Transposon mutagenesis in bacteria generally requires efficient delivery of a transposon suicide vector to allow the selection of relatively infrequent transposition events. We have developed an IS903-based transposon mutagenesis system for diverse gram-negative bacteria that is not limited by transfer efficiency. The transposon, IS903φkan, carries a cryptic kan gene, which can be expressed only after successful transposition. This allows the stable introduction of the transposon delivery vector into the host. Generation of insertion mutants is then limited only by the frequency of transposition. IS903φkan was placed on an IncQ plasmid vector with the transposase gene located outside the transposon and expressed from isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible promoters. After transposase induction, IS903φkan insertion mutants were readily selected in Escherichia coli by their resistance to kanamycin. We used IS903φkan to isolate three catalase-deficient mutants of the periodontal pathogen Actinobacillus actinomycetemcomitans from a library of random insertions. The mutants display increased sensitivity to hydrogen peroxide, and all have IS903φkan insertions within an open reading frame whose predicted product is closely related to other bacterial catalases. Nucleotide sequence analysis of the catalase gene (designated katA) and flanking intergenic regions also revealed several occurrences of an 11-bp sequence that is closely related to the core DNA uptake signal sequence for natural transformation of Haemophilus influenzae. Our results demonstrate the utility of the IS903φkan mutagenesis system for the study of A. actinomycetemcomitans. Because IS903φkan is carried on a mobilizable, broad-host-range IncQ plasmid, this system is potentially useful in a variety of bacterial species.
The bacterium Ornithobacterium rhinotracheale has been recognized as an emerging pathogen in poultry since about 10 years ago. Knowledge of this bacterium and its mechanisms of virulence is still very limited. Here we report the development of a transformation system that enables genetic modification of O. rhinotracheale. The system is based on a cryptic plasmid, pOR1, that was derived from an O. rhinotracheale strain of serotype K. Sequencing indicated that the plasmid consisted of 14,787 nucleotides. Sequence analysis revealed one replication origin and several rep genes that control plasmid replication and copy number, respectively. In addition, pOR1 contains genes with similarity to a heavy-metal-transporting ATPase, a TonB-linked siderophore receptor, and a laccase. Reverse transcription-PCR demonstrated that these genes were transcribed. Other putative open reading frames exhibited similarities with a virulence-associated protein in Actinobacillus actinomycetemcomitans and a number of genes coding for proteins with unknown function. An Escherichia coli-O. rhinotracheale shuttle plasmid (pOREC1) was constructed by cloning the replication origin and rep genes from pOR1 and the cfxA gene from Bacteroides vulgatus, which codes for resistance to the antibiotic cefoxitin, into plasmid pGEM7 by using E. coli as a host. pOREC1 was electroporated into O. rhinotracheale and yielded cefoxitin-resistant transformants. The pOREC1 isolated from these transformants was reintroduced into E. coli, demonstrating that pOREC1 acts as an independent replicon in both E. coli and O. rhinotracheale, fulfilling the criteria for a shuttle plasmid that can be used for transformation, targeted mutagenesis, and the construction of defined attenuated vaccine strains.
To develop targeted gene integration in the periodontal pathogen Actinobacillus actinomycetemcomitans, a ColE1-based, spectinomycin-resistant plasmid containing a segment of the leukotoxin gene was electroporated into strain JP2. In all of the stable spectinomycin-resistant transformants that arose, the plasmid had recombined into the genomic leukotoxin locus since ColE1-based vectors cannot replicate extrachromosomally in A. actinomycetemcomitans. Directed genomic integration was then used to construct a leukotoxin-negative strain by transforming the leukotoxin-producing strain JP2 with a ColE1-based plasmid containing an internal fragment of the leukotoxin gene. Cytotoxicity assays proved that these transformants had < 0.1% of the leukotoxin activity of the parental strain. These results demonstrate that integration-based approaches can be used for generating isogenic mutants in specific virulence genes in A. actinomycetemcomitans.
