An O-methylflavone from Artemisia afra kills non-replicating hypoxic Mycobacterium tuberculosis

Abstract

Ethnopharmacological relevance: African wormwood (Artemisia afra Jacq. ex Willd.) has been used traditionally in southern Africa to treat illnesses causing fever and was recently shown to possess anti-tuberculosis activity. As tuberculosis is an endemic cause of fever in southern Africa, this suggests that the anti-tubercular activity of A. afra may have contributed to its traditional medicinal use.

Aim of the study: Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), is a deadly and debilitating disease globally affecting millions annually. Emerging drug-resistant Mtb strains endanger the efficacy of the current therapies employed to treat tuberculosis; therefore, there is an urgent need to develop novel drugs to combat this disease. Given the reported activity of A. afra against Mtb, we sought to determine the mechanisms by which A. afra inhibits and kills this bacterium.

Materials and methods: We used transcriptomics to investigate the impact of Artemisia spp. extracts on Mtb physiology. We then used chromatographic fractionation and biochemometric analyses to identify a bioactive fractions of A. afra extracts and identify an active compound.

Results: Transcriptomic analysis revealed that A. afra exerts different effects on Mtb compared to A. annua or artemisinin, suggesting that A. afra possesses other phytochemicals with unique modes of action. A biochemometric study of A. afra resulted in the isolation of an O-methylflavone (1), 5-hydroxy-7-methoxy-2-(4-methoxyphenyl)chromen-4-one, which displayed considerable activity against Mtb strain mc²6230 in both log phase growth and metabolically downshifted hypoxic cultures.

Conclusions: The present study demonstrated that an O-methylflavone constituent of Artemisia afra explains part of the activity of this plant against Mtb. This result contributes to a mechanistic understanding of the reported anti-tubercular activity of A. afra and highlights the need for further study of this traditional medicinal plant and its active compounds.

Keywords: Artemisia afra; Biochemometrics; Hypoxia; Infectious disease; Metabolomics; Tuberculosis.

Figure 1.. A. afra, A. annua, and artemisinin have distinct transcriptomic impacts on Mtb.

Aerobically growing Mtb was treated with each extract or compound at lethal doses for four hours or at inhibitory and sub-inhibitory doses for 24 hours. Untreated cultures were harvested at the same two time-points and RNAseq was used to generate transcriptomic profiles. All conditions were tested in quadruplicate. A. PCA was done on the read count tables from each sample in each condition, revealing that each treatment clustered separately. B. Expression of each gene in each treatment was compared to that in the time-matched control, and differentially expressed genes were subject to Gene Set Enrichment Analysis. Gene sets were GO Biological Process gene lists obtained from AmiGO.(Carbon et al., 2009) “Activated” gene sets had higher expression in the treated samples compared to the controls while “suppressed” genes sets had lower expression. “GeneRatio” indicates the proportion of genes in the set that were differentially expressed in the indicated condition. “p.adjust” is the P value of the overrepresentation of genes within the set among the differentially expressed genes, after correction for multiple comparisons.

Figure 2.. Time-kill curves of Mtb treated with an A. afra extract and constituent fractions and subfractions.

In all cases, treatment was applied to liquid cultures at Day 0 and aliquots were plated on drug-free solid media at the indicated timepoints. Colonies were enumerated after 3–4 weeks of growth in order to quantify the number of colony forming units (CFU) that had survived drug treatment at each timepoint. All no-drug samples were given DMSO as a vehicle control. A. Mtb was exposed to crude DCM extract of A. afra and the three flash chromatography fractions with the lowest MICs (see Table 1). B. Mtb was exposed to crude DCM extract of A. afra and subfraction HAB9. No viable colonies were recovered from cultures treated with HAB9 after six days. The limit of detection is indicated with a dashed line. C. Mtb was sealed in vials with 17 mL of culture and 11 mL of headspace. The cultures became hypoxic approximately eight days after sealing as evidenced by methylene blue decoloration. After the cultures had been sealed for 14 days, subfraction HAB9 or the vehicle control DMSO were injected by needle to prevent introduction of oxygen. This was considered day 0 of treatment. No colonies were recovered from cultures treated with HAB9 after two or six days of treatment. The limit of detection is indicated with a dashed line.

Figure 3.. Biochemometric identification of 1.

(A) S-line plot illustrating the covariance (y-axis) and correlation (coloration) between features and classification of the OPLS-DA model. Greater values in the covariance and correlation represent features that hold a larger influence in the model generation and discrimination between active and inactive samples. Compound 1 is highlighted, along with ions from another A. afra subfraction (HAB4). (B) Extracted ion chromatogram of 1, m/z 299.0841 from subfraction HAB9.