Curcumin rescues Caenorhabditis elegans from a Burkholderia pseudomallei infection

Abstract

The tropical pathogen Burkholderia pseudomallei requires long-term parenteral antimicrobial treatment to eradicate the pathogen from an infected patient. However, the development of antibiotic resistance is emerging as a threat to this form of treatment. To meet the need for alternative therapeutics, we proposed a screen of natural products for compounds that do not kill the pathogen, but in turn, abrogate bacterial virulence. We suggest that the use of molecules or compounds that are non-bactericidal (bacteriostatic) will reduce or abolish the development of resistance by the pathogen. In this study, we adopted the established Caenorhabditis elegans-B. pseudomallei infection model to screen a collection of natural products for any that are able to extend the survival of B. pseudomallei infected worms. Of the 42 natural products screened, only curcumin significantly improved worm survival following infection whilst not affecting bacterial growth. This suggested that curcumin promoted B. pseudomallei-infected worm survival independent of pathogen killing. To validate that the protective effect of curcumin was directed toward the pathogen, bacteria were treated with curcumin prior to infection. Worms fed with curcumin-treated bacteria survived with a significantly extended mean-time-to-death (p < 0.0001) compared to the untreated control. In in vitro assays, curcumin reduced the activity of known virulence factors (lipase and protease) and biofilm formation. To determine if other bacterial genes were also regulated in the presence of curcumin, a genome-wide transcriptome analysis was performed on curcumin-treated pathogen. A number of genes involved in iron acquisition and transport as well as genes encoding hypothetical proteins were induced in the presence of curcumin. Thus, we propose that curcumin may attenuate B. pseudomallei by modulating the expression of a number of bacterial proteins including lipase and protease as well as biofilm formation whilst concomitantly regulating iron transport and other proteins of unknown function.

Keywords: Burkholderia pseudomallei; Caenorhabditis elegans; antibiotic resistance; bacterial attenuation; curcumin.

Figure 1

Natural products enhanced the survival of B. pseudomallei infected worms. The chart above compares the hits from the screen performed on 42 natural products (200 μg/mL). The data are plotted at the point where 50% of the untreated worms survived the infection. Natural products resulting in survival exceeding the untreated control were selected for further analysis. Results are expressed as mean ± SE from at least two independent assays.

Figure 2

Antimicrobial property of positive hits toward B. pseudomallei. (A) A clear zone of 30 mm was formed for the positive control (tetracycline) while no zone of inhibition was observed for the negative control (DMSO). No inhibition zones were formed for UE-09 and UE-11 implying that they did not exert antimicrobial effect on the bacteria. (B) The formation of a clear inhibition zone (9 mm) around the disc indicated susceptibility of the bacteria to UE-08.

Figure 3

UE-15 rescued C. elegans from B. pseudomallei infection. (A) The survival curves for worms treated with the 200 μg/ml of the 6 positive hits. Treatment with UE-09, UE-11, UE-14, UE-18, and UE-21 failed to enhance the survival of infected worms whereas the addition of UE-15 extended the lifespan when compared to the untreated worms. Graph shows the mean ± SD from a representative of three independent assays. In a pair-wise analysis using the log-rank test, differences between UE-15 treated and untreated samples were significant (p < 0.0001). (B) Chemical structure of UE-15 or curcumin, a polyphenol from C. longa.

Figure 4

Curcumin exhibited no antimicrobial effect on B. pseudomallei. (A) At concentrations ranging from 0.6 μM to 600 μM, curcumin did not affect bacterial growth as reflected by the insignificant decrease in CFU when compared to the untreated control. DMSO at a concentration of 11.01% was antibacterial and prevented further analysis with curcumin at 1200 μM. The positive control, tetracycline, significantly decreased the number of CFU from 2.3 μM onwards. The bar chart corresponded to mean ± SE from three independent assays. (B) Growing B. pseudomallei in the presence of 50 μM, 100 μM, and 543 μM curcumin did not affect pathogen growth when compared to the untreated control. The graph shows the mean ± SE from three independent assays. (C) Treatment of B. pseudomallei with EGTA affected bacterial growth in a dose dependent manner. At 5 mM EGTA, no significant reduction of growth was observed. However, treatment with 10 mM EGTA significantly restricted the growth of B. pseudomallei when compared to the untreated control. The graph shows the mean ± SE from two independent assays.

Figure 5

Curcumin inhibited the growth of Gram negative and positive bacteria. Antimicrobial effect of curcumin on (A) E. coli OP50, (B) E. faecalis, (C) S. aureus, and (D) MRSA. At 1200 μM curcumin, growth of (A) E. coli OP50 and (B) E. faecalis was reduced when compared to the untreated control. On the other hand, the growth of (C) S. aureus and (D) MRSA was inhibited by 600 μM curcumin and more prominent at 1200 μM.

Figure 6

Curcumin targets B. pseudomallei to rescue C. elegans from infection. The chart above demonstrates worm survival following pre-treatment of B. pseudomallei with curcumin where the lifespan of worms was extended compared to the untreated control. The graph shows the mean ± SD from at least two independent assays. In a pair-wise analysis using the log-rank test, differences between treatment and control were significant.

Figure 7

Effect of curcumin on B. pseudomallei virulence factors. In the hemolysin test, α hemolysis occurred in both (A) untreated and (B) curcumin-treated bacteria implying curcumin did not target B. pseudomallei hemolysin. In the lipase test, the presence of lipase is indicated by the presence of orange-red fluorescing colonies under UV light. The orange-red fluorescence was stronger in the untreated control (C) when compare to the curcumin-treated cells (D). In the protease test, the formation of a large halo was observed in untreated cells (E) whilst a much smaller halo was observed in curcumin-treated bacteria (F). Biofilm formation was also reduced following curcumin treatment (G,H). The bar chart represents mean ± SD from two independent assays.

Figure 8

Functional classification of curcumin regulated B. pseudomallei genes. A total of 88 genes were differentially regulated by curcumin with 76 genes up-regulated whilst 12 down-regulated. Bars indicate number of genes in each group that were significantly regulated by curcumin. Genes were divided into functional categories based on Comprehensive Microbial Resources (CMR) annotations.

Figure 9

The effect of curcumin on B. pseudomallei siderophore. (A) The CAS solution changes color from blue to orange upon curcumin treatment whereas the mixture for untreated control remained blue. (B) The amount of siderophore detected was significantly higher in curcumin-treated bacteria than in the untreated control. The bar chart represents mean ± SE from two independent assays.