The Antibacterial and Anti-Eukaryotic Type VI Secretion System MIX-Effector Repertoire in Vibrionaceae

Abstract

Vibrionaceae is a widespread family of aquatic bacteria that includes emerging pathogens and symbionts. Many Vibrionaceae harbor a type VI secretion system (T6SS), which is a secretion apparatus used to deliver toxins, termed effectors, into neighboring cells. T6SSs mediate both antibacterial and anti-eukaryotic activities. Notably, antibacterial effectors are encoded together with a gene that encodes a cognate immunity protein so as to antagonize the toxicity of the effector. The MIX (Marker for type sIX effectors) domain has been previously defined as a marker of T6SS effectors carrying polymorphic C-terminal toxins. Here, we set out to identify the Vibrionaceae MIX-effector repertoire and to analyze the various toxin domains they carry. We used a computational approach to search for the MIX-effectors in the Vibrionaceae genomes, and grouped them into clusters based on the C-terminal toxin domains. We classified MIX-effectors as either antibacterial or anti-eukaryotic, based on the presence or absence of adjacent putative immunity genes, respectively. Antibacterial MIX-effectors carrying pore-forming, phospholipase, nuclease, peptidoglycan hydrolase, and protease activities were found. Furthermore, we uncovered novel virulence MIX-effectors. These are encoded by “professional MIXologist” strains that employ a cocktail of antibacterial and anti-eukaryotic MIX-effectors. Our findings suggest that certain Vibrionaceae adapted their antibacterial T6SS to mediate interactions with eukaryotic hosts or predators.

A novel class of polymorphic toxins in Bacteroidetes

“…When the downstream-encoded toxin was predicted to mediate antibacterial activity, we also found an adjacent gene encoding a predicted immunity protein. A similar phenomenon, in which N-terminal secretion or delivery domains either carry the toxin domain in the same polypeptide chain or are encoded by an adjacent gene, was previously observed in T6SS effectors containing delivery domains such as MIX and FIX (21,30). It is possible that in such cases when the toxin is encoded by a separate gene, the protein product of the toxin gene interacts with the adjacently encoded TANFOR-containing protein.…”

A comparative genomics methodology reveals a widespread family of membrane-disrupting T6SS effectors

“…For cytosolic expression of effectors in E. coli, genes were inserted into pBAD, in-frame with the C-terminal Myc-6xHis tag. For periplasmic expression, genes were inserted into the Kan R pPER5/Myc-His vector 17 (hereafter named pPER5), a pBAD derivative in which the PelB signal sequence was inserted at the 5′ of the multiple cloning site. The accession numbers of the amplified effectors were WP_015297525.1 (Tme1) from isolate BB22OP, WP_047706523.1 (Tme2) from isolate T9109, and NP_232421.1 (VasX), a Vibrio cholerae pore-forming effector from V. cholerae V52.…”

“…The T6SS effector arsenal is dynamic. Isolates of the same bacterial species carry different T6SS effector repertoires [17][18][19][20][21] . This diversity has been associated with the horizontal transfer of effector genes, and it may also have arisen from effector gene duplications 19,22 .…”

“…They may also fail to identify effectors that are expressed and delivered in low amounts under the examined experimental conditions. Computational approaches, including analyses of genes found inside T6SS clusters and auxiliary modules 14,20,[26][27][28][29] , genes encoding proteins with T6SS marker domain (e.g., MIX and FIX) 17,23,26 , or genes that neighbor T6SS adapters (e.g., DUF4123, DUF1795, and DUF2169) 13,15,19,[30][31][32] , have resulted in the discovery of many effector families that have proven useful in preliminary analyses of new bacterial genomes 33 . However, such bioinformatic approaches may fail to identify "orphan effectors" that do not neighbor any known T6SS component on the genome, and that do not contain any known T6SS-associated domain.…”

“…The expect value threshold was set to 10 −5 . In addition, RPS-BLAST was used to identify the MIX 17,23 and FIX 26 domains.…”