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Mucin promotes rapid surface motility in Pseudomonas aeruginosa.

Yeung AT, Parayno A, Hancock RE - MBio (2012)

Bottom Line: In this study, we added mucin to swimming media and found that it promoted the ability of P. aeruginosa to exhibit rapid surface motility.Interestingly, bacterial cells at the thick edge appeared piled up and lacked flagella, while cells at the motility center had flagella.Our data from various genetic and phenotypic studies suggest that mucin may be promoting a modified form of swarming or a novel form of surface motility in P. aeruginosa.

View Article: PubMed Central - PubMed

Affiliation: Centre for Microbial Diseases and Immunity Research, University of British Columbia, Vancouver, British Columbia, Canada.

ABSTRACT

Unlabelled: An important environmental factor that determines the mode of motility adopted by Pseudomonas aeruginosa is the viscosity of the medium, often provided by adjusting agar concentrations in vitro. However, the viscous gel-like property of the mucus layer that overlays epithelial surfaces is largely due to the glycoprotein mucin. P. aeruginosa is known to swim within 0.3% (wt/vol) agar and swarm on the surface at 0.5% (wt/vol) agar with amino acids as a weak nitrogen source. When physiological concentrations or as little as 0.05% (wt/vol) mucin was added to the swimming agar, in addition to swimming, P. aeruginosa was observed to undergo highly accelerated motility on the surface of the agar. The surface motility colonies in the presence of mucin appeared to be circular, with a bright green center surrounded by a thicker white edge. While intact flagella were required for the surface motility in the presence of mucin, type IV pili and rhamnolipid production were not. Replacement of mucin with other wetting agents indicated that the lubricant properties of mucin might contribute to the surface motility. Based on studies with mutants, the quorum-sensing systems (las and rhl) and the orphan autoinducer receptor QscR played important roles in this form of surface motility. Transcriptional analysis of cells taken from the motility zone revealed the upregulation of genes involved in virulence and resistance. Based on these results, we suggest that mucin may be promoting a new or highly modified form of surface motility, which we propose should be termed "surfing."

Importance: An important factor that dictates the mode of motility adopted by P. aeruginosa is the viscosity of the medium, often provided by adjusting agar concentrations in vitro. However, the gel-like properties of the mucous layers that overlay epithelial surfaces, such as those of the lung, a major site of Pseudomonas infection, are contributed mostly by the production of the glycoprotein mucin. In this study, we added mucin to swimming media and found that it promoted the ability of P. aeruginosa to exhibit rapid surface motility. These motility colonies appeared in a circular form, with a bright green center surrounded by a thicker white edge. Interestingly, bacterial cells at the thick edge appeared piled up and lacked flagella, while cells at the motility center had flagella. Our data from various genetic and phenotypic studies suggest that mucin may be promoting a modified form of swarming or a novel form of surface motility in P. aeruginosa.

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Growth and bacterial cell counts of PA14 WT determined using various amino acids as sole nitrogen (N) sources. (A) P. aeruginosa bacteria were grown in liquid MSCFM supplemented with total amino acids (control) or single amino acids serving as the sole N source. Growth was measured at 37°C using a Tecan Spectrofluor Plus reader. (B) P. aeruginosa bacteria were spotted onto MSCFM agar-mucin plates supplemented with total amino acids (control) or a single sole N source (as indicated) and incubated at 37°C for 13 h. The entire surface motility colony was resuspended in 1× PBS and serially plated on LB agar plates. Results shown are means ± SD for at least three independent experiments with duplicates for each experiment. Asterisks indicate a statistically significant difference (P ≤ 0.05) between the sole nitrogen source and control as determined by Student’s t test.
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fig7: Growth and bacterial cell counts of PA14 WT determined using various amino acids as sole nitrogen (N) sources. (A) P. aeruginosa bacteria were grown in liquid MSCFM supplemented with total amino acids (control) or single amino acids serving as the sole N source. Growth was measured at 37°C using a Tecan Spectrofluor Plus reader. (B) P. aeruginosa bacteria were spotted onto MSCFM agar-mucin plates supplemented with total amino acids (control) or a single sole N source (as indicated) and incubated at 37°C for 13 h. The entire surface motility colony was resuspended in 1× PBS and serially plated on LB agar plates. Results shown are means ± SD for at least three independent experiments with duplicates for each experiment. Asterisks indicate a statistically significant difference (P ≤ 0.05) between the sole nitrogen source and control as determined by Student’s t test.

