Functional mapping of an oligomeric autotransporter adhesin of Aggregatibacter actinomycetemcomitans

Extracellular matrix protein adhesin A (EmaA) is a 202-kDa nonfimbrial adhesin, which mediates the adhesion of the oral pathogen Aggregatibacter actinomycetemcomitans to collagen. EmaA oligomers form surface antenna-like protrusions consisting of a long helical rod with an ellipsoidal ending. The functional analysis of in-frame emaA deletion mutants has located the collagen binding activity to the amino terminus of the protein corresponding to amino acids 70 to 386. The level of collagen binding of this deletion mutant was comparable to the emaA mutant strain. Transmission electron microscopy studies indicate that the first 330 amino acids of the mature protein form the ellipsoidal ending of the EmaA protrusions, where the activity resides. Amino acid substitution analysis within this sequence has identified a critical amino acid, which is essential for the formation of the ellipsoidal ending and for collagen binding activity.     

Investigation of the Three-Dimensional Architecture of the Collagen Adhesin EmaA of Aggregatibacter actinomycetemcomitans by Electron Tomography

The periodontal pathogen Aggregatibacter actinomycetemcomitans displays on the bacterial surface a nonfimbrial adhesin, EmaA, which is required for collagen binding. In this study, electron tomography was used to characterize the three-dimensional (3D) architecture of this adhesin. The antenna-like surface appendages, corresponding to EmaA, were found to be composed of an ellipsoidal domain capping a rod-like domain that adopts either a straight or a bent conformation at various positions along the length. The most common flexible point along the length of the EmaA appendage was localized 29.4 nm away from the distal end. One-fifth of the appendages were straight and the remaining showed angles distributed between 140° and 170° at this location. Deletion analysis mapped this bend to amino acids 611 to 640 of the protein sequence. The 3D structure of the collagen binding domain of EmaA was generated by alignment and averaging of 9 subvolumes of the adhesin extracted from tomograms. The structure contains three subdomains: a globular structure with a diameter of ∼5 nm and a cylindrical domain (∼4.4 nm by 5.8 nm) separated by a linker region with a diameter of ∼3 nm, followed by a cylindrical domain (∼4.6 nm by 6.6 nm). This is the first 3D structure of a trimeric autotransporter protein of A. actinomycetemcomitans.Bacterial adhesion to host receptors, a crucial step for colonization and infection, is mediated by fimbrial and nonfimbrial adhesins. These adhesins are proteinaceous appendages displayed on the surface of bacteria and contain the receptor binding domains. Aggregatibacter actinomycetemcomitans, a gram-negative, nonmotile bacterium is found associated with periodontal diseases and other extraoral infections (12, 23, 32, 40). When isolated from the oral cavity, the bacterium exists as a fimbriated form and switches to an afimbriated form upon planktonic subculturing (5, 14). A. actinomycetemcomitans fimbriae mediate the nonspecific adherence of the bacterium to abiotic and organic surfaces and decorate the bacterial surface with long fibrils of 5 to 7 nm in diameter (14, 15). In addition to fimbriae, nonfimbrial adhesins, which mediate the specific binding to host cells and tissues, have been identified in this bacterium (1, 6, 19, 27, 29). Among these nonfimbrial adhesins, only the extracellular matrix protein adhesin A, EmaA, has been visualized forming structures on the bacterial surface by transmission electron microscopy (29).EmaA is an outer membrane collagen adhesin unique to A. actinomycetemcomitans; however, orthologous proteins exist in other bacterial genera (13, 18, 21, 26, 33, 38). The protein is encoded by a 6-kb gene present in all A. actinomycetemcomitans strains investigated (36). Genetic heterogeneity within the gene exists between different strains, which are based on the serotype of the organism. Based on this heterogeneity, two molecular forms of the protein have been identified: a full-length and an intermediate form. The prototypic or full-length protein exists as a 202-kDa protein and shares 75% amino acid homology with the intermediate form. The intermediate protein form (173 kDa) contains an in-frame 279-amino-acid deletion but maintains collagen binding activity and surface appendages similar to the prototypic form (36).EmaA is associated with the binding of A. actinomycetemcomitans to both isolated acid-soluble collagen and collagen found in tissues (19, 29, 35, 39). The specificity of EmaA for collagen was demonstrated using a rabbit cardiac valve tissue model (35). Valves with an intact endothelium bound equal amounts of the wild type or emaA isogenic mutants. Removal of the endothelium by trypsin treatment, thereby exposing the underlying collagen, did not affect the level of binding of the mutant. However, the number of wild-type bacteria bound to the exposed collagen was five times the number of mutant bacteria. This represents a 10-fold increase with respect to the number of bacteria bound to the endothelium. The role of EmaA as a virulence determinant in A. actinomycetemcomitans infection was demonstrated in a rabbit endocarditis infection model, in which the wild-type bacterium outcompeted the binding of the mutant 10-fold (35).Sequence analysis indicates that EmaA belongs to the Oca (oligomeric coiled-coil adhesin) family of autotransporter adhesins (19). Multimers of EmaA oligomerize to form appendages on the bacterial surface and are visible as long rods or antenna-like structures capped by an ellipsoidal domain (29). A strong correlation exists between the translocated region of the protein (head and stalk domains) and the structural features. The head domain, consisting of amino acids 70 to 386, forms the ellipsoidal ending of the appendage, which is essential for collagen binding, while amino acids 387 to 1900 form the stalk domain (39).Contained within the translocation domain of EmaA are three “neck” sequences, which are conserved in the Oca family protein members (21, 29, 33). These sequences are considered to stabilize the oligomer and transition between β-rolls and coiled-coil regions of the molecule (21, 26). In the EmaA sequence, two “neck” sequences are found within the first 628 amino acids of the protein sequence (19, 29). The third sequence is located in the stalk domain adjacent to the carboxy-terminal membrane anchor domain, which comprises amino acids 1901 to 1965 (19, 29). The membrane anchor domains of three or four monomers are proposed to form β-barrels that are required for pore formation and protein translocation (18, 29, 37).The translocated domain of EmaA has been subjected to a two-dimensional (2D) study by transmission electron microscopy, and the overall dimensions of the EmaA appendages have been determined by the analysis of a large number of micrographs (29). The ellipsoidal ending shows diameters of 2.8 by 4.6 nm, and the stalk domain, which is at least 150 nm long, has a diameter of 4.1 nm. Several conformations of the stalk domain were present in the micrographs: either straight or containing a bend at 29.2 nm from the distal end. This bend position was correlated with amino acids localized between the first two neck sequences (29).In this study, electron tomography was used to characterize the 3D structure of the EmaA appendages of A. actinomycetemcomitans in situ. The functional domain of EmaA was found to be composed of three distinct subdomains followed by a long stalk domain. Distinct regions of the molecule were identified that provide flexibility for the molecule and allow for the deformation or bending of the adhesin. A correlation between these flexible regions and specific amino acids in the sequence was ascertained.     

