Connections between Exoproteome Heterogeneity and Virulence in the Oral Pathogen Aggregatibacter actinomycetemcomitans

Aggregatibacter actinomycetemcomitans is a Gram-negative bacterial pathogen associated with severe periodontitis and nonoral diseases. Clinical isolates of A. actinomycetemcomitans display a rough (R) colony phenotype with strong adherent properties. Upon prolonged culturing, nonadherent strains with a smooth (S) colony phenotype emerge. To date, most virulence studies on A. actinomycetemcomitans have been performed with S strains of A. actinomycetemcomitans, whereas the virulence of clinical R isolates has received relatively little attention. Since the extracellular proteome is the main bacterial reservoir of virulence factors, the present study was aimed at a comparative analysis of this subproteome fraction for a collection of R isolates and derivative S strains, in order to link particular proteins to the virulence of A. actinomycetemcomitans with serotype b. To assess the bacterial virulence, we applied different infection models based on larvae of the greater wax moth Galleria mellonella, a human salivary gland-derived epithelial cell line, and freshly isolated neutrophils from healthy human volunteers. A total number of 351 extracellular A. actinomycetemcomitans proteins was identified by mass spectrometry, with the S strains consistently showing more extracellular proteins than their parental R isolates. A total of 50 known extracellular virulence factors was identified, of which 15 were expressed by all investigated bacteria. Importantly, the comparison of differences in exoproteome composition and virulence highlights critical roles of 10 extracellular proteins in the different infection models. Together, our findings provide novel clues for understanding the virulence of A. actinomycetemcomitans and for development of potential preventive or therapeutic avenues to neutralize this important oral pathogen. IMPORTANCE Periodontitis is one of the most common inflammatory diseases worldwide, causing high morbidity and decreasing the quality of life of millions of people. The bacterial pathogen Aggregatibacter actinomycetemcomitans is strongly associated with aggressive forms of periodontitis. Moreover, it has been implicated in serious nonoral infections, including endocarditis and brain abscesses. Therefore, it is important to investigate how A. actinomycetemcomitans can cause disease. In the present study, we applied a mass spectrometry approach to make an inventory of the virulence factors secreted by different clinical A. actinomycetemcomitans isolates and derivative strains that emerged upon culturing. We subsequently correlated the secreted virulence factors to the pathogenicity of the investigated bacteria in different infection models. The results show that a limited number of extracellular virulence factors of A. actinomycetemcomitans have central roles in pathogenesis, indicating that they could be druggable targets to prevent or treat oral disease

TTT - Cure diagram of an anhydride-cured epoxy system including gelation, vitrification, curing kinetics model and monitoring of the glass transition temperature

The curing reaction of an epoxy resin [diglycidyl ether of bisphenol A (DGEBA)] combined with a methyl-hexahydrophtalic anhydride (MHHPA) hardener and a benzyldimethylamine (BDMA) accelerator was studied over a temperature range of 60140°C to build its isothermal time-temperature-transformation (TTT)cure diagram. This includes gelation and vitrification measurements using rheological measurement techniques, monitoring of the glass transition temperature and the reaction kinetics by differential scanning calorimetry, and determination of the following critical glass transition temperatures: Tg0, GelTg, and Tg.. A new curing kinetics model, based on the Sesta´k-Berggren model, was developed including both chemicaland diffusion-controlled stages of reaction. Then, the TTTcure diagram was built by numerical integration of the kinetics model, and good agreement was obtained by comparison with experimental data. Additionally, for the first time, this model takes into account the incomplete cure, which occurs when the thermosetting system is cured below its ultimate glass transition temperature, leading to a more realistic description of the cure evolution after vitrification