The Vibrio cholerae Type Six Secretion System Is Dispensable for Colonization but Affects Pathogenesis and the Structure of Zebrafish Intestinal Microbiome

ABSTRACT

Zebrafish (Danio rerio) are an attractive model organism for a variety of scientific studies, including host-microbe interactions. The organism is particularly useful for the study of aquatic microbes that can colonize vertebrate hosts, including Vibrio cholerae, an intestinal pathogen. V. cholerae must colonize the intestine of an exposed host for pathogenicity to occur. While numerous studies have explored various aspects of the pathogenic effects of V. cholerae on zebrafish and other model organisms, few, if any, have examined how a V. cholerae infection alters the resident intestinal microbiome and the role of the type six secretion system (T6SS) in that process. In this study, 16S rRNA gene sequencing was utilized to investigate how strains of V. cholerae both with and without the T6SS alter the aforementioned microbial profiles following an infection. V. cholerae infection induced significant changes in the zebrafish intestinal microbiome, and while not necessary for colonization, the T6SS was important for inducing mucin secretion, a marker for diarrhea. Additional salient differences to the microbiome were observed based on the presence or absence of the T6SS in the V. cholerae utilized for challenging the zebrafish hosts. We conclude that V. cholerae significantly modulates the zebrafish intestinal microbiome to enable colonization and that the T6SS is important for pathogenesis induced by the examined V. cholerae strains. Furthermore, the presence or absence of T6SS differentially and significantly affected the composition and structure of the intestinal microbiome, with an increased abundance of other Vibrio bacteria observed in the absence of V. cholerae T6SS.

INTRODUCTION

Vibrio cholerae, the organism responsible for the disease cholera, is a Gram-negative aquatic bacterium belonging to the class Gammaproteobacteria (1–3). Humanity is currently experiencing its seventh cholera pandemic, which continues to ravage the developing world (3). The major human virulence factors largely responsible for this ongoing crisis, namely, cholera toxin (CT) and toxin coregulated pilus (TCP), are the most well-known of the pandemic-capable O1/O139 strains of V. cholerae, and they have been studied extensively in mammalian animal models to uncover their pathogenic mechanisms (4–7). However, CT and TCP are specific to O1/O139 pandemic V. cholerae and are rarely present in the genomes of the hundreds of other V. cholerae serogroups, which are generally referred to as “non-O1/O139” or environmental V. cholerae. Nearly all V. cholerae (both O1/O139 and non-O1/O139) bacteria possess other virulence traits, such as hemolysins, additional toxins (e.g., RTX, cholix, and MakA), adhesin proteins, and various secretion systems, including the type six secretion system (T6SS), which has become a greater topic of research in recent years (8–18).

V. cholerae utilizes a T6SS, which is wide spread among Gram-negative bacteria (∼25% of species). All strains of V. cholerae possess the T6SS; however, it is not functional in every instance (17). The T6SS is unusual in that it is one of the few bacterial secretion systems that can deliver effectors or toxins directly into both other bacteria and eukaryotic cells (19, 20). The T6SS shares a great deal of similarity with the puncturing device of bacteriophages, and the system itself is believed to have been acquired from bacteriophages (21). One of the first bacterial species in which the system was observed was a non-O1/O139 strain of V. cholerae, V52, which was isolated from Sudan in 1968. All non-O1/O139 strains of V. cholerae are apparently able to utilize their T6SS, and the system is constitutively active (8). For the O1/O139 strains, however, the T6SS is much more tightly regulated, with quorum sensing playing a major role in the regulatory process (unphosphorylated LuxO results in higher HapR and lower c-di-GMP levels, which causes higher levels of T6SS activity), particularly in aquatic environments (21–24). The regulation of the T6SS (for all serogroups) in a host is less well understood. Studies have shown that mucins, large glycoproteins found in the intestines of numerous vertebrates, activate production of the T6SS. Furthermore, the magnitude of bacterial killing through the T6SS is regulated by host bile acids, of which some can decrease the power of the V. cholerae T6SS (18, 25). In the case of O1 classical biotype strains, however, the T6SS is apparently nonfunctional. For the majority of the O1 El Tor biotype strains, the T6SS is produced only under certain circumstances, as follows: when there is a large density of V. cholerae; V. cholerae is attached to chitin; and there are limited carbon sources, a high salt concentration, a high-viscosity liquid, or mucin proteins present (18, 23, 26, 27). The more recent waves of El Tor V. cholerae, however, such as the strain responsible for the outbreak in Haiti (H1), have T6SSs with heightened activity compared with those of their fellow El Tor counterparts (28, 29).

The T6SS has a structure that is akin to a spear or harpoon, thus serving as a weapon for bacteria that possess it and allowing them to attack and potentially eliminate nearby cells, both prokaryotic and eukaryotic, via the injection of effectors (21). Numerous proteins are involved in the construction and utilization of the T6SS, with the protein Hcp forming the body of the harpoon being projected from the attacking cell, while other proteins such as VasK are essential components of the inner membrane complex (30, 31). While there are numerous cellular effectors, the vast majority are antibacterial and tend to do one of two things, namely, target peptidoglycan or act as lipases/esterases. V. cholerae has 4 different known effectors that target prokaryotic cells (depending on the strain), with at least 105 known variations of these 4 effectors. In addition to the prokaryotic effectors, there are also strict eukaryotic effectors and dual-purpose effectors, which act on both prokaryotic and eukaryotic organisms. The effector VgrG1 strictly targets eukaryotic organisms by attacking actin cross-linking domains, causing disruptions in the cell membrane. The effector VasX causes pore formation in both prokaryotic and eukaryotic organisms (21, 32, 33).

In V52, the strain of V. cholerae in which the T6SS was initially discovered, the four effectors are the lipase TseL, the membrane-disrupting VasX, the peptidoglycan-degrading VgrG-3, and the hydrolase TseH, which also degrades peptidoglycan. All of these effectors are situated at the distal end of the Hcp tube, which impales target cells. The immunity proteins for these four effectors (which protect from attack by sister cells) are tsiV1, tsiV2, tsiV3, and tsiH, respectively (21, 33). Other V. cholerae strains possess variations of these effectors (with different names); however, there are also other strains that lack some of them. For example, non-O1/O139 strain AM-19226 appears to lack a pore-forming effector, but it still possesses a lipase (A33_1346) and peptidoglycan-degrading effectors (A33_A0310 and VCB_00278) in its two auxiliary and main clusters. Few eukaryotic effectors have been found that are specific to the T6SS; however, those that are specific induce actin cross-linking or microtubule-dependent internalization (a slow and disordered endocytosis process) (33, 34).

