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Mycofabricated biosilver nanoparticles interrupt Pseudomonas aeruginosa quorum sensing systems.

Singh BR, Singh BN, Singh A, Khan W, Naqvi AH, Singh HB - Sci Rep (2015)

Bottom Line: Transcriptional studies demonstrated that mfAgNPs reduced the levels of LasIR-RhlIR.Further genes quantification analyses revealed that mfAgNPs significantly down-regulated QS-regulated genes, specifically those encoded to the secretion of virulence factors.The results clearly indicated the anti-virulence property of mfAgNPs by inhibiting P. aeruginosa QS signaling.

View Article: PubMed Central - PubMed

Affiliation: Centre of Excellence in Materials Science (Nanomaterials), Z.H. College of Engineering &Technology, Aligarh Muslim University, Aligarh-202002, India.

ABSTRACT
Quorum sensing (QS) is a chemical communication process that Pseudomonas aeruginosa uses to regulate virulence and biofilm formation. Disabling of QS is an emerging approach for combating its pathogenicity. Silver nanoparticles (AgNPs) have been widely applied as antimicrobial agents against human pathogenic bacteria and fungi, but not for the attenuation of bacterial QS. Here we mycofabricated AgNPs (mfAgNPs) using metabolites of soil fungus Rhizopus arrhizus BRS-07 and tested their effect on QS-regulated virulence and biofilm formation of P. aeruginosa. Transcriptional studies demonstrated that mfAgNPs reduced the levels of LasIR-RhlIR. Treatment of mfAgNPs inhibited biofilm formation, production of several virulence factors (e.g. LasA protease, LasB elastrase, pyocyanin, pyoverdin, pyochelin, rhamnolipid, and alginate) and reduced AHLs production. Further genes quantification analyses revealed that mfAgNPs significantly down-regulated QS-regulated genes, specifically those encoded to the secretion of virulence factors. The results clearly indicated the anti-virulence property of mfAgNPs by inhibiting P. aeruginosa QS signaling.

No MeSH data available.


Related in: MedlinePlus

Assessment of anti-QS activity of mfAgNPs.(A) A bioindicator strain C. violaceum 12472 was used for examining anti-QS activity. Treatments include (a) solvent vehicle (H2O), (b-d) various mfAgNPs concentrations including 5, 10, and 25 μg/mL. (e) 10 μg/mL of HF. (f) 10 μg/mL of GNM. Inhibition of violacein pigment production (e.g. a colorless and opaque halo zone) indicates anti-QS effect (red arrows), while a clear and transparent zone shows cidal effect (yellow arrows). (B) Inhibition of violacein production at different mfAgNPs concentrations (0, 5, 10, and 25 μg/mL) was quantified spectrophotometrically with OD at A585 nm. Growth curves of (C) CV026 and (D) PAO1 at different concentrations of mfAgNPs for 25 h. Error bars indicate the SD of 6 measurements. (E) Examination the effect of mfAgNPs on PAO1 cell viability using CLSM. For this, cells were grown in the presence of (a) untreated control, (b) solvent vehicle (H2O), (c and d) 25 and 50 μg/mL of mfAgNPs for 24 h and stained with SYTO-9 green for live cells and PI red for dead cells.
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f3: Assessment of anti-QS activity of mfAgNPs.(A) A bioindicator strain C. violaceum 12472 was used for examining anti-QS activity. Treatments include (a) solvent vehicle (H2O), (b-d) various mfAgNPs concentrations including 5, 10, and 25 μg/mL. (e) 10 μg/mL of HF. (f) 10 μg/mL of GNM. Inhibition of violacein pigment production (e.g. a colorless and opaque halo zone) indicates anti-QS effect (red arrows), while a clear and transparent zone shows cidal effect (yellow arrows). (B) Inhibition of violacein production at different mfAgNPs concentrations (0, 5, 10, and 25 μg/mL) was quantified spectrophotometrically with OD at A585 nm. Growth curves of (C) CV026 and (D) PAO1 at different concentrations of mfAgNPs for 25 h. Error bars indicate the SD of 6 measurements. (E) Examination the effect of mfAgNPs on PAO1 cell viability using CLSM. For this, cells were grown in the presence of (a) untreated control, (b) solvent vehicle (H2O), (c and d) 25 and 50 μg/mL of mfAgNPs for 24 h and stained with SYTO-9 green for live cells and PI red for dead cells.

