Staphylococcal Protein Secretion and Envelope Assembly

ABSTRACT

The highly cross-linked peptidoglycan represents the rigid layer of the bacterial envelope and protects bacteria from osmotic lysis. In Gram-positive bacteria, peptidoglycan also functions as a scaffold for the immobilization of capsular polysaccharide, wall teichoic acid (WTA), and surface proteins. This chapter captures recent development on the assembly of the envelope of Staphylococcus aureus including mechanisms accounting for immobilization of molecules to peptidoglycan as well as hydrolysis of peptidoglycan for the specific release of bound molecules, facilitation of protein secretion across the envelope and cell division. Peptidoglycan, WTA and capsular polysaccharide are directly synthesized onto undecaprenol. Surface proteins are anchored by Sortase A, a membrane-embedded transpeptidase that scans secreted polypeptides for the C-terminal LPXTG motif of sorting signals. The resulting acyl enzyme intermediate is resolved by lipid II, the undecaprenol-bound peptidoglycan precursor. While these pathways share membrane diffusible undecaprenol, assembly of these molecules occurs either at the cross-walls or the cell poles. In S. aureus, the cross-wall represents the site of de novo peptidoglycan synthesis which is eventually split to complete the cell cycle yielding newly divided daughter cells. Peptidoglycan synthesized at the cross-wall is initially devoid of WTA. Conversely, lipoteichoic acid (LTA) synthesis which does not require bactoprenol is seemingly restricted to septal membranes. Similarly, S. aureus distinguishes two types of surface protein precursors. Polypeptides with canonical signal peptides are deposited at the cell poles, whereas precursors with conserved YSIRK-GXXS motif signal peptides traffic to the cross-wall. A model for protein trafficking in the envelope and uneven distribution of teichoic acids is discussed.

The cell wall envelope of Staphylococcus aureus is a surface organelle with anchored proteins, teichoic acids, and polysaccharides that enable bacteria to promote specific interactions with their environment (1). During colonization and invasion of their mammalian hosts, secreted and cell wall-anchored surface proteins fulfill a wide spectrum of functions, including bacterial adhesion to host cells or tissues, evasion of innate host defenses, and diversions of host adaptive immune responses (2). Secreted proteins trafficking in and out of the cell wall envelope promote both the persistent colonization of mammalian hosts and the pathogenesis of specific disease states that are characteristically associated with S. aureus (2).

CELL WALL SYNTHESIS AND ENVELOPE ASSEMBLY

When analyzed by electron microscopy of thin-sectioned bacteria, staphylococci are spherical cells whose envelope is composed of a plasma membrane and surrounding cell wall layer (20 to 40 nm in diameter) (3). Cell walls are isolated by physical disruption of staphylococci and chemical treatments that remove secondary polymers, including wall teichoic acids (WTAs), polysaccharides, and proteins (4). The remaining structures, designated murein sacculi, retain the spherical shape of the bacteria and are composed of peptidoglycan, including glycan strands with a repeating disaccharide, →4(N-acetylmuramoyl-β-(1→4)-N-acetylglucosaminyl-β-(1→), abbreviated (MurNAc-GlcNAc)_n, and cross-linked wall peptides, l-alanyl-d-iso-glutaminyl-(pentaglycyl)l-lysyl-d-alanyl_n, abbreviated [Ala-iGln-(Gly₅)Lys-Ala]_n (5, 6). Wall peptides are tethered to glycan chains via amide bonds formed by the amino-group of l-alanyl and the carboxyl-group of MurNAc (6, 7). Wall peptides of neighboring peptidoglycan strands are cross-linked by amide bonds formed from the amino group of pentaglycyl and the carboxyl-group of d-alanyl (8). During peptidoglycan synthesis, the bactoprenol-linked intermediate, lipid II [C₅₅-(PO₄)₂-MurNac(-Ala-iGln-(NH₂-Gly₅)Lys-Ala-Ala-COOH)-GlcNAc], is translocated across the staphylococcal plasma membrane (9, 10). Penicillin binding protein 2 polymerizes lipid II substrate to generate glycan strands and cross-linked wall peptides via hydrolysis of its phosphodiester and Ala-Ala bonds (11–13).

