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.     

Aggregatibacter actinomycetemcomitans leukotoxin causes activation of lymphocyte function‐associated antigen 1

Repeats‐in‐toxin leukotoxin (LtxA) produced by the oral bacterium Aggregatibacter actinomycetemcomitans kills human leukocytes in a lymphocyte function‐associated antigen 1 (LFA‐1, integrin α_L/β₂)‐dependent manner, although the mechanism for this interaction has not been identified. The LtxA internalisation by LFA‐1‐expressing cells was explored with florescence resonance energy transfer (FRET) microscopy using a cell line that expresses LFA‐1 with a cyan fluorescent protein‐tagged cytosolic α_L domain and a yellow fluorescent protein‐tagged β₂ domain. Phorbol 12‐myristate 13‐acetate activation of LFA‐1 caused transient cytosolic domain separation. However, addition of LtxA resulted in an increase in FRET, indicating that LtxA brings the cytosolic domains closer together, compared with the inactive state. Unlike activation, this effect was not transient, lasting more than 30 min. Equilibrium constants of LtxA binding to the cytoplasmic domains of both α_L and β₂ were determined using surface plasmon resonance. LtxA has a strong affinity for the cytosolic domains of both the α_L and β₂ subunits (K_d = 15 and 4.2 nM, respectively) and a significantly lower affinity for the cytoplasmic domains of other integrin α_M, α_X, and β₃ subunits (K_d = 400, 180, and 230 nM, respectively), used as controls. Peptide fragments of α_L and β₂ show that LtxA binds membrane‐proximal domain of α_L and intermediate domain of β₂.     

Alteration in abundance of specific membrane proteins of Aggregatibacter actinomycetemcomitans is attributed to deletion of the inner membrane protein MorC

Aggregatibacter actinomycetemcomitans is an important pathogen in the etiology of human periodontal and systemic diseases. Inactivation of the gene coding for the inner membrane protein, morphogenesis protein C (MorC), results in pleotropic effects pertaining to the membrane structure and function of this bacterium. The role of this protein in membrane biogenesis is unknown. To begin to understand the role of this conserved protein, stable isotope dimethyl labeling in conjunction with MS was used to quantitatively analyze differences in the membrane proteomes of the isogenic mutant and wild‐type strain. A total of 613 proteins were quantified and 601 of these proteins were found to be equal in abundance between the two strains. The remaining 12 proteins were found in lesser (10) or greater (2) abundance in the membrane preparation of the mutant strain compared with the wild‐type strain. The 12 proteins were ascribed functions associated with protein quality control systems, oxidative stress responses, and protein secretion. The potential relationship between these proteins and the phenotypes of the MorC mutant strain is discussed.     

Purification of a fibrolast-inhibitory factor from Actinobacillus actinomycetemcomitans Y4

Abstract A factor showing inhibitory activity against human gingival fibrolasts was extracted from the cytosol fraction of Actinobacillus actinomycetemcomitans Y4. The activity markedly inhibited the proliferation of human gingival fibrolasts, but had no effect on cell viability or gross morphology. No such activity was found in cytosol fractions from either Porphyromonas gingivalis 381 or Escherichia coli HB101. The extract from A. actinomycetemcomitans Y4 was then purified by anion-exchange chromatography, hydroxyapatite chromatography and gel-filtration chromatography to give a single band on SDS-PAGE with an apparent molecular mass of 65 kDa. The purification ratio was 183-fold with a recovery rate of 5% compared with the crude extract (starting material) when the activity was assessed by direct cell counts.     

Interaction of the salivary low-molecular-weight mucin (MG2) with Actinobacillus actinomycetemcomitans

