Phage detection by a bacterial NLR-related protein is mediated by DnaJ

Aaron Whiteley — Tue, 04 Jun 2024 15:00:00 +0000

Phage detection by a bacterial NLR-related protein is mediated by DnaJ Aaron Whiteley Tue, 06/04/2024 - 09:00

Amy Conte , Madison Ruchel , Samantha Ridgeway , Emily Kibby , Toni Nagy , Aaron Whiteley

BioRxiv (2024). PubMed PMID: 38895412; PubMed Central PMCID: PMC11185742.

Abstract

Bacteria encode a wide range of antiphage systems and a subset of these proteins are homologous to components of the human innate immune system. Mammalian nucleotide-binding and leucine-rich repeat containing proteins (NLRs) and bacterial NLR-related proteins use a central NACHT domain to link detection of infection with initiation of an antimicrobial response. Bacterial NACHT proteins provide defense against both DNA and RNA phages. Here we determine the mechanism of RNA phage detection by the bacterial NLR-related protein bNACHT25 in E. coli. bNACHT25 was specifically activated by Emesvirus ssRNA phages and analysis of MS2 phage escaper mutants that evaded detection revealed a critical role for Coat Protein (CP). A genetic assay confirmed CP was sufficient to activate bNACHT25 but the two proteins did not directly interact. Instead, we found bNACHT25 requires the host chaperone DnaJ to detect CP. Our data suggest that bNACHT25 detects a wide range of phages by guarding a host cell process rather than binding a specific phage-derived molecule.

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Citation

Conte AN, Ridgeway SM, Ruchel ME, Kibby EM, Nagy TA, Whiteley AT. Phage detection by a bacterial NLR-related protein is mediated by DnaJ. bioRxiv. 2024 Jun 4;. doi: 10.1101/2024.06.04.597415. PubMed PMID: 38895412; PubMed Central PMCID: PMC11185742.

Conte AN, Ridgeway SM, Ruchel ME, Kibby EM, Nagy TA, ➤Whiteley, AT | BioRxiv 2024

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(p)ppGpp and c-di-AMP Homeostasis Is Controlled by CbpB in Listeria monocytogenes.

Anonymous — Tue, 25 Aug 2020 15:00:00 +0000

(p)ppGpp and c-di-AMP Homeostasis Is Controlled by CbpB in Listeria monocytogenes. Anonymous (not verified) Tue, 08/25/2020 - 09:00

Peterson BN , Young MKM , Luo S , Wang J , ➤Whiteley AT , Woodward JJ , Tong L , Wang JD , Portnoy DA

mBio 2020 Aug 25;11(4):e01625-20. doi: 10.1128/mBio.01625-20.

Abstract

The facultative intracellular pathogen Listeria monocytogenes, like many related Firmicutes, uses the nucleotide second messenger cyclic di-AMP (c-di-AMP) to adapt to changes in nutrient availability, osmotic stress, and the presence of cell wall-acting antibiotics. In rich medium, c-di-AMP is essential; however, mutations in cbpB, the gene encoding c-di-AMP binding protein B, suppress essentiality. In this study, we identified that the reason for cbpB-dependent essentiality is through induction of the stringent response by RelA. RelA is a bifunctional RelA/SpoT homolog (RSH) that modulates levels of (p)ppGpp, a secondary messenger that orchestrates the stringent response through multiple allosteric interactions. We performed a forward genetic suppressor screen on bacteria lacking c-di-AMP to identify genomic mutations that rescued growth while cbpBwas constitutively expressed and identified mutations in the synthetase domain of RelA. The synthetase domain of RelA was also identified as an interacting partner of CbpB in a yeast-2-hybrid screen. Biochemical analyses confirmed that free CbpB activates RelA while c-di-AMP inhibits its activation. We solved the crystal structure of CbpB bound and unbound to c-di-AMP and provide insight into the region important for c-di-AMP binding and RelA activation. The results of this study show that CbpB completes a homeostatic regulatory circuit between c-di-AMP and (p)ppGpp in Listeria monocytogenes

IMPORTANCE Bacteria must efficiently maintain homeostasis of essential molecules to survive in the environment. We found that the levels of c-di-AMP and (p)ppGpp, two nucleotide second messengers that are highly conserved throughout the microbial world, coexist in a homeostatic loop in the facultative intracellular pathogen Listeria monocytogenes Here, we found that cyclic di-AMP binding protein B (CbpB) acts as a c-di-AMP sensor that promotes the synthesis of (p)ppGpp by binding to RelA when c-di-AMP levels are low. Addition of c-di-AMP prevented RelA activation by binding and sequestering CbpB. Previous studies showed that (p)ppGpp binds and inhibits c-di-AMP phosphodiesterases, resulting in an increase in c-di-AMP. This pathway is controlled via direct enzymatic regulation and indicates an additional mechanism of ribosome-independent stringent activation.

Keywords

bacteria; cyclic dinucleotide; stringent response

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Phage detection by a bacterial NLR-related protein is mediated by DnaJ

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(p)ppGpp and c-di-AMP Homeostasis Is Controlled by CbpB in Listeria monocytogenes.

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