Mentions: We investigated the effects of different carbon and nitrogen sources on the ability of P. aeruginosa to propagate on the mucin plates. To examine the effects of different carbon sources on mucin-promoted motility, we excluded lactate from and replaced glucose in the MSCFM with equimolar amounts of each of the following carbon sources: α-ketoglutarate, succinate, fumarate, citrate, malate, glycerol, and mannitol (Fig. 6A). P. aeruginosa exhibited a statistically significant increase in mucin-promoted surface motility when glucose was replaced with citrate and a decrease in surface motility with succinate, even though those carbon sources supported similar growth rates (data not shown), indicating that growth differences could not explain these results. When the total free amino acids in the mucin-MSCFM plates were replaced with an equal concentration (19 mM) of NH4Cl as the sole nitrogen source, surface propagation was impaired by about 50% (Fig. 6B); in contrast, this concentration of NH4Cl completely eliminated swarming. We also examined the effects of individual amino acids serving as the sole nitrogen sources on the surface motility. The total free amino acids in the normal MSCFM were replaced with equimolar amounts of a single amino acid. When they were provided as the sole nitrogen source, we observed that many of the amino acids tested were able to support this form of surface motility (Fig. 6B). Replacement with glycine, methionine, valine, tryptophan, glutamine, isoleucine, and ornithine gave significantly weaker mucin-stimulated surface motility phenotypes, but the motility zones in these cases still had fold changes greater than 50% compared to the surface coverage of the motility zones in the presence of the total free amino acids in MSCFM. Growth experiments showed that P. aeruginosa exhibited moderate growth defects in liquid MSCFM when glutamine was provided as the sole nitrogen source while growth was greatly impaired in the presence of methionine, valine, tryptophan, isoleucine, glycine, or ornithine (Fig. 7A). Although the majority of amino acids that supported strong surface motility exhibited only little to moderate growth impairment (data not shown), leucine, phenylalanine, and threonine each caused significant growth impairments when provided as sole nitrogen sources but supported strong surface motilities (Fig. 6B and 7A). One possibility is that there were fewer bacterial cells in the motility zones with leucine, phenylalanine, or threonine than on normal MSCFM plates. To test this, we resuspended surface motility colonies grown on threonine, leucine, phenylalanine, or normal MSCFM in buffer and performed serial plating. Resultant bacterial cell counts revealed significantly lower numbers of bacterial cells from the surface motility colonies grown on leucine, phenylalanine, or threonine compared to normal MSCFM (Fig. 7B).


Mucin promotes rapid surface motility in Pseudomonas aeruginosa.

Yeung AT, Parayno A, Hancock RE - MBio (2012)

Growth and bacterial cell counts of PA14 WT determined using various amino acids as sole nitrogen (N) sources. (A) P. aeruginosa bacteria were grown in liquid MSCFM supplemented with total amino acids (control) or single amino acids serving as the sole N source. Growth was measured at 37°C using a Tecan Spectrofluor Plus reader. (B) P. aeruginosa bacteria were spotted onto MSCFM agar-mucin plates supplemented with total amino acids (control) or a single sole N source (as indicated) and incubated at 37°C for 13 h. The entire surface motility colony was resuspended in 1× PBS and serially plated on LB agar plates. Results shown are means ± SD for at least three independent experiments with duplicates for each experiment. Asterisks indicate a statistically significant difference (P ≤ 0.05) between the sole nitrogen source and control as determined by Student’s t test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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fig7: Growth and bacterial cell counts of PA14 WT determined using various amino acids as sole nitrogen (N) sources. (A) P. aeruginosa bacteria were grown in liquid MSCFM supplemented with total amino acids (control) or single amino acids serving as the sole N source. Growth was measured at 37°C using a Tecan Spectrofluor Plus reader. (B) P. aeruginosa bacteria were spotted onto MSCFM agar-mucin plates supplemented with total amino acids (control) or a single sole N source (as indicated) and incubated at 37°C for 13 h. The entire surface motility colony was resuspended in 1× PBS and serially plated on LB agar plates. Results shown are means ± SD for at least three independent experiments with duplicates for each experiment. Asterisks indicate a statistically significant difference (P ≤ 0.05) between the sole nitrogen source and control as determined by Student’s t test.
Mentions: We investigated the effects of different carbon and nitrogen sources on the ability of P. aeruginosa to propagate on the mucin plates. To examine the effects of different carbon sources on mucin-promoted motility, we excluded lactate from and replaced glucose in the MSCFM with equimolar amounts of each of the following carbon sources: α-ketoglutarate, succinate, fumarate, citrate, malate, glycerol, and mannitol (Fig. 6A). P. aeruginosa exhibited a statistically significant increase in mucin-promoted surface motility when glucose was replaced with citrate and a decrease in surface motility with succinate, even though those carbon sources supported similar growth rates (data not shown), indicating that growth differences could not explain these results. When the total free amino acids in the mucin-MSCFM plates were replaced with an equal concentration (19 mM) of NH4Cl as the sole nitrogen source, surface propagation was impaired by about 50% (Fig. 6B); in contrast, this concentration of NH4Cl completely eliminated swarming. We also examined the effects of individual amino acids serving as the sole nitrogen sources on the surface motility. The total free amino acids in the normal MSCFM were replaced with equimolar amounts of a single amino acid. When they were provided as the sole nitrogen source, we observed that many of the amino acids tested were able to support this form of surface motility (Fig. 6B). Replacement with glycine, methionine, valine, tryptophan, glutamine, isoleucine, and ornithine gave significantly weaker mucin-stimulated surface motility phenotypes, but the motility zones in these cases still had fold changes greater than 50% compared to the surface coverage of the motility zones in the presence of the total free amino acids in MSCFM. Growth experiments showed that P. aeruginosa exhibited moderate growth defects in liquid MSCFM when glutamine was provided as the sole nitrogen source while growth was greatly impaired in the presence of methionine, valine, tryptophan, isoleucine, glycine, or ornithine (Fig. 7A). Although the majority of amino acids that supported strong surface motility exhibited only little to moderate growth impairment (data not shown), leucine, phenylalanine, and threonine each caused significant growth impairments when provided as sole nitrogen sources but supported strong surface motilities (Fig. 6B and 7A). One possibility is that there were fewer bacterial cells in the motility zones with leucine, phenylalanine, or threonine than on normal MSCFM plates. To test this, we resuspended surface motility colonies grown on threonine, leucine, phenylalanine, or normal MSCFM in buffer and performed serial plating. Resultant bacterial cell counts revealed significantly lower numbers of bacterial cells from the surface motility colonies grown on leucine, phenylalanine, or threonine compared to normal MSCFM (Fig. 7B).

