The cell wall envelope of Gram-positive bacteria can be thought of as a surface organelle for the assembly of macromolecular structures that enable the unique lifestyle of each microorganism. Sortases - enzymes that cleave the sorting signals of secreted proteins to form isopeptide (amide) bonds between the secreted proteins and peptidoglycan or polypeptides - function as the principal architects of the bacterial surface. Acting alone or with other sortase enzymes, sortase construction leads to the anchoring of surface proteins at specific sites in the envelope or to the assembly of pili, which are fibrous structures formed from many protein subunits. The catalysis of intermolecular isopeptide bonds between pilin subunits is intertwined with the assembly of intramolecular isopeptide bonds within pilin subunits. Together, these isopeptide bonds endow these sortase products with adhesive properties and resistance to host proteases.

SummaryVegetative forms of Bacillus cereus are reported to form pili, thin protein filaments that protrude up to 1 mm from the bacterial surface. Pili are assembled from two precursor proteins, BcpA and BcpB, in a manner requiring a pilus-associated sortase enzyme (SrtD). Pili are also formed on the surface of Bacillus anthracis expressing bcpA-srtD-bcpB. BcpA is distributed throughout the entire pilus, whereas BcpB appears positioned at its tip. In agreement with the hypothesis for pilus assembly in Gram-positive bacteria, BcpA encompasses the YPK pilin motif and the LPXTG sorting signal, each of which is absolutely required for the incorporation of BcpA and BcpB into pili. In contrast to BcpB, which relies on the presence of BcpA for incorporation into pili, BcpA fibre assembly occurs even in the absence of BcpB. B. anthracis sortase A (srtA), but not sortase B (srtB) or C (srtC), is required for proper anchoring of pili to the bacterial envelope, suggesting that BcpA/BcpB pili are linked to peptidoglycan cross-bridges.

SummaryBacillus anthracis, the causative agent of anthrax, is a dangerous biological weapon, as spores derived from drug-resistant strains cause infections for which antibiotic therapy is no longer effective. We sought to develop an anti-infective therapy for anthrax and targeted CapD, an enzyme that cleaves poly-g-Dglutamate capsule and generates amide bonds with peptidoglycan cross-bridges to deposit capsular material into the envelope of B. anthracis. In agreement with the model that capsule confers protection from phagocytic clearance, B. anthracis capD variants failed to deposit capsule into the envelope and displayed defects in anthrax pathogenesis. By screening chemical libraries, we identified the CapD inhibitor capsidin, 4-[(4-bromophenyl)thio]-3-(diacetylamino)benzoic acid), which covalently modifies the active-site threonine of the transpeptidase. Capsidin treatment blocked capsular assembly by B. anthracis and enabled phagocytic killing of nonencapsulated vegetative forms.

Pilin precursors are the building blocks of pili on the surface of Gram-positive bacteria; however, the assembly mechanisms of these adhesive fibers are unknown. Here, we describe the chemical bonds that assemble BcpA pilin subunits on the surface of Bacillus cereus. Sortase D cleaves BcpA precursor between the threonine (T) and the glycine (G) residues of its LPXTG sorting signal and catalyzes formation of an amide bond between threonine (T) of the sorting signal and lysine (K) in the YPKN motif of another BcpA subunit. Three CNA B domains of BcpA generate intramolecular amide bonds, and one of these contributes also to pilus formation. Conservation of catalysts and structural elements in pilin precursors in Gram-positive bacteria suggests a universal mechanism of fiber assembly.CNA B domain ͉ LPXTG sorting signal ͉ sortase ͉ YPKN motif

There is increasing evidence that bacterial biofilms play a role in a variety of ocular infections. Bacterial growth is characterized as a biofilm when bacteria attach to a surface and/or to each other. This is distinguished from a planktonic or free-living mode of bacterial growth where these interactions are not present. Biofilm formation is a genetically controlled process in the life cycle of bacteria resulting in numerous changes in the cellular physiology of the organism, often including increased antibiotic resistance compared to growth under planktonic conditions. The presence of bacterial biofilms has been demonstrated on many medical devices including intravenous catheters, as well as materials relevant to the eye such as contact lenses, scleral buckles, suture material, and intraocular lenses. Many ocular infections often occur when such prosthetic devices come in contact with or are implanted in the eye. For instance, 56% of corneal ulcers in the United States are associated with contact lens wear. Bacterial biofilms may participate in ocular infections by allowing bacteria to persist on abiotic surfaces that come in contact with, or are implanted in the eye, and by direct biofilm formation on the biotic surfaces of the eye. An understanding of the role of bacterial biofilm formation in ocular infections may aid in the development of future antimicrobial strategies in ophthalmology. We review the current literature and concepts relating to biofilm formation and infections of the eye.