Periodontitis is associated with the presence of certain Gram-negative bacteria in the oral cavity, among these Actinobacillus actinomycetemcomitans. In order to determine which types of salivary components interact with A. actinomycetemcomitans two strains (HG 1175 and FDC Y4) were incubated with whole saliva and individual glandular secretions, viz. parotid, submandibular, and sublingual saliva. Immunochemical analysis by immunoblotting of bacteria-bound salivary proteins showed that IgA, the low-molecular mucin MG2, parotid agglutinin, and a 300 kDa sublingual and submandibular glycoprotein, were bound to the bacterial strains tested. In addition, adherence of A. actinomycetemcomitans to salivary proteins in a solid-phase was studied. After electrophoresis and transfer of salivary proteins to nitrocellulose membranes A. actinomycetemcomitans adhered only to MG2. In this assay periodate treatment, mild acid hydrolysis or neuraminidase digestion of the saliva glycoproteins abolished binding of two clinical isolates (HG 1175 and NY 664), suggesting that sialic acid residues on MG2 are involved in the binding. In contrast, adherence of the smooth laboratory strain Y4 was not affected by removal of sialic acid residues or even periodate treatment of MG2.Abbreviations S-IgA Secretory IgA - MG1 high-molecular-weight mucin - MG2 low-molecular-weight mucin - EP-GP extra parotid-glycoprotein - PRPs proline-rich proteins - SNA Sambucus nigra agglutinin - MAA Maackia amurensis agglutinin - PNA peanut agglutinin - UEA Ulex europaeus agglutinin     

Reconstitution and purification of cytolethal distending toxin of Actinobacillus actinomycetemcomitans

Cytolethal distending toxin (CDT) has been found in various pathogenic bacterial species and causes a cell distending and a G2 arrest against eukaryotic cells. All the cdtABC genes, which encode CDT, are known to be required for the CDT activities although the CDT holotoxin structure has not been elucidated. We cloned the cdtABC genes of Actinobacillus actinomycetemcomitans and constructed an Escherichia coli expression system for them. We found that crude extracts from six deletion mutants (delta cdtA, delta cdtB, delta cdtC, delta cdtBC, delta cdtAC, and delta cdtAB) of recombinant E. coli, which showed very weak or no detectable CDT activities, restored the CDT activities when pre-mixing and pre-incubation of them were performed in combinations to contain all the CdtA, CdtB, and CdtC proteins. These results indicate that all the Cdt proteins are required for the CDT activities. We also found that the chimera CdtB protein, CdtB-intein-CBD (chitin binding domain) like CdtB protein itself assembled with CdtA and CdtC. The reconstituted CDT containing the chimera CdtB protein was specifically extracted by chitin beads and the only CDT portion was isolated from the chitin beads by a cleavage reaction of the intein. The purified reconstituted-CDT was found to consist of CdtA, CdtB, and CdtC proteins, and showed appreciable CDT activities, indicating that the CDT holotoxin structure is the CdtABC complex. To our knowledge, this is the first report succeeded in complete purification of an active CDT and may offer useful tools for elucidation of the toxic mechanism of CDT.     

The regulatory effect of fermentable sugar levels on the production of leukotoxin by Actinobacillus actinomycetemcomitans

The relationship between sugar availability and RTX (repeats in toxin) cytotoxin (leukotoxin) production in the periodontopathic bacterium, Actinobacillus actinomycetemcomitans, was investigated using a chemostat. A. actinomycetemcomitans 301-b produced significant amounts of leukotoxin in anaerobic fructose-limited chemostat cultures at a dilution rate of 0.15 h⁻¹ and at pH 7.0. When the growth limitation was relieved by pulsing the cultures with 50 or 150 mM fructose (final concentrations), leukotoxin production immediately stopped and the amount of cellular leukotoxin decreased until the culture was returned to fructose-limited conditions. Leukotoxin synthesis was also repressed in the chemostat cultures by pulsing with glucose but not with the non-fermentable sugar analog, α-methyl-d-glucoside. Leukotoxin production was also repressed by fructose in chemostat cultures of ATCC 33384, which is generally recognized as a non-leukotoxin-producing or minimally leukotoxic strain.     

Molecular characterization and functional analysis of a secA gene homolog in Actinobacillus actinomycetemcomitans

Results of Southern blot analyses and polymerase chain reaction revealed that the Gram-negative pathogen, Actinobacillus actinomycetemcomitans, harbored DNA homologous to the secA gene of Escherichia coli. In E. coli, the secA gene product is essential for translocation of proteins across the inner membrane via the Sec system. This A. actinomycetemcomitans secA homolog was cloned and its nucleotide sequence determined. Amino acid sequence analysis of the cloned gene revealed significant homology to the SecA proteins of Haemophilus influenzae, E. coli, Caulobacter crescentus and Bacillus subtilis. Although the cloned gene did not complement a temperature sensitive mutation in the E. coli secA gene, strains harboring the cloned gene did produce a protein that cross-reacted with anti-SecA antibody. In addition, the cloned gene did restore sensitivity to sodium azide in an E. coli azide mutant. These data support the hypothesis that A. actinomycetemcomitans may use a system similar to the Sec system of E. coli to transport proteins across the cytoplasmic membrane, but suggest that the A. actinomycetemcomitans gene product may require genera-specific Sec proteins to complement some Sec mutations in E. coli.     

