Limits...
Global Regulator MorA Affects Virulence-Associated Protease Secretion in Pseudomonas aeruginosa PAO1.

Ravichandran A, Ramachandran M, Suriyanarayanan T, Wong CC, Swarup S - PLoS ONE (2015)

Bottom Line: Further investigations suggest that the MorA-mediated c-di-GMP signaling affects protease secretion largely at a post-translational level.We thus report c-di-GMP second messenger system as a novel regulator of T2SS function in P. aeruginosa.Given that T2SS is a central and constitutive pump, and the secreted proteases are involved in interactions with the microbial surroundings, our data broadens the significance of c-di-GMP signaling in P. aeruginosa pathogenesis and ecological fitness.

View Article: PubMed Central - PubMed

Affiliation: Metabolites Biology Lab, Department of Biological Sciences, National University of Singapore, Singapore 117543; Mechanobiology Institute, National University of Singapore, 5A Engineering Drive 1, Singapore 117411.

ABSTRACT
Bacterial invasion plays a critical role in the establishment of Pseudomonas aeruginosa infection and is aided by two major virulence factors--surface appendages and secreted proteases. The second messenger cyclic diguanylate (c-di-GMP) is known to affect bacterial attachment to surfaces, biofilm formation and related virulence phenomena. Here we report that MorA, a global regulator with GGDEF and EAL domains that was previously reported to affect virulence factors, negatively regulates protease secretion via the type II secretion system (T2SS) in P. aeruginosa PAO1. Infection assays with mutant strains carrying gene deletion and domain mutants show that host cell invasion is dependent on the active domain function of MorA. Further investigations suggest that the MorA-mediated c-di-GMP signaling affects protease secretion largely at a post-translational level. We thus report c-di-GMP second messenger system as a novel regulator of T2SS function in P. aeruginosa. Given that T2SS is a central and constitutive pump, and the secreted proteases are involved in interactions with the microbial surroundings, our data broadens the significance of c-di-GMP signaling in P. aeruginosa pathogenesis and ecological fitness.

Show MeSH

Related in: MedlinePlus

(A) MorA does not affect cellular levels of LasB. Top panel- Extracellular LasB from WT and morA KO culture supernatants at mid log and late log phases (SDS-PAGE). XcpQ mutant lacks functional T2SS and does not secrete any proteases; negative control. Bottom panel- LasB from cellular fractions of respective cultures immunoblotted using polyclonal antibody. Samples on both panels were loaded based on proteins from equal number of cells as described in methods. (B) Levels of T2SS machinery proteins remain unaltered. Immunoblots of the T2SS machinery component proteins from membrane fraction. Membrane proteins were loaded from equal number of bacterial cells. XcpY and XcpZ are inner membrane proteins while XcpP spans both the inner and outer membranes. Respective culture supernatants were loaded to compare secreted LasB levels. RNA pol- RNA polymerase from the cellular protein fraction was used as loading control.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4404142&req=5

pone.0123805.g004: (A) MorA does not affect cellular levels of LasB. Top panel- Extracellular LasB from WT and morA KO culture supernatants at mid log and late log phases (SDS-PAGE). XcpQ mutant lacks functional T2SS and does not secrete any proteases; negative control. Bottom panel- LasB from cellular fractions of respective cultures immunoblotted using polyclonal antibody. Samples on both panels were loaded based on proteins from equal number of cells as described in methods. (B) Levels of T2SS machinery proteins remain unaltered. Immunoblots of the T2SS machinery component proteins from membrane fraction. Membrane proteins were loaded from equal number of bacterial cells. XcpY and XcpZ are inner membrane proteins while XcpP spans both the inner and outer membranes. Respective culture supernatants were loaded to compare secreted LasB levels. RNA pol- RNA polymerase from the cellular protein fraction was used as loading control.

Mentions: Comparison of expression levels of genes encoding two key secreted proteins (LasB and CbpD) between P. aeruginosa PAO1 WT and morA KO revealed only a small increase (< 2-fold change of RNA levels; p-value<0.05) due to MorA loss (S3 Fig). It is to be noted that at LasB transcript levels were much lower in morA mutant than WT especially at late log phase beyond which secreted elastase was detected in the culture supernatants. At late log phase, the secreted protein levels (~60% increase; Fig 1B) and transcript levels (~30% increase) of CbpD due to morA mutation are not directly correlating. Further, unlike the extracellular protein levels, no significant change in the cell-associated levels of LasB was observed over time due to MorA loss (Fig 4A). Quantification of immunoblot using polyclonal LasB antibody on the cellular fraction confirmed that the difference due to morA mutation was less than 5%. A mutant defective in the T2SS outer membrane pore complex protein XcpQ, which is incapable of secretion [73, 74], had significant cellular accumulation, as expected (negative control) (Fig 4A). As there are small differences at RNA level too, a minor contribution of regulatory control at RNA level cannot be ruled out. However, as this does not proportionally reflect in protease production as seen in the cellular fractions, post-translational control seems to be the major control step for T2SS secretion by MorA.