Glycosylation of the Collagen Adhesin EmaA of Aggregatibacter actinomycetemcomitans Is Dependent upon the Lipopolysaccharide Biosynthetic Pathway

The human oropharyngeal pathogen Aggregatibacter actinomycetemcomitans synthesizes multiple adhesins, including the nonfimbrial extracellular matrix protein adhesin A (EmaA). EmaA monomers trimerize to form antennae-like structures on the surface of the bacterium, which are required for collagen binding. Two forms of the protein have been identified, which are suggested to be linked with the type of O-polysaccharide (O-PS) of the lipopolysaccharide (LPS) synthesized (G. Tang et al., Microbiology 153:2447-2457, 2007). This association was investigated by generating individual mutants for a rhamnose sugar biosynthetic enzyme (rmlC; TDP-4-keto-6-deoxy-d-glucose 3,5-epimerase), the ATP binding cassette (ABC) sugar transport protein (wzt), and the O-antigen ligase (waaL). All three mutants produced reduced amounts of O-PS, and the EmaA monomers in these mutants displayed a change in their electrophoretic mobility and aggregation state, as observed in sodium dodecyl sulfate (SDS)-polyacrylamide gels. The modification of EmaA with O-PS sugars was suggested by lectin blots, using the fucose-specific Lens culinaris agglutinin (LCA). Fucose is one of the glycan components of serotype b O-PS. The rmlC mutant strain expressing the modified EmaA protein demonstrated reduced collagen adhesion using an in vitro rabbit heart valve model, suggesting a role for the glycoconjugant in collagen binding. These data provide experimental evidence for the glycosylation of an oligomeric, coiled-coil adhesin and for the dependence of the posttranslational modification of EmaA on the LPS biosynthetic machinery in A. actinomycetemcomitans.The Gram-negative, nonmotile, microaerophilic, and oropharyngeal bacterium Aggregatibacter actinomycetemcomitans preferentially colonizes the subgingival region of the human oral cavity. This microorganism is implicated as the etiological agent of localized aggressive periodontitis (9, 13) and causes extraoral infections, including pneumonia, osteitis (30), and infective endocarditis (6). Recent studies also link this periodontal pathogen to cardiovascular diseases, such as atherosclerosis (20).Typical of Gram-negative bacteria, the outer membrane of A. actinomycetemcomitans possesses an asymmetric lipid-protein bilayer. The inner leaflet of the outer membrane is mainly phospholipids, and the outer leaflet consists of lipopolysaccharide (LPS), phospholipids, and proteins (4). LPS molecules are ubiquitously distributed on the outer membrane and are essential for maintaining the membrane integrity (3). Intact LPS molecules are also required for the assembly of some large outer membrane proteins (3, 18, 41). A typical LPS molecule is composed of hydrophobic lipid A, a nonrepeat core oligosaccharide, and a repeating O-antigen or O-polysaccharide (O-PS). The distal O-PS is a major antigen, stimulating the host immune response, and the basis for serotyping Gram-negative bacteria (36), including A. actinomycetemcomitans (32, 50).Six different serotypes (a to f) and the corresponding genetic loci have been identified for A. actinomycetemcomitans (19, 22, 27, 44, 50, 54, 55). Serotype b remains one of the common serotypes found in the human oral cavity (9, 13, 51). The serotype b O-PS of A. actinomycetemcomitans is encoded by an operon composed of 21 genes, which are responsible for the biosynthesis of the repeating trisaccharide unit of this particular serotype (53, 55). Each O-PS unit of serotype b contains a disaccharide backbone composed of d-fucose (d-Fuc) and l-rhamnose (l-Rha), linked by a nonreducing d-N-acetylgalactosamine (d-GalNAc) at the O-3 position of l-Rha (33) (Fig. ​(Fig.1A1A).Open in a separate windowFIG. 1.