Thermosensitive pilus production by FCT type 3 Streptococcus pyogenes controlled by Nra regulator translational efficiency

Abstract

Streptococcus pyogenes produces a diverse variety of pili in a serotype-dependent manner and thermosensitive expression of pilus biogenesis genes was previously observed in a serotype M49 strain.However, the precise mechanism and biological significance remain unclear. Herein, the pilus expression analysis revealed the thermosensitive pilus production only in strains possessing the transcriptional regulator Nra. Experimental data obtained for nra deletion and conditional nra-expressing strains in the background of an M49 strain and the Lactococcus heterologous expression system, indicated that Nra is a positive regulator of pilus genes and also highlighted the importance of the level of intracellular Nra for the thermoregulation of pilus expression. While the nra mRNA level was not significantly influenced by a temperature shift, the Nra protein level was concomitantly increased when the culture temperature was decreased. Intriguingly, a putative stem-loop structure within the coding region of nra mRNA was a factor related to the post-transcriptional efficiency of nra mRNA translation. Either deletion of the stemloop structure or introduction of silent chromosomal mutations designed to melt the structure attenuated Nra levels, resulting in decreased pilus production. Consequently, the temperature-dependent translational efficacy of nra mRNA influenced pilus thermoregulation, thereby potentially contributing to the fitness of nra-positive S. pyogenes in human tissues.

Genetics, Structure, and Function of Group A Streptococcal Pili

“…Temperature dependent regulation is important in FCT-3 strains. This is primarily governed by post-transcriptional control of nra mRNA translation via a stem loop structure in the coding region ( Nakata et al, 2020 ). The stem loop is positioned proximal to the ribosome border and is considered to be important for promoting translation, although other factors such as its distance from the translation start codon and other mRNA structures around the Shine and Dalgarno sequence may contribute.…”

“…Previous reports have indicated that both RofA and Nra can function as autoregulators ( Podbielski et al, 1999 ; Granok et al, 2000 ), and expression of pilus genes in FCT-3 strains is positively or negatively regulated by Nra in a strain-specific manner ( Podbielski et al, 1999 ; Luo et al, 2008 ). Recent studies of in-frame deletion mutants indicated that Nra acts as a positive regulator in several M3 strains and an M49 strain ( Calfee et al, 2018 ; Nakata et al, 2020 ). RofA has been reported to be a positive regulator for the protein F1 gene ( prtF 1) ( Fogg et al, 1994 ).…”

“…Moreover, the expression of FctA appeared to be bistable, as only some cells in S. pyogenes chains were FctA-positive in immunofluorescence experiments ( Nakata et al, 2009 ). Furthermore, decreasing the temperature to 25°C induced pilus production by the majority of cells ( Nakata et al, 2020 ). This bistabilty was later characterized in more detail for type 1 pilus genes from pneumococci and shown to depend on the positive regulator RlrA acting in a positive feedback loop on pilus genes ( Basset et al, 2011 , 2017 ).…”

“…Historically, GAS cultures for a T-typing test have been grown at 30°C ( Griffith, 1934 ). Thermosensitive pilus production occurs at the transcriptional level and the expression pattern is restricted to nra -positive FCT-3 strains ( Nakata et al, 2009 , 2020 ). The underlying mechanism involves post-transcriptional control of nra mRNA translation, namely promoting translation at low temperatures ( Figure 3 ).…”

“…The underlying mechanism involves post-transcriptional control of nra mRNA translation, namely promoting translation at low temperatures ( Figure 3 ). Of note, in a study that utilized an M3 and an M49 strain, introduction of silent base substitutions in the chromosome to melt the predicted stem loop structure located 23 bases downstream of the AUG start codon decreased detection of both the Nra protein and pili especially at low temperatures ( Nakata et al, 2020 ). Considering that temperature at the initial infection site is lower than the core body temperature and formation of mRNA stem-loop structure is influenced by temperature, we speculated that the predicted stem-loop structure is an mRNA thermometer within nra mRNA.…”