Recent studies have examined the role of the T6SS in various bacterial infections and colonization. Shigella and Salmonella infections have demonstrated microbe-microbe interactions that are dependent on the T6SS within the mouse intestine (35, 36). Additionally, members of the genus Bacteroides use their T6SS to maintain their presence in the mouse intestine by eliminating bacterial competition. For V. cholerae, studies have demonstrated in mouse models that the T6SS clears commensals in the intestine during colonization (37, 38). Another study by Logan et al. (17) examining V. cholerae in germfree zebrafish larvae found that the T6SS does not actually have to engage the resident bacteria directly to eliminate them. Rather, the T6SS is able to expel bacteria within the zebrafish intestine by modulating intestinal motility, causing increased movement to flush out the competing bacteria, which in this instance was Aeromonas veronii, a common commensal of the zebrafish intestine. The T6SS accomplishes this through its effector VgrG1, which can induce actin cross-linking in the intestinal epithelium. Logan et al. found that deletion of the VgrG1 effector caused no increase in the frequency or strength of zebrafish intestinal contractions in germfree fish that were or were not monocolonized with A. veronii. Yet, even with the deletion of the VgrG1 effector, V. cholerae could still engage and eliminate resident A. veronii (17). Furthermore, in a normal and healthy zebrafish (adult or juvenile), there is a far more diverse microbial community within the zebrafish intestine, which must be taken into account when considering the broader role of the T6SS in an infection (39).

While a number of different animal models have been used to study the pathogenicity of V. cholerae, many have limitations. Adult mammalian models require extensive physiological modification to be used as viable animal models, of which some include surgical modification (ileal loop models) or elimination of the resident intestinal microbiome (40, 41). Some of the best mammalian models are infant animals, which lack complex intestinal microbial communities and mature immune responses. Furthermore, the cycle of cholera disease can differ significantly from that observed in a human. For example, infant mice do not experience the watery diarrhea that humans experience, which is a hallmark of the cholera disease (40). To overcome these limitations, a V. cholerae model in fish, which are natural V. cholerae hosts, was developed. The zebrafish (Danio rerio) has proven to be a viable animal model for studying the entire life cycle of V. cholerae and other enteric pathogens (16, 42). The zebrafish digestive tract is similar to that of a human; however, there are a few notable differences. The most noteworthy of these differences, for our purposes, is that zebrafish lack the intestinal crypts found in the mammalian small intestine. Additionally, zebrafish do not have a dedicated acidified stomach. Even with these differences, the zebrafish has proven to be an extremely useful model for studying V. cholerae, as it is environmentally relevant; recapitulates the cholera disease model; does not need to be surgically modified or manipulated in anyway; and perhaps most importantly, it has an intact and fully developed intestinal microbiome that does not need to be altered to enable colonization and disease (16, 42–44).

While much progress has been made in defining the overall pathogenic cycle of V. cholerae and elucidating its primary virulence factors, not much is known about which intestinal bacteria V. cholerae is competing with. Additionally, little information is available about how a normal and intact host microbiome responds to a V. cholerae infection. In previous studies, we have examined the effects of housing environment on the zebrafish intestinal microbiome, as well as the pathogenicity cycle of V. cholerae in a zebrafish host (16, 39, 42, 43). The aim of this study was to investigate the potential role for the T6SS in both El Tor and non-O1/O139 V. cholerae during a cholera infection in adult zebrafish. Our results suggest that the T6SS is dispensable for intestinal colonization but its presence induces intestinal mucin secretion and significantly impacts the composition of the intestinal microbiome.

RESULTS

Killing assays verify the presence or absence of T6SS-mediated cell killing.

To verify the presence or absence of an active, functional T6SS in V. cholerae, in vitro killing assays were utilized. We also included a V52 ΔvasK mutant as an additional negative control, as this mutant will not form any of the T6SS machinery, as opposed to the Δhcp mutant which is still is able to form the inner membrane T6SS complex. As expected, the non-O1/O139 V. cholerae strain V52 displayed significant killing of the prey strain MG1655 Escherichia coli, whereas the V52 Δhcp and V52 ΔvasK strains did not (Fig. 1A). For the El Tor O1 V. cholerae strain N16961, the salinity of the LB was increased from the standard 85 mM to 340 mM to ensure activation of the T6SS, as unlike with non-O1/O139 strains, the system is activated only under select biological conditions (23). The increased salt concentration did not have a significant effect on the CFU count of the prey E. coli strain or the ability of V. cholerae V52 to eliminate prey bacteria. N16961 El Tor O1 V. cholerae N16961 displayed significant killing of the prey E. coli, while N16961 ΔvxrB did not (Fig. 1B) compared with E. coli on the high-salt LB, thus demonstrating the effect of the presence or absence of an active T6SS.

FIG 1.

Bacterial killing assay. CFU/ml levels of surviving prey E. coli after a 4-hour competition assay with select V. cholerae strains on normal (85 mM NaCl) LB (A) and high-salt (340 mM NaCl) LB (B). The data are the results of 5 to 6 individual experiments; n = 3 to 6 biological replicates per group. Statistical tests were performed with Dunnett’s multiple-comparison test on log-transformed data. Error bars display SEM. **, P ≤ 0.005; ***, P ≤ 0.0005.

V. cholerae can colonize the zebrafish intestine and persist in the aquatic environment with or without an active T6SS.

Having established the presence of an active or inactive V. cholerae T6SS in our strains, we next sought to examine whether the T6SS had a significant impact on V. cholerae colonization of the zebrafish intestine or survival in the tank water. After zebrafish were exposed to V. cholerae for 24 h, all strains utilized in this study were capable of colonizing (or persisting in) the zebrafish intestines with or without an active T6SS (Fig. 2A), and they retained viability in the autoclaved infection water (Fig. 2B).

FIG 2.

CFU and mucin levels following V. cholerae infections in a zebrafish host. (A) CFU/ml levels of V. cholerae in the zebrafish intestines. (B) CFU/ml levels in the infection water 24 hours postinfection. The data are the results of 2 to 8 individual experiments per sample group. Fish, n = 8 to 16; water, n = 2 to 6. Fifty-milliliter water samples were spun down and resuspended in 1 ml of PBS then analyzed for mucin concentrations using Schiff’s reagent (C) and Bradford protein assay reagent (D). n = 2 to 6. Statistical tests were performed using Dunnett’s multiple-comparison test (A and B), and Sidak’s multiple-comparison test (C and D) on log-transformed data. Tests conducted with Sidak’s multiple-comparison test: control versus V52; control versus V52 Δhcp; control versus N16961; control versus N16961 ΔvxrB; N16961 versus N16961 ΔvxrB; and V52 versus V52 Δhcp. Error bars display SEM. *, P ≤ 0.05; **, P ≤ 0.005; ***, P ≤ 0.0005; ****, P ≤ 0.0001.

