Identification of a glutathione transporter in A. actinomycetemcomitans

ABSTRACT Bacteria rely on extracellular chemical cues to sense their environment, interact with one another, and shape their chemical landscape. Understanding this biochemistry can help us elucidate the mechanisms by which bacterial interactions impact community function. By applying mass spectrometry to mono- and co-cultures of the oral pathogen Aggregatibacter actinomycetemcomitans and the oral commensal Streptococcus gordonii, we identified hundreds of extracellular small molecules produced by these bacteria. We discovered that A. actinomycetemcomitans secretes millimolar amounts of glutathione and identified a five-gene operon (called gttABCDE) that is important for maximum secretion. The metabolomics data set generated in this study provides a valuable data set for unraveling the importance of individual molecules for mediating polymicrobial interactions in the oral cavity. IMPORTANCE Microbes produce a large array of extracellular molecules, which serve as signals and cues to promote polymicrobial interactions and alter the function of microbial communities. This has been particularly well studied in the human oral microbiome, where key metabolites have been shown to impact both health and disease. Here, we used an untargeted mass spectrometry approach to comprehensively assess the extracellular metabolome of the pathogen Aggregatibacter actinomycetemcomitans and the commensal Streptococcus gordonii during mono- and co-culture. We generated and made publicly available a metabolomic data set that includes hundreds of potential metabolites and leveraged this data set to identify an operon important for glutathione secretion in A. actinomycetemcomitans.

