4 and JP2. Primers amplifying a specific 285-bp amplification product in the ltxA region of the leukotoxin gene and a specific 547-bp amplification product of 16 S rRNA were used to genetically confirm identification of the organisms. Primers amplifying sequences in the leukotoxin promoter region were used to identify the deletion. Species specific primers definitively identified all A. actinomycetemcomitans isolates. No isolates from army recruits or the reference strains displayed the deletion in the leukotoxin promoter region. However, in the periodontitis group, a 24-year-old individual from Ghana with localized juvenile periodontitis was identified with an intraoral infection with highly toxic A. actinomycetemcomitans (JP2-like). Present results confirm previous observations of absence of a highly toxic clone of A. actinomycetemcomitans among periodontally healthy and diseased Caucasians as well as a possible presence in localized juvenile periodontitis in individuals of African origin. Received: 31 August 1998 / Accepted: 3 August 1999     

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 promoter region occurred both in individuals having severe periodontitis and in adolescents revealing no evidence of destructive periodontal disease.     

Clonal diversity of Actinobacillus actinomycetemcomitans isolates from young adults with minimal periodontal disease

Actinobacillus actinomycetemcomitans is a major periodontal pathogen which is associated with both early-onset periodontitis and adult cases refractory to conventional periodontal therapy, although the organism has also been shown to be widely distributed among dentate healthy individuals. The observed disease status may be associated with a variation in virulence of different strains or clones. The aim of the present study was to analyse genotype distribution as assessed by an arbitrarily primed polymerase chain reaction (AP-PCR) among 51 isolates of A. actinomycetemcomitans recovered from more than 200 young adult recruits with no or minor periodontal disease. In addition, isolates from 25 periodontitis patients as well as reference strains were genotyped. Primers amplifying (i) a specific sequence in the ltxA region, (ii) a specific 16S rRNA sequence and (iii) sequences in the leukotoxin promoter region were used to verify species identity of the strains. Three random oligonucleotide primers were employed to analyse genomic polymorphisms of the organism by means of PCR. A total of 19 genotypes could be distinguished, which were grouped by cluster analysis into 5 major clusters based on genetic similarity and a complete linkage sort. Whereas 3 clusters assembled A. actinomycetemcomitans genotypes isolated from both healthy subjects and periodontitis patients, one cluster containing 4 different genotypes exclusively comprised isolates from healthy or gingivitis subjects. Another cluster with 2 genotypes consisted of strains originating from periodontitis patients (p < 0.05). One strain characterized by a specific 530 bp deletion in the promoter region of the leukotoxin region was identified in a Ghanese patient with localized juvenile periodontitis. It was concluded that there is considerable clonal diversity of A. actinomycetemcomitans strains isolated from healthy or periodontally diseased subjects, and that genetically closely related groups might be associated with health or disease.     

Characterization of Aggregatibacter actinomycetemcomitans strains in periodontitis patients in Germany

Objectives

Aggregatibacter actinomycetemcomitans strains of serotype b and with a deletion of 530?bp in the promoter region of the leukotoxin gene (JP2 clone) are known to be associated with severe periodontitis. Our study was aimed to detect virulence genes of A. actinomycetemcomitans strains obtained from patients living in four German cities with different proportions of immigrants.

Material and methods

Samples were obtained from severe periodontitis patients in Frankfurt, Hamburg, Leipzig, and Jena. Those being tested positive for A. actinomycetemcomitans were analyzed for serotypes, deletion in the promoter region of the leukotoxin gene, presence of cytolethal distending toxin encoding genes (cdtA, cdtB, and cdtC) and fibril gene1(flp-1).

Results

From all 99 A. actinomycetemcomitans-positive samples, the JP2 clone was found in two immigrants in Frankfurt. Seventy strains were tested positive for the cdtA, 52 for cdtB, and 92 for cdtC and flp-1 genes. Twenty-five strains belonged to serotype a, 22 to serotype b, 21 to serotype c, 31 to the others or could not be serotyped, respectively. The distribution of the serotypes differed between the cities. Further, differences regarding the serotypes were also significant between natives and immigrants.