V. cholerae strains lacking an active T6SS have reductions in diarrhea metrics.

While all strains of V. cholerae tested were able to colonize the zebrafish intestine, the question remained as to whether they were capable of causing diarrhea following gut colonization. Previous metrics for fish diarrhea were described that included measurements of excreted mucin, excreted protein, and increased optical density at 600 nm (OD₆₀₀) of the water (16, 45). Both wild-type (WT) strains of V. cholerae tested induced a significant increase in mucin secretion at 24 h postinfection, while the two T6SS mutants had reduced, but not significantly different, levels of mucin secretion compared with their wild-type counterparts (Fig. 2C). A Bradford protein assay was also utilized as a secondary diarrhea metric by testing for excreted proteins in the infection water, and the same trend was largely observed; significantly higher levels of excreted protein were detected from the zebrafish infected with either strain of wild-type V. cholerae, with those two groups yielding the highest protein levels. No difference was observed when comparing excreted protein levels from the zebrafish infected with N16961 ΔvxrB or V52 Δhcp mutant to the uninfected control fish, while a significant difference was observed when comparing V52 to its T6SS null counterpart (Fig. 2D). These findings strongly suggest that the T6SS is important for fish pathogenesis in terms of causing diarrhea.

V. cholerae infections induce significant changes in the zebrafish intestinal microbiome.

Having established that all strains of V. cholerae utilized in this study are capable of colonizing the zebrafish intestine, we next investigated if the colonization by these strains caused significant changes to the broader intestinal microbiome. Beginning with the α-diversity of the zebrafish intestinal microbiome, multiple groups of zebrafish showed significant differences in the overall richness (Chao index) of their intestinal microbiomes following infection with V. cholerae. Strain N16961 colonization induced significant decreases in overall amplicon sequence variant (ASV) richness of the microbiome, while colonization with strains N16961 ΔvxrB and V52 induced significant increases in richness. The heterogeneity (Shannon-Weiner and inverse Simpson indices) of the zebrafish intestinal microbiome also displayed mixed results. The absence of a functional N16961 T6SS did not affect the heterogeneity of the zebrafish intestinal microbiome (Fig. 3). However, infection with the V52 wild type led to an intestinal microbiome with greater heterogeneity than infection with its T6SS null mutant (Fig. 3).

FIG 3.

α-Diversity of the zebrafish intestinal microbiome following V. cholerae infections in a zebrafish host. α-Diversity of the zebrafish intestinal microbiome following a 24-hour V. cholerae infection presented as scatterplots. Con, uninfected control fish. Statistical tests were performed using generalized linear modeling (multiple comparisons of mean values, Tukey contrasts). *, P ≤ 0.05; **, P ≤ 0.005; ***, P ≤ 0.0005. Fish, n = 48 (control, n = 9; N16961, n = 11; N16961 and ΔvxrB, n = 8; V52, n = 8; V52 Δhcp, n = 12).

Next, we assessed whether V. cholerae colonization induced changes in β-diversity of the zebrafish intestinal microbiome. The addition of N16961 ΔvxrB changed the structure, but not the composition, of the zebrafish gut microbiome (Fig. 4A). Specifically, compared with negative controls, both N16961 and N16961 ΔvxrB led to a reduction in the relative abundance of Aeromonas sp., Fluviicola sp., and an unclassified Proteobacterium (Fig. 4B), while the addition of N16961 and N16961 ΔvxrB led to a greater relative abundance of Fictibacillus sp. and Vibrio ASVs, respectively (Fig. 4B). The addition of N16961 and N16961 ΔvxrB affected the composition and structure of the zebrafish gut microbiome in different ways. Compared with the gut microbiomes of fish inoculated with the N16961 wild type, those inoculated with N16961 ΔvxrB exhibited higher relative abundances of Vibrio sp., Shewanella sp., and Cetobacterium sp., and lower relative abundances of Aeromonas sp., Brevinema sp., Brucella sp., an unclassified Gammaproteobacteria, and unclassified Burkholderiaceae, Bradyrhizobium sp., and Fictibacillus sp. (Fig. 4B).

FIG 4.

β-Diversity of the zebrafish intestinal microbiome following V. cholerae infections in a zebrafish host. (A) β-Diversity of the zebrafish intestinal microbiome following a 24-hour N16961 or N16961 ΔvxrB V. cholerae infection expressed as PCoA plots based on Jaccard and Bray-Curtis dissimilarity indices. Statistical tests were one-way PERMANOVAs run using Adonis with the strata function controlling for collection date. (B) Linear discriminant analysis effect size (LEfSe) figures displaying which ASVs experienced significant shifts following the V. cholerae infections (again, controlling for sample collection date). All of the ASVs that had an LDA score greater than 2.5 are presented. Fish numbers: control, n = 9; N16961, n = 11; N16961, ΔvxrB n = 8.

Both V52 and the mutant V52 Δhcp led to changes in the composition and structure of the zebrafish gut microbiome (Fig. 5A). As with N16961 and its mutant, V52 and V52 Δhcp led to a reduction in the relative abundance of Aeromonas sp. and Fluviicola sp. (Fig. 5B). They additionally led to a reduction in the relative abundance of Sediminibacterium sp. and an increase in multiple Vibrio ASVs. The wild-type V52 further led to an increased relative abundance of Plesiomonas sp., an unclassified Vibrionaceae, and Cetobacterium sp., which are the same taxa that distinguished zebrafish gut microbiomes inoculated with V52 from those inoculated with V52 Δhcp (Fig. 5B). Notably, the deletion of the T6SS led to much greater abundance of Vibrio ASVs in infected fish than that in fish infected with WT V52, suggesting that the V. cholerae T6SS may be acting directly or indirectly on other Vibrio strains or species.

FIG 5.

β-Diversity of the zebrafish intestinal microbiome following V52 V. cholerae infections in a zebrafish host. (A) β-Diversity of the zebrafish intestinal microbiome following a 24-hour V52 or V52 Δhcp V. cholerae infection expressed as PCoA plots based on Jaccard and Bray-Curtis dissimilarity indices. Statistical tests were one-way PERMANOVAs run using Adonis with the strata function controlling for collection date. (B) LEfSe figures displaying which ASVs experienced significant shifts following the V. cholerae infections (again, controlling for sample collection date). All of the ASVs that had an LDA score greater than 2.5 are presented. Fish numbers: control, n = 9; V52, n = 8; V52 Δhcp, n = 12.

