Dual defensin strategy for targeting Enterococcus faecalis.

A leading cause of antibiotic-resistant hospital infection (1), Enterococcus faecalis, decorates its surface with proteins that contribute to its ability to colonize and survive. Several proteins, including Esp (2), aggregation substance (3), a collagen adhesin (4), and a pilin (5), correlate with a particular disease or exacerbate the course of infection in amodel system. In PNAS, Kandaswamy et al. (6) describe the targeting by human β-defensin 2 (hBD2) and resulting disorganization of the cellular machinery used by E. faecalis to secrete and anchor these proteins to the cell surface. In 1962, Cole and Hahn (7) showed that in dividing cells, new cell wall was deposited by the ovoid Gram-positive Streptococcus pyogenes exclusively near the growing septum, and not by intercalation into existing cell wall (Fig. 1A). Rather than directly labeling peptidoglycan, however, these investigators used fluorescent antibodies to label surface-localized antiphagocytic M protein, or the group C cell wall carbohydrate. The authors observed that new M protein (or group C carbohydrate) was inserted at or near the developing septum, and spread from there toward the midcell as new cell wall accrued (Fig. 1A). With the appearance of nucleotide sequence data, the steps between translation of a gene product and its occurrence attached to the Gram-positive bacterial surface could begin to be inferred. Schneewind et al. (8) identified a C-terminal sequence motif common to many protein precursors destined for attachment to the Gram-positive cell surface. The C-terminal amino acid sequence motif, LPXTG, together with a positively charged C-terminal tail, were found to be required to efficiently anchor protein A from Staphylococcus aureus to the cell surface. The enzyme that catalyzes this transpeptidation reaction was subsequently identified and termed “sortase” (9). In follow-up work, the laboratories of Lindahl and colleagues (10), Fischetti and colleagues (11), and Schneewind and colleagues (12) separately probed the additional role of an N-terminal secretion signal sequence with the motif YSIRK/GS in directing proteins to the septum of S. pyogenes (10, 11) and S. aureus (12). As shown in Fig. 1B, primary translation products with the YSIRK/ GS motif, such as the M protein precursor of S. pyogenes (10,11), or ClfA of S. aureus (12), are secreted selectively at or near the growing septum. In contrast, streptococcal protein F (SfbI) (10, 11) or S. aureus proteins including SasF (12), all of which lack the motif, are deposited into old cell wall (Fig. 1B). Interestingly, because the YSIRK/GS motif can be mutated (10) or scrambled (12) without affecting the pattern of surface distribution, the specific feature of YSIRK-containing signal peptides responsible for the localization pattern remains obscure. S. aureus mutants defective in expression of a group of proteins containing abortive infectivity (ABI) domains are also aberrant in surface localization of YSIRK-motif proteins (13), but the mechanism for this association remains to be determined. In parallel studies, Rosch and Caparon (14) dissected the process of secretion of the S. pyogenes exotoxin SpeB. During secretion, SpeB localizes to discrete foci near the septum, suggesting that protein secretion occurs Fig. 1. (A) New M protein deposited on the cell wall of M type 19 group A Streptococcus. Preexisting M protein was blocked using unlabeled antibody. After 30-min additional growth, fluorescein-labeled anti-M19 antibody stains the growing chain mainly near the septa of dividing cells. Blue-dashed cartoon lines depict approximate inferred cell boundaries, added for illustration (adapted from ref. 7). (Magnification, 2,100×.) (B) S. pyogenes, grown first in the presence of trypsin and pronase to removed preformed surface proteins, then subcultured for an additional 2 min before fixing and staining to label newly integrated cell wall proteins. Cells were stained with fluorescent antibody to M protein (red) and protein F (green), highlighting the differential initial localization of the YSIRK-signal peptide containing M protein at the septum (adapted from ref. 11). (C ) Transient focal location of sortase (red) at or near the septum (green) at early stages in cell division of S. pyogenes (adapted from ref. 15). In early phases of S. pyogenes growth, sortase largely localizes to an area coincident with the septum. At later stages in cell division, SrtA appears to distribute more evenly around the cell, essentially as found in the accompanying examination of sortase distribution in E. faecalis (6). Author contributions: M.S.G., F.L., and D.V.T. wrote the paper.