Isocitrate dehydrogenase mutation in Vibrio anguillarum results in virulence attenuation and immunoprotection in rainbow trout (Oncorhynchus mykiss)

The aquaculture industry now produces half of all fish intended for human consumption and employs millions of people worldwide [1]. Although the first value sale of harvested fish has increased by 267% between 2004 and 2014 to over US$160 billion, infectious diseases, especially those caused by Vibrio spp. including Vibrio anguillarum, still represent a major impediment to the production of fish [1]. V. anguillarum causes diseases in crustaceans and bivalves, and is the leading causative agent of vibriosis in finfish including salmon, rainbow trout, turbot, sea bass, sea bream, cod, eel, and ayu [2]. Infections by this bacterial species have resulted in severe economic losses to aquaculture industries worldwide [3].

V. anguillarum is an extracellular pathogen that invades its host fish through the intestine, skin or gills [4, 5]. Systemic infection by V. anguillarum usually causes fish to die within 1–4 days [6–9]. Chemotactic motility and the metalloprotease EmpA have been shown to be important virulence factors during the invasion stage while the siderophore anguibactin, flagellin subunits and lipopolysaccharides were shown to be important for persistence in the host during the post-invasion stage [2, 10]. Three secreted proteins that are cytotoxic against epithelial cells and erythrocytes have been characterized in V. anguillarum: the HlyA homolog Vah1, the phospholipase Plp, and the MARTX toxin RtxA [7, 9, 11]. Mutations in vah1 and/or plp resulted in slight attenuation against juvenile Atlantic salmon (Salmo salar); however, rtxA mutants were avirulent [7, 9, 11]. Additionally, a V. anguillarum mutant that lacks H-NS, a global transcriptional regulator that represses the transcription of vah1, plp, and rtxA, showed attenuation in virulence when injected intraperitoneally, suggesting that proper coordination of gene expression is an important factor during the post-invasion stage [8].

Since the 1980s, several bacterial species that are auxotrophic for aromatic compounds have been shown to be avirulent [12–16]. More recently, mutants that are hypothesized to experience growth defects in the nutrient limited environment inside a phagocyte have been characterized. In Salmonella enterica, an intracellular bacterial pathogen, some tricarboxylic acid (TCA) cycle mutant strains were avirulent and immunoprotective for subsequent wild-type S. enterica infection [17–21]. A functional fructose repressor (Cra) was also required for S. enterica infection [22]. Similar results have been observed for central metabolism mutants in other intracellular pathogens such as uropathogenic Escherichia coli (UPEC), Mycobacterium tuberculosis, and the facultative intracellular fish pathogen Edwardsiella ictaluri [23–27]. These observations demonstrate that central metabolism is important for pathogenesis by intracellular pathogens.

Accordingly, we hypothesized that mutations in central metabolism could interrupt the infection process of V. anguillarum in juvenile rainbow trout (Oncorhynchus mykiss). In this study, we identified and created six TCA cycle mutant strains plus one fructose metabolism mutant strain, and tested their virulence against juvenile rainbow trout using two infection methods, intraperitoneal (IP) injection and immersion. Further, the expression of each of the three hemolysin genes (vah1, plp, and rtxA) was examined to determine whether attenuation resulted from decreased virulence factor expression in these mutants. The growth rates and yield of each mutant strain in complex media were also determined. We specifically characterized the growth defect of the attenuated icd mutant. We also created, tested, and compared a restored wild type icd strain for virulence and growth to both the wild type and the icd mutant.

The tricarboxylic acid (TCA) cycle is involved in the generation of energy through the oxidation of acetate. TCA intermediates serve as precursor metabolites for the synthesis of amino acids and peptidoglycan. The M93Sm genome along with the published genomes of V. anguillarum strains 775, 96F, M3, NB10, RV22, 90-11-286 were examined for TCA cycle enzymes and the following genes were found: gltA, acnB, icd, sucAB, sucCD, sdhCDAB, frdABCD, fumA, and mdh (Fig. 1 and Table 3). Additionally, cra, which encodes the repressor of fructose metabolism in S. enterica and E. coli and has previously been shown to be essential for S. enterica virulence, is present in the V. anguillarum genomes [22].