WTAs and capsular polysaccharide are also synthesized as bactoprenol-linked intermediates (14, 15). Once translocated across the membrane, WTAs and capsular polysaccharides are anchored to the peptidoglycan scaffold (16, 17). Staphylococci replicate by forming cell division septa at midcell and by synthesizing peptidoglycan in the cross-wall compartment, which is bounded by the septal membranes of newly dividing staphylococci (3, 18). Once completed, cross-wall peptidoglycan is split to separate the dividing cells (3). Staphylococcal murein sacculi are not permeable for proteins and other large macromolecules. Cell division, assembly of secondary polymers, and the secretion of proteins across the bacterial cell wall requires specific murein hydrolases that modify murein sacculi, thereby enabling protein secretion across the cell wall envelope and the anchoring of secondary cell wall polymers to peptidoglycan (19, 20).

TEICHOIC ACID SYNTHESIS AND ENVELOPE ASSEMBLY

The envelope of S. aureus encompasses both polyribitol-phosphate and polyglycerol-phosphate polymers (21, 22). Polyribitol-phosphate [(Rib-PO₄)_n], also designated WTA, is synthesized in the bacterial cytoplasm from CDP-ribitol and tethered to its bactoprenol-linked carrier, thereby generating lipid III [C₅₅-(PO₄)₂-GlcNAc-ManNAc-(Gro-PO₄)₂-(Rib-PO₄)_11–40] (23, 24). Rib-PO₄ is polymerized via phosphodiester bonds engaging ribitol hydroxyls at O1 and O5; the remaining hydroxyl moieties (O2, O3, and O4) are variably substituted with α- or β-linked GlcNAc (25–27). Following translocation of lipid III across the membrane, LCP enzymes tether WTA to peptidoglycan, generating phosphodiester bonds between the O1 of GlcNAc and the O6 of MurNAc (28). The Rib-PO₄ polymer of anchored WTA is further modified via esterification with d-alanyl (29, 30). Peptidoglycan synthesized at the cross-wall is initially devoid of WTA (31) (Fig. 1). Upon cross-wall splitting and cell separation, cross-wall peptidoglycan is modified with WTA (31). LtaS polymerizes polyglycerol-phosphate [(Gro-PO₄)_n], also designated lipoteichoic acid (LTA), from phosphatidylglycerol, transferring the assembled Gro-PO₄ polymer onto its glycolipid anchor gentiobiosyldiacylglycerol: Glc-β-(1→6)-Glc-β-(1→3)-diacylglycerol [Glc₂-DAG] (32–34). The O2 hydroxyl of Gro-PO₄ is variably substituted with esterified d-alanyl (30, 35). LtaS and LTA synthesis are restricted to septal membranes and are required for staphylococcal cell division and growth (36) (Fig. 1). Thus, assembly sites of WTA and LTA synthesis are unevenly distributed in the envelope of dividing staphylococci.

FIGURE 1.

Schematic illustrating S. aureus cell division, PBP2-mediated synthesis of cross-wall peptidoglycan, LtaS-mediated synthesis of lipoteichoic acid (LTA) in septal membranes, and the trafficking of surface protein precursors with YSIRK-GXXS motif signal peptides (blue circles) to septal membranes. Following translocation, sortase A-mediated cell wall sorting, and incorporation into the cross-wall, the peptidoglycan layer is split and divided cells are separated, exposing cross-wall-incorporated proteins on the bacterial surface. Surface proteins with canonical signal peptides (red circles) are deposited into polar segments of the peptidoglycan layer.