The periodontal pathogen Actinobacillus actinomycetemcomitans possesses myriad virulence factors, among them the ability to adhere to and invade epithelial cells. Recent advances in the molecular manipulation of this pathogen and the sequencing of strain HK 1651 (http://www.genome.ou.edu/act.html) have facilitated examination of the genetics of its interaction with epithelial cells. The related gram-negative organism, Haemophilus influenzae, possesses autotransporter adhesins. A search of the sequence database of strain HK 1651 revealed a homologue with similarity in the pore-forming domain to that of the H. influenzae autotransporter, Hap. A. actinomycetemcomitans mutants deficient in the homologue, Aae, showed reduced binding to epithelial cells. A method for making A. actinomycetemcomitans SUNY 465 transiently resistant to spectinomycin was used with conjugation to generate an isogenic aae mutant. An allelic replacement mutant was created in the naturally transformable A. actinomycetemcomitans strain ATCC 29523. Lactoferrin, an important part of the innate host defense system, protects against bacterial infection by bactericidal and antiadhesion mechanisms. Lactoferrin in human milk removes or cleaves Hap and another autotransporter, an immunoglobulin A1 protease, from the surface of H. influenzae, thereby reducing their binding to epithelial cells. Human milk whey had similar effects on Aae from A. actinomycetemcomitans ATCC 29523 and its binding to epithelial cells; however, there was little effect on the binding of SUNY 465. A difference in the genetic structure of aae in the two strains, apparently due to the copy number of a 135-base repeated sequence, may be the cause of the differential action of lactoferrin. aae is the first A. actinomycetemcomitans gene involved in adhesion to epithelial cells to be identified.
tfoX (sxy) is a regulatory gene needed to turn on competence genes. Aggregatibacter (Actinobacillus) actinomycetemcomitans has a tfoX gene that is important for transformation. We cloned this gene on an IncQ plasmid downstream of the inducible tac promoter. When this plasmid was resident in cells of A. actinomycetemcomitans and tfoX was induced, the cells became competent for transformation. Several strains of A. actinomycetemcomitans, including different serotypes, as well as rough (adherent) and isogenic smooth (nonadherent) forms were tested. Only our two serotype f strains failed to be transformed. With the other strains, we could easily get transformants with extrachromosomal plasmid DNA when closed circular, replicative plasmid carrying an uptake signal sequence (USS) was used. When a replicative plasmid carrying a USS and cloned DNA from the chromosome of A. actinomycetemcomitans was linearized by digestion with a restriction endonuclease or when genomic DNA was used directly, the outcome was allelic exchange. To facilitate allelic exchange, we constructed a suicide plasmid (pMB78) that does not replicate in A. actinomycetemcomitans and carries a region with two inverted copies of a USS. This vector gave allelic exchange in the presence of cloned and induced tfoX easily and without digestion. Using transposon insertions in cloned katA DNA, we found that as little as 78 bp of homology at one of the ends was sufficient for that end to participate in allelic exchange. The cloning and induction of tfoX makes it possible to transform nearly any strain of A. actinomycetemcomitans, and allelic exchange has proven to be important for site-directed mutagenesis.
periodontitis; pathogen; USS; allelic exchange; site-directed mutagenesis; competence
The leukotoxin produced by Actinobacillus actinomycetemcomitans has been implicated in the etiology of juvenile periodontitis. To initiate a genetic analysis of the role of this protein in disease, we have cloned the leukotoxin gene in Escherichia coli. Recombinant colonies carrying toxin gene sequences were isolated by screening a genomic A. actinomycetemcomitans library with a DNA probe for the leukotoxin gene from a related bacterium, Pasteurella haemolytica. To demonstrate that the cloned A. actinomycetemcomitans DNA contained a functional leukotoxin gene, protein extracts of E. coli containing the A. actinomycetemcomitans clone were tested directly for leukotoxic activity against human cell lines in chromium release assays. A construct containing the entire cloned region produced a functional toxin. No cytotoxicity was seen when extracts from cells containing plasmids with deletions in the putative coding region were used. Furthermore, the toxin produced by the cloned gene has the same target cell specificity as the leukotoxin extracted directly from A. actinomycetemcomitans. These results indicate that sequences encoding a functional leukotoxin have been cloned and are expressed in E. coli. Southern blot analysis of DNA from leukotoxin-producing (Lkt+) and non-leukotoxin-producing (Lkt-) strains indicated that the Lkt- strain also contained a copy of the gene.
The first example of conjugal transfer of DNA from Escherichia coli to the periodontal pathogen Actinobacillus actinomycetemcomitans is presented. Derivatives of the incompatibility group P (IncP) plasmid RK2 successfully transferred from an E. coli donor to an A. actinomycetemcomitans recipient. The resulting A. actinomycetemcomitans transconjugants transferred the plasmids back to E. coli recipients. The IncP transfer functions were also used in trans to mobilize the IncQ plasmid pBK1 from E. coli to A. actinomycetemcomitans. The IncP and IncQ plasmids both transferred into A. actinomycetemcomitans at high frequencies (0.3 to 0.5 transconjugants per donor) and showed no gross deletions, insertions, or rearrangements. Determinations of MICs of various antibiotics for the A. actinomycetemcomitans transconjugant strains demonstrated the expression of ampicillin, chloramphenicol, and kanamycin resistance determinants.
