Deciphering the Antibacterial Mode of Action of Alpha-Mangostin on Staphylococcus epidermidis RP62A Through an Integrated Transcriptomic and Proteomic Approach

Abstract

Background: Alpha-mangostin (α-MG) is a natural xanthone reported to exhibit rapid bactericidal activity against Gram-positive bacteria, and may therefore have potential clinical application in healthcare sectors. This study sought to identify the impact of α-MG on Staphylococcus epidermidis RP62A through integrated advanced omic technologies. Methods: S. epidermidis was challenged with sub-MIC (0.875 μg/ml) of α-MG at various time points and the differential expression pattern of genes/proteins were analyzed in the absence and presence of α-MG using RNA sequencing and LC-MS/MS experiments. Bioinformatic tools were used to categorize the biological processes, molecular functions and KEGG pathways of differentially expressed genes/proteins. qRT-PCR was employed to validate the results obtained from these analyses. Results: Transcriptomic and proteomic profiling of α-MG treated cells indicated that genes/proteins affected by α-MG treatment were associated with diverse cellular functions. The greatest reduction in expression was observed in transcription of genes conferring cytoplasmic membrane integrity (yidC2, secA and mscL), cell division (ftsY and divlB), teichoic acid biosynthesis (tagG and dltA), fatty-acid biosynthesis (accB, accC, fabD, fabH, fabI, and fabZ), biofilm formation (icaA) and DNA replication and repair machinery (polA, polC, dnaE, and uvrA). Those with increased expression were involved in oxidative (katA and sodA) and cellular stress response (clpB, clpC, groEL, and asp23). The qRT-PCR analysis substantiated the results obtained from transcriptomic and proteomic profiling studies. Conclusion: Combining transcriptomic and proteomic methods provided comprehensive information about the antibacterial mode of action of α-MG. The obtained results suggest that α-MG targets S. epidermidis through multifarious mechanisms, and especially prompts that loss of cytoplasmic membrane integrity leads to rapid onset of bactericidal activity.

Keywords: LC-MS/MS; RNA-sequencing; alpha-mangostin; bactericidal; cytoplasmic membrane.

FIGURE 1

Interaction analysis of upregulated genes identified by RNA-sequencing. (A) Interactome map predicted using STRING_10.5. The network nodes represent the proteins encoded by the genes. Edges represent protein-protein associations with high confidence score. The associations are meant to be specific and meaningful with associated proteins jointly contributing to a shared function. This does not necessarily mean that the proteins are physically binding each other, (B) significant biological process and (C) molecular function of upregulated genes.

FIGURE 2

Significant GO annotation of downregulated genes identified by RNA-sequencing. (A) Interactome map predicted using STRING_10.5. The network nodes represent the proteins encoded by the genes. Edges represent protein–protein associations with high confidence score. The associations are meant to be specific and meaningful with associated proteins jointly contributing to a shared function. This does not necessarily mean that the proteins are physically binding each other and (B) biological process of downregulated genes.

FIGURE 3

The histogram summarizing the enriched KEGG pathways of differentially expressed genes identified using RNA-sequencing. (A) Upregulated pathways and (B) downregulated pathways upon α-MG treatment.

FIGURE 4

GO annotation of differentially expressed proteins of S. epidermidis upon α-MG treatment identified using LC-MS/MS analysis. Biological process (A,C) and molecular function (B,D).

FIGURE 5

KEGG pathway analysis of differentially expressed proteins identified using LC-MS/MS analysis.

FIGURE 6

The qRT-PCR validation of differentially expressed genes which are randomly selected based on transcriptomic and proteomic approaches. BRG stands for biofilm regulatory gene. The data represented the mean ± standard deviation of three independent experiments. The statistical significance of data was determined by unpaired Student’s t-test. ^∗P < 0.05, ^∗∗P < 0.005, ^∗∗∗P < 0.001.

FIGURE 7

Schematic representation of multifarious antibacterial mode of action for α-MG. (1) α-MG shows electrostatic interaction with negatively charged bacterial membrane, where it binds with the bacterial inner membrane (2) in which, the strong hydrophobic interaction with lipid alkyl chain of cytoplasmic membrane might be the major driving force for the rapid bactericidal property. Further, α-MG interacts with the transmembrane precursor proteins via hydrogen bonding. (3) The downregulation of YidC2, SecA, FtsY, and MscL has evidenced that α-MG thwart the cytoplasmic membrane integrity. (4) The downregulation of dltA inhibits D-alanylation of anomeric teichoic acids and hence, the development of resistance against α-MG has been aborted. (5) TagG, a part of two component ABC transporter which exports the anomeric polyglycerophosphate chains (TAs) from cytoplasm. Inhibition of tagG shows that teichoic acid biosynthetic pathway has also been affected, because tagG is localized in cytoplasmic membrane. This might possibly due to the loss of cytoplasmic membrane integrity which resulted in decreased export of teichoic acids from cytoplasm. (6) DNA replication and mismatch repair mechanism has been downregulated upon α-MG treatment. (7) α-MG also inhibited fatty acid biosynthesis pathway (8) α-MG also enhanced the expression of heat shock proteins. (9) α-MG has also shown to increase the genes involved in oxidative stress response. CPM, cytoplasmic membrane; PGN, peptidoglycan.