(A) O-PS structure of serotype b A. actinomycetemcomitans. (B) Silver-stained 5 to 15% polyacrylamide-SDS gel of serotype b LPS. A total of 1.0 ml of mid-logarithmic-phase cells were collected and lysed. Three lysates from each strain were combined and treated with proteinase K at 60°C for 60 min before electrophoresis, followed by silver staining. C, control: whole-cell lysate without proteinase K digestion; WT, wild type (VT1169); emaA, extracellular matrix protein adhesin A mutant; rmlC, rhamnose epimerase mutant; wzt, ATP-binding cassette sugar transport mutant. The dark brown staining of the high molecular weight (75,000 to 250,000) corresponds to polymerized O-PS.The assembly of LPS molecules in Gram-negative bacteria involve diverse enzymes and pathways due to the variation of the O-PS structures among different bacteria (36). RmlC (previously RfbD), Wzt (previously AbcA or RfbB), and WaaL are three enzymes involved in different stages of the LPS synthesis of some Gram-negative bacteria (7, 36, 37). A homologue of RmlC, TDP-4-keto-6-deoxy-d-glucose 3,5-epimerase, which is required for l-Rha synthesis, has been identified in A. actinomycetemcomitans (53, 55). Wzt is an ATP binding cassette (ABC) transporter that exports saccharide polymers from the cytoplasm to the periplasmic space (7, 36). A homologue of wzt was originally identified from a serotype b strain of A. actinomycetemcomitans, based on protein sequence identity with Aeromonas salmonicida (55). Kaplan et al. (19) later showed that a serotype f wzt mutant strain of A. actinomycetemcomitans produces less O-PS. WaaL, an O-antigen ligase found in Escherichia coli and Pseudomonas aeruginosa, ligates an undecaprenol pyrophosphate-linked oligo- or polysaccharide onto the lipid A-core oligosaccharide in the periplasm (1, 36). A putative O-antigen ligase is located in the chromosome of a serotype b A. actinomycetemcomitans strain (HK1651), based on sequence homology (Oralgen, Los Alamos, NM).Our earlier work suggested a correlation between the type of LPS molecule and the form of EmaA synthesized by A. actinomycetemcomitans (46). The EmaA of serotype b A. actinomycetemcomitans is a 202-kDa protein that forms the antennae-like appendages found on the surface of A. actinomycetemcomitans and is required for collagen binding (40). The appendages are composed of three EmaA monomers that oligomerize to form an ellipsoidal structure required for the collagen binding activity (56, 57). The ellipsoidal structure corresponds to the amino termini of the proteins and is located at the distal end of a long stalk domain that is attached to the outer membrane by the carboxyl termini (57). The carboxyl termini of the proteins assume β-barrel structures required for pore formation and translocation of the molecules through the outer membrane, similar to those of other type V_c autotransporter proteins (14). Recently, we have demonstrated that EmaA is important in the initiation of infective endocarditis in a rabbit model of infectious endocarditis (45).Two transposon mutant strains (rmlC and wzt) and a waaL mutant strain generated by site-directed insertional mutagenesis have been developed and characterized in this study. The rmlC mutant did not synthesize l-Rha and did not produce detectable O-PS. The wzt and waaL mutant strains synthesized less O-PS than the wild-type strain. Complementation of the mutant strains restored the production of the serotype b O-PS to wild-type levels. An increase in the electrophoretic mobility of the EmaA monomer was observed in all three mutants, which suggests the presence of carbohydrate. The EmaA mobility reverted to wild-type mobility upon complementation. The presence of carbohydrate associated with EmaA was confirmed by lectin blotting, and in vitro collagen binding assessment demonstrated that the glycoconjugant is important for the full function of this adhesin. The experimental data suggest that EmaA contains carbohydrate similar to that present in O-PS and is a substrate for the O-antigen ligase of the LPS biosynthetic pathway of A. actinomycetemcomitans.     