Variation of loop sequence alters stability of cytolethal distending toxin (CDT): crystal structure of CDT from Actinobacillus actinomycetemcomitans

Cytolethal distending toxin (CDT) secreted by Actinobacillus actinomycetemcomitans induces cell cycle arrest of cultured cells in the G2 phase. The crystal structure of the natural form of A. actinomycetemcomitans DCT (aCDT) has been determined at 2.4 A resolution. aCDT is a heterotrimer consisting of the three subunits, aCdtA, aCdtB, and aCdtC. Two crystallographically independent aCDTs form a dimer through interactions of the aCdtB subunits. The primary structure of aCDT has 94.3% identity with that of Haemophilus ducreyi CDT (hCDT), and the structure of aCDT is quite similar to that of hCDT reconstituted from the three subunits determined recently. However, the molecular packings in the crystal structures of aCDT and hCDT are quite different. A careful analysis of molecular packing suggests that variation of the amino acid residues in a nonconserved loop (181TSSPSSPERRGY192 of aCdtB and 181NSSSSPPERRVY192 of hCdtB) is responsible for the different oligomerization of very similar CDTs. The loop of aCdtB has a conformation to form a dimer, while the loop conformation of hCdtB prevents hCDT from forming a dimer. Although dimerization of aCDT does not affect toxic activity, it changes the stability of protein. aCDT rapidly aggregates and loses toxic activity in the absence of sucrose in a buffered solution, while hCDT is stable and retains toxic activity. Another analysis of crystal structures of aCDT and hCDT suggests that the receptor contact area is in the deep groove between CdtA and CdtC, and the characteristic "aromatic patch" on CdtA.     

Actinobacillus pleuropneumoniae haemolysin II is secreted from Escherichia coli by A. pleuropneumoniae pleurotoxin secretion gene products

Abstract Actinobacillus pleuropneumoniae serotype 2 secretes type II haemolysin and pleurotoxin activities. Here, the genes for type II haemolysin were cloned in Escherichia coli , but type II haemolysin antigen and haemolysin activity were only detected intracellularly and not exported to culture supernatant. It has been reported that the genes for type II haemolysin are not linked to functional secretion genes, while those for pleurotoxin are. In this report the means of secretion of type II haemolysis was examined by constructing a hybrid plasmid carrying the genes required for type II haemolysin expression, together with determinants which allow secretion of pleurotoxin and are linked to the pleurotoxin toxin genes. These genes facilitated the export of type II haemolysin from E. coli , and may perform this function in A. pleuropneumoniae .     

Isolation and characterization of hemolytic genes from Actinobacillus actinomycetemcomitans

Abstract Periodontopathic Actinobacillus actinomycetemcomitans produces hemolysin and other leukotoxins. In the present study, two distinct clones which lysed horse erythrocytes were isolated by screening genomic DNA libraries of A. actinomycetemcomitans ATCC 43718 on blood agar plates. DNA hybridization analysis indicates that there were two distinct hemolytic genes present. Sonicated extracts from both Escherichia coli clones possessed hemolytic activities on horse, sheep and human erythrocytes, but not those of rabbit. Rabbit antiserum to A. actinomycetemcomitans ATCC 43718 whole cells inhibited the hemolytic activities of these clones.     

Rhizobial secreted proteins as determinants of host specificity in the rhizobium–legume symbiosis

Rhizobia are Gram-negative bacteria than can elicit the formation of specialized organs, called root nodules, on leguminous host plants. Upon infection of the nodules, they differentiate into nitrogen-fixing bacteroids. An elaborate signal exchange precedes the symbiotic interaction. In general, both rhizobia and host plants exhibit narrow specificity. Rhizobial factors contributing to this specificity include Nod factors and surface polysaccharides. It is becoming increasingly clear that protein secretion is important in determining the outcome of the interaction as well. This paper discusses our current understanding of the symbiotic role played by rhizobial secreted proteins, transported both by secretion systems that are of general use, such as the type I secretion system, and by specialized, host-targeting secretion systems, such as the type III, type IV and type VI secretion systems.     

