innate immunity

Bacterial NLR-related proteins protect against phage

Anonymous — Mon, 08 May 2023 15:17:54 +0000

Bacterial NLR-related proteins protect against phage Anonymous (not verified) Mon, 05/08/2023 - 09:17

Emily Kibby Amy Conte A. Maxwell Burroughs Toni Nagy Alfredo Vargas Lindsay Whalen L. Aravind Aaron Whiteley

Cell (2023). https://doi.org/10.1016/j.cell.2023.04.015

BioRxiv Preprint, July 20 2022, https://www.biorxiv.org/content/10.1101/2022.07.19.500537v1

Abstract

Bacteria use a wide range of immune pathways to counter phage infection. A subset of these genes shares homology with components of eukaryotic immune systems, suggesting that eukaryotes horizontally acquired certain innate immune genes from bacteria. Here, we show that proteins containing a NACHT module, the central feature of the animal nucleotide-binding domain and leucine-rich repeat containing gene family (NLRs), are found in bacteria and defend against phages. NACHT proteins are widespread in bacteria, provide immunity against both DNA and RNA phages, and display the characteristic C-terminal sensor, central NACHT, and N-terminal effector modules. Some bacterial NACHT proteins have domain architectures similar to the human NLRs that are critical components of inflammasomes. Human disease-associated NLR mutations that cause stimulus-independent activation of the inflammasome also activate bacterial NACHT proteins, supporting a shared signaling mechanism. This work establishes that NACHT module-containing proteins are ancient mediators of innate immunity across the tree of life.

News and Commentaries

Ancient mediators of innate immunity [SBGrid]
Cross-kingdom defence proteins [Nature Reviews Microbiology]

Analysis of human cGAS activity and structure

Anonymous — Thu, 02 May 2019 06:00:00 +0000

Analysis of human cGAS activity and structure Anonymous (not verified) Thu, 05/02/2019 - 00:00

Zhou W ➤Whiteley AT Kranzusch PJ

Methods Enzymol. 2019;625:13-40. doi: 10.1016/bs.mie.2019.04.012. Epub 2019 May 2.

Abstract

Cyclic GMP-AMP synthase (cGAS) is an innate immune system enzyme responsible for recognition of double-stranded DNA aberrantly localized in the cell cytosol. cGAS binds DNA and is activated to catalyze production of the nucleotide second messenger 2'-5'/3'-5' cyclic GMP-AMP (2'3' cGAMP). In spite of a major role for cGAS in the cellular immune response, a complete understanding of cGAS biology has been limited by a lack of genetic tools to rapidly screen cGAS activity, instability of human cGAS-DNA interactions in vitro, and a previous absence of structural information for the human cGAS-DNA complex. Here we detail procedures to map the molecular determinants of cGAS activation and describe methods developed to prepare human cGAS-DNA crystals for structural analysis. Together with earlier systems established to study mammalian homologs of cGAS, these innovations provide a foundation to understand and therapeutically target human cGAS biology.

Bacterial cGAS-like enzymes synthesize diverse nucleotide signals

Anonymous — Fri, 01 Mar 2019 07:00:00 +0000

Bacterial cGAS-like enzymes synthesize diverse nucleotide signals Anonymous (not verified) Fri, 03/01/2019 - 00:00

➤Whiteley AT Eaglesham JB de Oliveira Mann CC Morehouse BR Lowey B Nieminen EA Danilchanka O King DS Lee ASY Mekalanos JJ* Kranzusch PJ*

*co-corresponding authors

Nature. 2019 Mar;567(7747):194-199. doi: 10.1038/s41586-019-0953-5. Epub 2019 Feb 20.

Abstract

Cyclic dinucleotides (CDNs) have central roles in bacterial homeostasis and virulence by acting as nucleotide second messengers. Bacterial CDNs also elicit immune responses during infection when they are detected by pattern-recognition receptors in animal cells. Here we perform a systematic biochemical screen for bacterial signalling nucleotides and discover a large family of cGAS/DncV-like nucleotidyltransferases (CD-NTases) that use both purine and pyrimidine nucleotides to synthesize a diverse range of CDNs. A series of crystal structures establish CD-NTases as a structurally conserved family and reveal key contacts in the enzyme active-site lid that direct purine or pyrimidine selection. CD-NTase products are not restricted to CDNs and also include an unexpected class of cyclic trinucleotide compounds. Biochemical and cellular analyses of CD-NTase signalling nucleotides demonstrate that these cyclic di- and trinucleotides activate distinct host receptors and thus may modulate the interaction of both pathogens and commensal microbiota with their animal and plant hosts.