Correlation of the amino-acid sequence and the 3D structure of the functional domain of EmaA from Aggregatibacter actinomycetemcomitans

Adhesion to collagen is an important virulence determinant for the periodontal pathogen Aggregatibacter actinomycetemcomitans. Binding to collagen is mediated by the extracellular-matrix protein adhesin-A (EmaA). EmaA is a homotrimeric autotransporter protein that forms flexible antenna-like appendages on the bacterium surface. An ellipsoidal structure at the distal end of the appendage, composed of three subdomains, contains the functional domain of the molecule. A correlation between amino-acid sequence and subdomain structure (SI and SII) was proposed based on an analysis of the volume/molecular weight ratio. EmaA from three mutant strains (deletions of amino-acids 70-206 and 70-386 and a substitution mutation G162S) has been studied by electron microscopy to test this hypothesis. 3D structures were analyzed using single-axis tilt tomography of negatively stained preparations of bacteria combined with subvolume averaging. Additionally, a large number of 2D images of the apical domain of the adhesins from the mutants were extracted from micrographs of the bacterial surface, aligned and classified. The combined data showed that amino-acids 70-206 localize to subdomain SI and 70-386 comprise subdomains SI and SII. Moreover, we showed that the substitution mutation G162S, which abolishes collagen binding activity, does not affect the overall structural integrity of the functional domain. However, the structure of subdomain SI in this mutant is slightly altered with respect to the wild-type strain. These data also have allowed us to interpret the architectural features of each subdomain of EmaA in more detail and to correlate the 3D structure of the functional domain of EmaA with the amino-acid sequence.     