The cell density-dependent control of gene expression is employed by many bacteria for regulating a variety of physiological functions, including the generation of bioluminescence, sporulation, formation of biofilms, and the expression of virulence factors. Although periodontal organisms do not appear to secrete acyl-homoserine lactone signals, several species, e.g., Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum, have recently been shown to secrete a signal related to the autoinducer II (AI-2) of the signal system 2 pathway in Vibrio harveyi. Here, we report that the periodontal pathogen Actinobacillus actinomycetemcomitans expresses a homolog of V. harveyi luxS and secretes an AI-2-like signal. Cell-free conditioned medium from A. actinomycetemcomitans or from a recombinant Escherichia coli strain (E. coli AIS) expressing A. actinomycetemcomitans luxS induced luminescence in V. harveyi BB170 >200-fold over controls. AI-2 levels peaked in mid-exponential-phase cultures of A. actinomycetemcomitans and were significantly reduced in late-log- and stationary-phase cultures. Incubation of early-log-phase A. actinomycetemcomitans cells with conditioned medium from A. actinomycetemcomitans or from E. coli AIS resulted in a threefold induction of leukotoxic activity and a concomitant increase in leukotoxin polypeptide. In contrast, no increase in leukotoxin expression occurred when cells were exposed to sterile medium or to conditioned broth from E. coli AIS−, a recombinant strain in which luxS was insertionally inactivated. A. actinomycetemcomitans AI-2 also induced expression of afuA, encoding a periplasmic iron transport protein, approximately eightfold, suggesting that LuxS-dependent signaling may play a role in the regulation of iron acquisition by A. actinomycetemcomitans. Finally, A. actinomycetemcomitans AI-2 added in trans complemented a luxS knockout mutation in P. gingivalis by modulating the expression of the luxS-regulated genes uvrB and hasF in this organism. Together, these results suggest that LuxS-dependent signaling may modulate aspects of virulence and the uptake of iron by A. actinomycetemcomitans and induce responses in other periodontal organisms in mixed-species oral biofilm.
In several bacterial species, iron availability in host tissues is coordinated with the expression of virulence determinants through the fur gene product. Initial experiments showed that a cloned Escherichia coli fur gene probe hybridized to Southern blots of Actinobacillus actinomycetemcomitans strain JP2 (serotype b) chromosomal DNA. The A. actinomycetemcomitans fur gene was then cloned utilizing partial functional complementation of the fur mutant in E. coli strain H1780. Analysis of the cloned DNA sequence revealed a 438-bp open reading frame with a deduced 146-amino-acid sequence exhibiting 80% identity to Haemophilus influenzae Fur and 62% identity to E. coli Fur. The pUC Aafur gene probe (generated from JP2 serotype b) hybridized to representatives from all five A. actinomycetemcomitans serotypes as well as to two strains derived from monkeys, suggesting that fur is widely distributed in A. actinomycetemcomitans. Open reading frames having >70% identity with the E. coli and H. influenzae flavodoxin and gyrase A genes, respectively, were found. Expression of the A. actinomycetemcomitans fur gene product repressed fiu expression and siderophore production in E. coli. A gel shift assay demonstrated that the expressed A. actinomycetemcomitans Fur protein bound the bacterial fur consensus sequence. Further characterization of the fur gene product in A. actinomycetemcomitans may improve our understanding of its role in the pathogenesis of periodontal disease and may lead to specific therapeutic modalities.
Actinobacillus actinomycetemcomitans is a member of the family Pasteurellaceae and a major causative agent of periodontitis. While several genera from this family are known to be competent for transformation, A. actinomycetemcomitans has yet to be fully characterized. Here we show that the competence of A. actinomycetemcomitans is remarkably similar to that of Haemophilus influenzae. In addition to having a similar frequency of transformation as H. influenzae, A. actinomycetemcomitans competence could also be induced at least 100-fold by cyclic AMP, suggesting that, as in H. influenzae, at least some competence genes are regulated by catabolite repression. Even more intriguing was the discovery of a putative A. actinomycetemcomitans DNA uptake signal sequence (USS) virtually identical to the USS of H. influenzae. Moreover, we provide evidence that this sequence functions in the same capacity as that from H. influenzae; the sequence appears to be required and sufficient for DNA uptake in a variety of assays. Finally, we have taken advantage of this system to develop a simple, highly efficient competence-based method for generating site-directed mutations in the wild-type fimbriated A. actinomycetemcomitans.
