Most of these technologies have been utilized to identify in vivo induced genes required
by S. pyogenes to grow under various conditions. These have helped to further improve
knowledge about how S. pyogenes reacts with the human host from both pharyngitis and deep tissue infections. Below represents a summary of the important findings learned about this human pathogen utilizing these technologies.
The STM method has recovered a total of 29 genes shown to be required for S. pyogenes
in vivo growth (239). Six of the genes identified have also been previously identified
including hasA, mga, smeZ, amrA, and two sil genes, lending support to the validity of the other genes. Genes identified covered a range of categories including transport, regulators, cellular processes, and secreted proteins. Some of the interesting genes involved in virulence included transport. The first was a putative macrolide efflux pump (mefE) that was shown to be involved in biofilm formation but did not provide any antibiotic resistance (239, 263). Another was an ABC transporter, salT, which is part of the salivaricin bacteriocin locus. Interestingly, S. pyogenes does not produce the bacteriocin and so the function of the transporter remains unknown. Other putative transporters and cytosolic proteins of unknown function were also identified showing there are still many genes for which we have yet to learn the function (239, 263).
RNA-seq has been performed on S. pyogenes twice, both involved in the identification of small RNA (sRNA). Tesorero et al. was able to find a small RNA with a link to acid stress during growth and infection by identifying the mRNA involved (264). Another group, Deltcheva et al., identified a novel sRNA that forms part of the complex involved in the processing of pre-crRNA (CRISPR RNA; clustered, regularly interspaced short palindromic repeats), short RNA elements that interfere with exogenous DNA elements (265).
Microarrays have probably given the most information and have been used in many models spanning ex vivo [human blood (266) and saliva (267)], in vitro (a range of temperatures) (268), and in vivo [mouse soft tissue infection models (269, 270) and a cynomolgus macaque model of pharyngitis (271)]. The first microarray chip for S.
pyogenes was created incorporating 92% of the known ORFs in strain SF370 and used to determine how temperature regulated gene expression. It was shown that 9% of the represented genes expressed a change when grown at 29°C, with a large number of those genes from the extracellular proteome (268). When using ex vivo experimentation, the strain and the technology changed. The microarray utilized shorter oligonucleotides of a higher density, creating much greater coverage of the genome, and was designed to cover six different S. pyogenes strains. Using this microarray technology, the two component system sptR/S was found to play a key role for persistence in saliva and was controlling genes involved with nutrient acquisition, response to oxidative stress, and evasion of innate and acquired immune responses (267, 272). When S. pyogenes was exposed to blood, the greatest change in gene expression was seen at the 30 minutes (min) period with 76% of the genome demonstrating a difference in expression. The streptokinase gene, ska, was upregulated along with adhesins such as emm1, collagen-binding proteins, and capsule; all of this indicating that mga was an important factor. S. pyogenes also changed the expression of many genes involved with metabolic functions, through the covR/S two-component system, in order to adapt to its new environment (266).
Numerous in vivo studies have also been performed to analyze gene expression at the genome level. From the mouse model of soft tissue infection, it was proposed that S.
pyogenes goes through a three-step process in order to establish itself in a host:
establishment, adaptation, and dissemination. These steps involve the orchestration of a number of genes at each stage. Establishment implicated the activation of genes involved in adherence and evasion of the immune system. Adaptation involved continued immune evasion as well as aggregate formation and rapid replication. Finally, the dissemination stage included nutrient acquisition along with tissue breakdown and shedding (269). Experimental pharyngitis using cynomolgus macaques also identified three separated phases of disease including colonization, acute, and asymptomatic. Colonization and inflammation was associated with the expression of SAgs and the different phases of diseases were associated with the regulators from two two-component systems (covR/S
and spy0680/spy0681). covR/S was not expressed during the colonization phase, but was
turned on during the acute phase, and repressed during the asymptomatic phase.
Realizing that two-component systems were important for the survival of the organism, the mouse abscess model was used with two-component system knockout mutants. The
spy0680/M5005_spy0681 knockout showed significantly larger abscess sizes compared to
the wild-type, while the other strains showed no difference. This was not surprising as it is a known repressor and has been shown to be active during the asymptomatic phase of experimental pharyngitis in cynomolgus macaques (270).
Utilizing microarray data from nine strains isolated from patients before 1987, clusters were created composed of three invasive strains and six pharyngeal strains. Based on genome expression differences of ~10% they were divided into the pharyngeal transcriptome profile (PTP) and the invasive transcriptome profile (ITP). The distinction between the two genomes was found to be a seven bp insertion creating a truncated covS, the histidine kinase of the covR/S two-component system (273). Later work showed that SpyCEP, a protease that increases expression in the covR/S truncation, was able to cleave chemokines associated with neutrophil activation, explaining their absence from sites of infection (274). Important work also asked how S. pyogenes was changing from a metabolic perspective, and how this changed pathogenicity. It was found that catabolite control protein A (CcpA) controlled virulence factors as well as carbohydrate utilization genes, having a severe affect on mouse oropharynx colonization (275). Further work was done to examine the maltose repressor (MalR), a surface carbohydrate binding protein. A malR knockout showed a significant reduction in colonization, but no lack of invasive disease, demonstrating how carbon sources can determine pathogenesis (276). Future work continues to use the same type of gene chip covering research in many different areas with to increase our overall knowledge of S. pyogenes.
Finally, utilizing the IVIAT method, 16 genes were initially identified in S. pyogenes, after which three were identified to be truly upregulated in vivo, as determined by qRT- PCR analysis. These genes included coaA, a putative pantothenate kinase, pbp1A, a putative penicillin binding protein, and tdcF, a hypothetical protein (261). coaA catalyzes the first step in the biosynthetic pathway leading to coenzyme A, essential in the metabolism of fatty acids, carbohydrates, and amino acids (261). Penicillin-binding proteins are essential for cell morphology and are typically involved in peptidoglycan
synthesis, but pbp1A was also found to be important for the resistance of phagocytosis in
S. agalactiae (261). tdcF encodes a hypothetical protein with 60% homology to a protein
in Vibrio vulnificus that was found to have a translation initiation inhibitor function.
These two genes may have a similar function found in S. pyogenes (261).