Alla Volgina

Characterization of point mutations in the cdtA gene of the cytolethal distending toxin of Actinobacillus actinomycetemcomitans

The Cdt is a family of gram‐negative bacterial toxins that typically arrest eukaryotic cells in the G0/G1 or G2/M phase of the cell cycle. The toxin is a heterotrimer composed of the cdtA, cdtB and cdtC gene products. Although it has been shown that the CdtA protein subunit binds to cells in culture and in an enzyme‐linked immunosorbent assay (CELISA) the precise mechanisms by which CdtA interacts with CdtB and CdtC has not yet been clarified. In this study we employed a random mutagenesis strategy to construct a library of point mutations in cdtA to assess the contribution of individual amino acids to binding activity and to the ability of the subunit to form biologically active holotoxin. Single unique amino acid substitutions in seven CdtA mutants resulted in reduced binding of the purified recombinant protein to Chinese hamster ovary cells and loss of binding to the fucose‐containing glycoprotein, thyroglobulin. These mutations clustered at the 5′‐ and 3′‐ends of the cdtA gene resulting in amino acid substitutions that resided outside of the aromatic patch region and a conserved region in CdtA homologues. Three of the amino acid substitutions, at positions S165N (mutA81), T41A (mutA121) and C178W (mutA221) resulted in gene products that formed holotoxin complexes that exhibited a 60% reduction (mutA81) or loss (mutA121, mutA221) of proliferation inhibition. A similar pattern was observed when these mutant holotoxins were tested for their ability to induce cell cycle arrest and to convert supercoiled DNA to relaxed and linear forms in vitro. The mutations in mutA81 and mutA221 disrupted holotoxin formation. The positions of the amino acid substitutions were mapped in the Haemophilus ducreyi Cdt crystal structure providing some insight into structure and function.

Localization of Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Subunits during Intoxication of Live Cells

ABSTRACT The cytolethal distending toxin (Cdt), produced by some clinically important Gram-negative bacterial species, is related to the family of AB-type toxins. Three heterologous proteins (CdtA, CdtB, and CdtC) and a genotoxin mode of action distinguish the Cdt from others in this toxin class. Crystal structures of several species-specific Cdts have provided a basis for predicting subunit interactions and functions. In addition, empirical studies have yielded significant insights into the in vivo interactions of the Cdt subunits. However, there are still critical gaps in information about the intoxication process. In this study, a novel protein tagging technology was used to localize the subunits in Chinese hamster ovary cells (CHO-K1). A tetracysteine motif was engineered in each subunit, and in subunits with mutations in predicted functional domains, to permit detection with the fluorescein arsenical hairpin binding (FlAsH) dye Lumio green. Live-cell imaging, in conjunction with confocal microscopy, was used to capture the locations of the individual subunits in cells intoxicated, under various conditions, with hybrid heterotrimers. Using this approach, we observed the following. (i) The CdtA subunit remains on the cell surface of CHO cells in association with cholesterol-containing and cholesterol-depleted membrane. (ii) The CdtB subunit is exclusively in the cytosol and, after longer exposure times, localizes to the nucleus. (iii) The CdtC subunit is present on the cell surface and, to a greater extent, in the cytosol. These observations suggest that CdtC, but not CdtA, functions as a chaperone for CdtB entry into cells.

Evaluation of the humoral immune response to the cytolethal distending toxin of Aggregatibacter actinomycetemcomitans Y4 in subjects with localized aggressive periodontitis

INTRODUCTION Cytolethal distending toxin (Cdt) is potentially one of several virulence factors of Aggregatibacter actinomycetemcomitans, the prime etiological agent of localized aggressive periodontitis (LAP). Little is known regarding the Cdt-specific antibody response in humans. The current study is a quantitative and qualitative evaluation of the toxin-specific antibody response in a cohort of LAP patients and age-, race- and sex-matched controls. METHODS Ninety-five subjects provided a total of 692 serum samples. Sera were analysed by enzyme-linked immunosorbent assays to determine the titers of antibody against the intact Cdt holotoxin as well as the individual subunit proteins (CdtA, CdtB, and CdtC). Neutralization of growth inhibition mediated by Cdt was evaluated in a modified colony-forming assay using Chinese hamster ovary cells. RESULTS Fourteen of the 95 subjects exhibited significant serum Cdt-binding activity. There were no differences in the percentages of seropositive individuals or in the mean antibody titers between the control and LAP groups. Binding activity was detected against each of the three Cdt subunit proteins in all of the positive samples. Neutralization of Cdt-mediated growth inhibition was detected in samples from all of the seropositive subjects (range 20-75%). CONCLUSIONS Cdt, a recently identified A. actinomycetemcomitans virulence factor, is capable of inducing a neutralizing antibody response indicating that the toxin is produced during natural infection of humans. The failure of a vast majority (20 of 23) of the LAP subjects to mount a significant anti-Cdt response may in part explain their relative susceptibility to the disease.

