Project Grant R21AI175964

DESCRIBING THE STABLE, NON-COVALENT BCLA-BXPB ATTACHMENT IN B. ANTHRACIS SPORES - PROJECT SUMMARY BACILLUS ANTHRACIS IS A GRAM-POSITIVE SOIL BACTERIUM THAT FORMS SPORES WHEN STARVED FOR NUTRIENTS AND CONTACT WITH THESE SPORES CAUSES ANTHRAX IN ANIMALS AND HUMANS. B. ANTHRACIS SPORES ARE SURROUNDED BY THREE PROTECTIVE LAYERS, THE OUTERMOST OF WHICH IS A LOOSELY FITTING EXOSPORIUM. THE EXOSPORIUM PLAYS KEY ROLES IN SPORE SURVIVAL AND DISEASE PROGRESSION. OVER THE PAST TWO DECADES, THERE HAS BEEN SIGNIFICANT PROGRESS IN IDENTIFYING THE PROTEINS THAT COMPRISE THE EXOSPORIUM AND THEIR FUNCTIONS, HOWEVER, THE ASSEMBLY PROCESS IS POORLY UNDERSTOOD. THIS PROPOSAL IS DESIGNED TO ELUCIDATE MAJOR COMPONENTS OF EXOSPORIUM ASSEMBLY. THE EXOSPORIUM IS A BIPARTITE STRUCTURE CONSISTING OF A PARACRYSTALLINE BASAL LAYER AND AN EXTERNAL HAIR- LIKE NAP. EACH FILAMENT OF THE NAP IS FORMED SOLELY BY A TRIMER OF THE COLLAGEN-LIKE GLYCOPROTEIN BCLA. IN CONTRAST, THE BASAL LAYER CONTAINS ~25 DIFFERENT PROTEINS. ONE OF THESE PROTEINS CALLED BXPB IS REQUIRED FOR THE ATTACHMENT OF NEARLY ALL BCLA IN THE EXOSPORIUM. BCLA ATTACHMENT OCCURS THROUGH AND REQUIRES ONLY ITS 38- RESIDUE AMINO-TERMINAL DOMAIN (NTD), WHICH IS PROTEOLYTICALLY PROCESSED DURING SPORULATION TO REMOVE RESIDUES 1-19. CRYO-ELECTRON MICROGRAPHS REVEAL THAT EACH FILAMENT OF THE NAP-THROUGH BCLA RESIDUES 20-38-IS ATTACHED TO A BASAL LAYER SURFACE PROTRUSION THAT APPEARS TO BE A TRIMER OF BXPB. WHEN EXTRACTED FROM SPORES, BCLA AND BXPB ARE PRESENT PRIMARILY IN >250-KDA COMPLEXES, THE STABILITY OF WHICH SUGGESTED THAT THE TWO PROTEINS ARE ATTACHED THROUGH A COVALENT BOND. RECENT STUDIES FROM THIS LAB HAVE DEMONSTRATED THAT COMPLEXES BETWEEN PURIFIED BXPB AND BCLA RESIDUES 20-38, THAT ARE AS STABLE AS BCLA-BXPB COMPLEXES FOUND IN SPORES, CAN BE FORMED IN VITRO. THESE COMPLEXES DO NOT CONTAIN COVALENTLY CROSS-LINKED PEPTIDES, INDICATING THAT BCLA-BXPB ATTACHMENT IS NONCOVALENT. FURTHERMORE, WE RECENTLY DETERMINED THE CRYSTAL STRUCTURE OF BXPB TRIMERS, WITH MONOMERS THAT ARE ALL B-STRAND WITH CONNECTING LOOPS. THE ORIENTATION OF THREE OF THESE LOOPS SUGGEST THAT THEY CAN INTERACT WITH AND ENTRAP THE BCLA NTD. THE PRIMARY GOAL OF THIS STUDY IS TO USE STRUCTURAL AND GENETIC METHODS TO DESCRIBE THE AMINO ACID CONTACTS THAT ACCOUNT FOR THE STABLE BCLA-BXPB ATTACHMENT. STRUCTURAL TOOLS INCLUDE X-RAY CRYSTALLOGRAPHY, FOCUSING ON A BCLA NTD-BXPB COMPLEX, AND HYDROGEN DEUTERIUM EXCHANGE BY MASS SPECTROMETRY TO REVEAL CONTACTS BETWEEN THE BCLA NTD AND BXPB IN SOLUTION. TARGETED MUTAGENESIS OF BXPB AND THE BCLA NTD WILL REVEAL SPECIFIC ROLES FOR INDIVIDUAL AMINO ACIDS IN THE ATTACHMENT PROCESS. RELATED STUDIES WILL EXAMINE THE MECHANISM OF BXPB ATTACHMENT TO AND STABILIZATION OF THE BASAL LAYER SCAFFOLD AND THE REQUIREMENT FOR BCLA NTD CLEAVAGE IN BCLA-BXPB COMPLEX FORMATION. THE EXPECTED OUTCOME IS A DETAILED MODEL FOR BCLA-BXPB ATTACHMENT AND INSERTION INTO THE EXOSPORIUM. THIS STUDY WILL FURTHER IMPACT THE FIELD AS THIS MODEL IS LIKELY TO BE SHARED BY MANY OTHER SPORE-FORMING BACTERIA, INCLUDING IMPORTANT BACILLUS AND CLOSTRIDIUM PATHOGENS.