When in a nutrient limited environment, bacteria must be able to synthesize any essential metabolites that are not freely available in order to grow. Previous studies have shown that mutations in central metabolism genes result in attenuation of virulence in several intracellular pathogens including S. enterica, uropathogenic E. coli (UPEC), M. tuberculosis and E. ictaluri [17–19, 21, 23–26]. These observations suggest that central metabolism is necessary for these intracellular pathogens to function inside the nutrient-limited environment of the phagosome; however, V. anguillarum is not an intracellular pathogen. While some studies have suggested that V. anguillarum can survive internally in fish epithelial cells and CHSE cells (derived from pooled embryonic cells from Oncorhynchus tshawytscha), more recent studies have demonstrated that V. anguillarum actively evades phagocytosis by fish epithelial cells and cannot survive for 24 h in macrophages [35–38]. In this study, fish were infected with V. anguillarum strains by either of two methods: intraperitoneal injection or immersion with both methods resulting in a similar percent survival when fish were challenged with M93Sm (20% for injection, see Fig. 2a and b; 0%~30% for immersion, see Fig. 3). Only the icd mutant had a statistically significant higher level of survival compared to the wild type, 100% for IP injection (Fig. 2a and b) and 90% for immersion (Fig. 3). It is not thought that reversion of the merodiploid to a wild type phenotype caused the other metabolism mutants to be virulent because chloramphenicol resistant colonies were isolated from the organs of dead fish. IP injection bypasses the need for invasion. No mortalities resulted from IP injection with the icd mutant indicating that icd is required for V. anguillarum persistence and growth in fish tissues. Rainbow trout infected with the icd mutant via immersion and subsequently challenged with the M93Sm wild type showed 100% survival (Fig. 4) demonstrating that the icd mutant had immunoprotective effects and elicited an adaptive immune response. Moreover, as a proof of concept, the data suggest that an icd deletion mutant could be the basis for a live attenuated vaccine against V. anguillarum infection.

Our observation that a knockout of the icd gene results in attenuation of virulence raises the question of whether expression of required virulence genes is significantly reduced in the mutant and, therefore, results in attenuation. We previously identified and characterized three hemolysin/cytolysin genes and their encoded proteins secreted by V. anguillarum: plp, vah1 and rtxA [7, 9, 11]. While mutations in plp and vah1 have modest effects on virulence against fish epithelial cells and fish, a knockout mutation in rtxA is avirulent in fish [7, 9, 11]. All metabolism mutants exhibited no significant declines in the expression of three hemolysins (Fig. 5) and most of the mutants exhibited increased expression. Accordingly, the icd mutant is not attenuated by the lack of hemolysin production because the decrease in rtxA and vah1 expression was not significant; however, future studies could examine the expression of other virulence factors. It is unclear why expression of plp and vah1 is increased in the metabolism mutants. Minato et al. [39] demonstrated the accumulation of acetyl-CoA in Vibrio cholerae central metabolism mutants resulted in an increased expression of its virulence gene activator ToxT. It is possible that accumulation or depletion of certain metabolites in V. anguillarum could increase hemolysin/cytolysin expression. Expression of hlyU, the positive regulator of the both the vah1 plp gene cluster and the rtxA gene cluster, was examined and shown to be up-regulated in the sucA and mdh mutants (data not shown) [40]. However, the increased expression of hlyU may not be the sole explanation for the increased expression of plp and vah1 because an increase in expression of rtxA should have also occurred.