Conclusions

The JP2 clone is not spread within the Caucasian inhabitants in German cities. The serotypes distribution seems to be influenced by the numbers of immigrants in the cities.

Clinical Relevance

Patients originated from North Africa should be especially screened for the presence of the deletion in the ltx promoter region.     

Humoral immune response to Aggregatibacter actinomycetemcomitans leukotoxin

Brage M, Holmlund A, Johansson A. Humoral immune response toAggregatibacter actinomycetemcomitans leukotoxin. J Periodont Res 2011; 46: 170–175. © 2010 John Wiley & Sons A/S Background and Objective: Periodontal disease is an inflammatory condition caused by bacterial infections that result in loss of the tooth supporting tissue. The periodontal pathogens produce virulence factors with capacity to affect the host immune response. Aggregatibacter actinomycetemcomitans is a periodontal pathogen that produces a leukotoxin that specifically affects human leukocytes. The aims of the present study were to examine the presence and function of systemic antibodies to the leukotoxin. Material and Methods: One hundred and ninety‐seven middle‐aged (57 ± 5 years) Swedes with well‐documented periodontal status and medical factors related to cardiovascular diseases were studied. These data have been published previously. The serum samples were examined for the presence of leukotoxin antibodies by western blot and the capacity to neutralize leukotoxicity in an activity assay with leukotoxin and cultured leukemic cells. Results: The results showed a high prevalence (57%) of antibodies against A. actinomycetemcomitans leukotoxin in the analyzed population. These antibodies were correlated with leukotoxin neutralizing capacity as well as with the ELISA titers of A. actinomycetemcomitans‐specific IgA and IgG. In addition, high levels of leukotoxin antibodies were correlated with increasing age, but not with periodontal disease parameters or cardiovascular risk factors. Conclusion: Systemic antibodies against A. actionmycetemcomitans leukotoxin were common in this adult Swedish population. These antibodies might contribute to limit the systemic effects of the infection.     

Multilevel modeling of gingival bleeding on probing in young adult carriers of non-JP2-like strains of Aggregatibacter actinomycetemcomitans

The influence of Aggregatibacter actinomycetemcomitans on inflammation in subjects with gingivitis has not been studied in great detail. Seventeen healthy young adults with plaque-induced gingivitis or localized mild chronic periodontitis harboring cultivable numbers of A. actinomycetemcomitans were thoroughly examined. Samples of subgingival plaque were obtained from mesial surfaces of all teeth present. In addition, 12 oral mucosal surfaces and unstimulated saliva were sampled. Species identity, presence of the leukotoxin gene, and absence of a specific 530 b deletion in the leukotoxin promoter region indicating non-JP2-like strains were assessed by polymerase chain reaction. Based on a multilevel random intercept model adjusted for probing depth, age, and smoking status, the odds of bleeding on probing was increased by a factor of 1.89 (1.09–3.29, p = 0.024) if, in addition to plaque, A. actinomycetemcomitans could be recovered from the site. At a site without visible supragingival plaque but with cultivable numbers of subgingival A. actinomycetemcomitans the odds ratio of bleeding on probing was 3.37 (0.86–13.2, p = 0.081). Simulating variance partition coefficients revealed that between 1–2% (a clean, shallow site without A. actinomycetemcomitans; a deep site covered by plaque containing A. actinomycetemcomitans) and 6–7% (a moderately deep site with neither visible plaque nor cultivable A. actinomycetemcomitans) of the residual variance was attributable to differences between subjects. The present cross-sectional study indicates that non-JP2-like strains of A. actinomycetemcomitans may enhance gingival bleeding tendency even in the absence of clinically visible supragingival plaque.     

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.     

