Biology subjects

Nicastro, G. G.

Publications and source records attributed to Nicastro, G. G..

8 recordsLinked to original sources

bioRxiv2026

An RNase Toxin Hijacks Elongation Factor-Tu to Cleave the Ribosomal Sarcin-Ricin Loop

Ribosome-targeting toxins typically recognize and attack their substrates directly. Here, we provide the functional characterization of SRLase1, a widespread member of a recently identified RNase superfamily, and show that it cleaves the sarcin-ricin loop (SRL) of 23S rRNA. Unlike previously described ribosome-targeting toxins, SRLase1 requires the host elongation factor Tu (EF-Tu) for activity. EF-Tu normally engages the ribosome as part of the aminoacyl-tRNA delivery cycle, but SRLase1 instead exploits this factor through a mechanism incompatible with canonical ternary-complex formation. This host-factor-assisted mechanism reveals a distinct strategy for translation inhibition, in which a toxin repurposes a conserved component of the elongation machinery to access a vulnerable ribosomal target. Homologs of SRLase1 are widespread across bacterial phyla and are frequently encoded in conflict-associated genomic loci. Together, these findings define a widespread family of SRL-targeting toxins, expand the mechanistic repertoire of ribosomal antibacterial toxins, and demonstrate how conserved components of the host translation machinery can be co-opted to promote toxin activity.

microbiology↗

bioRxiv2026

A structure-guided classification framework reveals the diversity and catalytic architecture of BECR ribonuclease

Microorganisms across all domains of life engage in molecular conflict, deploying toxins to inhibit competitors or respond to biological threats. Among these, ribonuclease toxins are particularly widespread and diverse. A substantial fraction is associated with the BECR fold, a compact /{beta} architecture that supports RNase activity despite extensive divergence. Although several canonical members are well characterized, many BECR-fold proteins remain difficult to identify because of low sequence similarity, variation in catalytic residues, and structural elaborations that obscure evolutionary relationships. The growing availability of high-confidence protein structure predictions provides an opportunity to reassess this deeply divergent protein landscape. Here, we integrate iterative profile-HMM searches, profile-similarity networks, structural analyses, active-site mapping, and genomic context to examine BECR proteins across the tree of life. Our analysis resolves an expanded BECR-fold landscape comprising canonical BECR and BECR-like superfamilies, refines the organization of canonical BECR proteins and identifies previously unrecognized families. We further validate BECR-Tox2 as a toxin neutralized by a cognate immunity protein and show that its homologs occur in both Menshen-like anti-phage systems and polymorphic toxin loci. Together, these findings expand and clarify the BECR-fold landscape and provide a framework for identifying and interpreting highly divergent proteins of this fold.

microbiology↗

bioRxiv2026

A methylome-derived m6-dAMP trigger assembles a PUA-Cal-HAD immune filament that depletes dNTPs to abort phage infection

Bacteria must distinguish phage attack from normal homeostatic processes, yet the danger signals that trigger many defence systems remain unknown. Here, we show that a PUA-Calcineurin-CE-HAD module from Escherichia coli ECOR28 confers broad anti-phage protection by binding Dam-methylated deoxyadenosine monophosphate (m6-dAMP) generated during phage-induced chromosome degradation. Ligand binding converts a preassembled PUA-Calcineurin-CE hexamer loaded with six HAD phosphatases into a polymerising filament. The filament acts as a high-flux dNTP sink through a two-enzyme cascade: HAD first dephosphorylates dATP to dADP, and Calcineurin-CE then converts dADP to dAMP. dNTP collapse halts phage replication and enforces abortive infection. Multiple mobile-element DNA mimic proteins block filament assembly, revealing a direct phage counter-defence. More broadly, our findings extend a conserved, cross-kingdom paradigm of immune filament assembly to nucleotide-depletion antiviral defence and suggest modified-nucleotide sensing by related PUA-Calcineurin-CE modules as a widespread, underappreciated bacterial strategy.

microbiology↗

bioRxiv2025

The Lipocone Superfamily: A Unifying Theme In Metabolism Of Lipids, Peptidoglycan And Exopolysaccharides, Inter-Organismal Conflicts And Immunity

Wnt proteins are critical signaling molecules in developmental processes across animals. Despite intense study, their evolutionary roots have remained enigmatic. Using sensitive sequence analysis and structure modeling, we establish that the Wnts are part of a vast assemblage of domains, the Lipocone superfamily, defined here for the first time. It includes previously studied enzymatic domains like the phosphatidylserine synthases (PTDSS1/2) and the TelC toxin domain from Streptococcus intermedius, the enigmatic VanZ proteins, the animal Serum Amyloid A (SAA) and a further host of uncharacterized proteins in a total of 30 families. Although the metazoan Wnts are catalytically inactive, we present evidence for a conserved active site across this superfamily, versions of which are consistently predicted to operate on head groups of either phospholipids or polyisoprenoid lipids, catalyzing transesterification and phosphate-containing head group cleavage reactions. We argue that this superfamily originated as membrane proteins, with one branch (including Wnt and SAA) evolving into diffusible versions. By comprehensively analyzing contextual information networks derived from comparative genomics, we establish that they act in varied functional contexts, including regulation of membrane lipid composition, extracellular polysaccharide biosynthesis, and biogenesis of bacterial outer-membrane components, like lipopolysaccharides. On multiple occasions, members of this superfamily, including the bacterial progenitors of Wnt and SAA, have been recruited as effectors in biological conflicts spanning inter-organismal interactions and anti-viral immunity in both prokaryotes and eukaryotes. These findings establish a unifying theme in lipid biochemistry, explain the origins of Wnt signaling and provide new leads regarding immunity across the tree of life. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/632903v3_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@d3bf0dorg.highwire.dtl.DTLVardef@1a3f929org.highwire.dtl.DTLVardef@3b20b9org.highwire.dtl.DTLVardef@1e16029_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2024

Genome-directed study uncovers a family of phospholipases and highlights the diversity of Salmonella T6SS effectors.

