P values among experiments were calculated using a two-tailed T-test. prepared to study the functional role of PhtD and PhtE-specific Fabs in blocking adherence. The addition of 1 1 g of IgG Fab from adult sera led to a 34% reduction (p= 0.002) and from children a 20% (p= 0.023) reduction in D562 epithelial cells with adherent pneumococci. In purified IgG from adult sera, the depletion of PhtD and PhtE specific Fab from total IgG Fab resulted in a significant increase in the number of D562 epithelial cells with adherent pneumococci (p=0.005 for PhtD and p=0.024 for PhtE). We conclude that antibody directed to PhtD and PhtE are adhesins of pneumococci, if raised by vaccination, may function to prevent pneumococcal adherence to human airway epithelial cells. == Introduction == Streptococcus pneumoniae(pneumococci) is usually associated with disease in a variety of host sites including septicemia, meningitis, pneumonia, sinusitis and pneumonia, resulting in high levels of morbidity and Capromorelin mortality. Risk groups for disease include young children, the elderly, and those with immunodeficiencies (1). Nasopharyngeal colonization by pneumococci represents the first step in pathogenesis, allowing the potential to seed the blood, brain, lungs, sinuses and middle ear (2). Colonization of the nasopharynx by pneumococci requires adherence of the bacteria to the epithelial cells of the upper respiratory tract and this process is usually mediated by cell wall associated surface proteins of pneumococci such as PsaA, CbpA, PavA, PsrR and others (3,4,5,6). When pneumococci have colonized the nasopharynx, subsequent viral upper respiratory contamination and associated generation of proinflammatory cytokines dramatically upregulate bacterial adherence receptors such as PAF-r and polymeric IgG receptor on host epithelial cells (7,8,9). Since colonization is the initial step in pathogenesis of pneumococcal Capromorelin infections, vaccination to prevent colonization is being sought as a potential strategy to prevent pneumococcal Capromorelin infections. The currently licensed pneumococcal conjugate vaccine has Capromorelin been successful in preventing pneumococcal colonization by strains of the organism expressing capsular polysaccharide specific to the serotypes in the vaccine. However alternative of strains expressing non-vaccine serotypes has occurred followed by the occurrence of disease associated with non-vaccine serotypes (10,11,12). PhtD and PhtE belong to the well-conserved Pht protein family expressed by pneumococci (13). They are surface uncovered proteins characterized mainly by a histidine triad motif. In animal models both of these proteins have been shown to afford protection against sepsis, pneumonia and colonization (14,15,16,17). PhtD and PhtE elicit antibody in children and adults in response Mouse monoclonal to RTN3 to natural contamination (18,19). The function of these proteins has not been explored in great detail; nevertheless, they are considered to play an instrumental role in the pneumococcal pathogenic process (20). The present study was undertaken to better understand the role of PhtD and PhtE in pneumococcal adherence and the ability of human antibody directed to these proteins to prevent adherence to human airway epithelial cells. Since Pht protein family members are a part of a complex operon system, it becomes difficult to assess their adherence attribute by merely comparing the binding of isogenic mutant strains to human epithelial cells (21). We have recently discovered that pneumococci tend to form bacterial aggregates in the Capromorelin presence of serum or purified IgG (Khanet al, under review). Therefore prior research showing function of antibody to adhesins likely was due in whole or in part to an aggregation of the bacteria and not a measure of adhesion blocking by antibodies against specific adhesins. In the view of the difficulties defined above, we have used a circulation cytometry- based approach to define the role of pneumococcal PhtD and PhtE as adhesins. To accomplish our aims, isogenic mutants of wild type TIGR4 pneumococci lacking expression of PhtD and PhtE were constructed, and PhtD and PhtE were expressed on the surface of a heterologousE.