Unique Features of Alarmone Metabolism in Clostridioides difficile

Abstract

Despite the role of the stringent response in antibiotic survival and recurrent infections, it has been a challenging target for antibacterial therapies because it is so ubiquitous. This is an especially relevant consideration for the treatment of Clostridioides difficile infection (CDI), as exposure to broad-spectrum antibiotics that harm commensal microbes is a major risk factor for CDI.

Metabolic Promiscuity of an Orphan Small Alarmone Hydrolase Facilitates Bacterial Environmental Adaptation

“…The presence of pGpp suggests that X. campestris pv. campestris RSHs may be able to produce pGpp, as recently reported in Clostridiodes difficile ( 19 ), or that it contains Nudix hydrolases that can convert (p)ppGpp to pGpp, similar to NahA in B. subtilis ( 12 ). Importantly, the Δ sah mutant showed elevation of pGpp ( Fig.…”

“…Alarmone synthetases and hydrolases belong to the RelA/SpoT homologs (RSHs) superfamily ( 16 ), which is further classified into multidomain “long” RSHs or single-domain homologs known as small alarmone synthetases (SASs), or small alarmone hydrolases (SAHs) ( 10 , 15 ). Long RSHs include the synthetase RelA ( 1 ) and the bifunctional synthetase and hydrolase Rel ( 17 ) and SpoT ( 18 ), which produce (p)ppGpp and in some cases pGpp ( 19 ). Examples of SASs include SasA (RelP), SasB (RelQ), and RelV ( 20 – 23 ), which produce (p)ppGpp and in some cases pGpp ( 24 ) or (p)ppApp ( 25 ).…”

Sound the (Smaller) Alarm: The Triphosphate Magic Spot Nucleotide pGpp

“…Pyrophosphotransfer to mono-, di-, and triphosphate adenosine and guanosine molecules has been documented for decades, but only (p)ppGpp was consistently detected in vivo and conclusively linked to a phenotype, and the small amounts of pGpp detected in some assays were easy to dismiss as artifacts or unimportant ( 41 ). It has recently been confirmed that some synthetase enzymes are capable of utilizing GMP as a substrate to synthesize pGpp directly ( 13 , 33 , 37 , 39 , 40 , 45 to 47 ). In addition, hydrolase enzymes can selectively remove 5′-phosphates from longer guanosine alarmones ( 29 , 33 , 43 , 48 to 50 ).…”

“…1 ). Most synthetase enzymes will utilize either GDP or GTP as the substrate but have a higher affinity for one of the nucleotides ( 13 , 34 to 40 ). As the ratio of GDP to GTP in bacterial cytoplasm is a dynamic parameter that changes quickly in response to nutrient conditions, the relative production of ppGpp and pppGpp could reflect substrate availability when alarmone synthesis is activated ( 26 , 29 ).…”

“…However, evidence quickly emerged that pGpp is an independently active regulator rather than a merely tolerable substitute for (p)ppGpp. Synthetases that generate pGpp preferentially or exclusively have been characterized, and putative effectors that bind pGpp preferentially or exclusively have been identified in pulldown assays ( 31 , 39 , 40 ).…”