Proteomic Profiling of Burkholderia thailandensis During Host Infection Using Bio-Orthogonal Noncanonical Amino Acid Tagging (BONCAT)

Abstract

Burkholderia pseudomallei and B. mallei are the causative agents of melioidosis and glanders, respectively, and are often fatal to humans and animals. Owing to the high fatality rate, potential for spread by aerosolization, and the lack of efficacious therapeutics, B. pseudomallei and B. mallei are considered biothreat agents of concern. In this study, we investigate the proteome of Burkholderia thailandensis, a closely related surrogate for the two more virulent Burkholderia species, during infection of host cells, and compare to that of B. thailandensis in culture. Studying the proteome of Burkholderia spp. during infection is expected to reveal molecular mechanisms of intracellular survival and host immune evasion; but proteomic profiling of Burkholderia during host infection is challenging. Proteomic analyses of host-associated bacteria are typically hindered by the overwhelming host protein content recovered from infected cultures. To address this problem, we have applied bio-orthogonal noncanonical amino acid tagging (BONCAT) to B. thailandensis, enabling the enrichment of newly expressed bacterial proteins from virtually any growth condition, including host cell infection. In this study, we show that B. thailandensis proteins were selectively labeled and efficiently enriched from infected host cells using BONCAT. We also demonstrate that this method can be used to label bacteria in situ by fluorescent tagging. Finally, we present a global proteomic profile of B. thailandensis as it infects host cells and a list of proteins that are differentially regulated in infection conditions as compared to bacterial monoculture. Among the identified proteins are quorum sensing regulated genes as well as homologs to previously identified virulence factors. This method provides a powerful tool to study the molecular processes during Burkholderia infection, a much-needed addition to the Burkholderia molecular toolbox.

Keywords: BONCAT; Burkholderia; host infection; intracellular pathogen; orthogonal amino acid labeling; protein enrichment; proteome profiling; quorum sensing (QS).

Figure 1

MetRS^NLL incorporates azidonorleucine (Anl) in place of methionine (Met). (Left) Structures of methionine (Met) and azidonorleucine (Anl). The engineered version of E. coli MetRS^NLL encodes three point mutations in the Met-binding pocket of methionyl-tRNA synthetase conferring high and preferential affinity for Anl resulting in loading of Anl onto Met-tRNA and into newly synthesized proteins in the place of Met. (Right) Copper catalyzed cycloaddition reaction of Anl-labeled protein with alkyne conjugated to biotin/fluorescent tag resulting in covalently tagged protein.

Figure 2

Expression of MetRS^NLL in B. thailandensis (Bt) leads to incorporation of Anl into Bt proteins. (A) Map of MiniTn7-kan-MetRS^NLL plasmid used for integration of MetRS^NLL into the genome of B. thailandensis strain E264. E. coli MetRS^NLL gene, optimized for expression in Burkholderia spp., is constitutively expressed using the P_S12 promoter. PC_S12 promoter drives the constitutive expression of kanamycin resistance used for selection of transformed bacteria. (B) Scheme of Tn7 transposon attachment sites downstream of glucosamine-6-phosphate synthetase genes 1 and 2 (glmS1/2), each located on one of the two B. thailandensis chromosomes, allowing for site-specific directional transposition of genes into B. thailandensis genome. FRT, flippase recognition target sites for flippase-mediated excision of FRT flanked DNA; P_S12, B. thailandensis ribosomal protein S12 gene promoter; PC_s12, B. cenocepacia rpsL promoter; T₀T₁, transcriptional terminator; Tn7L and Tn7R, left and right transposase recognition sites of Tn7 transposon; R6K_⋎ori, origin of replication; oriT, conjugal origin of transfer. Black arrows indicate genes and their transcriptional orientations (MetRS^NLL, methionyl-tRNA synthetase; kan, kanamycin resistance; glmS, glucosamine-6 phosphate synthetase). (C) Growth curves of wild type Bt and Bt-MetRS^NLL cultured in LB broth with or without azidonorleucine (Anl). Optical density (OD₆₀₀) measurements of cultures were taken during log phase. Each growth curve represents two biological replicates. (D) Survival of bacteria (wild-type Bt and Bt-MetRS^NLL strains) assessed by infecting human epithelial cells (A549) at MOI 10. Cultures were grown in DMEM supplemented with or without Anl. At 2 h hpi, cells were washed with PBS to remove unassociated extracellular bacteria. Cell layers were washed and lysed with saponin at 6 and 24 hpi and bacterial counts determined by plating culture dilutions on LB agar. Colony forming units (CFUs) were normalized to the respective MOI for each strain. (E) Incorporation of Anl in Bt-MetRS^NLL proteins was confirmed by subjecting lysates of Bt or Bt-MetRS^NLL cultured in broth with or without Anl to click reaction using biotin-alkyne. Biotinylated proteins were detected by Western blot stained using streptavidin-HRP. Primary goat anti-Burkholderia antibodies and secondary donkey anti-goat antibodies conjugated to HRP were used to stain the blot as a loading control. (F) Relative protein quantity in lysates from E was assessed by loading the same amounts of lysates onto SDS-PAGE gel and detecting total protein using Sypro Ruby stain.

