Subinhibitory concentration of kanamycin induces the Pseudomonas aeruginosa type VI secretion system

Abstract

Pseudomonas aeruginosa is a Gram-negative bacterium found in natural environments including plants, soils and warm moist surfaces. This organism is also in the top ten of nosocomial pathogens, and prevalent in cystic fibrosis (CF) lung infections. The ability of P. aeruginosa to colonize a wide variety of environments in a lasting manner is associated with the formation of a resistant biofilm and the capacity to efficiently outcompete other microorganisms. Here we demonstrate that sub-inhibitory concentration of kanamycin not only induces biofilm formation but also induces expression of the type VI secretion genes in the H1-T6SS cluster. The H1-T6SS is known for its role in toxin production and bacterial competition. We show that the antibiotic induction of the H1-T6SS only occurs when a functional Gac/Rsm pathway is present. These observations may contribute to understand how P. aeruginosa responds to antibiotic producing competitors. It also suggests that improper antibiotic therapy may enhance P. aeruginosa colonization, including in the airways of CF patients.

Figure 1. Induction of biofilm formation in P. aeruginosa by subinhibitory concentrations of antibiotics.

(A) P. aeruginosa PAK biofilm formation upon addition of kanamycin. Top panel shows staining of biofilm biomass with crystal violet in glass test tubes. The concentration of kanamycin (in µg/ml) is indicated above each tube. Biofilm production is quantified by measuring the optical density (OD₆₀₀) of the dissolved crystal violet (middle panel). Error bars show standard deviation. The kanamycin concentration used is indicated below each bar. * indicates significantly higher biofilm levels compared to PAK incubated without antibiotic (Student’s T Test, P<0.05). Planktonic growth (OD₆₀₀) of PAK with increasing kanamycin concentration is shown over a ten hour period (lower panel). (B) Quantification of biofilm formation by PAK grown with increasing concentrations of tobramycin (top), gentamycin (middle) and tetracycline (lower). The concentration of each antibiotic used is indicated below each bar.

Figure 2. Analysis of c-di-GMP levels in P. aeruginosa PAK strains.

Relative fluorescence values are shown for strains carrying the c-di-GMP reporter plasmid (cdrA-gfp promoter fusion) [28]. Levels are shown for wild type PAK, PAK incubated with kanamycin at 30 µg/ml, and a control strain (PAKΔretS) known to have elevated c-di-GMP levels [35]. RFU indicates arbitrary relative fluorescence units corrected for optical density. Error bars show standard deviation.

Figure 3. Kanamycin induces components of the H1-T6SS in P. aeruginosa PAK.

(A) Genetic organisation of the H1-T6SS cluster. The PAKΔH1-T6SS mutant has a deletion encompassing the hsiA1 to vgrG1a genes as indicated by the hatched box. (B) Western blot of PAK whole cell extracts from cells incubated with increasing concentrations of kanamycin, indicated in µg/ml below the blots. Cell extracts were probed with anti-VgrG1a (upper blot) and Hcp1 (lower blot). The expected position of the proteins of interest is indicated on the right of the blot, and molecular weight markers (in kDa) are indicated on the left. Cell extract from the PAKΔH1-T6SS mutant is included as a control to positively identify VgrG1a and Hcp1, which are encoded within the H1-T6SS cluster and thus absent in this strain (right hand lane).

Figure 4. Kanamycin induction of H1-T6SS requires a functional LadS/Gac/Rsm pathway.

(A) Schematic representation of the Gac/Rsm signalling cascade which antagonistically controls the phenotypic switches biofilm/motility and T6SS/T3SS. RetS, GacS and LadS are inner membrane located sensors, GacA is a cytoplasmic response regulator, RsmYZ are small RNAs and RsmA a translational repressor. Pointed arrows indicate a positive effect, while blocked arrows indicate a negative effect. Dashed lines indicate an indirect interaction. An unknown activating signal is represented by the lightning bolt. (B) Planktonic growth of PAK and isogenic mutants in components of the LadS/Gac/Rsm cascade with and without kanamycin at a concentration of 30 µg/ml. The black arrow indicates sampling time for cells and supernatants used in Figure 4D. (C) Biofilm formation upon addition of kanamycin to PAK and PAK ΔrsmYZ, as indicate by the key below the graph. (D) Western blot of whole cell extracts from PAK and isogenic deletion mutants lacking genes encoding components of the regulatory pathway as indicated above the blot. Strains were incubated with or without kanamycin at 30 µg/ml as indicated below the blot. Cell extracts were probed with anti-VgrG1a (upper blot) and Hcp1 (lower blot). The expected positions of the proteins of interest are indicated on the right of the blot, and molecular weight markers (in kDa) are shown on the left.