Selective retention and degradation of molecules with a single mutant α1(I) chain in the Brtl IV mouse model of OI

We investigated the secretion, matrix incorporation and interactions of molecules with one and two mutant alpha1(I) collagen chains in the Brtl IV murine model for Osteogenesis Imperfecta, carrying a Gly-349 to Cys substitution in one col1a1 allele. We detected a significant deviation from the expected 25 and 50% content of the molecules with no (37-46%) and one (26-40%) mutant chains in skin and bone as well as in fibroblast and osteoblast cell culture media. Steady-state labeling with (35)S-Cys demonstrated incomplete secretion of the mutant collagen in cell culture, particularly molecules containing one mutant chain. Pulse and pulse-chase experiments revealed slower secretion of the latter. An enlargement of endoplasmic reticulum in skin fibroblasts from Brtl IV mice, clearly visible by electron microscopy, supported the abnormal secretion identified by biochemical studies. We observed increased susceptibility of molecules with one mutant chain to proteolytic degradation in vitro, but we did not detect significant selective degradation in cell culture media. Mutant collagen molecules incorporated from the media into newly deposited fibers and into fully crosslinked and mature matrix in the same ratio as they were secreted. Specific labeling of reactive -SH demonstrated that about half of the Cys349-SH groups in the mutant molecules were exposed and potentially available for aberrant interactions with other molecules inside or outside the cells. Based on these and our previous findings, we argue that the outcome in Brtl IV may be significantly affected by cellular stress and malfunction caused by the retention and degradation of newly synthesized mutant collagen.     

High-level production of Serratia proteamaculans metalloprotease using a recombinant ABC protein exporter-mediated secretion system in Pseudomonas fluorescens

Serratia proteamaculans metalloprotease (SPP) was successfully secreted by a heterologous ABC protein exporter, the Pseudomonas fluorescens TliDEF, in recombinant host strains. Escherichia coli and P. fluorescens cells containing the SPP-encoding gene showed the extracellular protease activity only when the TliDEF-encoding gene cluster was coexpressed. Recombinant P. fluorescens produced an approximately 34.8-fold higher amount of extracellular SPP than did E. coli. The use of a more nutrient-rich medium and controlled dissolved oxygen conditions was effective in increasing SPP secretion in P. fluorescens batch fermentation (an 8.7-fold increase from 41.8 U/mL to 365.2 U/mL). Therefore, SPP, which could not be secreted without an ABC protein exporter, was produced in large quantities by applying the heterologous TliDEF exporter in P. fluorescens. The results also suggest that the use of the ABC protein exporter in P. fluorescens could be an efficient production platform for an industrially promising type I secretion pathway-dependent enzyme.     

YKL-40 secreted from adipose tissue inhibits degradation of type I collagen

Obesity is considered a chronic low-grade inflammatory status and the stromal vascular fraction (SVF) cells of adipose tissue (AT) are considered a source of inflammation-related molecules. We identified YKL-40 as a major protein secreted from SVF cells in human visceral AT. YKL-40 expression levels in SVF cells from visceral AT were higher than in those from subcutaneous AT. Immunofluorescence staining revealed that YKL-40 was exclusively expressed in macrophages among SVF cells. YKL-40 purified from SVF cells inhibited the degradation of type I collagen, a major extracellular matrix of AT, by matrix metalloproteinase (MMP)-1 and increased rate of fibril formation of type I collagen. The expression of MMP-1 in preadipocytes and macrophages was enhanced by interaction between these cells. These results suggest that macrophage/preadipocyte interaction enhances degradation of type I collagen in AT, meanwhile, YKL-40 secreted from macrophages infiltrating into AT inhibits the type I collagen degradation.     

Purification and phosphorylation of the effector protein NopL from Rhizobium sp. NGR234

Bacterial pathogens use type III secretion systems (TTSSs) to deliver virulence factors into eukaryotic cells. These effectors perturb host-defence responses, especially signal transduction pathways. A functional TTSS was identified in the symbiotic, nitrogen-fixing bacterium Rhizobium sp. NGR234. NopL (formerly y4xL) of NGR234 is a putative symbiotic effector that modulates nodulation in legumes. To test whether NopL could interact with plant proteins, in vitro phosphorylation experiments were performed using recombinant nopL protein purified from Escherichia coli as well as protein extracts from Lotus japonicus and tobacco plants. NopL serves as a substrate for plant protein kinases as well as purified protein kinase A. Phosphorylation of NopL was inhibited by the Ser/Thr kinase inhibitor K252a as well as by PD98059, a mitogen-activated protein (MAP) kinase kinase inhibitor. It thus seems likely that, after delivery into the plant cell, NopL modulates MAP kinase pathways.     