Bottom Line: In this study, we added mucin to swimming media and found that it promoted the ability of P. aeruginosa to exhibit rapid surface motility.Interestingly, bacterial cells at the thick edge appeared piled up and lacked flagella, while cells at the motility center had flagella.Our data from various genetic and phenotypic studies suggest that mucin may be promoting a modified form of swarming or a novel form of surface motility in P. aeruginosa.

View Article: PubMed Central - PubMed

Affiliation: Centre for Microbial Diseases and Immunity Research, University of British Columbia, Vancouver, British Columbia, Canada.

ABSTRACT

Unlabelled: An important environmental factor that determines the mode of motility adopted by Pseudomonas aeruginosa is the viscosity of the medium, often provided by adjusting agar concentrations in vitro. However, the viscous gel-like property of the mucus layer that overlays epithelial surfaces is largely due to the glycoprotein mucin. P. aeruginosa is known to swim within 0.3% (wt/vol) agar and swarm on the surface at 0.5% (wt/vol) agar with amino acids as a weak nitrogen source. When physiological concentrations or as little as 0.05% (wt/vol) mucin was added to the swimming agar, in addition to swimming, P. aeruginosa was observed to undergo highly accelerated motility on the surface of the agar. The surface motility colonies in the presence of mucin appeared to be circular, with a bright green center surrounded by a thicker white edge. While intact flagella were required for the surface motility in the presence of mucin, type IV pili and rhamnolipid production were not. Replacement of mucin with other wetting agents indicated that the lubricant properties of mucin might contribute to the surface motility. Based on studies with mutants, the quorum-sensing systems (las and rhl) and the orphan autoinducer receptor QscR played important roles in this form of surface motility. Transcriptional analysis of cells taken from the motility zone revealed the upregulation of genes involved in virulence and resistance. Based on these results, we suggest that mucin may be promoting a new or highly modified form of surface motility, which we propose should be termed "surfing."

Importance: An important factor that dictates the mode of motility adopted by P. aeruginosa is the viscosity of the medium, often provided by adjusting agar concentrations in vitro. However, the gel-like properties of the mucous layers that overlay epithelial surfaces, such as those of the lung, a major site of Pseudomonas infection, are contributed mostly by the production of the glycoprotein mucin. In this study, we added mucin to swimming media and found that it promoted the ability of P. aeruginosa to exhibit rapid surface motility. These motility colonies appeared in a circular form, with a bright green center surrounded by a thicker white edge. Interestingly, bacterial cells at the thick edge appeared piled up and lacked flagella, while cells at the motility center had flagella. Our data from various genetic and phenotypic studies suggest that mucin may be promoting a modified form of swarming or a novel form of surface motility in P. aeruginosa.

Show MeSH
Related in: MedlinePlus