A New Contact Killing Toxin Permeabilizes Cells and Belongs to a Broadly Distributed Protein Family

• Published in: mSphere 2021-08, Vol.6 (4), p.e0031821
• Main Authors: Crisan, Cristian V, Chandrashekar, Harshini, Everly, Catherine, Steinbach, Gabi, Hill, Shannon E, Yunker, Peter J, Lieberman, Raquel R, Hammer, Brian K
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - genetics; Bacterial Toxins - classification; Bacterial Toxins - genetics; Bacterial Toxins - metabolism; Escherichia coli - genetics; Interspersed Repetitive Sequences; Microbial Genetics; Multigene Family; Research Article; Type VI Secretion Systems - metabolism; Vibrio cholerae - classification; Vibrio cholerae - genetics
• ISSN: 2379-5042; 2379-5042
• DOI: 10.1128/mSphere.00318-21

Vibrio cholerae is an aquatic Gram-negative bacterium that causes severe diarrheal cholera disease when ingested by humans. To eliminate competitor cells in both the external environment and inside hosts, V. cholerae uses the type VI secretion system (T6SS). The T6SS is a macromolecular contact-dependent weapon employed by many Gram-negative bacteria to deliver cytotoxic proteins into adjacent cells. In addition to canonical T6SS gene clusters encoded by all sequenced V. cholerae isolates, strain BGT49 encodes another locus, which we named auxiliary (Aux) cluster 4. The Aux 4 cluster is located on a mobile genetic element and can be used by killer cells to eliminate both V. cholerae and Escherichia coli cells in a T6SS-dependent manner. A putative toxin encoded in the cluster, which we name TpeV ( ype VI ermeabilizing ffector ), shares no homology to known proteins and does not contain motifs or domains indicative of function. Ectopic expression of TpeV in the periplasm of E. coli permeabilizes cells and disrupts the membrane potential. Using confocal microscopy, we confirm that susceptible target cells become permeabilized when competed with killer cells harboring the Aux 4 cluster. We also determine that , the gene located immediately downstream of , encodes an immunity protein that neutralizes the toxicity of TpeV. Finally, we show that TpeV homologs are broadly distributed across important human, animal, and plant pathogens and are localized in proximity to other T6SS genes. Our results suggest that TpeV is a toxin that belongs to a large family of T6SS proteins. Bacteria live in polymicrobial communities where competition for resources and space is essential for survival. Proteobacteria use the T6SS to eliminate neighboring cells and cause disease. However, the mechanisms by which many T6SS toxins kill or inhibit susceptible target cells are poorly understood. The sequence of the TpeV toxin that we describe here is unlike any previously described protein. We demonstrate that it has antimicrobial activity by permeabilizing cells, eliminating membrane potentials, and causing severe cytotoxicity. TpeV homologs are found near known T6SS genes in human, animal, and plant bacterial pathogens, indicating that the toxin is a representative member of a broadly distributed protein family. We propose that TpeV-like toxins contribute to the fitness of many bacteria. Finally, since antibiotic resistance is a critical global health threat, the discovery of new anti