ABSTRACT
SpuA is a large multimodular cell wall-attached enzyme involved in the degradation of glycogen by the pathogenic bacterium Streptococcus pneumoniae. The deletion of the gene encoding SpuA from the bacterium resulted in a strain with reduced competitiveness in a mouse model of virulence relative to the parent strain, linking the degradation of host-glycogen to the virulence of the bacterium. Through the combined use of X-ray crystallography, small-angle X-ray scattering, and inhibitor binding, the molecular features involved in substrate recognition by this complex protein are revealed. This uniquely illustrates the complexity of the active site, the conformational changes incurred during carbohydrate binding by this protein, and the interaction and cooperation of its composite modules during this process. New insight into the function of this particular pneumococcal virulence factor is provided along with substantial contributions to the nascent framework for understanding the structural and functional interplay between modules in multimodular carbohydrate-active enzymes.
Subject(s)
Bacterial Proteins/chemistry , Glycogen , Glycoside Hydrolases/chemistry , Multiprotein Complexes/chemistry , Pneumococcal Infections/microbiology , Recombinant Proteins/chemistry , Streptococcus pneumoniae , Virulence Factors/chemistry , Animals , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , Cell Line, Tumor , Cell Wall/chemistry , Cell Wall/metabolism , Crystallography, X-Ray , Glycogen/metabolism , Glycoside Hydrolases/genetics , Glycoside Hydrolases/metabolism , Humans , Lung/microbiology , Mice , Mice, Inbred Strains , Models, Molecular , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Pneumococcal Infections/pathology , Protein Binding , Protein Conformation , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Scattering, Small Angle , Streptococcus pneumoniae/chemistry , Streptococcus pneumoniae/metabolism , Streptococcus pneumoniae/pathogenicity , Virulence , Virulence Factors/genetics , Virulence Factors/metabolismABSTRACT
BACKGROUND: Chronic myocarditis is often initiated by viral infection, the most common of which is coxsackievirus infection. The precise mechanism by which viral infection leads to chronic autoimmune pathology is poorly understood, however it is clear that the early immune response plays a critical role. Previous results have shown that the inflammatory cytokine interleukin (IL)-6 is integral to the development of experimental-induced autoimmune myocarditis. However, the function of IL-6 during viral-mediated autoimmunity has yet to be elucidated. METHODS AND RESULTS: To address the requirement of IL-6 during disease induction, IL-6 deficient mice were infected with coxsackievirus B3 (CB3). Following infection, mice lacking IL-6 developed increased chronic autoimmune disease pathology compared to wild type controls without a corresponding change in the level of viral replication in the heart. This increase in disease severity was accompanied by elevated levels of TNF-alpha, MCP-1, IL-10, activated T cells and cardiac infiltrating macrophage/monocytes. Injection of recombinant IL-6 early following infection in the IL-6 deficient mice was sufficient to lower the serum cytokines TNF-alpha and IL-10 as well as the serum chemokines MCP-1, MIP-1beta, RANTES and MIG with a corresponding decrease in the chronic disease pathology strongly suggests an important regulatory role for IL-6 during the early response. CONCLUSIONS: While IL-6 plays a pathogenic role in experimental-induced autoimmune disease, its function following viral-induced autoimmunity is not reprised. By regulating the early immune response and thereby controlling the severity of chronic disease, IL-6 directs the outcome of chronic autoimmune myocarditis.
Subject(s)
Autoimmune Diseases/physiopathology , Coxsackievirus Infections/immunology , Interleukin-6/physiology , Myocarditis/physiopathology , Animals , Autoimmune Diseases/immunology , Chronic Disease , Coxsackievirus Infections/virology , Enterovirus B, Human/physiology , Interleukin-6/administration & dosage , Interleukin-6/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Myocarditis/immunology , Recombinant Proteins/administration & dosage , Severity of Illness Index , Virus ReplicationABSTRACT
The presence of a fucose utilization operon in the Streptococcus pneumoniae genome and its established importance in virulence indicates a reliance of this bacterium on the harvesting of host fucose-containing glycans. The identities of these glycans, however, and how they are harvested is presently unknown. The biochemical and high resolution x-ray crystallographic analysis of two family 98 glycoside hydrolases (GH98s) from distinctive forms of the fucose utilization operon that originate from different S. pneumoniae strains reveal that one enzyme, the predominant type among pneumococcal isolates, has a unique endo-beta-galactosidase activity on the LewisY antigen. Altered active site topography in the other species of GH98 enzyme tune its endo-beta-galactosidase activity to the blood group A and B antigens. Despite their different specificities, these enzymes, and by extension all family 98 glycoside hydrolases, use an inverting catalytic mechanism. Many bacterial and viral pathogens exploit host carbohydrate antigens for adherence as a precursor to colonization or infection. However, this is the first evidence of bacterial endoglycosidase enzymes that are known to play a role in virulence and are specific for distinct host carbohydrate antigens. The strain-specific distribution of two distinct types of GH98 enzymes further suggests that S. pneumoniae strains may specialize to exploit host-specific antigens that vary from host to host, a factor that may feature in whether a strain is capable of colonizing a host or establishing an invasive infection.
