Periodontitis is associated with a dramatic shift in the subgingival microflora towards predominantly gram negative organisms. Campylobacter rectus is unique among the gram negative periodontal pathogens in its expression of a 150 kd surface-layer (S-layer) protein. These abundant proteins form homogeneous arrays covering the surfaces of numerous bacteria and are required for bacterial pathogenesis. Thus , it is critical that S-layers are properly localized to the cell surface. There are only a few reports on the secretion mechanisms of S-layer proteins. Interestingly, unlike most other S-layer proteins, no amino acids are removed from the C. rectus S-layer protein (crsA) as it is transported to the cell surface. In addition, a homologue, crsD, to one of the three components of type I secretion systems is found in an operon upstream of the C. rectus crsA gene. This suggests that crsA is transported by a type I pathway. Most excitingly, the crsD operon contains another gene, crsC, whose protein has no known function. Analysis of the phenotype of a polar crsC mutant we constructed suggests that crsC is involved in crsA secretion, synthesis or degradation. We now propose to exploit the molecular genetic tools we have developed in order to determine the mechanisms used by C. rectus to transport its S-laver to the cell surface. It is our hypothesis that the crsA protein will be transported by a type l system. Importantly, we hypothesize that crsC will have a novel function in the type I secretion pathway perhaps as a chaperone. One goal of this proposal is to identify the C. rectus S-layer sequences that direct it to the cell surface using targeted mutagenesis and gene replacement technology. In addition, the genes required for crsA transport will be identified by cloning and sequencing the rest of the crsCD operon or by transposon mutagenesis. Importantly, the function of each putative transport gene will be characterized by making non-polar mutants. The resulting characterization of the crsC mutants will define, for the first time, the function of this novel protein in S-layer metabolism. Finally, the transport mutants will be examined for the possible mis-localization of non-S-layer proteins since there are two reports of type I transport pathways transporting more than one virulence protein.
