Note the C-terminal extension containing a cysteine residue. We draw the conclusion that Staphylococcal Protein A may be a crucial determinant in the colonization and immune evasion of ST infections, contributing to persistent spread in the hospital settings.
We investigated whether the absence of SpA is associated with genetic polymorphisms common to the SpA- subset of strains, and whether other changes in the virulence gene expression profile are associated with the SpA- phenotype, highlighting a profound interconnectedness of SpA and other immune evasion factors.
B Alignment of spa locus in the subset of strains. Although all the strains carried the spa locus data not shownnine of them resulted negative in Western blot analysis Figure 1B.
The collection was composed of strains, including commensal and clinical isolates from diverse niches and different geographic origin as well as some lab strains, and categorized by Multilocus sequence typing MLST Figure 1A.
In addition, to enable a directed immobilisation of the different proteins for binding studies, a codon for a unique cysteine residue was engineered into the C-termini of the different proteins.
The yield of scFv fragment after purification was approximately 2.
The genes comprised in Cluster 3 are highly upregulated as growth progresses. There are five main clusters representing different transcript kinetic profiles, which are represented in Supplementary Figure S1.Using a high-throughput qRT-PCR approach on a selected panel of virulence-related genes, we identified that the SpA- phenotype is associated with lower spa transcript levels and increased expression and production of capsule as well as other changes in the transcription of several key virulence factors. Prior to inoculation, mice were administered ampicillin all strains were ampicillin-resistant in their drinking water for 24 h and for the duration of the experiment in order to reduce interference from commensal bacterial flora. This is particularly evident when comparing the mean kinetic profile of N with the other strain Supplementary Figure S1 , cluster 4. The molecular mechanisms that determine the SpA- phenotypes were found to be different among the SpA- strains and involve both RNA translation in the three SpA- strains that lacked a functional spa RBS, and likely alterations in the transcriptional network. All sequences are given in one letter code. The immobilisations were performed according to the suppliers recommendations, employing the free thiol group of the cysteine in the C-termini of the fusion proteins. B SpA expression in a representative panel of strains. One of the causes of its success as a pathogen is the peculiar array of immune evasion factors through which the bacterium avoids host defenses, where the staphylococcal protein A SpA plays a major role thanks to its IgG binding activities. Three days after inoculation, eight mice per group were killed and evaluated for nasal carriage of S. The polymorphism of this region is based on the deletions or duplications of these repeats and punctual mutations. The superantigen activity of SpA leads to immunodominance, limiting host responses to other S. The reaction mixtures contained 30 pmol of each primer in a standard buffer Perkin-Elmer. Growth curves for each of the analyzed strains were performed to define the OD corresponding to the different growth phases. For each interaction, the obtained response was normalised with respect to the capability of the immobilised ligand to bind HSA injected at a single concentration. S27 isolate carries several SNPs across the sequence as well as a shorter X variable region.