PROTEIN SECRETION

The genome of S. aureus carries more than 1,300 genes with products encompassing predicted N-terminal signal peptides (37). When subjected to proteomic analysis, most of these gene products can be identified in the filtered supernatant of centrifuged S. aureus cultures (38). Genetic and biochemical experiments characterized the Sec secretion pathway in the model organism Escherichia coli (39–41). Elements of this pathway are conserved among all bacteria, including staphylococci (42). Some of the sec genes have also been characterized in S. aureus. Signal peptide-bearing precursors are translocated across the plasma membrane through the Sec translocon, a complex formed from three membrane proteins, SecY, SecE, and SecG (43). Protein translocation requires SecA, a cytoplasmic ATPase that pushes unfolded precursors through the translocon (44, 45). SecDF, a chaperone whose activity is sustained by the proton motive force, assists in the folding of translocated proteins (46, 47). Similar to E. coli, S. aureus secA is absolutely essential for bacterial growth and protein secretion, whereas secDF is not essential, providing auxiliary functions to protein secretion (48–50). Translocated precursors are cleaved by signal peptidase I, LepB in E. coli (51). S. aureus carries two signal peptidase I genes, spsA and spsB (52). SpsB, but not SpsA (it lacks a critical catalytic residue), can substitute for E. coli LepB, and expression of spsB is required for staphylococcal growth and the cleavage of secreted precursors in S. aureus (52). Suppressor mutations that induce expression of a putative ABC transporter (ABC1-3) can restore both growth and precursor secretion in S. aureus spsB mutants (53, 54). A physiological role has not yet been assigned to the putative ABC1-3 transporter. SagB glucosaminidase cleaves staphylococcal peptidoglycan to generate short glycan strands with variable lengths (MurNAc-GlcNAc)_3–10. S. aureus sagB mutants exhibit diminished growth, cross-wall separation defects during cell division, and reduced secretion of proteins into the extracellular medium (19). Thus, the generation of short glycan strands is a prerequisite for the transport of protein across the cell wall peptidoglycan. Three other murein hydrolases—autolysin (Atl amidase and glucosaminidase), Sle1, and LytN (CHAP domain amidases and endopeptidases)—contribute to cell division, cross-wall separation, and likely also to protein secretion across the staphylococcal cell wall envelope (55–59).

PROTEIN ANCHORING TO THE CELL WALL

Surface proteins of staphylococci are covalently linked to peptidoglycan (60). Following translocation of signal peptide-bearing precursors across the plasma membrane, the C-terminal end of surface proteins is cleaved between the threonyl (T) and the glycyl (G) of their LPXTG motif sorting signal by sortase A, which generates a thioester-linked acyl-enzyme intermediate (61–64) (Fig. 2). Nucleophilic attack of the peptidoglycan biosynthetic intermediate, i.e., of the amino-group of pentaglycyl within lipid II, resolves the sortase A acyl-enzyme, synthesizing an amide linkage between the carboxyl group of the C-terminal threonyl of surface proteins and the amino-group of pentaglycyl (65, 66). The surface protein-lipid II intermediate is subsequently incorporated into the peptidoglycan via penicillin binding protein 2-catalyzed transglycosylation and transpeptidation reactions (67) (Fig. 2). Sortase A and surface protein anchoring to the cell wall envelope occur both within the cross-wall compartment and within polar peptidoglycan (68, 69) (Fig. 1). Genome sequences of thousands of isolates reveal between 18 and 23 genes for sortase A-anchored surface proteins in S. aureus (70).

FIGURE 2.

