Biology subjects

Shalom, M.

Publications and source records attributed to Shalom, M..

4 recordsLinked to original sources

bioRxiv2026

Kingdom-wide Discovery of Type VI Secretion Effectors via AI Uncovers Three New Bacterial Toxins

The type VI secretion system (T6SS) is a molecular harpoon used by 19% of bacteria to inject toxic proteins into microbial or host cells. Despite its widespread distribution, many T6SS effector (T6Es) genes remain unidentified. Here, we developed an XGBoost classifier integrating 42 genetic and biochemical features to identify novel T6Es. Applying our model to 2,559 T6SS-encoding bacterial genomes revealed 19,958 T6E candidates. We validated our computational predictions and confirmed that three effector candidates, having new 3D structures, demonstrated cytotoxicity to bacteria and yeast, mostly in the bacterial periplasm. For two of these, cognate immunity proteins neutralizing toxicity have been validated. T6EC11, a new peptidoglycan degrading effector, quickly leads to spherical E. coli, with inner membrane separation, and gradual peptidoglycan degradation in a unipolar manner, demonstrating an intriguing moon-like structural phenotype. T6EC12, an effector toxic to bacteria and yeast, carries a conserved zinc-binding motif. Mutations the motif markedly reduced toxicity, suggesting that T6EC12 represents a novel metalloprotease. Our study expands the known T6E repertoire, and provides thousands of T6E predictions in all T6SS-encoding bacteria.

microbiology↗

bioRxiv2026

A modular platform for scalable recombinant production of highly toxic bacterial proteins

Bacterial protein toxins constitute a vast and largely untapped reservoir of antimicrobial activities with substantial therapeutic and biotechnological potential. However, their intrinsic toxicity frequently prevents stable recombinant expression in bacterial hosts, creating a major bottleneck for biochemical characterization, structural analysis, and development as antimicrobial agents. Here, we present a modular platform for the scalable recombinant production of highly toxic bacterial proteins based on transient intramolecular toxin neutralization. The strategy covalently links each toxin to its cognate immunity protein, promoting neutralization during biosynthesis while permitting recovery of the native toxin through site-specific proteolytic cleavage. Using this approach, we produced multiple previously intractable polymorphic toxin domains that could not be obtained using conventional inducible expression, toxin-immunity co-expression, or bacterial cell-free systems. We further streamlined the production workflow through intracellular protease-mediated cleavage, reducing the purification process from four steps to two and increasing protein recovery. To address cases in which native immunity proteins were insufficient, we incorporated computational protein design to engineer improved toxin-binding partners, enabling production of an additional toxin that remained refractory to the original platform. Purified toxins retained enzymatic activity following denaturation and refolding, confirming recovery of functional proteins and enabling identification of a previously uncharacterized nuclease activity. Together, these findings establish a scalable and adaptable microbial biotechnology platform for the production of intrinsically toxic proteins. The integration of transient intramolecular neutralization with computational engineering provides a route toward systematic production and characterization of toxic proteins for antimicrobial discovery, structural biology, protein engineering, and future biotechnological applications.

biochemistry↗

bioRxiv2023

A genomic and structural bioinformatic pipeline identifies candidate type VI secretion antibacterial effector-immunity pairs

Type VI secretion systems (T6SS) are common bacterial contractile injection systems that inject toxic "effector" proteins into neighboring cells. We bioinformatically investigated T6SS core proteins in 11,832 genomes of Gram negative bacteria. Comparison of T6SS core proteins that are covalently attached to toxic T6SS effector proteins (T6Es) versus those that are not revealed differences in phylogenetic distribution, physical properties, and genomic position. Using the data generated from our bioinformatic analysis, we developed a new genomic- and Alphafold2-based pipeline for discovery of putative T6Es. We experimentally validated the toxic and immunity activities of four putative antibacterial T6SS effector proteins and four cognate immunity genes from diverse species, respectively. We used Foldseek to predict possible mechanisms of action of the putative T6Es, which was much more effective than sequence-based methods. Evidence of the possible mechanisms of action of the putative T6Es was explored through fluorescence microscopy, where we observed cell wall-targeting, DNA degradation, and cell filamentation. This study shows how combining genomic data mining with new structure-based bioinformatic tools can facilitate identification of novel antibacterial toxins.

microbiology↗

bioRxiv2021

Systematic Discovery of Antimicrobial Polymorphic Toxins

Microbes employ toxins to kill competing microbes or eukaryotic host cells. Polymorphic toxins are proteins that encode C-terminal toxin domains. Here, we developed a computational approach to discover novel toxin domains of polymorphic toxins within 105,438 microbial genomes. We validated nine short novel toxins ("PTs") that cause bacterial or yeast cell death. The novel PTs are encoded by [~]2.2% of the sequenced bacteria, including numerous pathogens. We also identified five cognate immunity genes ("PIMs") that neutralize the toxins. Intriguingly, we observed an antifungal effect of the PTs against various pathogenic fungi. The toxins likely act as enzymes that cause severe damage to cell shape, membrane, and DNA. Finally, we solved the 3D structure of two PTs in complex with their PIMs, and showed that they function as novel DNAses. The new potent toxins likely play key roles in inter-microbial competition and can be utilized in various clinical and biotechnological applications.

microbiology↗