The ß-prism lectin domain of Vibrio cholerae hemolysin promotes self-assembly of the ß-pore-forming toxin by a carbohydrate-independent mechanism.

Vibrio cholerae cytolysin/hemolysin (VCC) is an amphipathic 65-kDa ß-pore-forming toxin with a C-terminal ß-prism lectin domain. Because deletion or point mutation of the lectin domain seriously compromises hemolytic activity, it is thought that carbohydrate-dependent interactions play a critical role in membrane targeting of VCC. To delineate the contributions of the cytolysin and lectin domains in pore formation, we used wild-type VCC, 50-kDa VCC (VCC(50)) without the lectin domain, and mutant VCC(D617A) with no carbohydrate-binding activity. VCC and its two variants with no carbohydrate-binding activity moved to the erythrocyte stroma with apparent association constants on the order of 10(7) M(-1). However, loss of the lectin domain severely reduced the efficiency of self-association of the VCC monomer with the ß-barrel heptamer in the synthetic lipid bilayer from ∼83 to 27%. Notably, inactivation of the carbohydrate-binding activity by the D617A mutation marginally reduced oligomerization to ∼77%. Oligomerization of VCC(50) was temperature-insensitive; by contrast, VCC self-assembly increased with increasing temperature, suggesting that the process is driven by entropy and opposed by enthalpy. Asialofetuin, the ß1-galactosyl-terminated glycoprotein inhibitor of VCC-induced hemolysis, promoted oligomerization of 65-kDa VCC to a species that resembled the membrane-inserted heptamer in stoichiometry and morphology but had reduced global amphipathicity. In conclusion, we propose (i) that the ß-prism lectin domain facilitated toxin assembly by producing entropy during relocation in the heptamer and (ii) that glycoconjugates inhibited VCC by promoting its assembly to a water-soluble, less amphipathic oligomer variant with reduced ability to penetrate the bilayer.

Structure of the Portal Complex from Staphylococcus aureus Pathogenicity Island 1 Transducing Particles In Situ and In Isolation.

Staphylococcus aureus is an important human pathogen, and the prevalence of antibiotic resistance is a major public health concern. The evolution of pathogenicity and resistance in S. aureus often involves acquisition of mobile genetic elements (MGEs). Bacteriophages play an especially important role, since transduction represents the main mechanism for horizontal gene transfer. S. aureus pathogenicity islands (SaPIs), including SaPI1, are MGEs that carry genes encoding virulence factors, and are mobilized at high frequency through interactions with specific "helper" bacteriophages, such as 80α, leading to packaging of the SaPI genomes into virions made from structural proteins supplied by the helper. Among these structural proteins is the portal protein, which forms a ring-like portal at a fivefold vertex of the capsid, through which the DNA is packaged during virion assembly and ejected upon infection of the host. We have used high-resolution cryo-electron microscopy to determine structures of the S. aureus bacteriophage 80α portal itself, produced by overexpression, and in situ in the empty and full SaPI1 virions, and show how the portal interacts with the capsid. These structures provide a basis for understanding portal and capsid assembly and the conformational changes that occur upon DNA packaging and ejection.

Structure of the portal complex from Staphylococcus aureus pathogenicity island 1 transducing particles in situ and in solution.

Staphylococcus aureus is an important human pathogen, and the prevalence of antibiotic resistance is a major public health concern. The evolution of pathogenicity and resistance in S. aureus often involves acquisition of mobile genetic elements (MGEs). Bacteriophages play an especially important role, since transduction represents the main mechanism for horizontal gene transfer. S. aureus pathogenicity islands (SaPIs), including SaPI1, are MGEs that carry genes encoding virulence factors, and are mobilized at high frequency through interactions with specific "helper" bacteriophages, such as 80α, leading to packaging of the SaPI genomes into virions made from structural proteins supplied by the helper. Among these structural proteins is the portal protein, which forms a ring-like portal at a fivefold vertex of the capsid, through which the DNA is packaged during virion assembly and ejected upon infection of the host. We have used high-resolution cryo-electron microscopy to determine structures of the S. aureus bacteriophage 80α portal in solution and in situ in the empty and full SaPI1 virions, and show how the portal interacts with the capsid. These structures provide a basis for understanding portal and capsid assembly and the conformational changes that occur upon DNA packaging and ejection.

Rheumatoid Arthritis-Associated Mechanisms of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans.