V. cholerae infections induce strain-specific changes in the zebrafish intestinal microbiome.

Since colonization with V. cholerae induced significant changes to the zebrafish microbiome in most instances, we next compared the different postinfection intestinal microbiomes to each other where applicable to assess ASV-level differences. Strain V52 displayed significant differences from N16961 in the postinfection composition of the intestinal microbiome and a near-significant difference in structure (P = 0.057) (Fig. 6A). V52-infected fish displayed higher relative abundances of Cetobacterium sp., Burkholderiaceae sp., Shewanella sp., and Cloacibacterium sp. than those of N16961-infected fish. Fish infected with N16961 V. cholerae displayed higher levels of Brucella sp., Brevinema sp., and Pseudomonas sp., along with others, than those of fish infected with V52 (Fig. 6B). Finally, a linear discriminant analysis effect size (LEfSe) comparison of the two different T6SS mutants N16961 ΔvxrB and V52 Δhcp revealed higher relative abundances of Shewanella sp. and Plesiomonas sp. in fish infected with N16961 ΔvxrB, whereas there were higher levels of Vibrio sp. and Aeromonas sp. in fish infected with V52 Δhcp (Fig. 6C).

FIG 6.

(Continued).

Fish infected with V52 and V52 Δhcp displayed higher bacterial abundance in the intestine.

To determine whether V. cholerae infection changed the overall bacterial load of the fish intestine, quantitative PCR (qPCR) was performed on the DNA extracted from the zebrafish intestinal homogenates. The intestines of fish infected with N16961 ΔvxrB, V52, and V52 Δhcp had higher overall bacterial loads than those of uninfected control fish (Fig. 6).

V. cholerae infection induced significant changes to the metabolomics profile of the zebrafish intestine.

As described above, V. cholerae infection induced significant changes to the zebrafish intestinal microbiome. These changes were likely to result in changes to the metabolites present in the intestine as well, which ultimately could select for or against certain bacterial species. To investigate changes to the metabolome, untargeted metabolomics analysis was performed on the zebrafish intestinal homogenates from uninfected control versus infected zebrafish. All of the V. cholerae infections induced significant differences to select compounds in the zebrafish intestines compared with uninfected control fish (see Fig. 8). Significant differences were also observed when comparing the metabolomics profiles of fish infected with WT V52 or N16961 to their T6SS null counterparts, when comparing the two T6SS null strains to one another, and when comparing fish infected with WT N1691 to those infected with V52. Control fish generally contained higher levels of some metabolites such as spermidine, benzoic acid, citramalic acid, taurocholic acid, oxoglutaric acid, and citric acid. Compared with the control population, fish infected with N16961 displayed significantly higher levels of some metabolites such as thymine, adenine, guanine, traumatin, 13-HOTE, cafestol, shikimic acid, jasmonic acid, traumatic acid, and cortisol (Fig. 7A); fish infected with V52 displayed significantly increased amino acid levels, including valine, proline, serine, and lysine (Fig. 7B); and fish infected with N16961 ΔvxrB or V52 Δhcp displayed higher levels of some metabolites such as valine, leucine, adenine, glutamine, proline, piperidine, guanosine, and propionic acid (Fig. 7C and D).

FIG 8.

(Continued).

FIG 7.

qPCR data of the bacterial load in each zebrafish intestine. Similarities in bacterial load, as assessed by 16S rRNA gene real-time quantitative PCR (qPCR) between zebrafish intestinal homogenates are displayed. Con, uninfected control fish. Statistical tests were performed with Dunn’s post hoc analysis and Bonferroni’s correction on log-transformed data. Error bars display SEM. *, P ≤ 0.05; **, P ≤ 0.005; ***, P ≤ 0.0005. Fish numbers: control, n = 10; N16961, n = 12; N16961 and ΔvxrB, n = 8; V52, n = 8; V52 Δhcp, n = 12.

Comparisons of the metabolomics profiles between fish infected with V. cholerae T6SS WT strains versus T6SS mutants revealed significant differences in metabolomics. Fish infected with N16961 displayed significantly higher levels of piperidine, creatine, fucose, pantetheine, uridine, and aldosterone, while fish infected with the N16961 T6SS null strain had significantly higher levels of propionic acid, alanine, glycerol, valine, pantothenic acid, gamma-glutamylleucine, phosphate, and adenosine (Fig. 7E). V52-infected fish had higher levels of jasmonic acid, pantetheine, citraconic acid, ascorbic acid, melatonin, estradiol, and N-acetyl-l-alanine, while fish infected with the T6SS null strain had higher levels of creatine, niacinamide isobutyric acid, capric acid, linolenic acid, linoelaidic acid, and dihydrotestosterone (Fig. 7F).

To assess the overall differences between the two parent strains used in this study, metabolomics comparisons were also done between V52 and N16961. Higher levels of propionic acid, piperidine, valine, proline, valine, threonine, leucine, glutamine, lysine, daucic acid, melatonin, adenosine, and uridine were observed in fish infected with V52, while higher levels of creatine, fucose, neocnidilide, glutamyltyrosine, glyceric acid, azelaic acid, and 2-nonenoic acid were observed in fish infected with N16961 (Fig. 7G). Lastly, a comparison of fish infected with N16961 ΔvxrB to fish infected with V52 Δhcp indicated higher levels of the metabolites jasmonic acid, traumatic acid, pantetheine, melatonin, estradiol, ascorbyl palmitate, and palmitoyl glucuronide in fish infected with N16961 ΔvxrB; and higher levels of spermidine, glycylleucine, methionine, taurine, salicylic acid, linoelaidic acid, and aldosterone in fish infected with V52 Δhcp (Fig. 7H). No significant correlations were found between the 15 most detected ASVs and the 15 most prominent metabolites (Mantel test; correlation, R = 0.03124; P = 0.3095). Quantities of all metabolites and statistical analyses are included as supplemental excel files (electrospray ionization + [ESI+] and ESI− untargeted metabolomics).

DISCUSSION

V. cholerae is able to persist in the aquatic environment and is capable of colonizing the zebrafish intestine, inducing pathological conditions akin to human cholera, including diarrhea. As described here, V. cholerae colonization of the zebrafish intestine did not require a functional T6SS. The presence of a functional T6SS was, however, associated with significant increases in mucin and protein excretion, which are metrics for fish diarrhea. This finding suggests that the T6SS may be a major player in the pathogenesis observed in fish infected with V. cholerae.

