The GacS sensor kinase controls phenotypic reversion of small colony variants isolated from biofilms of Pseudomonas aeruginosa PA14

Abstract

The GacS/GacA two-component regulatory system in pseudomonads regulates genes involved in virulence, secondary metabolism and biofilm formation. Despite these regulatory functions, some Pseudomonas species are prone to spontaneous inactivating mutations in gacA and gacS. A gacS(-) strain of Pseudomonas aeruginosa PA14 was constructed to study the physiological role of this sensor histidine kinase. This loss-of-function mutation was associated with hypermotility, reduced production of acylhomoserine lactones, impaired biofilm maturation, and decreased antimicrobial resistance. Biofilms of the gacS(-) mutant gave rise to phenotypically stable small colony variants (SCVs) with increasing frequency when exposed to silver cations, hydrogen peroxide, human serum, or certain antibiotics (tobramicin, amikacin, azetronam, ceftrioxone, oxacilin, piperacillin or rifampicin). When cultured, the SCV produced thicker biofilms with greater cell density and greater antimicrobial resistance than did the wild-type or parental gacS(-) strains. Similar to other colony morphology variants described in the literature, this SCV was less motile than the wild-type strain and autoaggregated in broth culture. Complementation with gacS in trans restored the ability of the SCV to revert to a normal colony morphotype. These findings indicate that mutation of gacS is associated with the occurrence of stress-resistant SCV cells in P. aeruginosa biofilms and suggests that in some instances GacS may be necessary for reversion of these variants to a wild-type state.

Phenotypic diversification in vivo: Pseudomonas aeruginosa gacS− strains generate small colony variants in vivo that are distinct from in vitro variants

“…A P. aeruginosa PA14 gacS 2 strain previously characterized by our laboratory was used to infect the rat prostate so that in vivo-derived SCVs produced by this mutant would be comparable with in vitro SCVs from the same parent (Davies et al, 2007). By using the rat prostate model in conjunction with the PA14 gacS 2 mutant we show that diversity generation can be critical for maintaining an infection in vivo.…”

“…Biofilm formation of gacS " SCVs from mucosal surface infections Biofilm formation has been shown to vary between WT, gacS 2 and in vitro-derived SCV P. aeruginosa morphotypes. IV-SCVs isolated from P. aeruginosa form exceptionally dense biofilms, whereas parental gacS 2 biofilms form very sparse and thin biofilms (Boles et al, 2004;Davies et al, 2007;Drenkard & Ausubel, 2002;Häussler et al, 2003). Thus, we hypothesized that dense biofilms would likewise be produced by the MS-SCVs isolated from the prostate.…”

“…Conversely, P. aeruginosa gacS 2 is particularly susceptible to antimicrobials (Davies et al, 2007). Thus, it was hypothesized that the MSSCVs isolated from the prostate would be more resistant to antimicrobial agents than PA14 WT or gacS 2 .…”

“…The presence of either of these variant types in sputa from CF patients correlates with decreased lung function and a poorer prognosis for the patient (Häussler et al, 1999;Pedersen et al, 1992;Schneider et al, 2008). Moreover, SCVs are better able to form biofilms, adhere to lung epithelia, resist antibiotic treatment and, in some cases, are more cytotoxic to macrophages than WT isolates (Davies et al, 2007;Drenkard & Ausubel, 2002;Häussler et al, 2003;von Götz et al, 2004). Together, these observations suggest that phenotypic variants play an important role in the disease process.…”

“…Pegs that were broken from the CBD were stained with 0.05 % (w/v) acridine orange (Sigma) for 5 min. The fluorescently labelled biofilms were examined by using a Leica DM IRE2 spectral confocal and multiphoton microscope with a Leica TCS SP2 acoustic optical beam splitter (Leica Microsystems), as described previously (Davies et al, 2007;Harrison et al, 2006). All images of biofilm slices were captured by using a 663 water-immersion objective.…”