Mentions: The possibility of QS attenuation by mfAgNPs was firstly investigated by disc diffusion anti-QS assay using a bio-indicator strain Chromobacterium violaceum 12472, which produces the AHL-regulated violet-colored ‘violacein’ pigment531. In this assay, the development of violacein represents AHL-dependent QS signaling, while the inhibition of violacein indicates the anti-QS activity via attenuation of AHL production. A concentration dependent inhibitory effect of the mfAgNPs on violacein production was observed. The highest inhibition was recorded at 25 μg/mL, while no activity was examined with 5 μg/mL (Fig. 3A-c,d). Control discs containing halogenated furanone (HF; C-30) and gentamycin (GMN) were included. As expected, a zone of growth inhibition was detected with GMN (Fig. 3A-e), while an opaque zone of QS inhibition was seen with the HF, and no inhibition was apparent with DW (Fig. 3A-f). However, 50 μg/mL of mfAgNPs and 25 μg/mL of SBH-synthesized AgNPs showed the growth inhibitory effect against C. violaceum (Supplementary figure 5), suggesting the surface modification of mfAgNPs by proteins of BRS-07. Similar results were observed in colorimetric measurement of violacein production as 100% inhibition was observed by 25 μg/mL of mfAgNPs (Fig. 3B). Next, we sought to examine the effect of mfAgNPs (25 μg/mL) on growth of C. violaceum CV26 and PAO1. The obtained results revealed that the cell densities of bacteria did not significantly differ between untreated control and cultures exposed to 25 μg/mL of mfAgNPs (Fig. 3C,D). In addition, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) toxicity assay was also employed against PAO1 and human epithelial cells which showed non-toxic effect of mfAgNPs (Supplementary figures 5C and D), as evident from the confocal laser scanning microscopy (CLSM) analysis of PAO1 (Fig. 3Ea–d). However, a strong toxicity effect was observed, when cells exposed to 50 μg/mL of mfAgNPs (Fig. 3E-d). The results obtained demonstrated that mfAgNPs could have interfered QS without any toxic effect via attenuation of AHL production.


Mycofabricated biosilver nanoparticles interrupt Pseudomonas aeruginosa quorum sensing systems.

Singh BR, Singh BN, Singh A, Khan W, Naqvi AH, Singh HB - Sci Rep (2015)

Assessment of anti-QS activity of mfAgNPs.(A) A bioindicator strain C. violaceum 12472 was used for examining anti-QS activity. Treatments include (a) solvent vehicle (H2O), (b-d) various mfAgNPs concentrations including 5, 10, and 25 μg/mL. (e) 10 μg/mL of HF. (f) 10 μg/mL of GNM. Inhibition of violacein pigment production (e.g. a colorless and opaque halo zone) indicates anti-QS effect (red arrows), while a clear and transparent zone shows cidal effect (yellow arrows). (B) Inhibition of violacein production at different mfAgNPs concentrations (0, 5, 10, and 25 μg/mL) was quantified spectrophotometrically with OD at A585 nm. Growth curves of (C) CV026 and (D) PAO1 at different concentrations of mfAgNPs for 25 h. Error bars indicate the SD of 6 measurements. (E) Examination the effect of mfAgNPs on PAO1 cell viability using CLSM. For this, cells were grown in the presence of (a) untreated control, (b) solvent vehicle (H2O), (c and d) 25 and 50 μg/mL of mfAgNPs for 24 h and stained with SYTO-9 green for live cells and PI red for dead cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4562228&req=5