News and Commentaries

Pyrimidines and Cyclic Trinucleotides Join the Second Messenger Symphony. [Cell host & microbe 2019]
Diverse bacterial nucleotide signals. [Science Signaling 2019]
Highlighted and discussed in This Week In Microbiology Podcast Episode #206

Links

➤Whiteley AT, Eaglesham JB, de Oliveira Mann CC, Morehouse BR, Lowey B, Nieminen EA, Danilchanka O, King DS, Lee ASY, Mekalanos JJ*, Kranzusch PJ* | Nature. 2019

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Structure of the Human cGAS-DNA Complex Reveals Enhanced Control of Immune Surveillance

Anonymous — Thu, 12 Jul 2018 06:00:00 +0000

Structure of the Human cGAS-DNA Complex Reveals Enhanced Control of Immune Surveillance Anonymous (not verified) Thu, 07/12/2018 - 00:00

Zhou W* ➤Whiteley AT* de Oliveira Mann CC Morehouse BR Nowak RP Fischer ES Gray NS Mekalanos JJ Kranzusch PJ

*These authors contributed equally to this work

Cell. 2018 Jul 12;174(2):300-311.e11. doi: 10.1016/j.cell.2018.06.026.

Abstract

Cyclic GMP-AMP synthase (cGAS) recognition of cytosolic DNA is critical for immune responses to pathogen replication, cellular stress, and cancer. Existing structures of the mouse cGAS-DNA complex provide a model for enzyme activation but do not explain why human cGAS exhibits severely reduced levels of cyclic GMP-AMP (cGAMP) synthesis compared to other mammals. Here, we discover that enhanced DNA-length specificity restrains human cGAS activation. Using reconstitution of cGAMP signaling in bacteria, we mapped the determinant of human cGAS regulation to two amino acid substitutions in the DNA-binding surface. Human-specific substitutions are necessary and sufficient to direct preferential detection of long DNA. Crystal structures reveal why removal of human substitutions relaxes DNA-length specificity and explain how human-specific DNA interactions favor cGAS oligomerization. These results define how DNA-sensing in humans adapted for enhanced specificity and provide a model of the active human cGAS-DNA complex to enable structure-guided design of cGAS therapeutics.

Comment in

Human cGAS Has a Slightly Different Taste for dsDNA. [Immunity. 2018]

Links

Zhou W*, ➤Whiteley AT*, de Oliveira Mann CC, Morehouse BR, Nowak RP, Fischer ES, Gray NS, Mekalanos JJ, Kranzusch PJ. | Cell. 2018

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Cyclic di-AMP is critical for Listeria monocytogenes growth, cell wall homeostasis, and establishment of infection

Anonymous — Tue, 28 May 2013 06:00:00 +0000

Cyclic di-AMP is critical for Listeria monocytogenes growth, cell wall homeostasis, and establishment of infection Anonymous (not verified) Tue, 05/28/2013 - 00:00 Witte CE ➤Whiteley AT Burke TP Sauer JD Portnoy DA Woodward JJ.

mBio. 2013 May 28;4(3):e00282-13. doi: 10.1128/mBio.00282-13.

Abstract

Listeria monocytogenes infection leads to robust induction of an innate immune signaling pathway referred to as the cytosolic surveillance pathway (CSP), characterized by expression of beta interferon (IFN-β) and coregulated genes. We previously identified the IFN-β stimulatory ligand as secreted cyclic di-AMP. Synthesis of c-di-AMP in L. monocytogenes is catalyzed by the diadenylate cyclase DacA, and multidrug resistance transporters are necessary for secretion. To identify additional bacterial factors involved in L. monocytogenes detection by the CSP, we performed a forward genetic screen for mutants that induced altered levels of IFN-β. One mutant that stimulated elevated levels of IFN-β harbored a transposon insertion in the gene lmo0052. Lmo0052, renamed here PdeA, has homology to a cyclic di-AMP phosphodiesterase, GdpP (formerly YybT), of Bacillus subtilis and is able to degrade c-di-AMP to the linear dinucleotide pApA. Reduction of c-di-AMP levels by conditional depletion of the di-adenylate cyclase DacA or overexpression of PdeA led to marked decreases in growth rates, both in vitro and in macrophages. Additionally, mutants with altered levels of c-di-AMP had different susceptibilities to peptidoglycan-targeting antibiotics, suggesting that the molecule may be involved in regulating cell wall homeostasis. During intracellular infection, increases in c-di-AMP production led to hyperactivation of the CSP. Conditional depletion of dacA also led to increased IFN-β expression and a concomitant increase in host cell pyroptosis, a result of increased bacteriolysis and subsequent bacterial DNA release. These data suggest that c-di-AMP coordinates bacterial growth, cell wall stability, and responses to stress and plays a crucial role in the establishment of bacterial infection.

Links

Witte CE, Whiteley AT, Burke TP, Sauer JD, Portnoy DA, Woodward JJ. | mBio. 2013

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