Sequence Analysis of the Variable Number Tandem Repeat in Staphylococcus aureus Protein A Gene - Bacterial Pathogenesis: Methods and Protocols - page 277

As is the case for MLST, spa typing provides objective information that is
easily scored and stored in a relational database. In MLST analysis, sequencing
results of seven gene fragments are analyzed, whereby each unique genetic
alteration (nucleotide change, deletions, or insertions), regardless of whether
it is a synonymous or non-synonymous change, is assigned an allelic number
(5,20) . The catenation of the allelic number for the seven gene fragments
defines the sequence type (ST); and the assignment of the allelic number and
ST is performed using a free web-based site (http://www.mlst.net) maintained
in London, England.
Analogous to the assignment of allelic numbers to genetic variants in MLST,
each repeat unit in the polymorphic X region of spa is given a unique identifier
( see Fig. 2 ). Based on two large international spa databases (eGenomics and
Ridom), there have been over 150 unique repeats identified to date. The alter-
ations in the repeats are primarily single nucleotide substitutions in the third
position (synonymous), more rarely codon insertions/deletions, and in each
case, the protein A sequence remains in-frame producing a functional protein.
Currently, of over 2100 different spa types from more than 30,000 S. aureus
isolates that have been identified in the eGenomics and Ridom databases, all
isolates contain at least one repeat unit and some strains contain up to 16 repeat
units. Conversely, there are no examples where the protein A gene is void of
repeats, where individual repeats are truncated, or where repeat sequences are
found out-of-frame. Although untested, this observation suggests a potential
biological significance underlying the intact, in-frame repeat sequences in the
polymorphic X region of the protein A gene. While protein A is a well-known
virulence factor in murine models of S. aureus infections including pneumonia
(36,37) , the biological function of the polymorphic X region is not well under-
stood. It is thought that the protein A domain encoded by the X region may
serve to extend the N-terminal immunoglobulin G binding portion of the protein
through the cell wall (38) . However, the biological function and significance
of the diverse repeat content and organization in isolates of S. aureus are not
clear.
As mentioned earlier, spa type determination is based on differences between
strains in the number and content of the VNTRs. Therefore, two strains with
the identical repeat sequence are assigned the same spa type (i.e., same repeat
content and organization) and considered genetically related; in short-term
investigations, identical spa types may be indicative of transmission events. As
spa typing does not have the resolving power of PFGE subtyping, identical
spa types can in some instances exhibit similar yet non-identical PFGE profiles
(33) . Nevertheless, identical spa types are strongly suggestive of clonality, and
different spa types that share a similar repeat motif may likewise be related
by descent. For example, spa types 1 and 7 share a near identical repeat motif