The analyses in this study focused on the effects of V. cholerae on the intestinal microbiome. Of the four V. cholerae strains tested here, three caused significant changes in the overall richness (Chao index) of the zebrafish intestinal microbiome, while a comparison of zebrafish infected with V52 and V52 Δhcp displayed significant differences between the two. Fish infected with V52 displayed higher levels of evenness in both indices, indicating the effect of the T6SS on the composition of the microbiota (Shannon-Weiner and inverse Simpson indices). When the β-diversity was analyzed, three of the four V. cholerae strains in this study induced significant structural changes to the zebrafish microbiome. Only one strain, however, namely, V52, caused significant compositional changes to the β-diversity of the zebrafish intestinal microbiome. A comparison of the postinfection zebrafish microbiomes revealed there were significant differences in the structures and compositions of the intestinal community based on the strain of V. cholerae utilized for infection, both for wild-type versus mutant counterparts and wild-type versus wild-type comparisons. Comparing the post-V52 infection zebrafish microbiome with the infected zebrafish revealed significant differences in composition between the two groups and nearly significant differences in structure. These strains are significantly different genetically, as N16961 is a pandemic El Tor strain and V52 is a non-O1/O139 strain that lacks the three El Tor pathogenicity islands (46).

Significant structural differences were also observed when comparing N16961 to its T6SS null strain, while the comparison of V52 to its T6SS null strain showed significant differences in composition (and nearly significantly different in structure). We have every reason to suspect that the N16961 T6SS is active within the zebrafish intestines based on the results from the mucin and Bradford assays, which displayed higher levels of mucin secretion in the wild-type-infected fish. Additionally, the results from the microbiome analyses display similar ASV patterns when comparing the wild-type to the T6SS null mutants, namely, reduced levels of Vibrio ASVs in the T6SS mutants. These observations suggest that while the T6SS has little observable effects on V. cholerae colonization, it does impact the composition and structure of the intestinal microbiota. Most striking was the observation that other vibrios are highly enriched when zebrafish are infected with the T6SS mutant strains, whereas the WT T6SS strains induced other changes to the microbiota. This result supports the hypothesis put forward by Pukatzki and colleagues that a major function of the V. cholerae T6SS is competition with other vibrio species (33).

Untargeted metabolomics analyses of the zebrafish intestines following V. cholerae infection revealed numerous metabolites that were modulated as a result of infection. In general, V. cholerae infection resulted in increased levels of amino acids in the intestine, while uninfected control fish had higher levels of spermidine, benzoic acid, oxoglutaric acid, citric acid, and taurocholic acid. The composition of metabolites impacts survival and growth of the intestinal microbiome and can select for or against certain members of the microbial community (47–49). It is unclear whether V. cholerae colonization directly causes these metabolomic changes or if directed competition between V. cholerae and certain members of the microbial community is the source of the observed changes. It is notable that the T6SS mutant strains differed from their WT parent strains in the metabolomics analyses, suggesting that competition via the T6SS impacts the composition of intestinal metabolites.

The finding that the T6SS was not essential for colonization of the zebrafish intestine was somewhat surprising. However, this finding is not unprecedented, as the study by Logan et al. (17) also noted that a T6SS null mutant was able to colonize the larval zebrafish intestine under germfree or limited competition conditions. These results suggest that in a zebrafish animal model, other virulence factors besides the T6SS play a more essential role in colonization. Since this colonization trend was observed in both El Tor and non-O1/O139 strains of V. cholerae, it suggests that a common shared virulence factor(s) is heavily involved in the colonization of the zebrafish intestine. It is also possible that, in an extended infection (beyond 24 hours), significant colonization defects would be observed in the V. cholerae strains lacking a T6SS. While the T6SS is not essential for the colonization of the zebrafish intestine, the significant differences noted in the zebrafish intestinal microbiome postinfection when comparing wild-type strains to their T6SS null mutant counterparts strains does reveal that the T6SS plays a role in the infection. Furthermore, the significant reduction in mucin and protein excretion by the fish that were colonized with the T6SS null mutants demonstrates that although the T6SS may not be essential for colonization, it is important for pathogenesis. The reason for this may lie in the ability of the T6SS null mutant strain to modulate the zebrafish intestine via actin cross-linking with its eukaryotic effectors, which induces intestinal secretion.

Both of the V. cholerae strains lacking the T6SS displayed significantly higher relative abundances of various Vibrio ASVs, suggesting other Vibrio bacteria, directly or indirectly, are a prime target for competition with the infecting V. cholerae. Furthermore, there were differences in which bacterial genera were eliminated by the T6SS in the N16961 O1 El Tor compared with the V52 O37 environmental strain. When comparing the postinfection bacterial population of the zebrafish intestine of N16961 to N16961 ΔvxrB, the LEfSe analysis indicated significantly higher relative abundances of Shewanella sp., Cetobacterium sp., and various Vibrio spp. in the N16961 T6SS null strain compared with those of the wild type. For V52, the LEfSe analysis displayed significantly higher levels of a select type of Vibrio spp. (ASV 8) in the zebrafish infected with V52 Δhcp compared with those infected with wild-type V52, which displayed higher levels of Cetobacterium, Vibrionaceae, and Shewanella species. This trend of selective elimination is again confirmed when comparing the postinfection bacterial populations of the fish receiving each T6SS null mutant, as fish that received N16961 ΔvxrB had significantly higher relative abundances of Shewanella sp., while fish that were infected with V52 Δhcp displayed significantly higher relative abundances of the aforementioned Vibrio sp. (ASV 8).

These differences suggest that different strains of V. cholerae directly target, indirectly eliminate, or out compete other residents of the microbiome. The differences in the ASV profiles also suggest that this elimination may be necessary to successfully colonize the host intestine. A number of the detected Gram-negative bacteria from our study, such as members of Aeromonas, Plesiomonas, Cetobacterium, Vibrionaceae, and Shewanella, are organisms commonly found in aquatic environments and/or associated with aquatic animals. The finding that most of these types of bacteria experienced the largest changes in relative abundance following a V. cholerae infection is expected, as they are major components of the fish microbiota. Members of other bacterial genera, however, such as those of Bradyrhizobium, Brevinema, and Burkholderiaceae, which are not commonly associated with aquatic environments or organisms, were also often greatly reduced in relative abundance following exposure to V. cholerae. Further analysis that would identify the particular species of bacteria being eliminated in V. cholerae infections would be especially useful, as it may help identify any species or strains that have the potential to provide a protective effect against V. cholerae infections.