The genetic determinants of the 120-kDa cytotoxin of Actinobacillus pleuropneumoniae serotype 2 were isolated from a lambda DNA library by a plaque immunoblot technique. Expression of the 120-kDa polypeptide was confirmed by Western immunoblot analysis of infected Escherichia coli cell lysates, which were shown to be toxic for porcine alveolar macrophages in vitro. The genetic determinants of the toxin were subcloned into the plasmid vector pUC18. This plasmid (pPTX1) directed the synthesis and secretion of the active 120-kDa cytotoxin in E. coli. The recombinant toxin was indistinguishable from native cytotoxin from A. pleuropneumoniae serotype 2 with respect to molecular size, reaction in Western blot analysis, heat lability, cytotoxic activity, and neutralization by serum antibody. A restriction endonuclease cleavage map of pPTX1 was prepared, and deletion mutants were used to locate the minimal region of DNA required for production of intracellular toxin; this gene was termed ptxA. Southern hybridization analysis with a 1.7-kb PvuII fragment located within the ptxA gene revealed sequences with a high degree of homology in serotype reference strains 2, 3, 4, 6, and 8. Other reference strains did not contain sequences that were recognized by this probe. However, related sequences (greater than 71% homology) were detected in Actinobacillus actinomycetemcomitans and A. equuli. Weak hybridization was observed between the ptxA probe and pLKT5, which carries the lktAC genes of Pasteurella haemolytica, and between the ptxA probe and pAPH1, which carries the structural gene for type II hemolysin from A. pleuropneumoniae. The isolation of the genetic determinants of this cytotoxin will enable investigations of the structure and organization of the ptx DNA region and further analysis of its role in the pathogenesis of pleuropneumonia.
Genetic analysis of an Actinobacillus actinomycetemcomitans population consisting of 88 clinically well characterized Finnish isolates performed by multilocus enzyme electrophoresis confirmed that the five serotypes divide into two phylogenetic lineages, one comprising serotypes b and c and one comprising serotypes a, d, and e. There was no association between any subpopulation and the periodontal health status of the subject from whom the isolates originated, suggesting that the role of A. actinomycetemcomitans in periodontitis is largely opportunistic in the population examined. Southern blot analyses of genomic DNA digested with each of the restriction endonucleases MspI, RsaI, and TaqI revealed extremely limited genetic polymorphism of the structural leukotoxin gene, ltxA, and its associated promoter. All isolates hybridized to a 530-bp DNA fragment derived from the promoter region of the leukotoxin gene operon of a minimally leukotoxic A. actinomycetemcomitans strain. Deletion of the 530-bp sequence has been associated with significantly increased toxin production detected among isolates from patients with juvenile periodontitis in North America but was detected neither among the 88 isolates in the present collection analyzed nor among more than 60 strains in another population of northern European A. actinomycetemcomitans isolates analyzed previously.
Actinobacillus actinomycetemcomitans, the etiologic agent of localized juvenile periodontitis, produces a potent leukotoxin that kills human neutrophils. The production of leukotoxin RNA can vary more than 50-fold among isolates of A. actinomycetemcomitans, and strains expressing high levels of leukotoxin RNA are most often found at sites of periodontal disease. To assess the relative contributions of transcription factors and promoter sequences in setting the disparate levels of leukotoxin RNA found, we have undertaken classical cis/trans analyses. First, the leukotoxin promoter regions from moderately leukotoxic (Y4) and minimally leukotoxic (ATCC 33384) strains of A. actinomycetemcomitans were cloned, sequenced, and compared with the previously sequences leukotoxin promoter region of the high-producer strain JP2. The Y4 and ATCC 33384 promoter regions each contain a 528-bp segment that is absent from JP2. Interestingly, the analysis of various deletion constructs in A. actinomycetemcomitans indicated that Y4, despite the large insertion, initiates leukotoxin RNA synthesis at the same promoter as JP2 does. To perform cis/trans analyses, these three leukotoxin promoter regions were cloned into a plasmid upstream of the reporter gene beta-galactosidase. Each plasmid was transformed into JP2, Y4, and ATCC 33384, and the beta-galactosidase levels were determined. The results indicated that the sequences responsible for down-regulating leukotoxin RNA levels in Y4 relative to JP2 are found within the transcribed region of the Y4 leukotoxin operon. Importantly, in ATCC 33384, strain-specific trans factors and promoter sequence differences are equally significant in determining the lower levels of leukotoxin RNA. We hypothesize that either strain ATCC 33384 has a negative regulatory protein (which is missing or mutated in JP2/Y4) or that JP2 and Y4 carry an activator that is missing or mutated in ATCC 33384.
