Regulation and Molecular Basis of Environmental Muropeptide Uptake and Utilization in Fastidious Oral Anaerobe Tannerella forsythia

Abstract

Tannerella forsythia is a Gram-negative oral anaerobe associated with periodontitis. This bacterium is auxotrophic for the peptidoglycan amino sugar N-acetylmuramic (MurNAc) and likely relies on scavenging peptidoglycan fragments (muropeptides) released by cohabiting bacteria during their cell wall recycling. Many Gram-negative bacteria utilize an inner membrane permease, AmpG, to transport peptidoglycan fragments into their cytoplasm. In the T. forsythia genome, the Tanf_08365 ORF has been identified as a homolog of AmpG permease. In order to confirm the functionality of Tanf_08365, a reporter system in an Escherichia coli host was generated that could detect AmpG-dependent accumulation of cytosolic muropeptides via a muropeptide-inducible β-lactamase reporter gene. In trans complementation of this reporter strain with a Tanf_08365 containing plasmid caused significant induction of β-lactamase activity compared to that with an empty plasmid control. These data indicated that Tanf_08365 acted as a functional muropeptide permease causing accumulation of muropeptides in E. coli and thus suggested that it is a permease involved in muropeptide scavenging in T. forsythia. Furthermore, we showed that the promoter regulating the expression of Tanf_08365 was activated significantly by a hybrid two-component system regulatory protein, GppX. We also showed that compared to the parental T. forsythia strain a mutant lacking GppX in which the expression of AmpG was reduced significantly attenuated in utilizing free muropeptides. In summary, we have uncovered the mechanism by which this nutritionally fastidious microbe accesses released muropeptides in its environment, opening up the possibility of targeting this activity to reduce its numbers in periodontitis patients with potential benefits in the treatment of disease.

Keywords: AmpG; GppX; Tannerella forsythia; muropeptides; peptidoglycan.

FIGURE 1

Reporter assay for detection of AmpG permease activity. The β-lactamase assay was performed on Escherichia coli strains BW25113 (parental strain), TP73 (ΔampD)/pNU305, AR74 (ΔampG/ΔampD)/pNU305/ pACY-AR1 (empty vector), and AR74/pNU305/pAC-Tanf_08635. Activity was determined by chromogenic nitrocefin substrate induced at 0, 30, 60 min and absorbance was measured at OD₄₈₆. Data are representative of three independent experiments with similar results. Each value represents the mean (±SD) of three values measured in one representative assay.

FIGURE 2

Analysis of AmpG and MurNAc utilization operon. RT-PCR analysis with (A) primer sets spanning adjacent genes (fragments a-j). Tanf_08345, xanthane lyase; Tanf_08350 (gtf), glycosyltransferase; Tanf_08355 (gtf), glycosyltransferase; Tanf_08360 (lytB), amidase enhancer precursor; Tanf_08365 (ampG), muropeptide permease; Tanf_08370 (ybbC), conserved hypothetical protein. (B) PCR products were separated on a 1% agarose gel. RNA samples with no reverse transcription reaction as template controls were run in lanes 1, genomic DNA as template in lanes 2, and cDNA as template for each primer set in lanes 3. MW; DNA ladder. (C) DNA sequence showing transcriptional start site determine by 5′RACE.

FIGURE 3

Reporter assay to assess GppX as a transcription regulator. The β-galactosidase assay was performed on E. coli strains MG1655 (positive control), pAC-lacTFggpΔHTH (negative control), pACYC184 (empty vector), and pAC-lac-gppX (in trans). Activity was determined by chromogenic O-nitrophenyl-β-D-galactoside (ONPG) substrate with, or without induction of isopropyl β-D-1-thiogalactopyranoside (IPTG) and measured using Miller Units. Each value represents the mean (±SD) of three values measured in one representative assay. Data are representative of three independent experiments with similar results, ^∗∗∗P < 0.001.

FIGURE 4

Growth of Tannerella forsythia on muropeptides. Growth of T. forsythia wild-type and ΔgppX in M9 liquid medium supplemented with 0.2% muropeptides or 0.2% MurNAc was measured at OD₆₀₀. Results of one out of three independent cultivations with similar outcome are given.

FIGURE 5

Schematic model of T. forsythia peptidoglycan recycling pathway. Abbreviations: anhMurNAc, anhydro-N-acetylmuramic acid; EPs, endopeptidases; GlcNAc, N-acetylglucosamine; GlcNAc-6-P, N-acetylglucosamine-6-phosphate; GlcN-6-P, glucosamine-6-phosphate; IM, inner cytoplasmic membrane; LTs, transglycosylases; MurNAc-6-P, N-acetylmuramic acid 6-phosphate; OM, outer membrane; PER, periplasmic space.