Surprisingly, the relative abundances of Aeromonas ASVs changed very little in the zebrafish intestine postinfection. Based on the aforementioned Logan et al. (17) study, which found that V. cholerae modulated the zebrafish intestine to expel the commensal bacteria, the small change in ASV abundances was an unexpected result. In our study, the Aeromonas genus was the second largest bacterial population in the zebrafish intestine. Moreover, Aeromonas sp. is vulnerable to attack by the T6SS of V. cholerae and likely other members of the Vibrio genus (the most detected bacterial population in our study) possessing T6SS. Since the Logan study was performed with a strain of V. cholerae not used in this study (C6706), however, this again displays a trend in which different V. cholerae strains seem to target different commensal bacteria in order to colonize. While these differences could be due to differences in various aspects of the T6SS between environmental and El Tor strains (i.e., effector production and system regulation and proximity to other bacteria encountered in the zebrafish gut), it is also possible that genetically diverse V. cholerae need to eliminate different bacterial competitors in order to colonize the intestine. Further study will be necessary to answer this question and to determine whether these commensal bacteria are being specifically targeted because of their ability to hinder V. cholerae colonization or if they are being randomly eliminated due to proximity and/or other virulence factors.

qPCR analysis of the bacterial load within the zebrafish intestines detected larger amounts of bacteria within the intestines of fish infected with N16961 ΔvxrB, V52, or V52 Δhcp. For the three strains which resulted in significantly higher bacterial loads, the reason could be in part due to the ability of these strains to multiply more rapidly within the zebrafish intestine. N16961, as a wild-type El Tor strain, is (likely) expending energy to fire its T6SS and to make numerous virulence factors, such as CT and TCP (although these human virulence factors have not been found to have roles in zebrafish colonization [42]), which could be slowing down its replication speed. Additionally, it is possible that for the infections resulting in a higher bacterial load, these V. cholerae strains are selectively competing with some members of the intestinal microbiome. Alternatively, other members of the resident microbiome are unaffected and may even potentially benefit from the infecting V. cholerae. A significant disruption in the host may cause cellular stress and the release of more nutrients into the intracellular space, which could allow some bacteria already present to proliferate. Lastly, the differences in bacterial loads could be a result of the variation in metabolites that were observed in the zebrafish intestines following infection. The metabolomics analysis offers some credence to this hypothesis, as it detected many essential amino acids in the zebrafish intestine following V. cholerae infection, along with other metabolites, such as shikimic acid, which can be used for the biosynthesis of aromatic amino acids. Additionally, the quantities of nucleotides such as adenine and guanine are generally elevated in the intestines of V. cholerae-infected zebrafish, which may be due to the death of host epithelial cells, which has been observed in V. cholerae infections (50). The metabolomics analysis also displayed metabolites such as isobutyric acid, a short-chain fatty acid; taurocholic acid, a bile acid; benzoic acid, which aids in digestion (51); and spermidine, a compound that has been shown to interfere with V. cholerae biofilm formation, which plays a crucial, but still enigmatic role in colonization and disease (52, 53). They were, unsurprisingly, higher in relative abundance in control fish than those infected with V. cholerae. This result is what we would expect as a large influx of V. cholerae would disrupt intestinal homeostasis, causing the depletion of metabolites through various means.

This study is the first to our knowledge to examine how the T6SS in both pandemic and nonpandemic V. cholerae affects the adult zebrafish intestinal microbiome. This study is also the first to examine the role of the T6SS in an adult zebrafish with an unmodified intestinal microbiome, demonstrating that T6SS-mediated killing of competitors is not required for colonization, but the T6SS is important for inducing pathogenesis and modulating members of the microbial community. Lastly, this study included a metabolomics analysis, examining how different strains of V. cholerae modulate the metabolomics profile of the zebrafish intestine. The specific interactions occurring at the microbial level remain to be elucidated. Pairing metabolomics data with an analysis that identifies the particular strains of bacteria present in the zebrafish intestines prior to V. cholerae infection would go a long way in helping to identify if the commensal bacteria being eliminated in the gut are being specifically targeted by the incoming V. cholerae in order to change the intestinal conditions to a state more favorable to the pathogen (i.e., whether eliminating particular types of bacteria ensures the availability of select metabolites favorable to V. cholerae).

MATERIALS AND METHODS

Bacterial strains and culture conditions.

Bacterial strains used in this study are listed in Table 1. All bacteria were cultured at 37°C in LB broth with streptomycin (100 μg/ml) for all V. cholerae strains or rifampacin (40 μg/ml) for E. coli strain MG1655. V. cholerae strain V52, T6SS mutant V52 Δhcp/hcp (referred to as V52 Δhcp from here on), and T6SS mutant V52 ΔvasK were kindly provided by Stefan Pukatzki. V. cholerae strain N16961 ΔvxrB (a T6SS mutant lacking a response regulator essential for the expression of several genes involved in the T6SS) was kindly provided by Tobias Doerr (31, 54).

TABLE 1.

Bacterial strains

Bacteria	Serogroup (if applicable)
E. coli MG1655	N/Aa
V. cholerae N16961	O1 El Tor
V. cholerae N16961 ΔvxrB	O1 El Tor
V. cholerae V52	O37
V. cholerae V52 Δhcp/hcp	O37
V. cholerae V52 ΔvasK	O37

^aN/A, not applicable.

Bacterial competition assays.

V. cholerae and E. coli MG1655 (prey) were cultured overnight on selective LB plates. The following day, colonies were harvested and resuspended in 2 ml of LB broth and diluted to an OD₆₀₀ of ∼1.0. The bacteria were then diluted to the volumes necessary for 10⁸ predator and 10⁷ prey bacteria. These calculated volumes were multiplied by 5, mixed together in an Eppendorf tube, centrifuged at 10,000 × g for 2 minutes, resuspended in 125 μl LB, spotted in duplicate at 25 μl onto plain LB, and incubated for 4 hours at 37°C. Immediately after starting the 4-hour incubation, the same volume of each predator and prey used for the competition assay was diluted to 1 ml with LB, serial diluted, spotted in 10-μl aliquots onto LB plates, and incubated at 37°C overnight to determine the starting CFU/ml for each strain. After the 4-hour incubation, the spots were harvested with a sterile loop and resuspended in 500 μl LB, serially diluted, plated onto selective LB plates overnight at 37°C, and counted the next day.

Zebrafish.

Adult, wild-type strain AB zebrafish were used in all experiments. The fish were housed in an Aquaneering aquatic housing system, with the tank water filtered by reverse osmosis and maintained at pH 7.0 to 7.5. Tank water was conditioned with Instant Ocean salt (Aquarium Systems, Mentor, OH) to a conductivity of 400 to 550 μS. The fish were fasted for at least 12 h prior to each experiment. All zebrafish were euthanized in 100 ml of 320 μg/ml Tricaine-S (tricaine methanesulfonate; MS-222; Western Chemical, Ferndale, WA) for a minimum of 25 min. All animal protocols were approved by the Wayne State University IACUC.

Inoculation of zebrafish and tank water via immersion.

