Investigating Peptidoglycan Recycling Pathways in Tannerella forsythia with N-Acetylmuramic Acid Bioorthogonal Probes

Abstract

The human oral microbiome is the second largest microbial community in humans, harboring over 700 bacterial species, which aid in digestion and protect from growth of disease-causing pathogens. One such oral pathogen, Tannerella forsythia, along with other species, contributes to the pathogenesis of periodontitis. T. forsythia is unable to produce its own N-acetylmuramic acid (NAM) sugar, essential for peptidoglycan biosynthesis and therefore must scavenge NAM from other species with which it cohabitates. Here, we explore the recycling potential of T. forsythia for NAM uptake with a bioorthogonal modification into its peptidoglycan, allowing for click-chemistry-based visualization of the cell wall structure. Additionally, we identified NAM recycling enzyme homologues in T. forsythia that are similar to the enzymes found in Pseudomonas putida. These homologues were then genetically transformed into a laboratory safe Escherichia coli strain, resulting in the efficient incorporation of unnatural NAM analogues into the peptidoglycan backbone and its visualization, alone or in the presence of human macrophages. This strain will be useful in further studies to probe NAM recycling and peptidoglycan scavenging pathways of T. forsythia and other cohabiting bacteria.

Keywords: N-acetylmuramic acid probes; bacterial cell-wall remodeling; bacterial pathogenesis; bioorthogonal; oral microbiome; peptidoglycan.

Figure 1:

Cell wall representation of Gram-negative oral pathogen, T. forsythia, with schematic model of NAM utilization for peptidoglycan biosynthesis. PG, peptidoglycan; PM, plasma membrane; LPS, lipopolysaccharide; OM, outer membrane. NAM is transported into the cytoplasm by MurT. NAM is hypothesized to go through two different routes: Direct utilization by AmgK/MurU to form UDP-NAM, or phosphorylated by MurK to form MurNAc-6P that can shuttle into glycolysis or theorized to undergo a dephosphorylation by an unknown enzyme to then serve as a substrate for AmgK/MurU. UDP-NAM is made into Park’s nucleotide and subsequently appended to the membrane and glycosylated by MurG. Lipid II is flipped across the membrane by MurJ where transglycosylases (TGs) and transpeptidases (TPs) act to form mature peptidoglycan.

Figure 2:. Labeling of wild-type T. forsythia with AzNAM and CuAAC.

A) Structures of NAM and AzNAM used to remodel the cells. B) Cells are able to be remodeled and fluorescently visualized following the CuAAC click reaction with Cy5-Alkyne and the AzNAM in the cell’s PG. NAM cells do not have the azide handle and therefore do not react with the fluorophores, leading to no fluorescence. Scale bars = 20 μm

Figure 3:. Incorporation of AzNAM probe at various concentrations in DH5α-Tf-KU and E. coli ΔMurQ-KU cells.

A) In DH5α-Tf-KU cells, AzNAM recovers cell growth at all concentrations tested for ~3 hours and 150 μM and above for at least 6 hours. B) In E. coli ΔMurQ-KU cells, AzNAM recovers cell growth at concentrations of 600 μM and above. All samples are treated with a lethal dose of fosfomycin and their respective compound at the concentration listed. Error bars represent the standard deviation of three biological replicates from one trial. The graph is representative of the average of triplicates from one of the three biological experiment replicates.

Figure 4:

A) Labeling of DH5α-Tf-KU utilizing CuAAC and SPAAC chemistries. DH5α-TfKU are remodeled by incorporating AzNAM in the cell wall with recycling enzymes AmgK and MurU. Bacteria are then fluorescently labeled using CuAAC or SPAAC to install a terminal or strained alkyne (Alk-488 or DBCO-488, respectively) fluorophore. NAM cells do not have the azide handle and therefore do not react with the fluorophores, leading to no fluorescence. Images were taken using confocal microscopy. Images are representative of a minimum of three fields viewed per replicate with at least two technical replicates, and experiments were conducted in at least three biological replicates. Scale bars = 10 μm B) Mass spectrometry verification of incorporation of AzNAM into the PG of DH5α-Tf-KU cells. Structure of one disaccharide fragment identified from digestion with lysozyme following remodeling with AzNAM.

Figure 5:

Flow cytometry verification of incorporation of AzNAM into the cell wall using CuAAC (A) and SPAAC (B) to fluorescently label DH5α-Tf-KU and EQKU cells. DH5α-Tf-KU and EQKU cells remodeled with AzNAM (orange and purple, respectively) both saw a shift in FITC compared to DH5α-Tf-KU and EQKU NAM controls (red and orange). Mean fluorescence intensity plots show this fluorescence intensity value is statistically significant (****p<0.0001). Data shown is representative of experiments performed in technical duplicate and biological triplicate. Statistically analyzed by ANOVA with Tukey’s test ****p<0.0001

Figure 6:

THP-1 macrophage cells invaded by remodeled DH5α-Tf-KU cells. DH5α-Tf-KU cells are remodeled by incorporating AzNAM bacterial cell wall precursor in the cell wall with recycling enzymes AmgK and MurU. The bacteria were invaded into THP-1 macrophage cells and fixed prior to the CuAAC click labeling (bacteria, green) and subsequent staining with Phallodin-TRITC (F-actin, purple) and DAPI (nucleus, blue). Images were taken on a Zeiss LSM800 Confocal Microscope with single plain snaps with averaging of 4. Images are representative of a minimum of three fields viewed per replicate with at least two technical replicates, and experiments were conducted in at least three biological replicates. (A) Scale bars = 10 μm (B) Scale bars = 5 μm