Figure 5. Impact of kanamycin on Hcp1 production in various P. aeruginosa backgrounds.

Western blot showing Hcp1 production in whole cell lysates of P. aeruginosa PAK, PAKΔretS, PAKΔladS and P. aeruginosa PA14. Each strain was grown with or without kanamycin at 30 µg/ml as indicated below the blots (+/-). The position of Hcp1 is indicated on the right of the blot, and molecular weight markers (in kDa) indicated on the left.

Figure 6. Effect of kanamycin on expression of PA0082 (hsiA1).

Levels of β-galactosidase activity in PAK carrying chromosomal transcriptional (A) and translational (B) fusions of the hsiA1 gene (PA0082) to lacZ [38] with increasing concentrations of kanamycin as indicated in µg/ml on the x axis. Both fusions introduced in a PAKΔrsmA mutant were included as a positive control showing increased transcription and translation in this background as shown previously [38]. The * indicates a reproducible statistically significant difference (Student’s T test, P<0.05) compared to incubation without kanamycin. (C) Western blot showing induction of Hcp1 production in the translational fusion strain under corresponding kanamycin concentrations.

Figure 7. Kanamycin represses the T3SS.

(A) Western blot analysis of whole cell extracts (left) and supernatant (SN) fractions (right) from P. aeruginosa PAK cells grown under increasing levels of kanamycin (µg/ml) as indicated below the blots. Upper blots are probed with anti-RNA polymlerase (RNAP), and lower blots probed with anti-PcrV. The expected position of these components is indicated on the left side of the blot, while molecular weight markers (in kDa) are shown on the right. (B) Effect of kanamycin on the activity of lacZ transcriptional fusions with exoT or pcrD promoter. β-galactosidase activity (Miller units) is shown for PAK strains carrying plasmid borne transcriptional fusions, incubated with increasing kanamycin concentration (µg/ml) indicated on the x-axis. * indicates a statistically significant difference in activity compared to the strain incubated without kanamycin. Error bars show standard deviation.

Figure 8. Kanamycin does not improve P. aeruginosa H1-T6SS mediated bacterial competition.

E. coli cells carrying the pCR2.1 vector were co-incubated with either P. aeruginosa PAK wild type (top row), or PAKΔretS (bottom row). The competition assay was carried out as previously described [32] on LB plates without antibiotics, or in the presence of kanamycin at a concentration of 50 µg/ml for five hours. Following competition cells were harvested and resuspended in LB broth, and a dilution series ranging from 10° to 10^-3 was plated in triplicate onto LB plates, as indicated in the white boxes in each corner. Competition assays performed without kanamycin are shown on the left, while those with kanamycin are on the right. Visualisation of a blue colour gives a qualitative indication of E. coli survival.

Figure 9. Kanamycin does not induce H1-T6SS-dependent secretion.

(A) Secretion assay performed on PAK cells incubated with increasing concentrations of kanamycin as indicated above the blot in µg/ml. Whole cell extracts are shown on the left of the blot, while supernatants are shown on the right. Blots have been probed with anti-PcrV (upper blot) and anti Hcp1 (lower blot). The expected location of each protein is indicated on the left. The PAKΔretS mutant strain, with a constitutively active H1-T6SS, is included as a positive control for secretion. * denotes that these samples have been diluted 1 in 10 with loading buffer to reduce the Hcp1 signal. (B) Western blot performed with anti-Tse3 antibodies on whole cell extracts prepared from PAK cells grown with increasing concentrations of kanamycin as indicated above the blots in µg/ml. PAKΔretS is included as control as it is known that Tse3 is induced in this background.