When using SDS-PAGE (SDS denatured polyacrylamide electrophoresis) to study the serum samples from scrub typhus convalescent patients at least 30 major proteins can be identified. Among these, a 56-kDa protein became the most widely studied antigen when using SDS-PAGE. It has subsequently also become the most studied for DNA sequence variation. This antigen is expressed mostly on the cell surface and contains both unique and cross-reactive epitopes. The 56-kDa protein is type specific to Orientia, and is the most plentiful cell-surface protein of O. tsutsugamushi. It has been determined that the protein consists of ~520 amino acids, although the protein is now known to vary from as small as ~500 to longer than 540 amino acids in different strains. The protein is usually considered to contain 4 variable domains (see figure below, from Kelly et al., 2009) and to be the primary antigen reflecting variation within the genus [Ohashi et al., 1990a].
Across most of its length, the protein sequence of the 56-kDa TSA shows no obvious homology or structural similarity to any other protein in the international nucleotide or protein databases, making it one of the most appealing targets for use in development of a vaccine against O. tsutsugamushi. A small portion of the carboxy-terminal end of the protein shows similarity to some outer-membrane proteins from the alphaproteobacteria. It is not clear what if any role the protein plays in the pathogenesis of O. tsutsugamushi, although it is thought that the protein acts as an adherent factor for adsorption of the bacteria to the host cell surface [Urakami, Tsuruhara, and Tamura 1983 ; Tamura 1988a] and may be a determinant of virulence for individual strains [Tamura, 1988b].
The nucleotide sequence of the 56-kDa TSA gene was one of the first DNA sequences from O. tsutsugamushi to be determined [Ohashi, 1990a; Stover et al., 1990b]. As nucleotide sequences from this gene have accumulated from many isolates, the gene has come to be recognized as providing the most useful single locus data for studies of genetic differentiation between Orientia strains. Other genes may be useful for studies of differentiation within Orientia strains, but to date, no other locus has been reported that contains levels of variation even close to those seen for the locus encoding the 56-kDa TSA protein. Thus, no other single locus seems to have the same potential of usefulness for fine-structure analysis of strain variation. However, the high variation seen in the 56-kDa TSA gene may actually obscure some of the underlying patterns of genetic relatedness among isolates, especially in light of the increasing evidence of high levels of genetic recombination in O. tsutsugamushi.
INTERSTRAIN VARIATION IN DNA SEQUENCE – 56kDa TYPE SPECIFIC ANTIGEN
As of January 2018, sequence information of the 56-kDa TSA gene has been deposited in the international DNA databases for >1990 isolates of O. tsutsugamushi (a few “standard” strains are represented more than once on the DNA databases). Sequences range in size from 149 to nucleotides 2314. Sequences that exceed ~1620 bases include portions of the flanking sequences of the gene. Details about the sequences for the 56kDa TSA gene are given in a supplemental page.