Functional definition of NrtR, a remnant regulator of NAD ⁺ homeostasis in the zoonotic pathogen Streptococcus suis

Abstract

NAD+ is an enzyme cofactor required for the 3 domains of life. However, little is known about the NAD+ biosynthesis and salvage pathways in the opportunistic pathogen Streptococcus suis. A genome‐wide search allows us to identify the NAD+ salvage pathway encoded by an operon of nadR‐pnuC‐nrtR (from SSU05_1973 to SSU05_1971 on the reverse strand) in the S. suis 05ZYH33 that causes streptococcal toxin shock–like syndrome. The regulator of this pathway is Nudix–related transcriptional regulator (NrtR), a transcription regulator of the Nudix family comprising an N‐terminal Nudix‐like effector domain, and a C‐terminal DNA‐binding winged helix‐turn‐helix–like domain. Intriguingly, the S. suis NrtR naturally contains a single amino acid substitution (K92E) in the catalytic site of its Nudix domain that renders it catalytically inactive but does not influence its ability to bind DNA. Despite its lack of enzymatic activity, DNA‐binding activity of NrtR is antagonized by the effector ADP‐ribose. Furthermore, nrtR knockout in S. suis serotype 2 reduces its capacity to form biofilms and attenuates its virulence in a mouse infection model. Genome mining indicates that nrtR appears in a strain‐specific manner whose occupancy is correlated to bacterial infectivity. Unlike the paradigmatic member of NrtR family having 2 unrelated functions (Nudix hydrolase and DNA binding), S. suis 2 retains a single regulatory role in the modulation of NAD+ salvage. This control of NAD+ homeostasis contributes to S. suis virulence.—Wang, Q., Hassan, B. H., Lou, N., Merritt, J., Feng, Y. Functional definition of NrtR, a remnant regulator of NAD+ homeostasis in the zoonotic pathogen Streptococcus suis. FASEB J. 33, 6055–6068 (2019). http://www.fasebj.org

A single regulator NrtR controls bacterial NAD+ homeostasis via its acetylation

“…A similar scenario in which the S. suis NrtR has an inactive Nudix hydrolase domain (Wang et al, 2019) allowed us to further hypothesize that most members of the Nudix-related regulator family might initially recruit ancient Nudix hydrolase as a signaling module, and then un-necessitate its enzymatic function while retaining its regulatory role as an evolutionary relic (Wang et al, 2019). The diversity of genomic organization of nrtR and its neighboring regulatory loci highlights that this gene is being subjected to dynamic domestication (Rodionov et al, 2008b).…”

“…We identified a 23-bp NrtR-binding palindrome conservatively located between the nrtR and nadA/B/C operons in mycobacteria (Figure 1C). The sequence of the NrtR-binding motif in Mycobacterium species [5′-GTTTTCGA-N7-TCGAAAAC-3′] is significantly different from that in Streptococcus [5′-ATA-N-TTTA-N3-TAAAA-N2-ATA-3′] (Wang et al, 2019) (Figure 1B). This may reflect the fact that these two NrtR homologs are not functionally exchangeable.…”

“…The diversity of genomic organization of nrtR and its neighboring regulatory loci highlights that this gene is being subjected to dynamic domestication (Rodionov et al, 2008b). Evidently, the nrtR is integrated into the ‘ nadR-pnuC-nrtR ’ cluster in the human pathogen S. suis 2, assuring a regulated salvage/recycling pathway (Rodionov et al, 2008b; Wang et al, 2019). By contrast, the nrtR on the opposite strand is adjacent to an operon of nadA/B/C (the intergenic region of which contains a NrtR-recognizable site; Figures 1A and 4A), guaranteeing the control of de novo NAD + synthesis (Figure 1D–E).…”

“…In fact, three types of regulatory systems have been described for NAD + biosynthesis and/or salvage. In addition to the two well-known regulatory proteins (NadR [Gerasimova and Gelfand, 2005; Raffaelli et al, 1999] and NiaR [Rodionov et al, 2008a]), a family of N udix- r elated t ranscriptional r egulators (NrtR) was initially proposed via bioinformatics (Rodionov et al, 2008b) and recently validated in Streptococcus suis (Wang et al, 2019). The paradigm NadR protein of Enterobacteriaceae is unusual in that it has three different functional domains (Grose et al, 2005): i) the N-terminal transcriptional repressor domain (Grose et al, 2005; Penfound and Foster, 1999); the central domain of a weak adenylyltransferase (Raffaelli et al, 1999; Grose et al, 2005), and the C-terminal domain of nicotinamide ribose kinase (Kurnasov et al, 2002; Grose et al, 2005).…”

“…NadR is a NAD + liganded regulator (Penfound and Foster, 1999), whereas NiaR is a nicotinic acid-responsive repressor in most species of Bacillus and Clostridium (Rodionov et al, 2008a). Although the prototypic NrtR possesses dual functions (Nudix-like hydrolase and DNA-binding/repressor) (Huang et al, 2009; Rodionov et al, 2008b), the NrtR homolog in S. suis seems to be an evolutionarily remnant regulator that lacks enzymatic activity (Wang et al, 2019). The phylogeny of NrtR suggests that it is widely distributed across diversified species (Huang et al, 2009; Rodionov et al, 2008b; Wang et al, 2019), and that its regulation of central NAD + metabolism contributes to the virulence of an opportunistic pathogen, Pseudomonas aeruginosa (Okon et al, 2017).…”