The leukotoxin produced by Actinobacillus actinomycetemcomitans has been implicated in the etiology of localized juvenile periodontitis. To initiate a genetic analysis into the role of this protein in disease, we have cloned its gene, lktA. We now present the complete nucleotide sequence of the lktA gene from A. actinomycetemcomitans. When the deduced amino acid sequence of the leukotoxin protein was compared with those of other proteins, it was found to be homologous to the leukotoxin from Pasteurella haemolytica and to the alpha-hemolysins from Escherichia coli and Actinobacillus pleuropneumoniae. Each alignment showed at least 42% identity. As in the other organisms, the lktA gene of A. actinomycetemcomitans was linked to another gene, lktC, which is thought to be involved in the activation of the leukotoxin. The predicted LktC protein was related to the leukotoxin/hemolysin C proteins from the other bacteria, since they shared a minimum of 49% amino acid identity. Surprisingly, although actinobacillus species are more closely related to pasteurellae than to members of the family Enterobacteriaciae, LktA and LktC from A. actinomycetemcomitans shared significantly greater sequence identity with the E. coli alpha-hemolysin proteins than with the P. haemolytica leukotoxin proteins. Despite the overall homology to the other leukotoxin/hemolysin proteins, the LktA protein from A. actinomycetemcomitans has several unique properties. Most strikingly, it is a very basic protein with a calculated pI of 9.7; the other toxins have estimated pIs around 6.2. The unusual features of the A. actinomycetemcomitans protein are discussed in light of the different species and target-cell specificities of the hemolysins and the leukotoxins.
Actinobacillus acrinomycetemcomirans isolates from periodontal pockets were examined for restriction fragment-length polymorphism using a characterized 4.7-kb DNA probe. A total of 6 patterns of RFLP was found in 133 isolates originating from 12 subjects. No relatedness was found between RFLP types and serotypes. Different periodontal sites within the same subject and different individuals within the same family sometimes showed only one type of A. actinomycetemcomitans RFLP. When members among the same family showed 2 RFLP types, children were always infected with the A. acfinomycefemcomitans strains found in at least one of the parents. These findings support the concept of familial spread of A. actinomycetemcomitans. A. actinomycetemcomitans RFLP type B, corresponding to reference strain JP2, seems to be particularly virulent, as indicated from the presence of RFLP type B in 3 subjects who converted from a healthy periodontal state to localized juvenile periodontitis. RFLP type B was not detected in any of the 21 A. acrinomycetemcomitans-infected patients with adult periodontitis. The RFLP method seems to be useful in determining the epidemiology and possibly the potential virulence of periodontal strains of A. actinomycetemcomitans.
juvenile periodontitis; epidemiology; virulence; restriction fragment-length polymorphism; DNA probes; Actinobaccillus actinomycetemcomitans
Actinobacillus actinomycetemcomitans is an important pathogen in the etiology of severe periodontitis. For epidemiological studies on the prevalence of certain pathogenic clones and transmission of this bacterium, adequate typing methods are necessary. The purpose of this study was to compare six different typing methods for A. actinomycetemcomitans. Five reference strains and 27 fresh clinical isolates from periodontitis patients were used. Serotyping showed 12 serotype a strains, 13 type b strains, 6 type c strains, and 1 nontypeable strain. Biotyping on the basis of the fermentation of mannose, mannitol, and xylose resulted in six biotypes. Antibiogram typing was evaluated by measuring the inhibition zones of seven antibiotics in agar diffusion tests. With this method eight main types which could be further differentiated into 15 subtypes were found. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of outer membrane proteins were similar among all isolates tested. Restriction endonuclease analysis (REA) of whole chromosomal DNA resulted in five main types. These five main types were further differentiated into 24 subtypes on the basis of DNA fragment differences in the high-molecular-weight region. Hybridization of DNA fragments with ribosomal DNA (ribotyping) resulted in 22 to 24 different types, depending on the restriction endonuclease used. Ribotype patterns were easy to interpret and provided an univocal distinction between different strains compared with REA results. When applied to epidemiologically related isolates, all methods were able to discriminate two clonal types among five isolates from five children from one family. We conclude that serotyping, biotyping, and outer membrane patterns were reproducible but had a low discriminatory potential. REA and ribotyping were reproducible and gave the highest number of distinct types. When the DNA typing methodis were compared, all strains tested could be distinguished. These findings confirm the heterogeneity found within the species A. actinomycetemcomitans.