Regulation of Aggregatibacter (Actinobacillus) actinomycetemcomitans leukotoxin secretion by iron

The gram-negative oral and systemic pathogen Aggregatibacter (Actinobacillus) actinomycetemcomitans produces a leukotoxin (LtxA) that is a member of the RTX (repeats in toxin) family of secreted bacterial toxins. We have recently shown that LtxA has the ability to lyse erythrocytes, which results in a beta-hemolytic phenotype on Columbia blood agar. To determine if LtxA is regulated by iron, we examined beta-hemolysis under iron-rich and iron-limiting conditions. Beta-hemolysis was suppressed in the presence of FeCl3. In contrast, strong beta-hemolysis occurred in the presence of the iron chelator deferoxamine. We found that secretion of LtxA was completely inhibited by free iron, but expression of ltxA was not regulated by iron. Free chromium, cobalt, and magnesium did not affect LtxA secretion. Other LtxA-associated genes were not regulated by iron. Thus, iron appears to play an important role in the regulation of LtxA secretion in A. actinomycetemcomitans in a manner independent of gene regulation.     

Repetitive architecture of the Haemophilus influenzae Hia trimeric autotransporter

The Hia autotransporter of Haemophilus influenzae belongs to the trimeric autotransporter subfamily and mediates bacterial adherence to the respiratory epithelium. In this report, we show that the structure of Hia is characterized by a modular architecture containing repeats of structurally distinct domains. Comparison of the structures of HiaBD1 and HiaBD2 adhesive repeats and a nonadhesive repeat (a novel fold) shed light on the structural determinants of Hia adhesive function. Examination of the structure of an extended version of the Hia translocator domain revealed the structural transition between the C-terminal translocator domain and the N-terminal passenger domain, highlighting a highly intertwined domain that is ubiquitous among trimeric autotransporters. Overall, this study provides important insights into the mechanism of Hia adhesive activity and the overall structure of trimeric autotransporters.     

Proteomics of Protein Secretion by Aggregatibacter actinomycetemcomitans

The extracellular proteome (secretome) of periodontitis-associated bacteria may constitute a major link between periodontitis and systemic diseases. To obtain an overview of the virulence potential of Aggregatibacter actinomycetemcomitans, an oral and systemic human pathogen implicated in aggressive periodontitis, we used a combined LC-MS/MS and bioinformatics approach to characterize the secretome and protein secretion pathways of the rough-colony serotype a strain D7S. LC-MS/MS revealed 179 proteins secreted during biofilm growth. Further to confirming the release of established virulence factors (e.g. cytolethal distending toxin [CDT], and leukotoxin [LtxA]), we identified additional putative virulence determinants in the secretome. These included DegQ, fHbp, LppC, Macrophage infectivity protein (MIP), NlpB, Pcp, PotD, TolB, and TolC. This finding indicates that the number of extracellular virulence-related proteins is much larger than previously demonstrated, which was also supported by in silico analysis of the strain D7S genome. Moreover, our LC-MS/MS and in silico data revealed that at least Type I, II, and V secretion are actively used to excrete proteins directly into the extracellular space, or via two-step pathways involving the Sec/Tat systems for transport across the inner membrane, and outer membrane factors, secretins and auto-transporters, respectively for delivery across the outer membrane. Taken together, our results provide a molecular basis for further elucidating the role of A. actinomycetemcomitans in periodontal and systemic diseases.     

