Insight into the resilience and susceptibility of marine bacteria to T6SS attack by Vibrio cholerae and Vibrio coralliilyticus

Abstract

The type VI secretion system (T6SS) is a nanomachine capable of killing adjacent microbial cells in a contact-dependent manner. Due to limited studies, relatively little is known about the range of marine bacteria that are susceptible to T6SS attack. Here, 15 diverse marine bacterial isolates from the phyla Bacteroidetes and Ɣ-Proteobacteria were challenged against the marine bacterium and human pathogen, Vibrio cholerae, which has a well described T6SS. V. cholerae killed several of the tested Ɣ-Proteobacteria, including members of the orders Vibrionales, Alteromonadales, Oceanospirillales, and Pseudomonadales. In contrast, V. cholerae co-existed with multiple Bacteroidetes and Ɣ-Proteobacteria isolates, but was killed by Vibrio coralliilyticus. Follow-up experiments revealed that five V. coralliilyticus strains, including known coral and shellfish pathogens survived the T6SS challenge and killed V. cholerae. By using predicted protein comparisons and mutagenesis, we conclude that V. coralliilyticus protected itself in the challenge by using its own T6SS to kill V. cholerae. This study provides valuable insight into the resilience and susceptibility of marine bacteria to the V. cholerae T6SS, and provides the first evidence for a functional T6SS in V. coralliilyticus, both of which have implications for human and ocean health.

Disease Diagnostics and Potential Coinfections by Vibrio coralliilyticus During an Ongoing Coral Disease Outbreak in Florida

“…In particular, T3SS and T6SS are important secretion systems for pathogenesis, with their needle-like delivery of toxins and enzymes through host membranes (Costa et al, 2015). The presence of an active T6SS in V. coralliilyticus was confirmed in a previous study (Guillemette et al, 2020). Lastly, all the V. coralliilyticus genomes contained several genes for multidrug export proteins (emrB, mepA), multidrug resistance proteins (mdtABCEGHKLMN, mexAB, norM), multidrug transporter (emrE), and putative multidrug resistance protein (emrK), which may contribute to antibiotic resistance in V. coralliilyticus.…”

“…In addition to protease activity, extracellular polysaccharide (EPS) production, as a proxy for biofilm formation, was also measured (Figure 7B). In comparison to the control, the OCN008 vcpR strain that has enhanced EPS production (Guillemette et al, 2020), only the Atlantic strain MmMcT2-4 had comparable EPS production (Dunnett's multiple comparison test, p = 0.16, n = 4).…”

“…The readings at 440 nm were standardized to culture density and presented as a ratio of OD 440nm to OD 600nm . A mutant, OCN008 vcpAB, with clean deletions of the major V. coralliilyticus proteases, genes vcpA and vcpB, was used as a negative control (Guillemette et al, 2020). The mean OD 400nm /OD 600nm ratio for each strain at logarithmic and stationary phase was compared at every time point using a 2-wayANOVA based on strain and time with a post-hoc Tukey's multiple comparisons test.…”

“…For other pathogenic vibrios like V. cholera or V. parahaemolyticus, biofilm production has been linked to virulence or host colonization (Silva and Benitez, 2016;Aagesen et al, 2018). To measure extracellular polysaccharide (EPS) production, indicative of biofilm formation, a modified crystal violet protocol was used based on previous studies (O'Toole, 2011; Guillemette et al, 2020). For each strain, an overnight SWB culture was diluted 1:1000 in fresh SWB, and then 1 mL aliquots of the diluted cultures were transferred to a 24-well plate (n = 4 for each strain).…”

“…After incubation, 500 µl of solution from each well was transferred to a new 24-well plate and the absorbance read at 550 nm using a plate reader. A mutant, OCN008 vcpR, with a deletion of the regulator vcpR and upregulated EPS production was used as a positive control (Guillemette et al, 2020). The mean OD 550nm for each strain was compared using a one-way ANOVA with a post-hoc Tukey's multiple comparisons test.…”