Streptococcus pneumoniae enolase is important for plasminogen binding despite low abundance of enolase protein on the bacterial cell surface

Abstract

Enolase represents one of the anchorless surface proteins of Streptococcus pneumoniae and has previously been identified as a plasminogen-binding protein, endowing this pathogen with host proteolytic activity. In this study the mAb 245,C-6 (IgG1) was produced in a BALB/c mouse after immunizing with a protein fraction from S. pneumoniae. The mAb reacted with recombinant pneumococcal enolase both under non-denaturing and denaturing conditions. The epitope for the mAb was mapped to residues 55 DKSRYGGLG 63 of pneumococcal enolase using a peptide array. By applying the previously reported structure of enolase, this epitope was localized in a surface-exposed loop in each of the monomers of the octameric enolase. Previous immunoelectron microscopic studies, using polyclonal rabbit antibodies against enolase, depicted enolase on the cell surface but did not quantify the amount of surface-exposed enolase on viable pneumococci. Here, flow cytometry revealed no binding of mAb 245,C-6 to viable pneumococci, including TIGR4 and its non-encapsulated isogenic mutant, and only a minor increase of fluorescence intensity was measured when the polyclonal anti-enolase antibodies were used. In contrast, control antibodies recognizing the choline-binding proteins (CBPs) PspA and PspC showed high reactivities. The non-encapsulated TIGR4 did not show increased levels of antibody binding for mAb 245,C-6 or polyclonal anti-enolase antibodies, but revealed increased binding of polyclonal antibodies reacting with PspA or PspC. These results suggest that, compared to other surface-exposed proteins such as CBPs, the amount of enolase under the selected conditions is low. Flow cytometry, however, with FITC-labelled plasminogen demonstrated that the amount of surface-exposed enolase is important for plasminogen binding and, therefore, is also important for pneumococcal pathogenesis. INTRODUCTIONStreptococcus pneumoniae is an important human pathogen causing a variety of diseases including pneumonia, meningitis, sepsis and otitis media. A prerequisite for invasive pneumococcal diseases (infections) is the ability of the bacteria to colonize the mucosal surface and penetrate the mucosal barriers. During these processes binding of bacterial factors such as adhesins are essential for the interactions with receptors on host cells. Among the bacterial factors that mediate direct adherence to human cells are phosphorylcholine (Cundell et al., 1995) and the choline-binding protein PspC, also known as SpsA and CbpA (Brooks-Walter et al., 1999;Hammerschmidt et al., 1997;Rosenow et al., 1997). The PspC protein binds to the secretory component of the polymeric Ig receptor or secretory IgA and this interaction mediates transmigration of pneumococci through mucosal epithelial cells (Elm et al., 2004;Zhang et al., 2000). In addition, PspC and the PspClike protein Hic have been shown to interact with complement factor H (Dave et al., 2004;Janulczyk et al., 2000). Many pathogenic bacteria also produce receptors for Abbreviations: GAPDH, glyceraldehyde-3-phos...

Cytosolic Proteins Contribute to Surface Plasminogen Recruitment ofNeisseria meningitidis

“…However, it is well known from other bacterial pathogens that plasminogen receptors can be present at the cell surface at very low levels, as plasminogen is highly abundant in many body fluids (serum concentrations, ϳ1.6 to 2 mol/liter, roughly corresponding to 100 to 200 mg/liter) (2,12,35,39). The presence of pneumococcal enolase, which has been shown to act as the major plasminogen receptor of S. pneumoniae, at the surfaces of these bacteria has been studied in detail (29). Comparing these data, N. meningitidis plasminogen receptors are present at the meningococcal surface in at least the same quantity as pneumococcal enolase (29).…”

“…The presence of pneumococcal enolase, which has been shown to act as the major plasminogen receptor of S. pneumoniae, at the surfaces of these bacteria has been studied in detail (29). Comparing these data, N. meningitidis plasminogen receptors are present at the meningococcal surface in at least the same quantity as pneumococcal enolase (29). The overall number of enolase, DnaK, and peroxiredoxin molecules was calculated in a quantitative ELISA to be approximately 10,000.…”

Genomic and Surface Proteomic Analysis of the Canine Pathogen Staphylococcus pseudintermedius Reveals Proteins That Mediate Adherence to the Extracellular Matrix

“…The identification of intracellular proteins on the cell surface is not unexpected, and numerous studies of S. aureus have reported a similar phenomenon (10,16,46,53,58). In some cases, alternative functions associated with the cell surface have been described for cytoplasmic proteins, including GAPDH (26,52), enolase (4,23), transcription elongation factors (1,24), and chaperone proteins GroES and DnaK (20,57). In addition, ribosomal proteins are commonly identified on the bacterial cell surface and can exhibit a high degree of immunogenicity for humans (25,48).…”

Localization of PcsB of Streptococcus pneumoniae and Its Differential Expression in Response to Stress

“…Likewise, when strain WU2 was analyzed, 5.68% of cells were positive, and 59.57% of the cells of its nonencapsulated mutant, JD908, were positive. PcsB at the plasma membrane is detectable by flow cytometric analysis, although the capsule and probably to a lesser degree other structures at the surface of the cell impede antibodies from interacting with PcsB, leading to higher detection on nonencapsulated than on encapsulated cells, as has also been seen with PspA and PspC (9).…”