Effects of low-dose doxycycline on cytokine secretion in human monocytes stimulated with Aggregatibacter actinomycetemcomitans

Doxycycline is an antibiotic used in the treatment of a variety of inflammatory conditions, including periodontitis. Apart from its antimicrobial properties, this drug also has independent anti-inflammatory effects at sub-antimicrobial doses. The present study aimed to investigate the effects of low-doses of doxycycline (LDD) on cytokine production by human monocytic cells challenged with the periodontal pathogen Aggregatibacter actinomycetemcomitans, for up to 6 h. The simultaneous regulation of 12 cytokines were measured by a Human Cytokine Array Kit. To validate the array findings, selected cytokines were also measured by enzyme-linked immunosorbant assay (ELISA). A. actinomycetemcomitans stimulated the production of tumor necrosis factor (TNF)-α, interleukin (IL)-1α, IL-1β, IL-6 and IL-8 by the cells after 6 h of challenge, and doxycycline significantly inhibited this effect. The kinetics of this regulation demonstrated an early (within 2 h) and significant (P<0.05) inhibition of pro-inflammatory cytokines, with a mild (0.5-fold) up-regulation of the anti-inflammatory cytokine IL-10. The results indicate that LDD acts as an anti-inflammatory agent in human monocytic cells stimulated with A. actinomycetemcomitans. This model provides clear evidence that some of the clinically proven benefits of LDD may be related to its ability to regulate inflammatory mediator release by monocytic cells. This property may contribute to the clinically proven benefits of this antibiotic as an adjunctive treatment for periodontitis.     

Proteomics of Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles

Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive forms of periodontitis and with endocarditis. Outer membrane vesicles (OMVs) released by this species have been demonstrated to deliver effector proteins such as cytolethal distending toxin (CDT) and leukotoxin (LtxA) into human host cells and to act as triggers of innate immunity upon carriage of NOD1- and NOD2-active pathogen-associated molecular patterns (PAMPs). To improve our understanding of the pathogenicity-associated functions that A. actinomycetemcomitans exports via OMVs, we studied the proteome of density gradient-purified OMVs from a rough-colony type clinical isolate, strain 173 (serotype e) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This analysis yielded the identification of 151 proteins, which were found in at least three out of four independent experiments. Data are available via ProteomeXchange with identifier PXD002509. Through this study, we not only confirmed the vesicle-associated release of LtxA, and the presence of proteins, which are known to act as immunoreactive antigens in the human host, but we also identified numerous additional putative virulence-related proteins in the A. actinomycetemcomitans OMV proteome. The known and putative functions of these proteins include immune evasion, drug targeting, and iron/nutrient acquisition. In summary, our findings are consistent with an OMV-associated proteome that exhibits several offensive and defensive functions, and they provide a comprehensive basis to further disclose roles of A. actinomycetemcomitans OMVs in periodontal and systemic disease.     

The conserved extension of the Hbp autotransporter signal peptide does not determine targeting pathway specificity

Autotransporters (ATs) of Gram-negative bacteria are often produced with an unusual signal peptide that carries a conserved N-terminal extension. Using combined in vitro and in vivo approaches we show that the extension of the AT hemoglobin protease (Hbp) does not affect targeting of Hbp via the SRP-pathway, suggesting that the extension is not involved in targeting pathway selection.     

The Haemophilus influenzae Hia autotransporter contains an unusually short trimeric translocator domain

Gram-negative bacterial autotransporter proteins are a growing group of virulence factors that are characterized by their ability to cross the outer membrane without the help of accessory proteins. A conserved C-terminal beta-domain is critical for targeting of autotransporters to the outer membrane and for translocation of the N-terminal "passenger" domain to the bacterial surface. We have demonstrated previously that the Haemophilus influenzae Hia adhesin belongs to the autotransporter family, with translocator activity residing in the C-terminal 319 residues. To gain further insight into the mechanism of autotransporter protein translocation, we performed a structure-function analysis on Hia. In initial experiments, we generated a series of in-frame deletions and a set of chimeric proteins containing varying regions of the Hia C terminus fused to a heterologous passenger domain and discovered that the final 76 residues of Hia are both necessary and sufficient for translocation. Analysis by flow cytometry revealed that the region N-terminal to this shortened translocator domain is surface localized, further suggesting that this region is not involved in beta-barrel formation or in translocation of the passenger domain. Western analysis demonstrated that the translocation-competent regions of the C terminus migrated at masses consistent with trimers, suggesting that the Hia C terminus oligomerizes. Furthermore, fusion proteins containing a heterologous passenger domain demonstrated that similarly small C-terminal regions of Yersinia sp. YadA and Neisseria meningitidis NhhA are translocation-competent. These data provide experimental support for a unique subclass of autotransporters characterized by a short trimeric translocator domain.     