Human immunoglobulin G2 (IgG2) responses are gamma interferon (IFN-γ) dependent, and monocyte-derived dendritic cells (mDCs) promote IgG2 production. DCs spontaneously emerge from monocytes in cultures prepared from localized aggressive periodontitis (LagP) patients, and these patients have high levels of IgG2 that is reactive with Actinobacillus actinomycetemcomitans. These results prompted the hypothesis that an interaction between mDCs and A. actinomycetemcomitans promotes IFN-γ production, and IFN-γ is known to promote both immunopathology and protective IgG2. A. actinomycetemcomitans induced mDCs to produce interleukin-12 (IL-12), and the addition of A. actinomycetemcomitans and DCs to cultured peripheral blood lymphocytes elicited high levels of IFN-γ within just 24 h. In contrast, IL-4 was not detectable although DC-derived IL-10 production was apparent. A. actinomycetemcomitans-stimulated macrophages prepared from the same monocytes lacked the ability to induce IL-12 or IFN-γ responses. NK cells of the innate immune system were the primary source of this early IFN-γ, although CD8 T cells also contributed some. The NK cell-derived IFN-γ was IL-12 dependent, and A. actinomycetemcomitans-DC interactions were Toll-like receptor 4 dependent. A. actinomycetemcomitans and A. actinomycetemcomitans lipopolysaccharide (LPS) were more potent than Escherichia coli and E. coli LPS in the ability to induce DC IL-12 and IFN-γ. The ability of A. actinomycetemcomitans-stimulated DCs to induce NK cells to rapidly produce IFN-γ in the absence of detectable IL-4 suggests their potential for skewing responses toward Th1. This may help explain the presence of Th1-associated cytokines in gingival crevicular fluid (GCF) from LagP patients and the high levels of IgG2 in their serum and GCF that is reactive with A. actinomycetemcomitans.
We report the evidence for apoptosis in J774.1 cells by the periodontopathic bacterium Actinobacillus actinomycetemcomitans, suggesting that the ability of A. actinomycetemcomitans to promote apoptosis might be important in the initiation and development of periodontitis. In this study, we examined the role of macrophage CD14, anchored by a glycerophosphatidylinositol tail, in the induction of apoptosis by A. actinomycetemcomitans infection by using the parent J774.1 cells and CD14-defective mutant (LR-9) cells. A small number of A. actinomycetemcomitans Y4 cells inside the LR-9 cells compared with the number in J774.1 cells was detected by confocal scanning microscopy. We found that LR-9 cells showed a weak cytotoxic effect after being infected with A. actinomycetemcomitans Y4. Apoptotic cell death of LR-9 cells infected with A. actinomycetemcomitans Y4, compared with that of the parent J774.1 cells was almost undetectable, as shown by the proportion of fragmented DNA in agarose gel electrophoresis and by the terminal deoxynucleotidyl transferase-mediated dUTP end-labeling method. Flow cytometric cell cycle analysis of J774.1 cells infected with A. actinomycetemcomitans Y4 revealed the increased percentage of apoptotic cells with hypodiploid DNA. However, LR-9 cells infected with A. actinomycetemcomitans Y4 showed no increase in population of apoptotic nuclei compared with the noninfected cells. These findings suggest that the CD14 molecules may contribute to the phagocytosis of A. actinomycetemcomitans by J774.1 cells and regulate, at least in part, apoptotic cell death of macrophages infected with A. actinomycetemcomitans.
Members of the family Pasteurellaceae are classified in part by whether or not they require an NAD supplement for growth on laboratory media. In this study, we demonstrate that this phenotype can be determined by a single gene, nadV, whose presence allows NAD-independent growth of Haemophilus influenzae and Actinobacillus pleuropneumoniae. This gene was cloned from a 5.2-kb plasmid which was previously shown to be responsible for NAD independence in Haemophilus ducreyi. When transformed into A. pleuropneumoniae, this cloned gene allowed NAD-independent growth on complex media and allowed the utilization of nicotinamide in place of NAD on defined media. Sequence analysis revealed an open reading frame of 1,482 bp that is predicted to encode a protein with a molecular mass of 55,619 Da. Compared with the sequence databases, NadV was found to have significant sequence homology to the human pre-B-cell colony-enhancing factor PBEF and to predicted proteins of unknown function identified in the bacterial species Mycoplasma genitalium, Mycoplasma pneumoniae, Shewanella putrefaciens, Synechocystis sp., Deinococcus radiodurans, Pasteurella multocida, and Actinobacillus actinomycetemcomitans. P. multocida and A. actinomycetemcomitans are among the NAD-independent members of the Pasteurellaceae. Homologues of NadV were not found in the sequenced genome of H. influenzae, an NAD-dependent member of the Pasteurellaceae, or in species known to utilize a different pathway for synthesis of NAD, such as Escherichia coli. Sequence alignment of these nine homologues revealed regions and residues of complete conservation that may be directly involved in the enzymatic activity. Identification of a function for this gene in the Pasteurellaceae should help to elucidate the role of its homologues in other species.
