The surface-associated elongation factor Tu is concealed for antibody binding on viable pneumococci and meningococci

Abstract

Proteome analyses revealed that elongation factor-Tu (EF-Tu) is associated with cytoplasmic membranes of Gram-positive bacteria and outer membranes of Gram-negative bacteria. It is still debatable whether EF-Tu is located on the external side or the internal side of the membranes. Here, we have generated two new monoclonal antibodies (mAbs) and polyclonal rabbit antibodies against pneumococcal EF-Tu. These antibodies were used to investigate the amount of surface-exposed EF-Tu on viable bacteria using a flow cytometric analysis. The control antibodies recognizing the pneumococcal surface protein A and phosphorylcholine showed a significant binding to viable pneumococci. In contrast, anti-EF-Tu antibodies did not recognize pneumococcal EF-Tu. However, heat killing of pneumococci lacking capsular polysaccharides resulted in specific antibody binding to EF-Tu and, moreover, increased the exposure of recognized phosphorylcholine epitopes. Similarly, our EF-Tu-specific antibodies did not recognize EF-Tu of viable Neisseria meningitidis. However, pretreatment of meningococci with ethanol resulted in specific antibody binding to EF-Tu on outer membranes. Importantly, these treatments did not destroy the membrane integrity as analysed with control mAbs directed against cytoplasmic proteins. In conclusion, our flow cytrometric assays emphasize the importance of using viable bacteria and not heat-killed or ethanol-treated bacteria for surface-localization experiments of proteins, because these treatments modulate the cytoplasmic and outer membranes of bacteria and the binding results may not reflect the situation under physiological conditions.

Enhanced Adhesion of Campylobacter jejuni to Abiotic Surfaces Is Mediated by Membrane Proteins in Oxygen-Enriched Conditions

“…This was expected as methods used for membrane fractionation do not separate exclusively membrane proteins [37], [38]. In fact, some of the predicted cytoplasmic proteins have already been described in membrane analysis such as the chaperone proteins GroEL, DnaJ, DnaK, [39] or the elongation factor EF-Tu [39]–[43]. Apart from the localization of intrinsic or secreted proteins being well predicted by the PSORTb algorithm due to their specific structure (signal peptide, transmembrane alpha helices, beta-barrel proteins, hydrophobicity, motif), the extrinsic proteins associated with the surface of the membranes could not be so easily predicted.…”

Identification of high immunoreactive proteins fromStreptococcus agalactiaeisolates recognized by human serum antibodies

“…Elongation factor Tu is also present in cytoplasmic fraction of bacterial cells. It is one of the most abundant proteins in prokaryotes, representing even 10% of the total amount of proteins in Escherichia coli (Kolberg et al ., ). High cytoplasmic content of elongation factor raises the question whether detection of this protein in bacterial membrane preparations might represent a contamination by cytosolic one during their preparation.…”

“…We found four immunoreactive proteins present in all 60 strains tested: enolase, dehydrogenase, trigger factor, and elongation factor Tu. All of these proteins are cytoplasmic protein and belong to cell surface‐associated or surface‐exposed proteins (Pancholi & Fischetti, ; Severin et al ., ; Kolberg et al ., ; Feng et al ., ; Sharma et al ., ).…”

Mutation of PEB4 alters the outer membrane protein profile ofCampylobacter jejuni

“…CmeC is the outer membrane component of the CmeABC efflux pump, which is responsible for the intrinsic resistance of Campylobacter to a variety of antimicrobial agents (Lin et al ., 2002). Although Ef‐Tu is primarily cytoplasmic and involved in protein translation, it has been identified in the outer membrane of several bacterial species, including E. coli and Mycoplasma pneumoniae (Berrier et al ., 2000; Pancholi & Chhatwal, 2003; Prokhorova et al ., 2006; Kolberg et al ., 2008). In our previous C. jejuni whole‐cell proteomic study, Ef‐Tu was less abundant overall in the cj0596 mutant (Rathbun et al ., 2009).…”