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Abstract: The tumoricidal activity of human α-lactalbumin complexes, such as HAMLET and its α-helical domain with sodium oleate, is well-documented. However, the potential of bacterial α-helical proteins to form analogous anticancer complexes remains unexplored. In the current study, we demonstrate that α-helical proteins of bacterial origin can form tumoricidal complexes with sodium oleate. Using non-hemolytic toxin A (NheA), an inactive component of the native tripartite (NheABC) toxin complex from Bacillus thuringiensis, we show that NheA, upon mixing with sodium oleate (NheA-O), forms potent tumoricidal complexes against colorectal cancer cells. The NheA-O complex binds to the plasma membrane of tumor cells, disrupting the function of cellular organelles and ultimately causing cell death. Mechanistically, NheA-O induces ACSL4 and suppresses GPX4 expression, which ultimately leads to the accumulation of lipid peroxidation, following suppression of β-catenin signaling. The suppression of β-catenin signaling and its target proteins ultimately leads to suppression of colorectal cancer tumorigenesis. Functionally, NheA-O inhibits tumor cell migration, spheroid formation, clonogenic potential, ATP production and induces lipid peroxidation. These findings establish that bacterial α-helical proteins, like their human counterparts, can be engineered to form tumoricidal complexes with sodium oleate. Our work highlights NheA-O as a novel candidate that causes activation of ferroptosis-like cell death in target cancer cells, leading to intracellular organelles dysfunction. Moreover, NheA-O activity synergizes with RSL3, and NheA-O mediated cell death is antagonized by Fer-1, indicating the role of NheA-O in inducing ferroptosis-like cell death. Overall, these results describe NheA-O as a novel therapeutic agent to combat tumorigenesis by targeting tumor cell membrane and proteasomal degradation of GPX4 to trigger ferroptosis-like cell death and expands the paradigm of tumoricidal protein-lipid complexes functionality across biological kingdoms. (Figure presented.)

Filifactor alocis is an emerging oral pathogen, and approximately 50% of known F.alocis strains encode and express a Repeats-in-Toxin (RTX) protein, FtxA. FtxAappears to be associated with both progress and severity of periodontal disease.Mechanisms are not yet known but could be linked to increased loads of F. alocisin ftxA-positive strains. Here, we investigated mechanistic correlations based onFtxA-activity, as present in F. alocis cells and extracellular vesicles and as arecombinant protein, exploiting THP-1 macrophage-like cells. For this, we usedthe ftxA-expressing strain, ATCC 35896 (ftxA+), and F. alocis 148B-17U (ftxA−),which naturally lacks the ftxA gene. Using RNA sequencing analysis (RNA-Seq) andcytokine array analysis, we have pinpointed a role of FtxA in shifting host responsetoward immunosuppression, also inhibiting apoptosis and immune cellrecruitment, and with a potential role in downregulating mitochondrial andoxidative phosphorylation pathways. Such role(s) could provide a plausibleexplanation why FtxA is associated with progress and severity of periodontaldisease, and further studies on FtxA-host cell interactions might reveal novelpotential therapeutic targets.

Pore-forming toxins (PFTs) are recognized as major virulence factors produced by both Gram-positive and Gram-negative bacteria. While the effects of PFTs have been extensively investigated using mammalian cells as a model system, their interactions with the environmental host, Acanthamoeba castellanii remains less understood. This study employed high-throughput image screening (HTI), advanced microscopy, western blot analysis, and cytotoxicity assays to evaluate the impact of PFT-producing bacterial species on their virulence against A. castellanii. Our unbiased HTI data analysis reveals that the cyst induction of A. castellanii in response to various bacterial species does not correlate with the presence of PFT-producing bacteria. Moreover, A. castellanii demonstrates resistance to PFT-mediated cytotoxicity, in contrast to mammalian macrophages. Notably, Vibrio anguillarum and Ralstonia eutropha triggered a high frequency of cyst formation and cytotoxicity in infected A. castellanii. In summary, our findings reveal that A. castellanii exhibits a unique resistance to PFTs, unlike mammalian cells, suggesting its potential ecological role as a reservoir for diverse pathogenic species and its influence on their persistence and proliferation in the environment. (Figure presented.)