Surface protein anchoring to the cell wall of S. aureus. (A) Primary structure of the precursor for staphylococcal protein A (SpA) with an N-terminal signal peptide, signal peptidase (SpsB) cleavage site, five immunoglobulin binding domains (IgBDs), region X (Xr), LysM domain, and C-terminal LPXTG-motif sorting signal. Cell wall-anchored SpA is tethered to the envelope via an amide bond between the carboxyl-group of its C-terminal threonyl (T in the LPXTG motif) and the amino group of the pentaglycine cross-bridge within peptidoglycan. Released nSpA encompasses murein tetrapeptide-tetraglycyl [l-Ala-d-iGln-(SpA-Gly₅)l-Lys-d-Ala-Gly₄] linked to the C-terminal threonyl of the surface protein. (B) Cell wall sorting pathway of surface proteins in S. aureus. Following Sec-mediated translocation of surface protein precursors across the membrane, sortase A cleaves the LPXTG-motif of the C-terminal sorting signal between the threonyl (T) and the glycyl (G) residues and forms a thioester bond between its active site cysteinyl and the carboxyl-group of the C-terminal threonyl. The sortase acyl-enzyme intermediate is relieved by the nucleophilic attack of lipid II, thereby generating surface protein linked to peptidoglycan precursor, which is subsequently incorporated by penicillin binding protein 2 into the cell wall peptidoglycan. Through the action of murein hydrolases, surface protein is released from the cell wall into the extracellular milieu.

PROTEIN SECRETION INTO THE CROSS-WALL COMPARTMENT

Thirteen S. aureus surface protein genes encode precursors with YSIRK-GXXS signal peptides, and their products are secreted across septal membranes into the cross-wall compartment (69, 71). Septal secretion promotes abundant deposition and broad distribution of these proteins over the staphylococcal surface (68). In contrast, surface proteins secreted via canonical signal peptides are deposited with low abundance into polar peptidoglycan (69). In addition to sortase A-anchored surface proteins, four other staphylococcal precursors carry YSIRK-GXXS signal peptides: the LytN murein hydrolase (55), the giant protein Ebh (72), and two glycerol-ester hydrolases, GehA and GehB (73). Septal secretion of LytN into the cross-wall promotes cleavage of peptidoglycan and the separation of cells that have completed the division process (55). The 10,421-residue Ebh precursor comprises an N-terminal YSIRK-GXXS signal peptide, a hyperosmolarity resistance domain (residues 179 to 2530), 7 repeats of the 54-residue FIVAR domain (possibly associated with polysaccharide binding), 50 repeats of the 123-residue FIVAR-GA domain, 7 repeats of DUF1542 (a 72-residue domain of unknown function), a putative SMC domain (structural maintenance of chromosomes; residues 8976 to 9576), a transmembrane domain (residues 10227 to 10247), and a positively charged cytoplasmic domain (72). Mutations that abolish ebh expression increase staphylococcal cell size and perturb the integrity of the cell wall envelope, suggesting that Ebh is a size determinant for the staphylococcal murein sacculus (72). GehA and GehB are secreted as 72-kDa pro-proteins with 250-residue N-terminal domains that are thought to assist in the folding of C-terminal glycerol-ester hydrolase domains (74). GehA cleaves the ester bonds of short-chain fatty acids within acyl-glycerol, whereas GehB hydrolyzes preferentially long-chain fatty acid esters (75–77). Following cell division, GehB pro-protein is released into the extracellular medium and cleaved by aureolysin, a cysteine protease that is secreted via the canonical Sec pathway (75). A specific role for GehA and GehB during staphylococcal cell division has thus far not been revealed.

During secretion, the precursor of SpA (staphylococcal protein A) is cleaved at Gly¹³, i.e., between the conserved (underlined) elements of its YSIRKLG¹³VGIAS motif (50). After translocation across the plasma membrane, SpsB signal peptidase cleaves SpA at Ala³⁷, thereby removing the N-terminal hydrophobic signal peptide (78). Translocated SpA is retained within the secretory pathway by its C-terminal sorting signal and is subsequently anchored to the cell wall envelope (79). Amino acid substitutions in the YSIRK-GXXS motif perturb both the proteolysis and the secretion of SpA precursors, suggesting that cleavage of the YSIRK-GXXS motif is a prerequisite for septal secretion (50). SecA ATPase and SecDF foldase promote the secretion of precursor proteins at septal and polar membranes (50). In contrast, LtaS and LTA synthesis are required for septal, but not polar, secretion in S. aureus (50). Thus, synthesis of LTA within septal membranes represents a unique trait of staphylococcal biology that directs precursors with YSIRK-GXXS signal peptides into the cross-wall compartment (Fig. 1). It is not yet clear, however, whether the abundance of LTA, the depletion of phosphatidylglycerol, i.e., the lipid substrate of LtaS, or the accumulation of di-acylglycerol, i.e., the byproduct of LTA synthesis, make specific contributions toward septal secretion. It is also conceivable that septal secretion of GehA and GehB may contribute to the specific features of septal membranes that support trafficking of YSIRK-GXXS precursors into the cross-wall compartment.