Bacterial cultures were incubated with aeration in LB broth at 37°C for 16 to 18 h. Cells were subsequently washed once and then diluted to the correct concentration in sterile 1× phosphate-buffered saline (PBS). Bacterial cell densities ranged from 10⁷ to 10⁹ per beaker (∼5 × 10⁴ to 5 × 10⁶ CFU/ml). Four zebrafish per group were placed into a 400-ml beaker with a perforated lid containing 200 ml of sterile infection water (autoclaved tank water). One milliliter of bacterial inoculum then was added to the beaker with fish. The control groups included fish that were exposed to 1 ml of 1× PBS only. Each beaker was placed into a glass-front incubator set at 28°C for the duration of the experiment (24 hours).

Intestinal colonization assessment.

After the specified time point (24 hours), fish were euthanized as described above. The entire intestinal tract of each fish was aseptically excised; placed into homogenization tubes (2.0-ml screw-cap tubes; Sarstedt, Numbrecht, Germany), with 1.5 g of 1.0-mm glass beads (BioSpec Products, Inc. Bartlesville, OK) and 1 ml of 1× PBS; and held on ice. Homogenization tubes were loaded into a Mini-Beadbeater-24 (BioSpec Products, Inc. Bartlesville, OK) and shaken at maximum speed for two 1-min cycles, with the samples being incubated for 1 min on ice after both the first and last cycles. Intestinal homogenates from each fish were diluted and plated for enumeration onto LB agar plates that contained 100 μg/ml streptomycin and were incubated overnight at 37°C.

Processing fish infection water.

A total of 50 ml of fish infection water was extracted, in duplicate, and put into two 50-ml conical tubes. Tubes were centrifuged at 10,700 × g for 10 min at 4°C, and the supernatant was decanted, being careful not to disturb the pellet. Each pellet was resuspended in 1 ml of 1× PBS. One of the 50-ml conical tubes was used for bacterial enumeration and microbial analysis, while the other was used for mucin and protein quantification.

DNA extraction.

A total of 500 μl of the obtained zebrafish intestinal homogenate was harvested for DNA extraction using a DNeasy Powersoil kit (Qiagen,; Valencia, CA) following the manufacturer’s instructions. The cutoff for low DNA recovery yield for samples was 1 ng/μl. Background technical controls (i.e., blank DNA extraction kits) did not yield detectable 16S rRNA gene PCR products.

DNA sequencing.

DNA isolated from individual fish and water samples was used to generate 16S rRNA gene libraries. Illumina MiSeq sequencing was performed at Michigan State University using methods described previously (55–57). The V4 region of the bacterial 16S rRNA gene was targeted for sequencing using the primers 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). All raw 16S rRNA gene sequence data were processed using the Divisive Amplicon Denoising Algorithm (DADA2, v1.12) in R (v 3.5.1) (https://www.R-project.org), as previously described (58–60). DADA2 is a model-based approach for correcting amplicon errors without constructing operational taxonomic units. This method analyzed the 16S rRNA gene amplicon sequence variants (ASVs), defined by 100% sequence similarity, based on the online MiSeq protocol (https://benjjneb.github.io/dada2/tutorial.html) with minor modifications. These modifications included allowing truncation lengths of 250 bp and 150 bp and a maximum number of expected errors of 2 bp and 7 bp for forward and reverse reads, respectively. Sample inference allowed for pooling of samples, which in turn allowed for increased power to detect rare variants. Additionally, samples in the resulting sequence table were pooled prior to removal of chimeric sequences. Sequences were then classified using the silva_nr_v132_train_set database with a minimum bootstrap value of 80%. Two fish samples were ultimately excluded from analysis for insufficient sequence yield (one from the control group and one from the N16961-infected group). The remaining samples (48 fish) were subsampled to the number of sequences in the least represented sample (4,771 sequences), and a consensus taxonomy was determined for each of the ASVs (n = 1,037; with 131 singletons). Good’s coverage values averaged 99.0% ± 0.6% (± standard deviation) for the fish intestine, indicating that there was thorough sample coverage in this study. Taxonomic designations, raw ASV count data, and Good’s coverage values for each sample are provided as supplemental data.

α-Diversity analysis (i.e., diversity within the microbiome).

The Chao1 index (here referred to as Chao) was utilized to estimate how many different bacterial ASVs were present in a given microbiome sample (i.e., richness). The nonparametric Shannon-Wiener and inverse Simpson indices were calculated to indicate both richness and evenness of microbiome samples (i.e., heterogeneity) (61, 62). Due to a batch effect that arose from collecting the fish over the course of several months, generalized linear modeling (GLM) was performed on log-transformed zebrafish intestinal diversity index data to evaluate variation in α-diversity using the car, nlme, lme4, and multcomp packages in R (63–66). The values of the indices were log transformed before analysis to normalize the data, as the residuals need to be normally distributed if tests requiring normality are to be used (67). The GLM output included post hoc comparisons evaluated by multiple comparisons of mean values, with Tukey contrasts.

β-Diversity analysis (i.e., diversity between two or more microbiomes).

Microbiome composition (i.e., membership) and structure (i.e., membership plus member relative abundances) were characterized using Jaccard and Bray-Curtis ecological similarity indices, respectively. Principal-coordinate analysis (PCoA) plots were used to visualize variation in microbiomes among samples, and the effects of V. cholerae infections on microbiome composition and structure were evaluated using permutation multivariate analysis of variance (PERMANOVA or nonparametric multivariate analysis of variance [NPMANOVA]) (68). The adonis function in the Vegan package of R (https://www.rdocumentation.org/packages/vegan/versions/2.4-2) (69) was used to control for fish batch effect (i.e., by the strata command) to account for the collection date differences in the fish intestine. Raw P values are presented for all multivariate permutation tests (999 permutations). No Bonferroni corrections were applied for β-diversity analyses, as they can be overly conservative when used with permutation tests (70). Linear discriminant analysis effect size (LEfSe) was conducted to identify ASVs that were differentially relatively abundant among the sample types (71). These LEfSe analyses were controlled for batch effect using the ID (i.e., collection time) parameter. For figures illustrating LEfSe results, all the ASVs that had a linear discriminant analysis (LDA) score of >2.5 were presented.

Microtiter periodic acid-Schiff's reagent (PAS) assay.

A microtiter PAS assay was performed as described in Mitchell et al. (16). A total of 1 ml of a 50% (wt/vol) periodic acid (Sigma-Aldrich, St. Louis, MO) stock solution was made which can be stored protected from light at 4°C for up to 1 week. A 96-well plate (Costar 3361; Corning, Corning, NY) was loaded with 100 μl/well of the blank (1× PBS), mucin standards (see below), and samples in triplicate. A volume of 50 μl/well of fresh 0.1% periodic acid solution (10 μl of the 50% periodic acid stock added to 5 ml of 7% acetic acid, used immediately after making) was added and mixed by pipetting. The plate was covered in plastic wrap and incubated at 37°C for 1 to 1.5 h. After incubation, the plate was cooled to room temperature (∼5 minutes) before adding 100 μl/well Schiff’s reagent (84655; Sigma-Aldrich) and mixed with a pipette. The plate was again covered in plastic wrap and placed on a rocker or shaker for 15 to 40 min or until sufficient color developed. Absorbance was read at 560 nm using a plate reader (Tecan SpectraFluor plus; Mannedorf, Switzerland).