f3: Assessment of anti-QS activity of mfAgNPs.(A) A bioindicator strain C. violaceum 12472 was used for examining anti-QS activity. Treatments include (a) solvent vehicle (H2O), (b-d) various mfAgNPs concentrations including 5, 10, and 25 μg/mL. (e) 10 μg/mL of HF. (f) 10 μg/mL of GNM. Inhibition of violacein pigment production (e.g. a colorless and opaque halo zone) indicates anti-QS effect (red arrows), while a clear and transparent zone shows cidal effect (yellow arrows). (B) Inhibition of violacein production at different mfAgNPs concentrations (0, 5, 10, and 25 μg/mL) was quantified spectrophotometrically with OD at A585 nm. Growth curves of (C) CV026 and (D) PAO1 at different concentrations of mfAgNPs for 25 h. Error bars indicate the SD of 6 measurements. (E) Examination the effect of mfAgNPs on PAO1 cell viability using CLSM. For this, cells were grown in the presence of (a) untreated control, (b) solvent vehicle (H2O), (c and d) 25 and 50 μg/mL of mfAgNPs for 24 h and stained with SYTO-9 green for live cells and PI red for dead cells.
Mentions: The possibility of QS attenuation by mfAgNPs was firstly investigated by disc diffusion anti-QS assay using a bio-indicator strain Chromobacterium violaceum 12472, which produces the AHL-regulated violet-colored ‘violacein’ pigment531. In this assay, the development of violacein represents AHL-dependent QS signaling, while the inhibition of violacein indicates the anti-QS activity via attenuation of AHL production. A concentration dependent inhibitory effect of the mfAgNPs on violacein production was observed. The highest inhibition was recorded at 25 μg/mL, while no activity was examined with 5 μg/mL (Fig. 3A-c,d). Control discs containing halogenated furanone (HF; C-30) and gentamycin (GMN) were included. As expected, a zone of growth inhibition was detected with GMN (Fig. 3A-e), while an opaque zone of QS inhibition was seen with the HF, and no inhibition was apparent with DW (Fig. 3A-f). However, 50 μg/mL of mfAgNPs and 25 μg/mL of SBH-synthesized AgNPs showed the growth inhibitory effect against C. violaceum (Supplementary figure 5), suggesting the surface modification of mfAgNPs by proteins of BRS-07. Similar results were observed in colorimetric measurement of violacein production as 100% inhibition was observed by 25 μg/mL of mfAgNPs (Fig. 3B). Next, we sought to examine the effect of mfAgNPs (25 μg/mL) on growth of C. violaceum CV26 and PAO1. The obtained results revealed that the cell densities of bacteria did not significantly differ between untreated control and cultures exposed to 25 μg/mL of mfAgNPs (Fig. 3C,D). In addition, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) toxicity assay was also employed against PAO1 and human epithelial cells which showed non-toxic effect of mfAgNPs (Supplementary figures 5C and D), as evident from the confocal laser scanning microscopy (CLSM) analysis of PAO1 (Fig. 3Ea–d). However, a strong toxicity effect was observed, when cells exposed to 50 μg/mL of mfAgNPs (Fig. 3E-d). The results obtained demonstrated that mfAgNPs could have interfered QS without any toxic effect via attenuation of AHL production.

Bottom Line: Transcriptional studies demonstrated that mfAgNPs reduced the levels of LasIR-RhlIR.Further genes quantification analyses revealed that mfAgNPs significantly down-regulated QS-regulated genes, specifically those encoded to the secretion of virulence factors.The results clearly indicated the anti-virulence property of mfAgNPs by inhibiting P. aeruginosa QS signaling.

View Article: PubMed Central - PubMed

Affiliation: Centre of Excellence in Materials Science (Nanomaterials), Z.H. College of Engineering &Technology, Aligarh Muslim University, Aligarh-202002, India.

ABSTRACT
Quorum sensing (QS) is a chemical communication process that Pseudomonas aeruginosa uses to regulate virulence and biofilm formation. Disabling of QS is an emerging approach for combating its pathogenicity. Silver nanoparticles (AgNPs) have been widely applied as antimicrobial agents against human pathogenic bacteria and fungi, but not for the attenuation of bacterial QS. Here we mycofabricated AgNPs (mfAgNPs) using metabolites of soil fungus Rhizopus arrhizus BRS-07 and tested their effect on QS-regulated virulence and biofilm formation of P. aeruginosa. Transcriptional studies demonstrated that mfAgNPs reduced the levels of LasIR-RhlIR. Treatment of mfAgNPs inhibited biofilm formation, production of several virulence factors (e.g. LasA protease, LasB elastrase, pyocyanin, pyoverdin, pyochelin, rhamnolipid, and alginate) and reduced AHLs production. Further genes quantification analyses revealed that mfAgNPs significantly down-regulated QS-regulated genes, specifically those encoded to the secretion of virulence factors. The results clearly indicated the anti-virulence property of mfAgNPs by inhibiting P. aeruginosa QS signaling.

No MeSH data available.


Related in: MedlinePlus