Bacterial warfare is a common and ancient phenomenon in nature, where bacterial species use strategies to inhibit the growth or kill competitors. This involves the production and deployment of antibacterial toxins that disrupt essential cellular processes in target cells. The continuous arms race in which bacteria acquire new toxin and immunity proteins to promote increased adaptation to their environment is responsible for the diversification of this toxin repertoire. Here, we deployed in-silico strategies to analyze 10,000 genomes and identify effectors secreted via the type VI secretion system of Salmonella. We identified 128 candidates, which are widespread in a vast array of Salmonella serovars and other bacterial species. Tox-Act1 is among the most frequent candidates and was selected for in-depth characterization. Tox-Act1 contains a permuted NlpC/P60 papain-like catalytic core characteristic of lipid-targeting members rather than the typical peptidases or amidases. Evolutionary analysis revealed the relationship of Tox-Act1 with acyltransferases. Biochemical assays with purified toxin and lipidomics of intoxicated cells showed that Tox-Act1 exhibits phospholipase activity, cleaving off acyl groups from phosphatidylglycerol and phosphatidylethanolamine. In addition, we demonstrate that Tox-Act1 is secreted in a T6SS-dependent manner and provide a competitive advantage during colonization of the gut of infected mice. This work broadens our understanding of toxin domains and provides the first direct characterization of a lipid-targeting NlpC/P60 domain in biological conflicts.

microbiology↗

bioRxiv2021

Antibacterial T6SS effectors with a VRR-Nuc domain induce target cell death via DNA Double-Strand Breaks

The T6SS (Type VI secretion System) secretes antibacterial effectors into target competitors. Salmonella spp. encode five phylogenetically distinct T6SSs. Here we characterize the function of the SPI-22 T6SS of S. bongori, showing that it has antibacterial activity. We identify a group of antibacterial T6SS effectors (TseV1-4) containing an N-terminal PAAR-like domain and a C-terminal VRR-Nuc domain encoded next to cognate immunity proteins that contain the DUF3396 domain (TsiV1-4). TseV2 and TseV3 are toxic when expressed in Escherichia coli and bacterial competition assays confirm that TseV2 and TseV3 are secreted by the SPI-22 T6SS. Phylogenetic analysis reveals that TseV1-4 are evolutionarily related to enzymes involved in DNA repair. TseV2 and TseV3 maintained the ability to bind DNA, but instead cause specific DNA double-strand breaks and induce the SOS response in target cells. The crystal structure of the TseV3:TsiV3 complex reveals that the immunity protein likely blocks the effector interaction with the DNA substrate. These results expand our knowledge on the function of Salmonella pathogenicity islands, the evolution of toxins used in biological conflicts, and the endogenous mechanism regulating the activity of these toxins.

microbiology↗

bioRxiv2020

A Superfamily of T6SS Antibacterial Effectors Displaying L,D-carboxypeptidase Activity Towards Peptidoglycan

Type VI secretion systems (T6SSs) are contractile nanomachines used by bacteria to inject toxic effectors into competitors. The identity and mechanism of many effectors remain unknown. We characterized a Salmonella SPI-6 T6SS antibacterial effector called Tae5STM (type VI amidase effector 5). Tae5STM is toxic in target-cell periplasm and is neutralized by its cognate immunity protein (Tai5STM). Microscopy analysis revealed that cells expressing the effector stop dividing and lose cell envelope integrity. Bioinformatic analysis uncovered similarities between Tae5STM and the catalytic domain of L,D-transpeptidase. Point mutations on conserved catalytic histidine and cysteine residues abrogated toxicity. Biochemical assays revealed that Tae5STM displays L,D-carboxypeptidase activity, cleaving peptidoglycan tetrapeptides between meso-diaminopimelic acid3 and D-alanine4. Phylogenetic analysis showed that Tae5STM homologs constitutes a new superfamily of T6SS-associated amidase effectors distributed among -, {beta}- and {gamma}-proteobacteria. This work expands our current knowledge about bacterial effectors used in interbacterial competition.

microbiology↗

bioRxiv2017

c-di-GMP-linked phenotypes are modulated by the interaction between a diguanylate cyclase and a polar hub protein

c-di-GMP is a major player in the decision between biofilm and motile lifestyles. Several bacteria present a large number of c-di-GMP metabolizing proteins, thus a fine-tuning of this nucleotide levels may occur. It is hypothesized that some c-di-GMP metabolizing proteins would provide the global c-di-GMP levels inside the cell whereas others would maintain a localized pool, with the resulting c-di-GMP acting at the vicinity of its production. Although attractive, this hypothesis has yet to be proven in Pseudomonas aeruginosa. We found that the diguanylate cyclase DgcP interacts with the cytosolic region of FimV, a peptidoglycan-binding protein involved in type IV pilus assembly. Moreover, DgcP is located at the cell poles in wild type cells, but scattered in the cytoplasm of cells lacking FimV. Overexpression of DgcP leads to the classical phenotypes of high c-di-GMP levels (increased biofilm and impaired motilities) in the wild-type strain, but not in a AfimV background. Therefore, our findings strongly suggest that DgcP is regulated by FimV and may provide the local c-di-GMP pool that can be sensed by other proteins at the cell pole, bringing to light a specialized function for a specific diguanylate cyclase.

microbiology↗