SUMMARY The cell wall envelopes of gram-positive bacteria represent a surface organelle that not only functions as a cytoskeletal element but also promotes interactions between bacteria and their environment. Cell wall peptidoglycan is covalently and noncovalently decorated with teichoic acids, polysaccharides, and proteins. The sum of these molecular decorations provides bacterial envelopes with species- and strain-specific properties that are ultimately responsible for bacterial virulence, interactions with host immune systems, and the development of disease symptoms or successful outcomes of infections. Surface proteins typically carry two topogenic sequences, i.e., N-terminal signal peptides and C-terminal sorting signals. Sortases catalyze a transpeptidation reaction by first cleaving a surface protein substrate at the cell wall sorting signal. The resulting acyl enzyme intermediates between sortases and their substrates are then resolved by the nucleophilic attack of amino groups, typically provided by the cell wall cross bridges of peptidoglycan precursors. The surface protein linked to peptidoglycan is then incorporated into the envelope and displayed on the microbial surface. This review focuses on the mechanisms of surface protein anchoring to the cell wall envelope by sortases and the role that these enzymes play in bacterial physiology and pathogenesis.

Staphylococcus aureus infection is a frequent cause of sepsis in humans, a disease associated with high mortality and without specific intervention. When suspended in human or animal plasma, staphylococci are known to agglutinate, however the bacterial factors responsible for agglutination and their possible contribution to disease pathogenesis have not yet been revealed. Using a mouse model for S. aureus sepsis, we report here that staphylococcal agglutination in blood was associated with a lethal outcome of this disease. Three secreted products of staphylococci - coagulase (Coa), von Willebrand factor binding protein (vWbp) and clumping factor (ClfA) – were required for agglutination. Coa and vWbp activate prothrombin to cleave fibrinogen, whereas ClfA allowed staphylococci to associate with the resulting fibrin cables. All three virulence genes promoted the formation of thromboembolic lesions in heart tissues. S. aureus agglutination could be disrupted and the lethal outcome of sepsis could be prevented by combining dabigatran-etexilate treatment, which blocked Coa and vWbp activity, with antibodies specific for ClfA. Together these results suggest that the combined administration of direct thrombin inhibitors and ClfA-antibodies that block S. aureus agglutination with fibrin may be useful for the prevention of staphylococcal sepsis in humans.

Staphylococcus aureus is an important human pathogen that causes skin and soft tissue abscesses. Abscess formation is not unique to staphylococcal infection and purulent discharge has been widely considered a physiological feature of healing and tissue repair. Here we present a different view, whereby S. aureus deploys specific virulence factors to promote abscess lesions that are distinctive for this pathogen. In support of this model, only live S. aureus are able to form abscesses, requiring genes that act at one or more of four discrete stages during the development of these infectious lesions. Protein A and coagulases are distinctive virulence attributes for S. aureus, and humoral immune responses specific for these polypeptides provide protection against abscess formation in animal models of staphylococcal disease.

SummaryStaphylococcus aureus encodes the specialized secretion system Ess (ESAT-6 secretion system). The ess locus is a cluster of eight genes (esxAB, essABC, esaABC) of which esxA and esxB display homology to secreted ESAT-6 proteins of Mycobacterium tuberculosis. EsxA and EsxB require EssA, EssB and EssC for transport across the staphylococcal envelope. Herein, we examine the role of EsaB and EsaC and show that EsaB is a negative regulator of EsaC. Further, EsaC production is repressed when staphylococci are grown in broth and increased when staphylococci replicate in serum or infected hosts. EsaB is constitutively produced and remains in the cytoplasm whereas EsaC is secreted. This secretion requires an intact Ess pathway. Mutants lacking esaB or esaC display only a small defect in acute infection, but remarkably are unable to promote persistent abscesses during animal infection. Together, the data suggest a model whereby EsaB controls the production of effector molecules that are important for host pathogen interaction. One such effector, EsaC, is a secretion substrate of the Ess pathway and implements its pathogenic function during infection.

Staphylococcus aureus is the most frequent cause of bacteremia and hospital-acquired infection, however a vaccine that prevents staphylococcal disease is currently not available. Two sortase-anchored surface proteins, IsdA and IsdB, have been identified as subunit vaccines that, following active immunization, protect experimental animals against intravenous challenge with staphylococci. Here we investigate the molecular basis of this immunity and report that, when passively transferred to naïve mice, purified antibodies directed against IsdA or IsdB protected against staphylococcal abscess formation and lethal intravenous challenge. When added to mouse blood, IsdA- or IsdB-specific antibodies did not promote rapid opsonophagocytic killing of wild-type staphylococci. Antibodies directed against IsdA interfered with heme-binding and IsdB antibodies perturbed the ability of this surface protein to bind hemoglobin. As the structural genes for isdA and isdB are required for heme-iron scavenging during the pathogenesis of infection, we hypothesize that IsdA and IsdB antibodies may at least in part provide protection against staphylococci by interfering with the pathogen's heme-iron scavenging mechanisms.