Figure 3

Anl-labeling of Bt-MetRS^NLL during infection is bacteria-specific and allows for in-situ fluorescent detection of host-associated bacteria. (A) Human epithelial cells (A549) were infected at MOI 100 and cultured for 18 hrs in DMEM media supplemented with or without 1 mM Anl. Lysates from infected and uninfected monolayers were subjected to click chemistry using alkyne conjugated to biotin. Biotin-tagged proteins in cell lysates were detected by Western blotting with streptavidin-HRP. As a loading control, human GAPDH was detected using primary rabbit anti-GAPDH antibodies and secondary goat anti-rabbit antibodies conjugated to HRP. (B) A549 cells were infected at an MOI of 100 with Bt-MetRS^NLL bacteria and grown in media supplemented with 1 mM Anl for 6 hrs. Infected cells were fixed and stained with Alexa Fluor 594-wheat germ agglutinin (WGA) conjugate to visualize host cell membranes (red). Cells were subjected to click chemistry using Alexa Fluor 488 conjugated to alkyne to tag Anl-labeled proteins (green). Host cell nuclei were stained using 6-diamidino-2-phenylindole (DAPI) (blue). White arrow indicates bacteria. Fluorescent signal was visualized using fluorescence microscopy; 100x magnification was used for all images. Scale bars indicate the distance of 10 μm.

Figure 4

Anl-labeled proteins expressed by Bt-MetRS^NLL can be purified from bacterial lysates. (A) Bt-MetRS^NLL bacteria were grown for 4 hrs in DMEM media with or without Anl. Bacterial lysates were subjected to cycloaddition reaction using biotin-conjugated alkyne and biotin-tagged proteins were purified using magnetic streptavidin beads. Input (I), unbound (U), and eluate (E) samples obtained by affinity purification were analyzed by Western blotting using streptavidin-HRP to visualize biotin-tagged proteins. Primary goat anti-Burkholderia antibodies and secondary donkey anti-goat antibodies conjugated to HRP were used to determine relative bacterial protein abundance in samples. (B) Venn diagram representing the number of proteins identified by mass spectrometry in input and eluate fractions from Anl-labeled bacterial lysates and an eluate derived from unlabeled bacterial lysate.

Figure 5

The enrichment of Anl-labeled Bt-MetRS^NLL from infected host cells via affinity purification. (A) A549 cells were infected with Bt-MetRS^NLL bacteria at MOI 50 and grown for 18 hrs in DMEM supplemented with Anl. Cell lysates were biotinylated via cycloaddition and subjected to affinity purification using streptavidin beads. Western blotting with streptavidin-HRP was used to detect tagged proteins in input (I), unbound (U), and eluate (E) fractions. The eluate fraction is concentrated 10x relative to the input. Primary rabbit anti-GAPDH antibodies and secondary goat anti-rabbit antibodies conjugated to HRP were used to assess the relative amount of host protein in samples. (B) Venn diagram representing bacterial proteins identified by mass spectrometry in input and eluate fractions that were derived from infected host cells. “Eluate (culture)” corresponds to bacterial proteins identified in lysates of bacteria grown in culture.

Figure 6

Spectral counts provide a highly reproducible relative quantitation of protein levels. (A) Spectral counts of biological replicates are highly correlated (average correlation = 0.97; average correlation between log-transformed spectral counts = 0.85). (B) Spectral counts of cultured vs. infection show substantial correlation as well (average = 0.77; average correlation between log-transformed spectral counts = 0.67). (C) Adjacent genes on the same operon show similar fold changes in protein levels, as calculated by DESeq2 (correlation = 0.53). (D) Volcano plot of the DESeq2 results, showing 125 proteins differentially expressed by at least 2-fold, with a FDR-adjusted p-value of 0.05 or better (shown in orange).

Figure 7

Protein overexpression in host-associated bacteria is correlated with gene expression of regulons involved in virulence. (A) Proteins encoded by genes upregulated by quorum sensing systems in B. thailandensis also tend to be overexpressed in host-associated bacteria. Here we show the genes differentially expressed when adding all three quorum sensing molecules to a mutant unable to produce them. (B) Proteins encoded by genes upregulated in an smcR deletion mutant also tend to be overexpressed in host-associated bacteria. (C) Proteins encoded by genes upregulated in the presence of urate also tend to be overexpressed in host-associated bacteria. (D) Genes upregulated in an mftR deletion mutant show no clear protein expression pattern in host-associated bacteria.

Figure 8

The distribution of recovered proteins reflect the genome distribution well. Blue circles: all 5561 coding sequences from the B. thailandensis E264 genome. Orange plus signs: the 1171 proteins detected in the culture and infection biological replicates with at least 2 peptides in any one sample.