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Naturally occurring mutations in regulatory proteins among streptococcus pyogenes isolates from distinct human infections
Publication . Pato, Catarina Teresa Condinho; Ramirez, Mário Nuno Ramos de Almeida
Streptococcus pyogenes (Group A Streptococcus, GAS) is among the most prevalent bacterial pathogens of humans and it is responsible for a wide range of infections, from pharyngitis and impetigo to life-threatening conditions such as necrotizing fasciitis and streptococcal toxic shock syndrome. However, until today, despite many years of research, there is still no consensus regarding which are the genotypic or phenotypic characteristics that confer to an isolate a certain tissue preference or a more invasive potential. The nasopharyngeal mucosa and skin are considered the primary sources of isolates responsible for invasive infections. This suggests a further ability of these isolates to invade and survive in deeper tissues. In the last years, spontaneous mutations occurring in the covRS two-component regulatory system have been considered a possible explanation for the transition from localized to systemic infection. It is estimated that this system controls directly or indirectly the expression of 10-15% of the GAS genome. As a consequence of these mutations, among the distinct patterns of expression of several virulence factors, the downregulation of the extracellular cysteine protease SpeB, has been considered crucial for the switch to a hypervirulent phenotype. The downregulation of SpeB has also been described as a consequence of mutations in a stand-alone transcriptional regulator named RopB. However, contrasting results were reported whether covRS mutations were more prone to occur in certain lineages, namely those more frequently associated with invasive infections or if they occur in a similar proportion among invasive and non-invasive isolates. To address these questions, in the present thesis, we determined the sequence of the covRS and ropB of 191 isolates from invasive infection and pharyngitis and evaluated the production of SpeB, as well of NAD glycohydrolase (NADase) and streptolysin S (SLS), which are two virulence factors supposed to be under the influence of CovRS. Moreover, skin and soft tissue infections (SSTI) are frequently considered the focal points for the development of invasive disease. However, most of the knowledge about GAS skin isolates is from studies that intended to find differences between isolates recovered from invasive and non-invasive infections generically. The majority of these studies use isolates recovered from SSTI but mostly from pharyngitis and compared those together against the isolates recovered from invasive infections, resulting in few data regarding isolates recovered from SSTI. Therefore, we characterized by multiple typing methods a total of 320 isolates from SSTI recovered in Portugal and performed the comparison with invasive isolates recovered during the same period which were previously characterized. All SSTI isolates were also tested for SpeB activity and for those without detectable SpeB activity we determined the sequence of covRS and ropB genes. Overall, we found that isolates with null covS alleles, which are predicted to eliminate the protein function have a significant association with invasive infections comparative with isolates from pharyngitis and SSTI. Additionally, none of these isolates, as expected, had SpeB activity, and, with few exceptions, they showed an increased activity of both NADase and SLS that could explain their potentially higher invasiveness. Even so, this mechanism was found to be uncommon, corresponding to only 10% of invasive isolates, which could be due to an overall fitness cost of these mutations. Moreover, null covS alleles were not more prevalent among isolates from clones frequently associated with invasive infection such as emm1 and emm64 and instead they were distributed throughout diverse genetic backgrounds. The few exceptions regarded the levels of NADase and SLS points to the complexity of the regulatory networks among distinct GAS lineages. Additionally, no null covR alleles were detected in our isolates and ropB null alleles were found in a low fraction of GAS isolates and were not associated with any infection type. Regarding SpeB activity, it was detected in a similar proportion in isolates recovered from the different sources, and therefore its absence was not associated to any type of infection, suggesting that its abrogation cannot by itself explain the higher ability of certain clones to cause invasive disease. Among SSTI isolates, we found that emm89 type isolates were the most prevalent and were significantly associated with these infections when compared with invasive isolates. In contrast, emm1, emm3, and emm64 isolates were associated with invasive infections. Within emm89 isolates, SSTI were only associated to those that lack the hasABC locus, corresponding to a recently emerged acapsular clade (clade 3) that also carries a variant of the ngs-ifs-slo locus. These results suggest that for some unknown reason these isolates may have an increased potential to cause SSTI. As a consequence of known differences in the emm-type between isolates causing these two types of infections, we also found significant associations between the ability to bind to different host proteins. This ability was presumed by inferring the emm-cluster through the emm-type results. The emm-cluster is a recent classification based on the entire sequence of the emm gene (M protein) where each cluster shares binding motifs to host proteins and other structural properties. Therefore, the ability to bind fibrinogen and albumin were significantly associated with invasive isolates, whereas the ability to bind to C4BP and IgG were associated with SSTI isolates. Differences in the presence of superantigen (SAg) genes, SAg profiles and in the distribution of sequence types (ST) determined by Multilocus Sequence Typing (MLST) were also noted. The possible impact of these differences in the ability of the isolates to cause these distinct infections remains to be clarified. Moreover, within each emm type the same MLST defined lineages and SAg profiles could be found in both types of infection, questioning the possibility that these characteristics dictate the tissue tropism of each isolate. In summary, the results described in this thesis indicate that isolates responsible for SSTI are genetically distinct from those recovered from normally sterile sites and while some GAS clones have more capacity to invade deeper tissues, others are more prone to cause SSTI. Moreover, the significant presence of null covS mutations among invasive isolates and the fact that no association was observed regarding the absence of SpeB activity in isolates from different types of infections, suggests that the role of spontaneous mutations impairing the CovRS activity is probably related with the regulation of others virulence factors under its control in addition to SpeB.
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Fundação para a Ciência e a Tecnologia
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SFRH
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SFRH/BD/81329/2011