Patients with juvenile periodontitis frequently have elevated levels of serum immunoglobulin A (IgA) antibodies to antigens of Actinobacillus actinomycetemcomitans. IgA occurs in two subclasses, IgA1 and IgA2, and in monomeric and polymeric forms. Because IgA1 is susceptible to cleavage by IgA1 proteases produced by microorganisms found at mucosal sites and in the gingival crevice, we wished to determine the IgA subclass distribution of antibodies to antigens of A. actinomycetemcomitans. The molecular form was examined because it may indicate the origin of the IgA and because the form differs in acute and chronic infections. There is also evidence that monomeric and polymeric IgA have different biological functions. Serum was taken from patients with juvenile periodontitis before and at intervals during and after initiation of therapy. IgA subclass distribution was determined against a sonic extracts of A. actinomycetemcomitans ATCC 2952a (serotype b) by using monoclonal anti-subclass reagents in an enzyme-linked immunosorbent assay. To determine the molecular form of the antibodies, sera were separated by high-performance liquid chromatography on a size-exclusion column. Fractions were assayed for antibody activity by the enzyme-linked immunosorbent assay, and described above. The results of the subclass analysis of the sera indicated that while both IgA1 and IgA2 antibodies to A. actinomycetemcomitans sonic extract are often found before, during, and after treatment, IgA1 antibodies dominated the response. There was a predominance of monomeric IgA1 antibodies to A. actinomycetemcomitans sonic extracts in most samples before, during, and after treatment. The monomeric form is consistent with what is seen in other chronic infections. The predominance of IgA1 antibodies implies that any protective effects of the IgA response to A. actinomycetemcomitans could be compromised by microbial IgA1 proteases.
The oral bacterium Actinobacillus actinomycetemcomitans is implicated as a causative agent of localized juvenile periodontitis (LJP). A. actinomycetemcomitans is classified into five serotypes (a to e) corresponding to five structurally and antigenically distinct O polysaccharide (O-PS) components of their respective lipopolysaccharide molecules. Serotype b has been reported to be the dominant serotype isolated from LJP patients. We determined the lipopolysaccharide O-PS structure from A. actinomycetemcomitans CU1000, a strain isolated from a 13-year-old African-American female with LJP which had previously been classified as serotype b. The O-PS of strain CU1000 consisted of a trisaccharide repeating unit composed of l-rhamnose and 2-acetamido-2-deoxy-d-galactose (molar ratio, 2:1) with the structure →2)-α-l-Rhap-(1–3)-2-O-(β-d-GalpNAc)-α-l-Rhap-(1→. O-PS from strain CU1000 was structurally and antigenically distinct from the O-PS molecules of the five known A. actinomycetemcomitans serotypes. Strain CU1000 was mutagenized with transposon IS903φkan, and three mutants that were deficient in O-PS synthesis were isolated. All three transposon insertions mapped to a single 1-kb region on the chromosome. The DNA sequence of a 13.1-kb region surrounding these transposon insertions contained a cluster of 14 open reading frames that was homologous to gene clusters responsible for the synthesis of A. actinomycetemcomitans serotype b, c, and e O-PS antigens. The CU1000 gene cluster contained two genes that were not present in serotype-specific O-PS antigen clusters of the other five known A. actinomycetemcomitans serotypes. These data indicate that strain CU1000 should be assigned to a new A. actinomycetemcomitans serotype, designated serotype f. A PCR assay using serotype-specific PCR primers showed that 3 out of 20 LJP patients surveyed (15%) harbored A. actinomycetemcomitans strains carrying the serotype f gene cluster. The finding of an A. actinomycetemcomitans serotype showing serological cross-reactivity with anti-serotype b-specific antiserum suggests that a reevaluation of strains previously classified as serotype b may be warranted.
A stable shuttle vector which replicates in Escherichia coli and Clostridium perfringens was constructed by ligating a 3.6-kilobase (kb) fragment of plasmid pBR322 with C. perfringens plasmid pHB101 (3.1 kb). The marker for this shuttle plasmid originated from the 1.3-kb chloramphenicol resistance gene of plasmid pHR106. The resulting shuttle vector, designated pAK201, is 8 kb in size and codes for resistance to 20 micrograms of chloramphenicol per ml in both E. coli and C. perfringens. Following shuttle vector construction in E. coli, plasmid pAK201 was transformed into E. coli HB101 and C. perfringens ATCC 3624A, using intact cell electroporation. The transformation frequencies were 10(6) and 10(4) transformants per microgram of DNA in E. coli and C. perfringens, respectively. Restriction enzyme analysis of the chimera isolated from transformants of both microorganisms suggested that the plasmids were identical. Reciprocal transformation experiments in E. coli and C. perfringens indicated no difference in transformation frequency. Plasmid pAK201 was stable in C. perfringens following repeated transfer in the absence of chloramphenicol pressure. The restriction map of plasmid pAK201 shows six unique cut sites which should be useful for future genetic analysis and C. perfringens gene library construction.