S treptococci including Streptococcus gordonii are found in biofilms throughout the human oral cavity (1).Within these communities, S. gordonii produces two extrac ellular metabolites, L-lactic acid and hydrogen peroxide (H 2 O 2 ), at millimolar concen trations.These molecules have been shown to mediate chemical interactions with neighboring oral bacteria.Among the most understood S. gordonii-interacting partners is the oral pathogen Aggregatibacter actinomycetemcomitans, and these interactions lead to more severe disease than mono-infections (2)(3)(4).This enhanced pathogenesis has been attributed to the fact that L-lactic acid is the preferred carbon source of A. actinomycetemcomitans (5), and its production by S. gordonii during co-culture provides A. actinomycetemcomitans with a carbon and energy source that allows this relatively slow-growing bacterium to avoid competition with other oral bacteria (2,5,6).S. gordonii-produced H 2 O 2 also plays a role in pathogenesis by inducing production of the A. actinomycetemcomitans outer membrane protein ApiA (3), which protects A. actinomycetemcomitans from complement killing.H 2 O 2 also plays a role in controlling the biogeography (7) of A. actinomycetemcomitans-S. gordonii co-infections by inducing dispersin B in A. actinomycetemcomitans, which degrades the biofilm matrix (4).
While L-lactate and H 2 O 2 are important metabolic cues mediating interactions between A. actinomycetemcomitans and S. gordonii, recent genomic work indicates that additional chemical interactions likely occur (8,9).Here, we used a discovery-based liquid chromatography-mass spectrometry (LC-MS) approach to identify new extracellu lar chemical cues produced by A. actinomycetemcomitans and S. gordonii.For these experiments, A. actinomycetemcomitans VT1169 and S. gordonii DL1.1 (ATCC 49818) were grown planktonically in mono-and co-culture in a chemically defined medium (CDM) (Fig. 1A).CDM is a MOPS-buffered, defined media containing over 60 components, including amino acids, vitamins, and nucleic acids with glucose as the primary energy source (6,10).Cell-free supernatants were harvested from exponentially growing monoand co-cultures at 3 and 5 hours and analyzed using LC-MS.
The metabolomics data arising from these LC-MS experiments are metabolite features, each containing a mass-to-charge ratio (m/z) and retention time.In addition, fragmentation (tandem) spectra are acquired using a data-dependent acquisition mode.The spectral features (unique retention time and m/z pairs) are grouped based on adducts and isotopes and can be identified by matching fragmentation patterns to known molecules in spectral databases.XCMS (https://xcmsonline.scripps.edu/) is a metabolomics analysis platform that extracts the abundances of detected features in mass spectrometry data sets by calculating area under the peak of each chromatogram, which can then be analyzed using a variety of statistical methods.Metabolite features were initially extracted using Compound Discoverer.We further sorted all detected features by P-value and fold-change using XCMS and performed identification via spectral matching to an in-house database and METLIN Metabolite and Chemical Entity Database (11).Selectively filtering all spectral features that showed a significant (P < 0.05) increase in abundance compared to uninoculated CDM yielded a total of 1,304 and 2,138 features at 3 and 5 hours, respectively, with 863 features shared between the timepoints (Fig. 1B and C). S. gordonii produced more unique features in mono-culture compared to A. actinomycetemcomitans, with over twice as many (551 vs 242) at the 5-hour timepoint (Fig. 1C).A significant number of features were produced only in co-culture, with 29.2% (381) and 26.8% (574) of total features found in 3-and 5-hour co-cultures, respectively (Fig. 1B and C).These results indicate that both S. gordonii and A. actinomycetemcomitans produce hundreds of extracellular metabolites during growth in CDM, many of which are only produced in mono-or co-culture.
From this expansive data set, we selected several molecules for further confirmation by matching retention times to known molecules and/or creating mirror plots of fragmentation spectra using the National Institute of Standards and Technology and Global Natural Products Social Molecular Networking (https://metabolomicsusi.ucsd.edu/dashinterface)databases (Table S1).Based on our interest in glutathione (GSH) (12) and the fact that GSH is a key modulator of virulence in several bacteria (13), we focused on this molecule.GSH is a low-molecular-weight thiol-containing tripeptide (l-γ-glutamyl-l-cysteinyl-glycine) that protects cells from a variety of stresses (13,14).GSH was detected in A. actinomycetemcomitans mono-and co-culture supernatants (Table S1).Although the retention time of the analytical standard of GSH matched that from the supernatants, the signal was near the detection limit.Therefore, we confirmed the presence of GSH and compared its abundances across conditions using a fluorometric assay.GSH was present in 5-hour A. actinomycetemcomitans mono-cultures at 425 µg/mL (1.4 mM) and in 5-hour co-cultures at 343 µg/mL (1.1 mM) but not detectable in S. gordonii mono-cultures (Fig. 2A).Thus, high level of extracellular GSH is produced during A. actinomycetemcomitans mono-culture and co-culture growth in CDM.
Synthesis of GSH in A. actinomycetemcomitans is proposed to occur in a single step via the bifunctional GSH synthase GshF (15).While the biosynthesis of GSH has been studied in A. actinomycetemcomitans, transport mechanisms are not known.To identify the mechanism of GSH export, we searched the A. actinomycetemcomitans genome for homologs of genes encoding peptide transporters in Escherichia coli.This analysis yielded a putative five-gene A. actinomycetemcomitans operon containing genes ACT74_06145 to ACT74_06165, which putatively encode proteins with homology to E. coli OppABCDF (Fig. 2B).OppABCDF is an ATP-dependent oligopeptide permease that has been well characterized in Salmonella typhimurium (16), where it imports peptides containing two to five amino acids.Genes ACT74_06150, ACT74_06155, ACT74_06160, and ACT74_06165 also had homology to the ATP-dependent tetrameric E. coli gluta thione transporter GsiABCD (17), albeit with lower overall identity (35%-47%).To test whether this operon was important for GSH secretion in A. actinomycetemcomitans, a transposon mutant containing an insertion in the A. actinomycetemcomitans homolog of oppD (ACT74_06160, gttD) was obtained from an ordered A. actinomycetemcomitans transposon mutant library (18).This mutant grew to similar yields as wild-type (WT) A. actinomycetemcomitans but secreted less GSH (Fig. 2C) (18).Expression of gttD in trans using the plasmid pJAK16 (19,20) significantly increased the extracellular level of GSH in the gttD mutant, although not to the level observed in WT A. actinomycetemcomitans (Fig. 2C).This is likely due to either polar effects of the transposon on downstream genes or suboptimal expression of ACT74_06160.These data, along with the fact that transpo son mutants in the A. actinomycetemcomitans oppB and oppC homologs (ACT74_06150 and ACT74_06155, respectively) also reduced extracellular glutathione levels (Fig. S1), designated the ACT74_06145 to ACT74_06165 operon as gttABCDE (glutathione transporter A-E).
To assess whether gttD is critical for S. gordonii-A.actinomycetemcomitans co-cul ture, we grew these bacteria in mono-and co-culture and then quantified bacterial numbers.Both WT A. actinomycetemcomitans and the gttD mutant grew to similar levels in mono-culture, and viable cells decreased to below the limit of detection in co-culture (Fig. S2), indicating that gttD plays no role in A. actinomycetemcomitans co-culture survival.S. gordonii numbers increased by over 10-fold during co-culture with WT A. actinomycetemcomitans compared to mono-culture, likely due to the ability of A. actinomycetemcomitans to detoxify S. gordonii-produced H 2 O 2 (3,4,8).However, this increase in S. gordonii numbers was not observed during co-culture with the A. actinomycetemcomitans gttD mutant (Fig. 2D), indicating that gttD is critical for S. gordonii survival during in vitro co-culture.Since S. gordonii can protect host cells from pathogenic bacteria (21), we propose that extracellular glutathione mayalso be beneficial to the host.Ultimately, this study provides a robust metabolomics data set for studying polymicro bial interactions in the future.

FIG 1 A
FIG 1 A. actinomycetemcomitans and S. gordonii produce hundreds of extracellular metabolites.(A) A. actinomycetemcomitans and S. gordonii mono-culture growth in CDM (n = 6).Error bars are standard deviation.(B-C) Mass spectrometry spectral features significantly enriched (P < 0.05) in mono-and co-culture growth at (B) 3 and (C) 5 hours.

FIG 2 A
FIG 2 A. actinomycetemcomitans secretes GSH, and gttD is necessary for maximum secretion.(A) Quantification of extracellular GSH using a fluorometric assay in A. actinomycetemcomitans and S. gordonii mono-and co-cultures after 5 hours of growth.S. gordonii did not produce detectable levels of GSH.(B) Glutathione transporter identified in A. actinomycetemco mitans, including percent identity to OppABCDF from E. coli MG1655.Operon gttABCDE corresponds to loci ACT74_06145 to ACT74_06165 in strain VT1169.(C) Extracellular glutathione produced by wild-type A. actinomycetemcomitans (Aa), the gttD transposon mutant (Aa gttD -), and the genetically complemented Aa gttD − (Aa gttD − + gttD).(D) S. gordonii CFUs during monoand co-culture with Aa or Aa gttD − .*P < 0.05 using unpaired t-test.