Statins in lymphangioleiomyomatosis. Simvastatin and atorvastatin induce differential effects on tuberous sclerosis complex 2-null cell growth and signaling.

Mutations of the tumor suppressor genes tuberous sclerosis complex (TSC)1 and TSC2 cause pulmonary lymphangioleiomyomatosis (LAM) and tuberous sclerosis (TS). Current rapamycin-based therapies for TS and LAM have a predominantly cytostatic effect, and disease progression resumes with therapy cessation. Evidence of RhoA GTPase activation in LAM-derived and human TSC2-null cells suggests that 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor statins can be used as potential adjuvant agents. The goal of this study was to determine which statin (simvastatin or atorvastatin) is more effective in suppressing TSC2-null cell growth and signaling. Simvastatin, but not atorvastatin, showed a concentration-dependent (0.5-10 μM) inhibitory effect on mouse TSC2-null and human LAM-derived cell growth. Treatment with 10 μM simvastatin induced dramatic disruption of TSC2-null cell monolayer and cell rounding; in contrast, few changes were observed in cells treated with the same concentration of atorvastatin. Combined treatment of rapamycin with simvastatin but not with atorvastatin showed a synergistic growth-inhibitory effect on TSC2-null cells. Simvastatin, but not atorvastatin, inhibited the activity of prosurvival serine-threonine kinase Akt and induced marked up-regulation of cleaved caspase-3, a marker of cell apoptosis. Simvastatin, but not atorvastatin, also induced concentration-dependent inhibition of p42/p44 Erk and mTORC1. Thus, our data show growth-inhibitory and proapoptotic effects of simvastatin on TSC2-null cells compared with atorvastatin. These findings have translational significance for combinatorial therapeutic strategies of simvastatin to inhibit TSC2-null cell survival in TS and LAM.

Role of aromatic amino acids in receptor binding activity and subunit assembly of the cytolethal distending toxin of Aggregatibacter actinomycetemcomitans.

ABSTRACT The periodontal pathogen Aggregatibacter actinomycetemcomitans produces a cytolethal distending toxin (Cdt) that inhibits the proliferation of oral epithelial cells. Structural models suggest that the CdtA and CdtC subunits of the Cdt heterotrimer form two putative lectin domains with a central groove. A region of CdtA rich in heterocyclic amino acids (aromatic patch) appears to play an important role in receptor recognition. In this study site-specific mutagenesis was used to assess the contributions of aromatic amino acids (tyrosine and phenylalanine) to receptor binding and CdtA-CdtC assembly. Predominant surface-exposed aromatic residues that are adjacent to the aromatic patch region in CdtA or are near the groove located at the junction of CdtA and CdtC were studied. Separately replacing residues Y105, Y140, Y188, and Y189 with alanine in CdtA resulted in differential effects on binding related to residue position within the aromatic region. The data indicate that an extensive receptor binding domain extends from the groove across the entire face of CdtA that is oriented 180° from the CdtB subunit. Replacement of residue Y105 in CdtA and residues Y61 and F141 in CdtC, which are located in or at the periphery of the groove, inhibited toxin assembly. Taken together, these results, along with the lack of an aromatic amino acid-rich region in CdtC similar to that in CdtA, suggest that binding of the heterotoxin to its cell surface receptor is mediated predominantly by the CdtA subunit. These findings are important for developing strategies designed to block the activity of this prominent virulence factor.