Global Regulator MorA Affects Virulence-Associated Protease Secretion in Pseudomonas aeruginosa PAO1.

Ravichandran A, Ramachandran M, Suriyanarayanan T, Wong CC, Swarup S - PLoS ONE (2015)

(A) MorA does not affect cellular levels of LasB. Top panel- Extracellular LasB from WT and morA KO culture supernatants at mid log and late log phases (SDS-PAGE). XcpQ mutant lacks functional T2SS and does not secrete any proteases; negative control. Bottom panel- LasB from cellular fractions of respective cultures immunoblotted using polyclonal antibody. Samples on both panels were loaded based on proteins from equal number of cells as described in methods. (B) Levels of T2SS machinery proteins remain unaltered. Immunoblots of the T2SS machinery component proteins from membrane fraction. Membrane proteins were loaded from equal number of bacterial cells. XcpY and XcpZ are inner membrane proteins while XcpP spans both the inner and outer membranes. Respective culture supernatants were loaded to compare secreted LasB levels. RNA pol- RNA polymerase from the cellular protein fraction was used as loading control.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4404142&req=5

pone.0123805.g004: (A) MorA does not affect cellular levels of LasB. Top panel- Extracellular LasB from WT and morA KO culture supernatants at mid log and late log phases (SDS-PAGE). XcpQ mutant lacks functional T2SS and does not secrete any proteases; negative control. Bottom panel- LasB from cellular fractions of respective cultures immunoblotted using polyclonal antibody. Samples on both panels were loaded based on proteins from equal number of cells as described in methods. (B) Levels of T2SS machinery proteins remain unaltered. Immunoblots of the T2SS machinery component proteins from membrane fraction. Membrane proteins were loaded from equal number of bacterial cells. XcpY and XcpZ are inner membrane proteins while XcpP spans both the inner and outer membranes. Respective culture supernatants were loaded to compare secreted LasB levels. RNA pol- RNA polymerase from the cellular protein fraction was used as loading control.
Mentions: Comparison of expression levels of genes encoding two key secreted proteins (LasB and CbpD) between P. aeruginosa PAO1 WT and morA KO revealed only a small increase (< 2-fold change of RNA levels; p-value<0.05) due to MorA loss (S3 Fig). It is to be noted that at LasB transcript levels were much lower in morA mutant than WT especially at late log phase beyond which secreted elastase was detected in the culture supernatants. At late log phase, the secreted protein levels (~60% increase; Fig 1B) and transcript levels (~30% increase) of CbpD due to morA mutation are not directly correlating. Further, unlike the extracellular protein levels, no significant change in the cell-associated levels of LasB was observed over time due to MorA loss (Fig 4A). Quantification of immunoblot using polyclonal LasB antibody on the cellular fraction confirmed that the difference due to morA mutation was less than 5%. A mutant defective in the T2SS outer membrane pore complex protein XcpQ, which is incapable of secretion [73, 74], had significant cellular accumulation, as expected (negative control) (Fig 4A). As there are small differences at RNA level too, a minor contribution of regulatory control at RNA level cannot be ruled out. However, as this does not proportionally reflect in protease production as seen in the cellular fractions, post-translational control seems to be the major control step for T2SS secretion by MorA.

Bottom Line: Further investigations suggest that the MorA-mediated c-di-GMP signaling affects protease secretion largely at a post-translational level.We thus report c-di-GMP second messenger system as a novel regulator of T2SS function in P. aeruginosa.Given that T2SS is a central and constitutive pump, and the secreted proteases are involved in interactions with the microbial surroundings, our data broadens the significance of c-di-GMP signaling in P. aeruginosa pathogenesis and ecological fitness.

View Article: PubMed Central - PubMed

Affiliation: Metabolites Biology Lab, Department of Biological Sciences, National University of Singapore, Singapore 117543; Mechanobiology Institute, National University of Singapore, 5A Engineering Drive 1, Singapore 117411.

ABSTRACT
Bacterial invasion plays a critical role in the establishment of Pseudomonas aeruginosa infection and is aided by two major virulence factors--surface appendages and secreted proteases. The second messenger cyclic diguanylate (c-di-GMP) is known to affect bacterial attachment to surfaces, biofilm formation and related virulence phenomena. Here we report that MorA, a global regulator with GGDEF and EAL domains that was previously reported to affect virulence factors, negatively regulates protease secretion via the type II secretion system (T2SS) in P. aeruginosa PAO1. Infection assays with mutant strains carrying gene deletion and domain mutants show that host cell invasion is dependent on the active domain function of MorA. Further investigations suggest that the MorA-mediated c-di-GMP signaling affects protease secretion largely at a post-translational level. We thus report c-di-GMP second messenger system as a novel regulator of T2SS function in P. aeruginosa. Given that T2SS is a central and constitutive pump, and the secreted proteases are involved in interactions with the microbial surroundings, our data broadens the significance of c-di-GMP signaling in P. aeruginosa pathogenesis and ecological fitness.

Show MeSH
Related in: MedlinePlus