The growth rate and final cell density was determined for all metabolism mutants grown in LB20 for 24 h. The slowest growing mutant, fumA, was as virulent as the wild type while the mutant with the lowest final cell density, icd, was attenuated suggesting that decreased final cell density results in a loss of pathogenicity against rainbow trout (Figs. 2, 6, Tables 4 and 5). When the mutation in icd was resolved, the restored icd strain demonstrated the wild type phenotype for both growth and pathogenicity (Additional file 3: Figure S3A and B and Fig. 8). While it is possible that the insertional mutation affected the expression of the two genes flanking icd (ribosomal large subunit pseudouridine synthase E (Accession number: WP_017043910.1) and cold shock domain protein CspD (Accession number: WP_013857087.1)), it is unlikely as neither gene is part of an operon that includes icd. Since isocitrate dehydrogenase catalyzes the formation of α-ketoglutarate (the immediate precursor of glutamate) from isocitrate, the icd mutant is auxotrophic for glutamate (Fig. 7a). Our data demonstrate that the icd mutant stops growing once exogenous glutamate or its derivatives are exhausted (Fig. 7b and c). The data also demonstrate the decreased growth yield was not do to a reduction of ATP production as addition of gluconate or succinate did not restore growth to a wild type cell density. It is interesting that the only other auxotrophic mutant, sucA, grows to a wild type cell density in LB20 and is as virulent as the wild type considering it cannot synthesize succinyl-CoA, a metabolite needed for the synthesis of lysine, methionine and diaminopimelic acid. Presumably, succinyl-CoA or its derivatives are not limiting in LB20 or in fish tissues. Furthermore, this also suggests that the icd mutant is primarily starved for glutamate and would not need to synthesize succinyl-CoA by metabolizing glutamate to α-ketoglutarate. We hypothesize that during infection the icd mutant is unable to obtain enough α-ketoglutarate derivatives to grow to a wild type cell density and, therefore, cannot reach a cell density necessary for a successful systemic infection. In support, it has previously been demonstrated that a V. anguillarum M93Sm mugA mutant that was unable to grow in salmon intestinal mucus was avirulent against Atlantic salmon [41]. Additionally, when V. anguillarum 775 was cured of its plasmid-encoded siderophore, the mutant was unable to sequester iron and exhibited decreased virulence [42, 43].

M93Sm is an O2α serotype and the presumed infection route is through the gastrointestinal tract as no necrotic skin lesions have ever been observed with this strain (unpublished data). The in vitro growth experiment (Table 5) suggests that there are not enough α-ketoglutarate derivatives in intestinal mucus to support the growth of the icd mutant to a wild type cell density even though it is the metabolite with the second highest concentration (3.03 mM) in rainbow trout mucus [44]. It should be noted that for in vitro growth experiments the concentrations of glutamate and glutamine in the mucus are not known and the growth conditions represent an ideal environment for growth; V. anguillarum does not have to evade the fish immune system or compete with commensal bacteria and it is not expected that the icd mutant will grow to the cell density shown in the in vitro growth experiments in the fish. As demonstrated by Muroga et al., [45] V. anguillarum found in the spleen and intestine of moribund fish challenged via immersion only reached a cell density of 4.0 × 10⁸ CFU/g and 2.5 × 10⁷ CFU/g respectively. Altinok et al. [46] showed a V. anguillarum succinate dehydrogenase mutant was avirulent against rainbow trout when injected at a dose at 10⁵ CFU. Similar to our results, the authors showed that the succinate dehydrogenase mutant grew to a cell density slightly lower than the wild type at 12 h; however, the authors failed to show the growth yield at 24 h. Further, the authors did not create a complement strain to demonstrate that the loss of virulence was solely do to mutating sdhB. Most importantly, the ATCC has redesignated their strain as a Pseudomonas species.

Seven V. anguillarum metabolism mutants were created and examined for pathogenicity against juvenile rainbow trout, hemolysin/cytolysin expression and growth in rich media. Of the central metabolism mutants, only the icd mutant showed strong attenuation in virulence, which did not result from a decrease in virulence factor expression. In addition, only the icd mutant had a final cell density that was lower than the wild type, which resulted from the inability to synthesize α-ketoglutarate and downstream metabolites. Taken together, the data suggest that during infection, if V. anguillarum is unable to synthesize essential molecules (e.g. α-ketoglutarate/2-oxoglutarate) and when those molecules or their derivatives (e.g. glutamate, glutamine) become limiting in the host, V. anguillarum will be unable to grow to a density necessary to sustain a systemic infection of the host.