Leukotoxic Activity of Aggregatibacter actinomycetemcomitans and Periodontal Attachment Loss

Aggregatibacter actinomycetemcomitans is a Gram-negative periodontitis-associated bacterium that expresses a toxin that selectively affects leukocytes. This leukotoxin is encoded by an operon belonging to the core genome of this bacterial species. Variations in the expression of the leukotoxin have been reported, and a well-characterized specific clonal type (JP2) of this bacterium with enhanced leukotoxin expression has been isolated. In particular, the presence of the JP2 genotype significantly increases the risk for the progression of periodontal attachment loss (AL). Based on these findings we hypothesized that variations in the leukotoxicity are linked to disease progression in infected individuals. In the present study, the leukotoxicity of 239 clinical isolates of A. actinomycetemcomitans was analysed with different bioassays, and the genetic peculiarities of the isolates were related to their leukotoxicity based on examination with molecular techniques. The periodontal status of the individuals sampled for the presence of A. actinomycetemcomitans was examined longitudinally, and the importance of the observed variations in leukotoxicity was evaluated in relation to disease progression. Our data show that high leukotoxicity correlates with an enhanced risk for the progression of AL. The JP2 genotype isolates were all highly leukotoxic, while the isolates with an intact leukotoxin promoter (non-JP2 genotypes) showed substantial variation in leukotoxicity. Genetic characterization of the non-JP2 genotype isolates indicated the presence of highly leukotoxic genotypes of serotype b with similarities to the JP2 genotype. Based on these results, we conclude that A. actinomycetemcomitans harbours other highly virulent genotypes besides the previously described JP2 genotype. In addition, the results from the present study further highlight the importance of the leukotoxin as a key virulence factor in aggressive forms of periodontitis.     

Characterization of a quinol peroxidase mutant in Aggregatibacter actinomycetemcomitans

Aggregatibacter actinomycetemcomitans is an oral pathogen causing localized aggressive periodontitis (LAP). Recently, we characterized for the first time a quinol peroxidase (QPO) that catalyzes peroxidase activity using quinol in the respiratory chain of A. actinomycetemcomitans for the reduction of hydrogen peroxide. In the present study, we characterized the phenotype of a QPO null mutant. The QPO null mutant shows an oxidative stress phenotype, suggesting that QPO plays a certain role in scavenging endogenously generated reactive oxygen species. Notably, we discovered that the QPO null mutant exhibits a production defect of leukotoxin (LtxA), which is a secreted bacterial toxin and is known to target human leukocytes and erythrocytes. This result suggests that QPO would be considered as a potential drug target to inhibit the expression of LtxA from A. actinomycetemcomitans for the treatment and prevention of LAP.     

Drosophila ia2 modulates secretion of insulin-like peptide

Islet antigen-2 (IA-2) is a major autoantigen in type I diabetes. To throw light on the function of IA-2 we examined the role of ia2, a Drosophila homologue, during Drosophila development. In situ hybridization showed that ia2 was expressed in the central nervous system and midgut region. The neuronal expression pattern of ia2 was very similar to that of IA-2 in mammals. Disruption of gut-specific ia2 expression by double stranded RNA interference (dsRNAi) resulted in defects in gut development, and this phenotype was rescued by overexpression of hexokinase. Until now the roles of IA-2 and hexokinase in insulin signaling have been described separately but we found that ia2 modulated the expression of both insulin and hexokinase. Moreover this modulation seems to be important for gut development during metamorphosis. As the pancreas develops from the gut during vertebrate development, our results suggest a possible role of IA-2 in insulin and hexokinase regulation.     