A novel insertion sequence increases the expression of leukotoxicity in Actinobacillus actinomycetemcomitans clinical isolates

BACKGROUND: The expression of leukotoxin varies among Actinobacillus actinomycetemcomitans strains and is dependent in part on the structure of the ltx promoter region. Highly leukotoxic strains, characterized by a 530 base pair (bp) deletion within the ltx promoter, have been associated with juvenile periodontitis in the United States and Europe. In the present study, we analyzed the ltx promoter structure to elucidate whether A. actinomycetemcomitans from Japanese periodontitis patients exhibits the highly toxic phenotype. METHODS: Forty-five A. actinomycetemcomitans strains, including 43 clinical isolates, the highly leukotoxic strain JP2, and a minimally leukotoxic strain 652 were used in the study. The ltx promoter structure was analyzed by polymerase chain reaction (PCR), with oligonucleotide primers focusing the ltx promoter region, and nucleotide sequencing. Leukotoxic activity was determined by trypan blue exclusion. Western blotting assay was performed to detect the level of leukotoxin polypeptide. RESULTS: A 495 bp PCR product was amplified from JP2, a 1025 bp product from 652 and 41 of the clinical isolates, and a 1926 bp product from the remaining two clinical isolates (AaIS1, AaIS2). Sequencing of the 1926 bp PCR fragment showed that it was similar to that of strain 652 but contained an 886 bp region that was identified as an insertion sequence (IS). Both AaIs strains expressed high levels of leukotoxicity, similar to strain JP2. In addition, a mutant (AaIS-) that had lost the IS element expressed a significantly lower level of leukotoxicity compared with AaIS strains. Furthermore, the levels of leukotoxin polypeptide expressed by these strains were consistent with their whole cell leukotoxicity. CONCLUSIONS: A. actinomycetemcomitans clinical strains which were isolated from Japanese periodontitis patients do not possess the 530 bp ltx promoter deletion. The results of this study suggest that a high level of leukotoxin expression correlates with the insertion of the transposable DNA element.     

Characterization and serotype distribution of Aggregatibacter actinomycetemcomitans isolated from a population of periodontitis patients in Spain

ObjectiveThere is no study characterizing the variability of Aggregatibacter actinomycetemcomitans isolates in periodontitis patients in Spain. It is therefore the aim of this investigation to study the serotype distribution of A. actinomycetemcomitans strains isolated from periodontitis patients in Spain. The polymorphism of the genes that codifies the leukotoxin and the operon of the cytolethal-distending toxin (cdt) will also be investigated.DesignFrom a total of 701 patients samples, 40 A. actinomycetemcomitans-positive periodontitis patients were included in the study (mean age 45.3, 62.5% females) and their clinical periodontal status was assessed. On average, 1–3 isolates from each patient were sub-cultured and characterized by PCR.ResultsUsing culture the prevalence of A. actinomycetemcomitans was 5.7%. The most frequent serotype was “b”, being 30 patients infected by a unique serotype, while 7 patients showed co-colonization, mostly with serotypes “a” and “b”. From the 79 pure isolates obtained, 24 were from serotype “a”, 30 from serotype “b”, 12 from serotype “c” and 4 from serotype “d”. Further characterization of these samples showed that none of these 79 isolates demonstrated the 530-bp deletion in the leukotoxin's promoter region that characterizes the JP2 strain. Conversely 65.8% of the isolates were cdt+.ConclusionsThe most common serotypes were “a” and “b”, being serotype “b” the most prevalent in mono-colonization, while serotypes “e” and “f” were not detected. In the majority of samples, operon that codifies the cdt (65.8%) and the genes responsible for the codification of leukotoxin (100%) were found. None of the isolates were JP2 strains.     

OCCURRENCE OF AGGREGATIBACTER ACTINOMYCETEMCOMITANS IN BRAZILIAN INDIANS FROM UMUTINA RESERVATION,MATO GROSSO,BRAZIL

A ggregatibacter actinomycetemcomitans is associated with periodontal disease, especially localized aggressive periodontitis, produces a potent leukotoxin and its distribution is influenced by ethnic characteristics of the population.

Objective:

Using culture and polymerase chain reaction (PCR) techniques, this study evaluated the occurrence of this microorganism and the distribution of leukotoxic strains isolated from Indians belonging to the Umutima Reservation, Mato Grosso, Brazil.

Material and Methods:

Forty-eight native Brazilians with gingivitis and 38 with chronic periodontitis, belonging to Umutina, Paresi, Bororo, Bakairi, Kayabi, Irantxe, Nambikwara and Terena ethnicities, were studied. Subgingival, supragingival and saliva samples of each patient were collected and transferred to VMGA III medium and to ultra pure Milli Q water. Bacteria were grown on TSBV agar and incubated in anaerobiosis (90% N₂ + 10% CO₂) at 37°C for 72 h. The presence of the ltx promoter was determined by PCR, and a 530 bp deletion in the promoter was evaluated by using specific primers.