Multivariate analyses of carbohydrate data from lipopolysaccharides of Actinobacillus (Haemophilus) actinomycetemcomitans, Haemophilus aphrophilus, and Haemophilus paraphrophilus

The taxonomic distinction between Actinobacillus (Haemophilus) actinomycetemcomitans and Haemophilus aphrophilus and the taxonomic distinction between H. aphrophilus and Haemophilus paraphrophilus have been questioned. This study was done to determine whether multivariate statistical analyses of carbohydrate data from lipopolysaccharides could be used to distinguish between these closely related species. Lipopolysaccharides were extracted with phenol-water and purified. Carbohydrates were assessed by using gas chromatography and gas chromatography-mass spectrometry after methanolysis and derivatization with trifluoroacetic acid anhydride. The lipopolysaccharides from all of the species contained rhamnose, fucose, galactose, glucose, L-glycero-D-mannoheptose, and glucosamine plus galactosamine, but in varying amounts. A. actinomycetemcomitans and H. paraphrophilus also contained D-glycero-D-mannoheptose, while H. aphrophilus did not. Sample- and variable-oriented principal-component analyses of the carbohydrate data clearly distinguished among A. actinomycetemcomitans, H. aphrophilus, and H. paraphrophilus. Soft independent modelling of class analogy showed that no sample in the A. actinomycetemcomitans class fell within the 95% confidence limits of the H. aphrophilus class. H. paraphrophilus fell outside both classes.     

Choline sulfatase from Ensifer (Sinorhizobium) meliloti: Characterization of the unmodified enzyme

Ensifer (Sinorhizobium) meliloti is a nitrogen-fixing α-proteobacterium able to biosynthesize the osmoprotectant glycine betaine from choline sulfate through a metabolic pathway that starts with the enzyme choline-O-sulfatase. This protein seems to be widely distributed in microorganisms and thought to play an important role in their sulfur metabolism. However, only crude extracts with choline sulfatase activity have been studied. In this work, Ensifer (Sinorhizobium) meliloti choline-O-sulfatase was obtained in a high degree of purity after expression in Escherichia coli. Gel filtration and dynamic light scattering experiments showed that the recombinant enzyme exists as a dimer in solution. Using calorimetry, its catalytic activity against its natural substrate, choline-O-sulfate, gave a k_cat=2.7×10^?1 s^?1 and a K_M=11.1 mM. For the synthetic substrates p-nitrophenyl sulfate and methylumbelliferyl sulfate, the k_cat values were 3.5×10^?2 s^?1 and 4.3×10^?2 s^?1, with K_M values of 75.8 and 11.8 mM respectively. The low catalytic activity of the recombinant sulfatase was due to the absence of the formylglycine post-translational modification in its active-site cysteine 54. Nevertheless, unmodified Ensifer (Sinorhizobium) meliloti choline-O-sulfatase is a multiple-turnover enzyme with remarkable catalytic efficiency.     

Purification and characterization of a novel ATP-independent type I DNA topoisomerase from a marine methylotroph

DNA topoisomerase is involved in DNA repair and replication. In this study, a novel ATP-independent 30-kDa type I DNA topoisomerase was purified and characterized from a marine methylotroph, Methylophaga sp. strain 3. The purified enzyme composed of a single polypeptide was active over a broad range of temperature and pH. The enzyme was able to relax only negatively supercoiled DNA. Mg(2+) was required for its relaxation activity, while ATP gave no effect. The enzyme was clearly inhibited by camptothecin, ethidium bromide, and single-stranded DNA, but not by nalidixic acid and etoposide. Interestingly, the purified enzyme showed Mn(2+)-activated endonuclease activity on supercoiled DNA. The N-terminal sequence of the purified enzyme showed no homology with those of other type I enzymes. These results suggest that the purified enzyme is an ATP-independent type I DNA topoisomerase that has, for the first time, been characterized from a marine methylotroph.