Mucin standards.

Mucin standards were made by suspending the appropriate amount of mucin from porcine stomach, as follows: type III (M1778; Sigma-Aldrich) in sodium acetate buffer (pH 5.5; 100 mM sodium acetate and 5 mM EDTA [pH 5.5], with glacial acetic acid), at 400 μg/ml, 300 μg/ml, 200 μg/ml, 150 μg/ml, 100 μg/ml, 75 μg/ml, 50 μg/ml, 25 μg/ml, and 10 μg/ml. The mixtures were stored at 4°C.

Protein assay.

One milliliter of the Pierce 660-nm protein assay reagent (Thermo Scientific; Waltham, MA) was combined with 67 μl of each protein standard or sample, mixed, and incubated at room temperature for 5 minutes. Absorbance was read at 660 nm on a spectrophotometer (Thermo Scientific) using double-distilled water (ddH₂O) as a blank.

Protein standards.

The following bovine serum albumin (BSA) standards were made from an 1,800-μg/ml BSA stock solution: 1,800 μg/ml, 1,000 μg/ml, 750 μg/ml, 500 μg/ml, 250 μg/ml, 125 μg/ml, 50 μg/ml, and 25 μg/ml. The solutions were stored at 4°C.

qPCR.

Total bacterial DNA abundance within samples was measured via amplification of the V1-V2 region of the 16S rRNA gene using the protocol of Dickson et al. (72) with minor modifications. These modifications included the use of a degenerative forward primer (27f-CM [5′-AGA GTT TGA TCM TGG CTC AG-3′]) and a degenerate probe containing locked nucleic acids (+) (BSR65/17 [5′-6-carboxyfluorescein (56FAM)-TAA + YA + C ATG + CA + A GT + C GA-black hole quencher 1 [BHQ1]-3′]). Each 20-μl reaction mixture contained 0.6 μM 27f-CM primer; 0.6 μM 357R primer (5′-CTG CTG CCT YCC GTA G-3′); 0.25 μM BSR65/17 probe; 10.0 μl of 2 TaqMan environmental master mix 2.0 (Life Technologies, Carlsbad, CA); and 3.0 μl of either purified DNA (diluted to 80 ng/μl when possible), elution buffer, or nuclease-free water. The total bacterial DNA qPCR was performed using the following conditions: 95°C for 10 min, followed by 45 cycles consisting of 94°C for 30 s, 50°C for 30 s, and 72°C for 30 s. Duplicate reactions were run for all samples. Raw amplification data were normalized to the ROX passive reference dye and analyzed using the online platform Thermo Fisher Cloud: Standard Curve (SR) 3.3.0-SR2-build15 with automatic threshold and baseline settings. Quantification cycle (C_q) values were calculated for samples based on the mean number of cycles required for normalized fluorescence to exponentially increase. After plotting a regression of log E. coli 16S rRNA gene copy number and C_q value for standard curves included in each qPCR run, the 16S rRNA gene copy number in zebrafish samples was calculated according to Gallup (73) using the equation X_o = E_AMP^{b −Cq}, where E_AMP is the exponential amplification value for the qPCR assay, calculated as E_AMP = 10^−1/m and b and m are the intercept and slope of the regression, respectively (59, 74).

Metabolomics.

Untargeted metabolomics was performed by Creative Proteomics. Briefly, zebrafish intestinal homogenates in 1× PBS were thawed, and 100-μl aliquots were extracted with 300 μl of 80% methanol. All samples were then kept at −40°C for 1 h. After that step, samples were vortexed for 30 s and centrifuged at 12,000 rpm and 4°C for 15 min. Finally, 200 μl of supernatant and 5 μl of DL-o-chlorophenylalanine (100 μg/ml) were transferred to a vial for liquid chromatography-mass spectrometry (LC-MS) analysis. Quality-control (QC) samples were used to evaluate the methodology. The same amount of extract was obtained from each sample and mixed as QC samples. The QC sample was prepared using the same sample preparation procedure. Ultraperformance liquid chromatography–time of flight mass spectrometry (UPLC-TOF-MS) separation was performed by an Ultimate 3000LC system combined with a Q Exactive MS instrument (Thermo Scientific) and screened with electrospray ionization MS (ESI-MS) (targeted MS/MS mode). The LC system is comprised of an Acquity UPLC HSS T3 column (100 by 2.1 mm, 1.8 μm) with the Ultimate 3000LC system. The mobile phase is composed of solvent A (0.05% formic acid-water) and solvent B (acetonitrile) with a gradient elution (0 to 1 min, 5% B;1 to 12 min, 5% to 95% B;12 to 13.5 min, 95% B;13.5 to 13.6 min, 95% to 5% B;13.6 to 16.0 min, 5% B). The flow rate of the mobile phase is 0.3 ml·min⁻¹. The column temperature is maintained at 40°C, and the sample manager temperature is set at 4°C. Mass spectrometry parameters in positive ion mode (ESI+; where the analyte is sprayed at low pH to encourage positive ion formation, generally detecting protonated and/or alkali adduct analyte molecules) and negative ion mode (ESI−; where the analysis is carried out well above a molecules isoelectric point to deprotonate the molecule) are listed as follows: ESI+: heater temp, 300°C; sheath gas flow rate, 45 arb; aux gas flow rate, 15 arb; sweep gas flow rate, 1 arb; spray voltage, 3.0 kV; capillary temp, 350°C; S-Lens radio frequency (RF) level, 30%; and ESI−: heater temp, 300°C, sheath gas flow rate, 45 arb; aux gas flow rate, 15 arb; sweep gas flow rate, 1 arb; spray voltage, 3.2 kV; capillary temp, 350°C; S-Lens RF level, 60%.

Statistical analysis.

Each experiment was performed a minimum of two times on separate occasions, unless otherwise specified in the figure legends. One-way and two-way analyses of variance (ANOVAs) with Tukey’s multiple-comparison test, Sidak’s multiple-comparison test, Dunnett’s multiple-comparison test against a control, Student’s t test, Mantel test, or Bonferroni’s correction were conducted as described in the figure legends. Analyses were performed using GraphPad Prism 7.0, past v4.02; Excel; and R software.

Data availability.

The data that support the findings of this study are available from the corresponding author (J.H.W.) by email, jwithey@med.wayne.edu, upon reasonable request.