Table 1.
Tryptophanase Sequences for Bioinformatic Analysis.
Table 2.
E.coli strains used and generated in this study.
Fig 1.
Blastocystis tryptophanase is conserved across subtypes.
A) BLAST multiple sequence alignment of the protein sequence of the most common isoform of TnaA from E. coli K12 and Blastocystis subtypes ST4, ST7, and ST1. Regions of homology between sequences are highlighted blue. Generated using Jalview [18] B) Phylogenetic tree of TnaA-expressing microorganisms, generated using PhyML [19]. Branches exclusive to Blastocystis subtypes are highlighted in red. Numerical values represent substitutions per site. For sequences used, see Table 1.
Fig 2.
BhTnaA contains unique regions not present in other tryptophanase proteins.
A) Tertiary structure of ST7-B BhTnaA generated via PHYRE2. Light blue represents area structurally similar to E. coli K12 TnaA. Domains A (purple), B (orange), and C (red) are loops not present in the tertiary structure of E. coli K12 TnaA. Highlighted in yellow is a putative hinge structure, while highlighted in green is a polar bond whose disruption is necessary for the functioning of the hinge. B) Output from the NCBI Conserved Domain Database (CDD)[27] when the protein sequence of ST7-B BhTnaA is provided. Pink represents sequences from the beta-eliminating lyase superfamily, green represents the TnaA superfamily, and blue represents TnaA-like sequences, including the AAT I superfamily. The red box outlines the approximate location of Domain C within the results.
Fig 3.
Blastocystis is capable of producing indole from tryptophan.
A) Indole test comparing indole production capability of Blastocystis subtypes (right-hand image) with bacterial and negative controls (left-hand image). Positive result for indole is indicated by the formation of a rosindole layer when combined with Kovac’s Reagent, for example in the E. coli control tube. B) Tabulation of the bacterial control and Blastocystis subtype results from Fig 2A, where + = indole-positive and— = indole-negative. C) Standard curve of the absorbance at 568nm of the rosindole layer created by reacting different quantities of indole with Kovac’s Reagent. D) Effect of increasing tryptophan concentration on indole production by Blastocystis ST7-B after a three-day cultivation period. Indole concentration was determined by measuring the absorbance of the rosindole layer, and applying the equation generated in Fig 2C.
Fig 4.
BhTnaA functions ideally at human body temperature and pH.
A and B) Simplified map of the BhTnaA insert within the pGEX-6P-1 vector. BhTnaA is tagged with GST, with a PreScission protease sequence for cleavage of the tag. C) Western blot of the purified and cleaved BhTnaA isolate. Used to demonstrate validity of the protein purification process. The TnaA band is equivalent to the mathematically-derived expected size of the protein. D) Hanes-Woolf plot generated via supplementing purified BhTnaA with increasing concentrations of tryptophan, and determining quantity of produced indole via absorbance (as in Fig 2C). Vmax is in units of μM product generated min-1. Km is in units of mM substrate. Curve calculated for the mean of the three data points shown at each tryptophan concentration. E) Effect of BhTnaA inactivation (dMut) on indole production. Production calculated as rosindole layer absorbance post-tryptophan supplementation and 72 hrs incubation. F and G) Optimal temperature and pH for purified BhTnaA activity. Experiment identical to Fig 3E with variation of either temperature during incubation, or pH of media. Error bars were calculated using Student’s t-test.
Fig 5.
BhTnaA preferentially performs the tryptophanase reverse reaction.
A) Standard curve of fluorescent signal generated by differing concentrations of tryptophan in solution. Tryptophan was dissolved in DMEM media, which was then assessed for fluorescence at 370 and 440nm. B) Hanes-Woolf plot generated via supplementing purified BhTnaA with increasing concentrations of indole, and determining quantity of produced tryptophan via fluorescence. Fluorescence was measured as in Fig 4A. Vmax is in units of μM product generated min-1. Km is in units of mM substrate.
Fig 6.
Indole and tryptophan have opposing effects on Blastocystis viability.
3*106 Blastocystis ST7-B were seeded in PBS supplemented with increasing concentrations of indole or tryptophan. A) Remaining number of live cells as determined by manual counting using a hemocytometer. Seed line indicates initial number of cells. B) Relative remaining proportion of live cells as determined by propidium iodide stain. Significance was calculated using a two-way ANOVA, with ** = p<0.01 and **** = p<0.0001. N = 3 for all data points.
Fig 7.
Blastocystis ST7-B does not convert tryptophan to indole readily.
A) Graph of tryptophan concentration post-24-hour incubation period when culturing Blastocystis and E. coli in PBS supplemented with tryptophan. B) Graph of indole concentration in the same cultures. Significance levels were calculated using a two-way ANOVA, with * = p<0.05, ** = p<0.01, and **** = p<0.0001. N = 3 for all columns.
Fig 8.
Blastocystis ST7-B produces tryptophan when supplemented with indole.
A) Graph of tryptophan concentration post-24-hour incubation period when culturing Blastocystis ST7 and E. coli in PBS supplemented with indole. B) Graph of indole concentration in the same cultures. Significance levels were calculated using a two-way ANOVA, with * = p<0.05, ** = p<0.01, and *** = p<0.001. N = 3 for all columns.
Fig 9.
Tryptophan and Indole may exist cyclically in a Blastocystis-infected gut.
Diagram of a hypothetical ‘tryptophan cycle’ between E. coli (upper) and Blastocystis spp. (lower) within the descending colon. Tryptophan is shown in orange, and indole is shown in blue. TnaA enzymes are depicted as hexagons, and E. coli tryptophan permease (TnaB) is depicted as a blue star. Extracellular tryptophan is taken up by E. coli using TnaB [43], and was processed into indole by TnaA before being secreted back into the environment. Blastocystis takes up and converts the indole back into tryptophan using BhTnaA, before secreting it. This tryptophan can then be re-metabolised by E. coli.