Surface hydrophilicity promotes bacterial twitching motility

Abstract

Twitching motility is a form of bacterial surface translocation powered by the type IV pilus (T4P). It is frequently analyzed by interstitial colony expansion between agar and the polystyrene surfaces of petri dishes. In such assays, the twitching motility of Acinetobacter nosocomialis was observed with MacConkey but not Luria-Bertani (LB) agar media. One difference between these two media is the presence of bile salts as a selective agent in MacConkey but not in LB. Here, we demonstrate that the addition of bile salts to LB allowed A. nosocomialis to display twitching. Similarly, bile salts enhanced the twitching of Acinetobacter baumannii and Pseudomonas aeruginosa in LB. These observations suggest that there is a common mechanism, whereby bile salts enhance bacterial twitching and promote interstitial colony expansion. Bile salts disrupt lipid membranes and apply envelope stress as detergents. Surprisingly, their stimulatory effect on twitching appears not to be related to a bacterial physiological response to stressors. Rather, it is due to their ability to alter the physicochemical properties of a twitching surface. We observed that while other detergents promoted twitching like bile salts, stresses applied by antibiotics, including the outer membrane-targeting polymyxin B, did not enhance twitching motility. More importantly, bacteria displayed increased twitching on hydrophilic surfaces such as those of glass and tissue culture-treated polystyrene plastics, and bile salts no longer stimulated twitching on these surfaces. Together, our results show that altering the hydrophilicity of a twitching surface significantly impacts T4P functionality.

Importance: The bacterial type IV pilus (T4P) is a critical virulence factor for many medically important pathogens, some of which are prioritized by the World Health Organization for their high levels of antibiotic resistance. The T4P is known to propel bacterial twitching motility, the analysis of which provides a convenient assay for T4P functionality. Here, we show that bile salts and other detergents augment the twitching of multiple bacterial pathogens. We identified the underlying mechanism as the alteration of surface hydrophilicity by detergents. Consequently, hydrophilic surfaces like those of glass or plasma-treated polystyrene promote bacterial twitching, bypassing the requirement for detergents. The implication is that surface properties, such as those of tissues and medical implants, significantly impact the functionality of bacterial T4P as a virulence determinant. This offers valuable insights for developing countermeasures against the colonization and infection by bacterial pathogens of critical importance to human health on a global scale.

Keywords: Acinetobacter; Pseudomonas aeruginosa; bile salts; detergents; hydrophilicity; surface property; twitching motility.

Fig 1

Bile salts enable Acinetobacter to twitch. (A) Bile salts allow A. nosocomialis M2 to twitch in LB media. The twitching motility of A. nosocomialis M2 was analyzed with MacConkey (MC) or Luria-Bertani media without or with 0.5% bile salts (+BSs), 1% lactose (+Lac), or 2% peptone (+Pep) with standard polystyrene petri dishes as described in Materials and Methods. Data shown are the averages from three biological experiments each performed in triplicate. Representative images of twitching motility are shown below their respective categories. (B) Bile salts provoke A. baumannii twitching in LB media. A. baumannii strains AB0057 and AYE were analyzed for twitching motility in LB agar without (−) or with (+) 0.5% bile salts on standard polystyrene petri dishes with A. nosocomialis (M2) as a control. Data shown are from three biological experiments, represented by different symbols, each performed in triplicate. The violin plot shows the frequency distribution curves of the data, where the horizontal line indicates the median. Single, double, and quadruple asterisks indicate two values are statistically different with P < 0.05, P < 0.01, and P < 0.0001, respectively.

Fig 2

Bile salts enhance P. aeruginosa twitching motility. (A) Bile salts increase P. aeruginosa twitching. Twitching motility of PAO1 was analyzed without (−) or with (+) 0.5% bile salts as in Fig. 1B with data similarly presented. Quadruple asterisks indicate two values are statistically different with P < 0.0001. (B) Dose effect of bile salts on PAO1 twitching. PAO1 twitching was analyzed with the standard polystyrene petri dish protocol as in panel A with varying concentrations (% [wt/vol]) of bile salts as indicated. Data presented are from three biological experiments, each performed in quadruplicate with data points from the same experiment represented by the same symbols in color and shape.

Fig 3

Detergents, but not antibiotics, promote P. aeruginosa twitching. (A) Effects of detergents. PAO1 twitching was analyzed with the standard petri dish protocol as in Fig. 2A with LB agar without modification (−) or with bile salts (BSs) (5 mg/mL), Triton X-100 (TX100) (75 µg/mL), Triton X-114 (TX114) (75 µg/mL), or SDS (850 µg/mL). (B) Effects of antibiotics. PAO1 twitching was analyzed as in (A) with ampicillin (Amp) (313 ng/mL), ciprofloxacin (Cipro) (31 ng/mL), gentamicin (Gent) (31 ng/mL), or polymyxin B (PB) (313 ng/mL). Data presented in both panels are from three biological experiments, each performed in triplicate. Data points from the same experiment are represented by the same symbols in color and shape. Results in both panels are from the same sets of biological replicates, and the controls (BSs and −) are from the same data sets as a result. Quadruple asterisks indicate two values are statistically different with P < 0.0001. Antibiotics resulted in values that are not significantly (ns) different with P > 0.05.

Fig 4

Glass surfaces increase P. aeruginosa twitching motility. (A) Glass surfaces stimulate PAO1 twitching motility, and bile salts no longer enhance it. The twitching motility of PAO1 and its isogenic pilA mutant was analyzed with polystyrene (PS) or glass microscope slides in LB without or with 0.5% bile salts (BSs) after 18 hours of incubation (see Materials and Methods). (B) Hydrophobic coating of glass reduces twitching motility. Experiments were performed as in A, except that the microscope slides were coated without or with polydimethylsiloxane (PDMS) (see Materials and Methods). Data presented in both panels are from three biological experiments each performed in triplicate. The data for the controls (PS and Glass) are the same controls in both panels as they are from the same sets of biological replicates. Quadruple asterisks indicating two values are statistically different with P < 0.0001.

Fig 5

TC-treated plates ameliorate twitching and abolish the effects of bile salts. (A) P. aeruginosa twitching on TC-treated polystyrene. The twitching motility of P. aeruginosa PAO1 and its isogenic pilA mutant strain was analyzed with six-well polystyrene plates (see Materials and Methods) either TC treated or non-treated in LB agar without or with 0.5% bile salts (BSs). (B) A. nosocomialis twitching on TC-treated polystyrene. The twitching motility of A. nosocomialis M2 and its isogenic pilA deletion mutant was analyzed as in panel A. Data presented in both panels are from three biological experiments each performed in triplicate. Quadruple asterisks indicate that two values are statistically different with P < 0.0001.