AHL-mediated quorum sensing regulation: Role in controlling cytotoxicity, T6SSs and CRISPR-Cas systems in Aliivibrio wodanis

Abstract

<p><i>Aliivibrio wodanis</i> has been associated with winter ulcer disease outbreaks. It has been reported that <i>A. wodanis</i> may act as a secondary pathogen in disease outbreaks and pathogenesis. The culture supernatant of <i>A. wodanis</i> causes a cytopathogenic effect (CPE) in various salmon cell lines. Moreover, in an Atlantic salmon bath challenge experiment, <i>A. wodanis</i> alone produces clinical symptoms in the fish. However, the contribution of <i>A. wodanis</i> to winter ulcer disease is not clear. Despite the knowledge to date and the research done in the field, there are still unanswered questions and a knowledge gap. Several <i>Vibrio</i> and <i>Aliivibrio</i> species use quorum sensing (QS) to regulate genes connected to host-pathogen interaction, virulence, survival, and adaptation mechanisms. In other aliivibrios, several QS systems, including the <i>ainS</i>/AinR and a transcriptional regulator <i>litR</i> (LuxR homologs), have been reported to regulate various phenotypic traits. <p>Moreover, temperature is an essential factor that governs the prevalence of bacteria in the environment and host and is a potent regulator of pathogenesis in many bacteria. Hence the thesis aimed to understand better the QS`s role and the effect of temperature in <i>A. wodanis</i>. Our study reveals that <i>ainS</i> autoinducer synthase is required to produce the Acyl-Homoserine Lactone (AHL) 3OHC10-HSL in <i>A. wodanis</i>. <p>Furthermore, we found that the 3OHC10-HSL production is cell density and temperature-dependent. The 3OHC10-HSL concentration was higher at 6°C, the temperature below the threshold temperature at which winter ulcer occurs, compared to 12°C. The results also showed that QS and temperature regulate various functions such as AHL production, motility, and production of proteases, hemolysin and siderophores. The cell culture study further revealed that cell density, QS and temperature influence the cytotoxicity in CHSE salmon cell lines. This suggests that <i>A. wodanis</i> produces cytolysins and cytotoxins that are implicated in cytotoxicity. <p>Bacteria use the Type VI secretion system (T6SS) for multiple functions like iron transport, interspecies competition, virulence, and niche adaptations. <i>A. wodanis</i> co-exists with the main pathogen M. viscosa in the infected fish during the winter ulcer disease outbreaks. In addition, the bacterium has been shown to hinder the growth and virulence of <i>M. viscosa</i>. The thesis further investigated the mechanisms by which <i>A. wodanis</i> may survive together with <i>M. viscosa</i> in skin ulcers and during winter ulcer outbreaks. We found that the <i>A. wodanis</i> genome encodes three T6SSs (T6SS1-T6SS3) and auxiliary clusters (Aux1-4); and several potential Type VI secretion system effectors (T6SEs). In addition, the <i>A. wodanis</i> genome is found to contain a type IF CRISPR-Cas system. This suggests that these two mechanisms may play a role during the survival, adaptation, and immune function of <i>A. wodanis</i> in its natural environment, inside the host or during its co-existence with <i>M. viscosa</i>. Next, we analyzed the genome-wide transcriptomics of <i>A. wodanis</i> and QS mutants <i>litR</i> and ainS mutants grown at different temperatures and cell densities. <p>We found that the genes involved in T6SSs, and CRISPR-Cas systems are regulated by cell density, temperature, and QS. The transcriptome analysis showed that the complete T6SS2 apparatus was less expressed in the <i>litR</i>/WT, suggesting <i>litR</i> regulates T6SS2 in <i>A. wodanis</i>. In this study, the transcriptome analysis demonstrated that deletion of <i>litR</i> decreased the hcp1 expression, a gene involved in bacterial competition and virulence in other bacteria. In addition to <i>litR</i> dependent expression, expression value of <i>hcp</i> decreased three times in <i>litR</i>/WT, HCD at 12°C when compared to 6°C and HCD. Our observation suggests that temperature 6°C and <i>litR</i> are crucial for expressing the genes related to virulence. <p>Understanding the strain diversity of the same species is important in exploring strategies to survive in a changing environment. Finally, we wanted to study the genomic similarity and variation between <i>A. wodanis</i> isolates by performing a pan-genome analysis of twenty-two <i>A. wodanis</i> isolates collected from various locations across Norway. The analysis revealed an open pan-genome with a wide inter-species diversity in <i>A. wodanis</i> genomes. We examined the phylogenetic relatedness between the isolates using single nucleotide variants (SNVs) and core genes. The phylogenetic trees were distributed into five groups, where Group 2, 4 and 5 encompassed conserved isolates. The accessory genomes (shell and cloud) accounted for about 73% of the total pan-genome suggesting the genomes have acquired most of the genes through horizontal gene transfer (HGT). Whole and cloud genome functional annotation revealed a larger number of genes related to functional families such as metabolism, signaling and cellular processes and genetic information processing, suggesting they are involved in energy metabolism and environmental interactions. By further analyzing the twenty-two <i>A. wodanis</i> genomes, we identified diverse CRISPR-Cas systems, spacers, and prophages. About 60% of isolates encoded a different CRISPR-Cas system compared to the reference strain. Like the reference strain, the other twenty-one <i>A. wodanis</i> isolates also encode multiple T6SSs where the T6SS2 and Aux-2 are either absent or showed differences in about 80% (18 out of 22) of isolates. In addition to the T6SSs and CRISPR-Cas, other elements relevant for adaptation, virulence, and survival potentials, such as virulence factors (VFs) and biosynthetic gene clusters (BGCs), were explored. We have found that the predicted VFs and BGCs were conserved between the <i>A. wodanis</i> isolates. <p>The results presented in this work yield knowledge about QS regulation of virulence, survival, and adaptation mechanisms in A. wodanis. This aids in understanding the mechanisms connected to co-existence of <i>A. wodanis</i> with M. viscosa and winter ulcer disease development and treatment.