Results:

A. actinomycetemcomitans was isolated from 8.33% of saliva, supragingival and subgingival samples from patients with gingivitis and from 18.42% of saliva and supragingival biofilm, and 26.32% subgingival biofilm from patients with chronic periodontitis. By PCR, the bacterial DNA was detected in 8.33% of saliva, supragingival and subgingival biofilms from patients with gingivitis and from 23.68% of saliva, 28.95% supragingival biofilm and 34.21% subgingival biofilm from patients with periodontitis. All strains were grouped as non-JP2 clones based on the absence of deletion in the leukotoxin promoter. Differences among the microbial and clinical parameters in patients were analyzed by using the Mann-Whitney, Chi-square or Fisher''s exact tests.

Conclusions:

The present results suggest that A. actinomycetemcomitans can be related to the attachment loss in this population, but the presence of minimally leukotoxic strains, as well as its role in the pathogenesis of the periodontitis in these native Brazilians need to be further investigated.     

Relationship of Serotype, leukotoxin gene type and lysogeny in Actinobbacillus actinomycetemcomitans to periodontal disease status

Actinobacillus actinomycetemcomitans has been associated with different forms of periodontities, particularly with localized juvenile periodontitis (LJP). The bacterium possesses several virulence factors which have been shown to interact with the hos immune system. Among these factors, leukotoxin, surface antigens (serotype) and bacteriophages have been suggested directly or indirectly to influence the course of infection. However, few studies have been able to show associations between these factors and periodontal disease, alonw or in combination. Thus, the purpose of the present study was to investigate possible correlations between periodontal disease status and selected virulence factors (serotype, presence of bacteriophages, and the presence of a 530 bp delection in the promotor region of the leukotoxin gene). 36 subjects took part in the study. Serotype c was the most frequently found serotype among periodontally affected subjects, although serotypes a and b were also present, 27 out of 36 strains harbored bacteriophages, and there was strong evidence that some of the bacteriophages were different from the previously characterized ΦA a Phage. actinomyceremcomitans the F-Fragment phage were more frequently associated with periodontal disease. Five strains, all serotyype b, 3 from LJP patients and 2 from healthy subjects, showed a 530-bp deletion in the promotor regiion of the leukotoxin gene.     

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.     

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.     

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.     

Actinobacillus actinomycetemcomitans induces lethal effects on the macrophage‐like human cell line U937

We examined the cytotoxicity in culture medium of Actinobacillus actinomycetemcomitans against the human monocyte‐macrophage‐like cell line U937 using the trypan blue exclusion test and WST‐1 test. We found that A. actinomycetemcomitans Y4 showed the highest cytotoxic activity among the three different serotype strains and the cytotoxic effects of both bacterial cells and culture supernatants in A. actinomycetemcomitans Y4 were stronger on phorbol‐12‐myristate 13‐acetate (PMA)‐induced U937 cells than uninduced U937 cells. Morphological changes in PMA‐induced U937 cells treated with culture supernatants differed from those treated with leukotoxin, and a difference in the susceptibility to 56 °C heat treatment was found between culture supernatants and leukotoxin. The cytotoxic activity by WST‐1 was determined more rapidly and strongly than that by trypan blue assay. These findings suggested that the cytotoxic effect of A. actinomycetemcomitans was influenced by the differentiation of U937 cells and may be more potent on the respiratory chain than the cell membrane.     

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.     

