Abstract
ABSTRACTVarious gram-negative species sequester host cytokines using outer membrane proteins or surface modulation by sulfated polysaccharides. An outer membrane lipoprotein (BilRI) of the periodontal pathogen Aggregatibacter actinomycetemcomitans binds several cytokines, including interleukin (IL)-8. Because IL-8 is positively charged at physiological pH, we aimed to determine whether IL-8 interacts with negatively charged lipopolysaccharide (LPS). Binding was investigated using electrophoretic mobility shift assays and microwell-based time-resolved fluorometric immunoassay. LPS from each tested strain of A. actinomycetemcomitans (N = 13), Pseudomonas aeruginosa (N = 1) and Escherichia coli (N = 1) bound IL-8. The Kd value of the A. actinomycetemcomitans LPS-IL-8 interaction varied between 1.2–17 μM irrespective of the serotype and the amount of phosphorus in LPS and was significantly lower than that of the BilRI-IL-8 interaction. Moreover, IL-8 interacted with whole A. actinomycetemcomitans cells and outer membrane vesicles. Hence, LPS might be involved in binding of IL-8 to the outer membrane of A. actinomycetemcomitans. This raises an interesting question regarding whether other gram-negative periodontal pathogens use LPS for IL-8 sequestering in vivo.
Highlights
Different gram-negative pathogens, including Pseudomonas aeruginosa, Neisseria meningitidis, Neisseria gonorrhoeae, Yersinia pestis, Escherichia coli and Aggregatibacter actinomycetemcomitans, can sequester host cytokines via either cytokine binding [1,2,3] or uptake [4,5]
The ability of A. actinomycetemcomitans LPS to interact with human cytokines was first tested using recombinant IL-8, IL-1β and interferon (IFN)-γ in electrophoretic mobility shift assay (EMSA)
Because IL-8 is a central chemokine in periodontitis, it was selected for further studies to determine the dissociation constants
Summary
Different gram-negative pathogens, including Pseudomonas aeruginosa, Neisseria meningitidis, Neisseria gonorrhoeae, Yersinia pestis, Escherichia coli and Aggregatibacter actinomycetemcomitans, can sequester host cytokines via either cytokine binding [1,2,3] or uptake [4,5] In this process, bacteria may utilize the outer membrane [1,4,5] and secreted [2] proteins or modify their surface by binding extracellular sulfated polysaccharides such as heparin, which interacts with the host signaling molecules [3]. A. actinomycetemcomitans strains can be divided into seven serotypes, namely, serotypes a through g, and nonserotypes [9,10,11,12,13], which lack the O-antigen Some serotypes, such as serotype b, are often associated with periodontitis and nonoral infections [14,15,16], there is no clear correlation between the virulence and the serotype of A. actinomycetemcomitans
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