The complete nucleotide sequence and genetic map of pVT745 are presented. The 25-kb plasmid was isolated from Actinobacillus actinomycetemcomitans, a periodontal pathogen. Two-thirds of the plasmid encode functions related to conjugation, replication, and replicon stability. Among potential gene products with a high degree of similarity to known proteins are those associated with plasmid conjugation. It was shown that pVT745 derivatives not only mobilized a coresident nontransmissible plasmid, pMMB67, but also mediated their own conjugative transfer to different A. actinomycetemcomitans strains. However, transfer of pVT745 derivatives from A. actinomycetemcomitans to Escherichia coli JM109 by conjugation was successful only when an E. coli origin of replication was present on the pVT745 construct. Surprisingly, 16 open reading frames encode products of unknown function. The plasmid contains a conserved replication region which belongs to the HAP (Haemophilus-Actinobacillus-Pasteurella) theta replicon family. However, its host range appears to be rather narrow compared to other members of this family. Sequences homologous to pVT745 have previously been detected in the chromosomes of numerous A. actinomycetemcomitans strains. The nature and origin of these homologs are discussed based on information derived from the nucleotide sequence.
Actinobacillus actinomycetemcomitans is recognized as a primary pathogen in localized juvenile periodontitis (LJP). Restriction fragment length polymorphisms (RFLP) within a collection of subgingival plaque isolates of this bacterium were identified and characterized as the first step in understanding the pathogenesis of LJP. Over 800 isolates, from members of 18 families (LJP families) with at least one member with active LJP or a documented history of the disease and one or more siblings, less than 13 years of age, having no clinical evidence of LJP and 32 healthy control subjects, were assigned to one of 13 distinct RFLP groups (II to XIV) by using a previously characterized 4.7-kb DNA probe cloned from the reference strain FDC Y4. Isolates belonging to RFLP groups II, IV, V, and XIII predominated subgingival sites in the subjects. Members of RFLP groups II, IV, VII, VIII, X, and XI were recovered only from LJP family subjects, while group XIII and XIV variants were found exclusively in healthy controls. A synthetic oligonucleotide, homologous to the 5' end of the leukotoxin gene (lktA), and the A. actinomycetemcomitans plasmid, pVT745, were tested for their abilities to subdivide the 13 RFLP groups. The leukotoxin probe specifically identified all RFLP group II variants because of the absence of a HindIII site in the upstream noncoding region of the lkt gene complex. The plasmid probe was not as selective but may be useful for identifying clinical isolates belonging to RFLP group I. The use of these probes for the identification of genetic variants of A. actinomycetemcomitans that may be preferentially colonize diseased and healthy subjects will facilitate the study of the role of this important pathogen in periodontal diseases.
Actinobacillus actinomycetemcomitans is a gram-negative, facultatively anaerobic bacterium which is associated especially with aggressive forms of periodontitis. Contradictory results on the localization of the A. actinomycetemcomitans serotype-specific antigen have been reported. The aim of the present study was to characterize the A. actinomycetemcomitans serotype d-specific antigen. The antigen was isolated by affinity chromatography. The affinity column was prepared from immunoglobulin G isolated from rabbit antiserum raised against A. actinomycetemcomitans serotype d. The isolated antigen was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and silver staining, all of which revealed a ladder-like structure typical for the O antigen of lipopolysaccharide (LPS). In a displacement enzyme-linked immunosorbent assay (ELISA), the isolated antigen displaced in a concentration-dependent manner the binding of the polyclonal rabbit antiserum raised against A. actinomycetemcomitans serotype d to the competing whole-cell serotype d antigen. The isolated antigen contained LPS, and an equal concentration of LPS isolated from A. actinomycetemcomitans serotype d gave a similar displacement curve in the ELISA. In order to test the immunogenic properties of the isolated antigen, it was used to immunize a rabbit. The antiserum raised against the isolated antigen displayed specificity in Western blotting and ELISA similar to that of antibody raised against LPS isolated from A. actinomycetemcomitans serotype d. In conclusion, our results show that the A. actinomycetemcomitans serotype d-specific antigen contains the O-antigenic structure of LPS.