Role of Intrachain Disulfides in the Activities of the CdtA and CdtC Subunits of the Cytolethal Distending Toxin of Actinobacillus actinomycetemcomitans

ABSTRACT The cytolethal distending toxin (Cdt) of Actinobacillus actinomycetemcomitans is an atypical A-B-type toxin consisting of a heterotrimer composed of the cdtA, cdtB, and cdtC gene products. The CdtA and CdtC subunits form two heterogeneous ricin-like lectin domains which bind the holotoxin to the target cell. Point mutations were used to study CdtC structure and function. One (mutC216F97C) of eight single-amino-acid replacement mutants identified yielded a gene product that failed to form biologically active holotoxin. Based on the possibility that the F97C mutation destabilized a predicted disulfide, targeted mutagenesis was used to examine the contribution of each of four cysteine residues, in two predicted disulfides (C96/C107 and C135/C149), to CdtC activities. Cysteine replacement mutations in two predicted disulfides (C136/C149 and C178/C197) in CdtA were also characterized. Flow cytometry and CHO cell proliferation assays showed that changing either C96 or C149 in CdtC to alanine abolished the biological activity of holotoxin complexes. However, replacing C107 or C135 in CdtC and any of the four cysteines in CdtA with alanine or serine resulted in only partial or no loss of holotoxin activity. Changes in the biological activities of the mutant holotoxins correlated with altered subunit binding. In contrast to elimination of the B chain of ricin, the elimination of intrachain disulfides in CdtC and CdtA by genetic replacement of cysteines destabilizes these subunit proteins but not to the extent that cytotoxicity is lost. Reduction of the wild-type holotoxin did not affect cytotoxicity, and the reduced form of wild-type CdtA exhibited a statistically significant increase in binding to ligand. A diminished role for intrachain disulfides in stabilizing CdtA and CdtC may have clinical relevance for the A. actinomycetemcomitans Cdt. The cdt gene products secreted by this pathogen assemble and bind to target cells in periodontally involved sites, which are decidedly reduced environments in the human oral cavity.

Functional and structural characterization of chimeras of a bacterial genotoxin and human type I DNAse

Chimeras composed of the cdtB gene of a novel bacterial genotoxin and the human type I DNAse I gene were constructed and their products characterized relative to the biochemical and enzymatic properties of the native proteins. The product of a cdtB/DNAse I chimera formed a heterotrimer with the CdtA and CdtC subunits of the genotoxin, and targeted mutations increased the specific activity of the hybrid protein. Expression of active chimeric gene products established that the CdtB protein is an atypical divalent cation-dependent endonuclease and demonstrated the potential for genetically engineering a new class of therapeutic agent for inhibiting the proliferation of cancer cells.

Urokinase-type plasminogen activator (uPA) is critical for progression of tuberous sclerosis complex 2 (TSC2)-deficient tumors

Lymphangioleiomyomatosis (LAM) is a fatal lung disease associated with germline or somatic inactivating mutations in tuberous sclerosis complex genes (TSC1 or TSC2). LAM is characterized by neoplastic growth of smooth muscle-α-actin–positive cells that destroy lung parenchyma and by the formation of benign renal neoplasms called angiolipomas. The mammalian target of rapamycin complex 1 (mTORC1) inhibitor rapamycin slows progression of these diseases but is not curative and associated with notable toxicity at clinically effective doses, highlighting the need for better understanding LAMs molecular etiology. We report here that LAM lesions and angiomyolipomas overexpress urokinase-type plasminogen activator (uPA). Tsc1−/− and Tsc2−/− mouse embryonic fibroblasts expressed higher uPA levels than their WT counterparts, resulting from the TSC inactivation. Inhibition of uPA expression in Tsc2-null cells reduced the growth and invasiveness and increased susceptibility to apoptosis. However, rapamycin further increased uPA expression in TSC2-null tumor cells and immortalized TSC2-null angiomyolipoma cells, but not in cells with intact TSC. Induction of glucocorticoid receptor signaling or forkhead box (FOXO) 1/3 inhibition abolished the rapamycin-induced uPA expression in TSC-compromised cells. Moreover, rapamycin-enhanced migration of TSC2-null cells was inhibited by the uPA inhibitor UK122, dexamethasone, and a FOXO inhibitor. uPA-knock-out mice developed fewer and smaller TSC2-null lung tumors, and introduction of uPA shRNA in tumor cells or amiloride-induced uPA inhibition reduced tumorigenesis in vivo. These findings suggest that interference with the uPA-dependent pathway, when used along with rapamycin, might attenuate LAM progression and potentially other TSC-related disorders.