The conserved carboxyl domain of MorC,an inner membrane protein of Aggregatibacter actinomycetemcomitans,is essential for membrane function

Morphogenesis protein C (MorC) of Aggregatibacter actinomycetemcomitans is important for maintaining the membrane morphology and integrity of the cell envelope of this oral pathogen. The MorC sequence and operon organization were found to be conserved in Gammaproteobacteria, based on a bioinformatic analysis of 435 sequences from representative organisms. Functional conservation of MorC was investigated using an A. actinomycetemcomitans morC mutant as a model system to express MorC homologs from four phylogenetically diverse representatives of the Gammaproteobacteria: Haemophilus influenzae, Escherichia coli, Pseudomonas aeruginosa, and Moraxella catarrhalis. The A. actinomycetemcomitans strains expressing the homologous proteins were assessed for sensitivity to bile salts, leukotoxin secretion, autoaggregation and membrane morphology. MorC from the most closely related organism (H. influenzae) was functionally identical to MorC from A. actinomycetemcomitans. However, the genes from more distantly related organisms restored some but not all A. actinomycetemcomitans mutant phenotypes. In addition, deletion mutagenesis indicated that the most conserved portion of the protein, the C‐terminus DUF490 domain, was necessary to maintain the integrity of the membrane. Deletion of the last 10 amino acids of this domain of the A. actinomycetemcomitans MorC protein was sufficient to disrupt membrane stability and leukotoxin secretion. The data suggest that the MorC sequence is functionally conserved across Gammaproteobacteria and the C‐terminus of the protein is essential for maintaining membrane physiology.     

Early-onset periodontitis in Morocco is associated with the highly leukotoxic clone of Actinobacillus actinomycetemcomitans

A particular clone (JP2) of Actinobacillus actinomycetemcomitans with increased leukotoxin production has been isolated from individuals with early-onset periodontitis (EOP). The aim of this study was to determine the frequency of carriers of this clone and its association with EOP in Moroccan schoolchildren. Of 217 plaque samples, 131 (60.4%) were culture-positive for A. actinomycetemcomitans. A total of 19 of these isolates had a 530-bp deletion in the leukotoxin promoter region characteristic of the JP2 clone. A strong association between the presence of A. actinomycetemcomitans with the 530-bp deletion and EOP was found (adjusted OR = 29.4; 95% Cl = 8.3 - 104.4; p < 0.0005), while no association could be demonstrated between the presence of A. actinomycetemcomitans without the deletion and EOP (adjusted OR = 1.3; 95% CI = 0.5 -2.9; p = 0.750). The study demonstrates that the endemic presence, in a human population, of the highly leukotoxic JP2 clone may result in an unusually high prevalence of EOP.     

Experimental periodontitis in mice selected for maximal or minimal inflammatory reactions: increased inflammatory immune responsiveness drives increased alveolar bone loss without enhancing the control of periodontal infection

Background and Objective: Inflammatory immune reactions that occur in response to periodontopathogens are thought to protect the host against infection, but may trigger periodontal destruction. However, the molecular and genetic mechanisms underlying host susceptibility to periodontal infection and to periodontitis development have still not been established in detail. Material and Methods: In this study, we examined the mechanisms that modulate the outcome of Aggregatibacter (Actinobacillus) actinomycetemcomitans‐induced periodontal disease in mice mouse strains selected for maximal (AIRmax) or minimal (AIRmin) inflammatory reactions. Results: Our results showed that AIRmax mice developed a more severe periodontitis than AIRmin mice in response to A. actinomycetemcomitans infection, and this periodontitis was characterized by increased alveolar bone loss and inflammatory cell migration to periodontal tissues. In addition, enzyme‐linked immunosorbent assays demonstrated that the levels of the cytokines interleukin‐1β, tumor necrosis factor‐α and interleukin‐17 were higher in AIRmax mice, as were the levels of matrix metalloproteinase (MMP)‐2, MMP‐13 and receptor activator of nuclear factor‐κB ligand (RANKL) mRNA levels. However, the more intense inflammatory immune reaction raised by the AIRmax strain, in spite of the higher levels of antimicrobial mediators myeloperoxidase and inducible nitric oxide synthase, did not enhance the protective immunity to A. actinomycetemcomitans infection, because both AIRmax and AIRmin strains presented similar bacterial loads in periodontal tissues. In addition, the AIRmax strain presented a trend towards higher levels of serum C‐reactive protein during the course of disease. Conclusion: Our results demonstrate that the intensity of the inflammatory immune reaction is associated with the severity of experimental periodontitis, but not with the control of A. actinomycetemcomitans periodontal infection, suggesting that the occurrence of hyperinflammatory genotypes may not be an evolutionary advantage in the complex host–pathogen interaction observed in periodontal diseases.     

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.