The objective of this chapter is to provide a detailed purification protocol for the surface-layer (S-layer) glycoproteins of the periodontal pathogen Tannerella forsythia. The procedure involves detergent based solubilization of the bacterial S-layer followed by cesium chloride gradient centrifugation and gel permeation chromatography. The protocol is suitable for the isolation of S-layer glycoproteins from T. forsythia strains with diverse O-glycan structures, and aid in understanding the biochemical basis and the role of protein O-glycosylation in bacterial pathogenesis.The type IX secretion system (T9SS) is a protein secretion system for gingipain proteases and is found on the cell surface of Porphyromonas gingivalis. Proteins secreted by T9SS contain a signal peptide, functional domains, an immunoglobulin (Ig)-like domain, and a C-terminal domain (CTD). Thirty genes on the P. gingivalis chromosome encode proteins that possess the CTD, which is important for T9SS-mediated translocation to the cell surface across the outer membrane. In T9SS mutant strains, proteins accumulate as precursors in the cell and therefore exhibit a phenotype similar to that of secreted protein-deficient mutants. Black pigment productivity and hemagglutination are phenotypic features of P. gingivalis associated with the activity of gingipains. In P. gingivalis T9SS mutants, unprocessed gingipains with high molecular weights accumulate in the cell, and colony pigmentation and hemagglutination are not observed in the same phenotype as a gingipain null mutant.The type IX secretion system (T9SS) is the most recently discovered secretion system in the gram-negative bacteria and is specific to the Bacteroidetes phylum. It is comprised of at least 19 proteins, which together allows for the secretion and cell surface attachment of a specific group of proteins (T9SS substrates), that harbor a signal sequence at the C-terminus. Here we describe the structural characterization of the PorK, **** and PorG components of the Porphyromonas gingivalis T9SS using electron microscopy and cross-linking mass spectrometry.Porphyromonas gingivalis is a gram-negative, rod-shaped, nonmotile bacterium belonging to the phylum Bacteroidetes. It produces abundant amounts of proteases in both cell-associated and secretory forms, including a group of cysteine proteases referred to as gingipains, which have attracted much attention due to their high proteolytic activity associated with pathogenicity. Gingipains are grouped into arginine (R)-specific (RgpA and RgpB) and lysine (K)-specific (Kgp) types. Both Rgp (collective term for RgpA and RgpB) and Kgp gingipains play crucial roles in the virulence of P. gingivalis, including the degradation of host periodontal tissues, disruption of host defense mechanisms, and loss of viability in host cells, such as fibroblasts and endothelial cells. In addition to their function in virulence, gingipains are also essential for the growth and survival of P. gingivalis in periodontal pockets through the acquisition of amino acids and heme groups. Furthermore, Rgp and Kgp gingipains are critical in processing fimbriae and several bacterial proteins that contribute to hemagglutination, coaggregation, and hemoglobin binding. This chapter describes the methods used to analyze gingipains.Porphyromonas gingivalis fimbriae play a critical role in colonization. Elucidation of the fimbrial structure in atomic detail is important for understanding the colonization mechanism and to provide means to combat periodontitis. X-ray crystallography is a technique that is used to obtain detailed information of proteins along with bound ligands and ions.  https://www.selleckchem.com/products/nvp-2.html  Crystallization of the protein of interest is the first step toward structure determination. Unfortunately it is not possible to predict the crystallization condition of a certain protein or even if the protein can be crystallized. Protein crystallization is, on the contrary, a matter of trial and error. However, the best strategy for success is to focus on the protein purification step to obtain a sample that is pure, stable, homogeneous and of high concentration. This chapter addresses general methods for crystallization of fimbrial proteins.Fimbriae of the periodontal pathogen Porphyromonas gingivalis mediate its colonization through associations with other bacteria and host tissues. P. gingivalis generally expresses two distinct fimbrial types, FimA and Mfa1. In P. gingivalis ATCC 33277, FimA fimbriae are present as long filaments easily detached from cells, whereas Mfa1 fimbriae are short filaments compactly bound to the cell surface. Because of this unique characteristic, FimA fimbriae have been selectively and easily isolated from the bacterial cell surface through mechanical shearing such as by pipetting and stirring. However, P. gingivalis ATCC 33277 harbors a mutation in the gene encode the fimbrial length regulator, FimB, and thus produces unusually long FimA fimbriae length. Hence, mechanical shearing to remove FimA is potentially applicable only for this type strain. Here we present protocols to purify intact Mfa1 fimbriae from a fimA-deficient mutant strain. Mfa1 fimbriae are purified from cell lysates, using a French pressure cell and through ion-exchange chromatography. The purity of Mfa1 fimbriae can be confirmed through sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and electron microscopy.Adhesive pili (or fimbriae) in bacteria are classified into five types, among which type V pili have been most recently described. Type V pili differ from other pili types with respect to transport mechanism, structure, and pilin synthesis. Genes of type V pili are restricted to the phylum Bacteroidetes. Protein subunits that compose type V pili are transported to the cell surface as lipoprotein precursors and then polymerized into a pilus through a strand-exchange mechanism, which is demonstrated by several experiments, including palmitic acid labeling and Cys-Cys cross-linking analysis. Here, we describe the use of these methods to analyze type V pili.Porphyromonas gingivalis, a significant periodontal pathogen, is known to possess genetic variations in relation to its virulence. Furthermore, fimbriae encoded by the fimA gene are involved in bacterial adherence to and invasion of host cells, and a known virulence factor of the bacterium. The fimA gene is classified into six variants (types I-V and Ib) and has been shown to be related to microbial virulence. Polymerase chain reaction (PCR) assay results are helpful to differentiate the genotypes, with fimA type-specific primer sets used for that have been developed by several researchers. Although room for improvement remains, fimA genotyping is expected to become a useful technique for periodontal examinations and diagnosis. In this chapter, currently available PCR methods to classify fimA genotypic variations of P. gingivalis are described.