Repurposing anti-diabetic drug "Sitagliptin" as a novel virulence attenuating agent in Serratia marcescens

Abstract

Background: Serratia marcescens is an emerging pathogen that causes a variety of health care associated infections. S. marcescens is equipped with an arsenal of virulence factors such as biofilm formation, swimming and swarming motilities, prodigiosin, protease and others which enable it to initiate and cause the infection. These virulence factors are orchestrated under the umbrella of an intercellular communication system named Quorum sensing (QS). QS allows bacterial population to synchronize the expression of virulence genes upon detection of a chemical signaling molecule. Targeting bacterial virulence is a promising approach to attenuate bacteria and enhances the ability of immune system to eradicate the bacterial infection. Drug repurposing is an advantageous strategy that confers new applications for drugs outside the scope of their original medical use. This promising strategy offers the use of safe approved compounds, which potentially lowers the costs and shortens the time than that needed for development of new drugs. Sitagliptin is dipeptidyl peptidase-4 (DPP-4) inhibitor, is used to treat diabetes mellitus type II as it increases the production of insulin and decreasing the production of glucagon by the pancreas. We aimed in this study to repurpose sitagliptin, investigating the anti-virulence activities of sitagliptin on S. marcescens.

Methods: The effect of sub-inhibitory concentrations of sitagliptin on virulence factors; protease, prodigiosin, biofilm formation, swimming and swarming motilities was estimated phenotypically. The qRT-PCR was used to show the effect of sitagliptin on the expression of QS-regulated virulence genes. The in-vivo protective activity of sitagliptin on S. marcescens pathogenesis was evaluated on mice.

Results: Sitagliptin (1 mg/ml) significantly reduced the biofilm formation, swimming and swarming motilities, prodigiosin and protease. The qRT-PCR confirmed the effect on virulence as shown by down regulating the expression of fimA, fimC, flhC, flhD, bsmB, rssB, rsmA, pigP, and shlA genes. Moreover, the in-vivo findings showed the efficient ability of sitagliptin to weaken S. marcescens pathogenesis.

Conclusion: Sitagliptin is a promising anti-virulence agent against S. marcescens that may be beneficial in the control of healthcare associated infections caused by S. marcescens.

Fig 1. Effect of sitagliptin (sub MIC) on growth of S. marcescens.

LB broth media containing 1 mg/ml of sitagliptin and control LB broth without sitagliptin were inoculated with an overnight culture from S. marcescens adjusted 0.5 McFarland Standard. After overnight incubation at 37°C, the optical densities of both cultures were measured at 600 nm. The Student's t-test was used to compare between sitagliptin treated and untreated cultures and the results were considered statistically significant when P values <0.05. No statistically significant difference between OD₆₀₀ of the sitagliptin treated and untreated cultures after overnight incubation in LB broth (P = 0.0749).

Fig 2. Biofilm inhibition of S. marcescens by sitagliptin.

S. marcescens was cultured in presence or absence of sitagliptin (1mg/ml) in polystyrene micro-titer plate, and incubated at 37°C either for 24 h for evaluation of biofilm formation. A) Biofilm forming cells were stained by crystal violet, 33% glacial acetic acid was added to solubilize the dye and optical density was measured at 590 nm. Assays were performed in triplicate and reduction in biofilm formation was calculated. The absorbance of sitagliptin treated S. marcescens were expressed as mean ± standard error of percentage change from untreated S. marcescens control. The Student's t-test was used to compare between sitagliptin treated and untreated cultures and the results were considered statistically significant when P < 0.05. Sitagliptin at 1 mg/ml significant reduced the biofilm formation (P < 0.0001) and the percentage biofilm inhibition reached about 56%. B) The formed S. marcescens biofilm on glass slides in presence or absence of sitagliptin (1 mg/ml) was stained with crystal violet. The slides were examined under the light microscope, (left) untreated S. marcescens and (right) sitagliptin treated S. marcescens biofilms.

Fig 3. Inhibition of S. marcescens swimming motility by sitagliptin.