RELEASE OF SURFACE PROTEINS FROM THE STAPHYLOCOCCAL ENVELOPE

During staphylococcal growth, cell wall-anchored surface proteins are released from the staphylococcal envelope into the extracellular milieu, where they fulfill specific functions (80). For example, release of SpA requires LytN CHAP amidase and LytM endopeptidase, releasing surface protein with murein tetrapeptide-tetraglycyl [l-Ala-d-iGln-(SpA-Gly₅)l-Lys-d-Ala-Gly₄] linked to the C-terminal threonyl (81) (Fig. 2). Surface proteins of staphylococci fulfill complex biological functions both on the bacterial surface and following their release from the peptidoglycan scaffold by murein hydrolases (82).

STAPHYLOCOCCAL PROTEIN A

All clinical isolates of S. aureus harbor the spa gene for staphylococcal protein A (SpA), which generates a precursor product composed of an N-terminal YSIRK/GXXS signal peptide followed by 4 or 5 immunoglobulin-binding domains (IgBDs), region X repeats (Xr), an LysM domain, and an LPXTG sorting signal (70, 83, 84). Following septal secretion and anchoring to cross-wall peptidoglycan, SpA is displayed on the bacterial surface (68). The IgBDs of SpA bind to the Fcγ domain of human and animal IgG, effectively blocking effector functions of antibodies, i.e., the binding of C1q complement or the engagement of Fc receptors, thereby inhibiting opsonophagocytic killing of staphylococci (85–87). SpA released from the bacterial envelope crosslinks V_H3 idiotype IgM B cell receptors and promotes the nonproductive proliferation of B cells and secretion of V_H3 idiotype antibodies that do not recognize staphylococcal antigens (88, 89). This B cell superantigen activity requires endocytosis of SpA crosslinked to B cell receptors, signaling via RIPK2 kinase, and CD4⁺ T helper cell functions (90). The B cell superantigen activity of SpA effectively blocks host adaptive immune responses and the establishment of protective immunity against S. aureus (85).

SURFACE PROTEINS AND HEME-IRON TRANSPORT

S. aureus encodes a second sortase gene, designated sortase B (srtB) (91). Sortase B cleaves the NPQTN-motif sorting signal of IsdC (iron-regulated surface determinant C) (92). Unlike sortase A, the sortase B acyl-enzyme intermediate is resolved by nucleophilic attack of the amino group of pentaglycyl within polymerized peptidoglycan, thereby anchoring of IsdC to peptidoglycan in the vicinity of the staphylococcal membrane (92). The sortase B anchoring mechanism enables S. aureus surface proteins to promote heme-iron scavenging from host hemoglobin and haptoglobin (93, 94). Initially, sortase A-anchored IsdB and IsdH retrieve heme-iron from host hemoproteins (95, 96). IsdA, another sortase A-anchored surface protein, captures the heme-iron from IsdB and IsdH for passage of the nutrient across the staphylococcal cell wall envelope and transfer to IsdC (97). IsdE lipoprotein and IsdF transporter are thought to capture heme-iron from IsdC for import into the bacterial cytoplasm (97, 98). IsdG and IsdI cleave the tetrapyrrol ring of heme to generate staphylobilin and iron for the assembly of iron-sulfur cluster proteins (99, 100). NEAT domains (near-iron transporter domains about 120 residues in length) promote the heme-iron binding and transfer reactions and are found in variable numbers within IsdA, IsdB, IsdC, IsdE, and IsdH (93, 101, 102).