Molecular components for the assembly of the Bacillus anthracis S-layer

Anthrax disease is an aggressive and lethal invasion of the mammalian host by the Grampositive bacteria, Bacillus anthracis. The pathogen lies in waiting in its metabolically inactive and resilient endospore form. Upon encounter of a mammalian host through an accessible entry point by consumption, inhalation, or break in the dermal layer, the endospore is revived to proliferate in its vegetative form. Within the host, the organism must defend itself from immune cells. B. anthracis achieves a successful infection by first, secreting lethal and edema toxins that inhibit and destroy phagocytic cells. Secondly, detection by immune cells is masked by the elaboration of a non-immunogenic, poly-gamma-D-glutamic acid capsule that is attached to the cell wall. Lastly, the ability to grow in long, filamentous chains is believed to create a hindrance to phagocytic clearance by the host. Evasion of host defenses enables B. anthracis to replicate to high titers and overwhelm the host, leading to death. Return of endospores to the environment, thus repeats the cycle.;The length of vegetative chains is controlled by proteins that attach to the surface of the cells. B. anthracis elaborates a paracrystalline lattice Surface layer (S-layer) comprised of two abundantly produced and secreted proteins, Sap and EA1. The function of these S-layer proteins is unknown, although S-layer proteins in other organisms have been proposed to contribute to virulence and to enhance survival under various environmental stresses. Attachment of S-layer proteins to the cell is mediated by a lectin-like interaction between the S-layer homology (SLH)- domain at the N-terminus of the proteins and a secondary cell wall polymer (SCWP) attached to the peptidoglycan. The unique ability of Sap and EA1 to self-assemble into a paracrystalline layer and form S layers is due to the presence of the crystallization domain. Bacillus S-layer associated proteins (BSLs) that are also deposited on the surface harbor domains for adhesion and iron-transport that aid in infection and nutrient uptake. These proteins harbor the SLHdomain for binding to the SCWP, but no crystallization domain and thus do not assemble into Slayers. Several BSL proteins harbor peptidoglycan hydrolase domains that have been demonstrated to separate chaining bacilli. The distribution of these proteins on the cell is dependent on the assembly of the S-layer proteins, Sap and EA1 -- a mechanism that is poorly understood. In the work that follows, we identify genes involved in the assembly of the S-layer that modify the secondary cell wall polysaccharide (Chapter IV) and aid in the efficient production of Sap and EA1 for assembly on the surface (Chapter II and III).;To identify genes that are involved in the assembly of the S-layer, a transposon screen for mutants that exhibit increased chain-lengths, a hallmark of S-layer disruption, identified three genes - an accessory secretion ATPase (secA2) and two S-layer assembly genes (slaP, slaQ). All three genes reside within the S-layer gene cluster that includes the genes for pyruvylation of the SCWP and the genes encoding the two S-layer proteins, sap and eag. In Chapter II and III, we describe the contribution of secA2, slaP, and slaQ in the assembly of the S-layer. Mutational lesions in secA2-slaP-slaQ abrogate the production and deposition of S-layer proteins, but not Slayer associated proteins or other secreted products. Loss of secA2 or slaP affects the deposition of the murein hydrolase, BslO to septal rings. Using translational hybrids to mCherry, we show that SlaQ is required for deposition of Sap, in a manner that is dependent on the presence of the crystallization domain, but not the signal peptide or SLH-domain. Instead, SecA2 and SlaP govern Sap deposition in a manner that requires the presence of the signal peptide and SLH domain.;In Chapter IV, we characterize the effect of mutational lesions in four predicted acetyltransferase genes encoded downstream of the S-layer gene cluster -- patA1B2-patA2B2. Mutations in these genes result in an exaggerated chain-length phenotype due to the inability to retain the murein hydrolase BslO in the envelope. Biochemical characterization of SCWP purified from wild-type and pat mutant bacteria suggests that the Pat enzymes acetylate the SCWP. Acetylation of SCWP is required for SLH binding as demonstrated by measuring the interaction of SLH-mCherry reporters to purified SCWP. Together, our studies extend the repertoire of genes and molecular requirements for S-layer assembly in B. anthracis..