Rheumatoid arthritis (RA) is an autoimmune disease of unknown etiology characterized by immune-mediated damage of synovial joints and antibodies to citrullinated antigens. Periodontal disease, a bacterial-induced inflammatory disease of the periodontium, is commonly observed in RA and has implicated periodontal pathogens as potential triggers of the disease. In particular, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans have gained interest as microbial candidates involved in RA pathogenesis by inducing the production of citrullinated antigens. Here, we will discuss the clinical and mechanistic evidence surrounding the role of these periodontal bacteria in RA pathogenesis, which highlights a key area for the treatment and preventive interventions in RA.

Rheumatoid Arthritis-Associated Autoimmunity Due to Aggregatibacter actinomycetemcomitans and Its Resolution With Antibiotic Therapy.

Background:Aggregatibacter actinomycetemcomitans (Aa) is a Gram-negative coccobacillus recognized as a pathogen in periodontitis and infective endocarditis. By producing a toxin (leukotoxin A, LtxA) that triggers global hypercitrullination in neutrophils, Aa has been recently linked to rheumatoid arthritis (RA) pathogenesis. Although mechanistic and clinical association studies implicate Aa infection in the initiation of autoimmunity in RA, direct evidence in humans is lacking. Case:We describe a 59-year-old man with anti-citrullinated protein antibody (ACPA)-positive RA who presented for evaluation of refractory disease. He was found to have Aa endocarditis. Following antibiotic treatment, joint symptoms resolved and ACPAs normalized. Given the implications for RA immunopathogenesis, we further investigated the bacterial, genetic and immune factors that may have contributed to the patient's clinical and autoimmune phenotypes. Methods:DNA was extracted from serum and used to amplify the Aa leukotoxin (ltx) promoter region by PCR, which was further analyzed by Sanger sequencing. High-resolution identification of HLA alleles was performed by sequenced based typing (SBT). TNF-α, IFN-γ, GM-CSF, IL-1ß, IL-6, IL-8, IL-17A, IL-18, IL-21, and IL-22 were quantified in serum by a multiplex immunoassay. IgG and IgA antibodies to Aa LtxA were assayed by ELISA. Results:Aa genotyping confirmed infection with a highly leukotoxic strain carrying a 530-bp ltx promoter deletion, shown to result in 10- to 20-fold higher bacterial expression of LtxA. Immuno-phenotyping showed high anti-LtxA antibodies, elevated cytokines implicated in RA pathogenesis (Th1/Th17), and specific host susceptibility conferred by three HLA alleles strongly linked to ACPAs and RA (DRB1*04:04, DRB1*15:01, and DPB1*04:01). One year after eradication of Aa, the patient remained free of arthritis and anti-CCP antibodies. Conclusion: In the context of genetic risk for RA, systemic subacute infection with a leukotoxic strain of Aa can drive ACPA production and a clinical phenotype similar to RA.

Vibrio cholerae hemolysin: The ß-trefoil domain is required for folding to the native conformation.

Vibrio cholerae cytolysin/hemolysin (VCC) is a 65 kDa ß-pore-forming toxin causing lysis and death of eukaryotic cells. Apart from the core cytolysin domain, VCC has two lectin domains with ß-trefoil and ß-prism folds. The ß-prism domain binds to cell surface carbohydrate receptors; the role of the ß-trefoil domain is unknown. Here, we show that the pro-VCC mutant without the ß-trefoil domain formed aggregates highly susceptible to proteolysis, suggesting lack of a properly folded compact structure. The VCC variants with Trp532Ala or Trp534Ala mutation in the ß-trefoil domain formed hemolytically inactive, protease-resistant, ring-shaped SDS-labile oligomers with diameters of ~19 nm. The Trp mutation induced a dramatic change in the global conformation of VCC, as indicated by: (a) the change in surface polarity from hydrophobic to hydrophilic; (b) movement of core Trp residues to the protein-water interface; and (c) decrease in reactivity to the anti-VCC antibody by >100-fold. In fact, the mutant VCC had little similarity to the wild toxin. However, the association constant for the carbohydrate-dependent interaction mediated by the ß-prism domain decreased marginally from ~3×108 to ~5×107 M-1. We interpret the observations by proposing: (a) the ß-trefoil domain is critical to the folding of the cytolysin domain to its active conformation; (b) the ß-prism domain is an autonomous folding unit.