LB agar plates with 0.3% agar with and without 1 mg/ml sitagliptin were prepared. The Student's t-test was used to compare between sitagliptin treated and untreated cultures and the results were considered statistically significant when P values < 0.05. A) 5μl from an overnight culture of S. marcescens (OD₆₀₀ 0.4) was inoculated into the center of the plates. B) The zones of swimming were measured and the experiment was repeated in triplicates, sitagliptin significantly inhibited the swimming motility S. marcescens (P < 0.0001) and the percentage of reduction reached about 81%.

Fig 4. Inhibition of S. marcescens swarming motility by sitagliptin.

LB agar plates with 0.5% agar with and without 1 mg/ml sitagliptin were prepared. The Student's t-test was used to compare between sitagliptin treated and untreated cultures and the results were considered statistically significant when P values < 0.05. A) 5μl from an overnight culture of S. marcescens (OD₆₀₀ 0.4) was inoculated into the center of the plates. B) The zones of swarming were measured and the experiment was repeated in triplicates, sitagliptin significantly inhibited the swarming motility of S. marcescens (P < 0.0001) and the percentage of inhibition reached about 85%.

Fig 5. Inhibition of prodigiosin pigment of S. marcescens by sitagliptin.

An optical density of overnight culture of S. marcescens was adjusted to 0.4 at 600nm, and inoculated in 2ml fresh LB broth at grown 28°C for 18 hr. The cells were collected and acidified to extract prodigiosin from cultures of S. marcescens in presence or absence of 1mg/ml sitagliptin. The growth of sitagliptin-treated and untreated cultures of S. marcescens was compared and the growth was not significantly affected by sitagliptin treatment. The experiment was made in triplicate, the absorbance was measured at 534 nm and the degree of inhibition was expressed as mean ± standard error of percentage change of prodigiosin production of sitagliptin treated S. marcescens from untreated S. marcescens control. Significantly, sitagliptin reduced the production of prodigiosin pigment of S. marcescens (P < 0.0001) with by about 65%.

Fig 6. Inhibition of protease activity of S. marcescens by sitagliptin.

A) S. marcescens overnight cultures in LB broth in the presence and absence of sitagliptin sub-MIC were adjusted to OD₆₀₀ 0.4, centrifuged at 10,000 rpm for 15 min and the protease activities were measured by adding the supernatants in 100 μl aliquots to the wells made in skim milk agar plates (5%). The diameters of the clear zones surrounding the wells in skim milk agar plate were measured. B) The diameters of the clear zones surrounding the growth were measured. The experiment was done in triplicate and the degree of inhibition was determined. The Student's t-test was used to compare between sitagliptin treated and untreated S. marcescens and the results were considered statistically significant when P values < 0.05. Sitagliptin significantly decreased the protease activity of S. marcescens (P < 0.0001) with by 47%.

Fig 7. Sitagliptin downregulated virulence genes of S. marcescens.

RNA was isolated from S. marcescens cultures treated and untreated with sitagliptin sub-MIC (OD₆₀₀ 0.4) to be used in cDNA synthesis. The cDNA was amplified by qRT-PCR and changes in the expression of each gene were normalized in relation to the mean critical threshold values of housekeeping gene rplU. Expression fold change in gene expression in sitagliptin-treated S. marcescens was calculated by the 2^-ΔΔCT method and compared to untreated bacteria. The data shown are the mean ± standard errors from three experiments. P<0.05 was considered significant using Student’s t-test. Sitagliptin significantly decreased the expression of genes fimA, fimC, flhC, flhD, bsmB, rssB, rsmA, pigP and shlA (P < 0.0001).

Fig 8. Sitagliptin protected mice from S. marcescens.

Five mouse groups of healthy mice comprising 5 mice each were used. Group 1, mice were intraperitoneally injected with sitagliptin-treated S. marcescens in sterile PBS, group 2 was injected with DMSO-treated bacteria, group 3 was injected with untreated bacteria, group 4 was injected with sterile PBS and group 5 was left un-inoculated. Mice survival in each group was recorded every day over 5-days, plotted using Kaplan-Meier method and significance (P < 0.05) was calculated using Log-rank test, GraphPad Prism 5. All mice in groups 4 and 5 (negative control groups) survived, while only 60% of mice survived (3 out of 5 mice) in the groups comprising the DMSO-treated bacteria or untreated bacteria. In contrast to untreated S. marcescens, all mice injected with sitagliptin-treated S. marcescens survived, showing 100% survival, conferring 40% protection (Log rank test for trend P = 0.02).