Identification of Ata, a multifunctional trimeric autotransporter of Acinetobacter baumannii

Acinetobacter baumannii has recently emerged as a highly troublesome nosocomial pathogen, especially in patients in intensive care units and in those undergoing mechanical ventilation. We have identified a surface protein adhesin of A. baumannii, designated the Acinetobacter trimeric autotransporter (Ata), that contains all of the typical features of trimeric autotransporters (TA), including a long signal peptide followed by an N-terminal, surface-exposed passenger domain and a C-terminal domain encoding 4 β-strands. To demonstrate that Ata encoded a TA, we created a fusion protein in which we replaced the entire passenger domain of Ata with the epitope tag V5, which can be tracked with specific monoclonal antibodies, and demonstrated that the C-terminal 101 amino acids of Ata were capable of exporting the heterologous V5 tag to the surface of A. baumannii in a trimeric form. We found that Ata played a role in biofilm formation and bound to various extracellular matrix/basal membrane (ECM/BM) components, including collagen types I, III, IV, and V and laminin. Moreover, Ata mediated the adhesion of whole A. baumannii cells to immobilized collagen type IV and played a role in the survival of A. baumannii in a lethal model of systemic infection in immunocompetent mice. Taken together, these results reveal that Ata is a TA of A. baumannii involved in virulence, including biofilm formation, binding to ECM/BM proteins, mediating the adhesion of A. baumannii cells to collagen type IV, and contributing to the survival of A. baumannii in a mouse model of lethal infection.     

Identification of a novel trimeric autotransporter adhesin in the cryptic genospecies of Haemophilus

Haemophilus biotype IV strains belonging to the recently recognized Haemophilus cryptic genospecies are an important cause of maternal genital tract and neonatal systemic infections and initiate infection by colonizing the genital or respiratory epithelium. To gain insight into the mechanism of Haemophilus cryptic genospecies colonization, we began by examining prototype strain 1595 and three other strains for adherence to genital and respiratory epithelial cell lines. Strain 1595 and two of the three other strains demonstrated efficient adherence to all of the cell lines tested. With a stably adherent variant of strain 1595, we generated a Mariner transposon library and identified 16 nonadherent mutants. All of these mutants lacked surface fibers and contained an insertion in the same open reading frame, which encodes a 157-kDa protein designated Cha for cryptic haemophilus adhesin. Analysis of the predicted amino acid sequence of Cha revealed the presence of an N-terminal signal peptide and a C-terminal domain bearing homology to YadA-like and Hia-like trimeric autotransporters. Examination of the C-terminal 120 amino acids of Cha demonstrated mobility as a trimer on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the capacity to present the passenger domain of the Hia trimeric autotransporter on the bacterial surface. Southern analysis revealed that the gene that encodes Cha is conserved among clinical isolates of the Haemophilus cryptic genospecies and is absent from the closely related species Haemophilus influenzae. We speculate that Cha is important in the pathogenesis of disease due to the Haemophilus cryptic genospecies and is in part responsible for the apparent tissue tropism of this organism.     

The essential role of IFN-gamma in the control of lethal Aggregatibacter actinomycetemcomitans infection in mice

Inflammatory immune reactions in response to periodontopathogens trigger periodontal destruction, but their role to protect the host against infection remains unknown. Thus, we examined the mechanisms by which IFN-gamma modulates the outcome of Aggregatibacter actinomycetemcomitans-induced periodontal disease in mice. Our results showed that IFN-gamma deficient mice developed less severe periodontitis in response to A. actinomycetemcomitans infection, characterized by significant lower alveolar bone loss and inflammatory reaction. However, the absence of IFN-gamma results in increased bacterial load in periodontal tissues and higher acute phase reaction, followed by a disseminated bacterial infection and mice death during the course of the disease. Such impaired host response was found to be associated with a reduction in the levels of inflammatory cytokines and chemokines and in the number of GR1+, F4/80+, CD4+ and CD8+ leukocytes in the diseased periodontium of IFN-gamma deficient mice. In addition, the levels of both antimicrobial mediators myeloperoxidase and inducible nitric oxide synthase were also found to be reduced in IFN-KO mice. Our results demonstrate for the first time that a periodontal infection may be lethal in an immunocompromised host. In addition, the mechanisms involved in IFN-gamma mediated cell migration to diseased periodontal tissues, and its essential role to control A. actinomycetemcomitans infection were clarified.