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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.

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Effect on protein secretion by MorA in P. aeruginosa is c-di-GMP signaling dependent.(A) Top panel—Total extracellular protein (ECP) from culture supernatants of P. aeruginosa strains loaded based on protein secreted from equal number of cells. Black arrow indicates the position of elastase (LasB) band. Bottom panel—Immunoblot of RNA polymerase (loading control) on cellular fractions of respective cultures in top panel. L-Ladder (Bio-Rad). (B) Percentage increase in levels of secreted LasB based on protein band quantification by densitometry. Band intensity values of MorA insertion (morA KO) and deletion (ΔmorA) mutants are compared with wildtype (WT) while those of strains expressing MorA with mutations in GGDEF and EAL motifs (represented as ΔmorA–pUG* and ΔmorA–pUE* respectively) are compared with complementation strain (ΔmorA-pU) containing full length morA expressed in ΔmorA background. Error bars represent mean +SE (n = 5). Student’s t-test, p-value<0.05.
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pone.0123805.g002: Effect on protein secretion by MorA in P. aeruginosa is c-di-GMP signaling dependent.(A) Top panel—Total extracellular protein (ECP) from culture supernatants of P. aeruginosa strains loaded based on protein secreted from equal number of cells. Black arrow indicates the position of elastase (LasB) band. Bottom panel—Immunoblot of RNA polymerase (loading control) on cellular fractions of respective cultures in top panel. L-Ladder (Bio-Rad). (B) Percentage increase in levels of secreted LasB based on protein band quantification by densitometry. Band intensity values of MorA insertion (morA KO) and deletion (ΔmorA) mutants are compared with wildtype (WT) while those of strains expressing MorA with mutations in GGDEF and EAL motifs (represented as ΔmorA–pUG* and ΔmorA–pUE* respectively) are compared with complementation strain (ΔmorA-pU) containing full length morA expressed in ΔmorA background. Error bars represent mean +SE (n = 5). Student’s t-test, p-value<0.05.

Mentions: Based on our previous findings from gene expression profiling of MorA mutant [57] and other reports showing evidence that c-di-GMP regulating proteins control bacterial secretion systems [45, 46, 52–55], we tested the effect of MorA on the P. aeruginosa secretome. Profiles of extracellular proteins of P. aeruginosa PAO1 WT and morA KO were compared. RNA polymerase α-subunit levels in cellular fractions from the same samples were used as controls for biomass. Strains lacking MorA had higher overall levels of extracellular proteins than WT in planktonic culture supernatants (Fig 1A). In order to quantitate the levels of extracellular proteins, densitometry analysis of individual bands from the protein profiles was performed. As shown in Fig 1B, there was at least 50% increase in secretion due to morA mutation in six out of eight proteins observed. Decrease in levels of extracellular proteins upon morA complementation (Fig 2) further validated that changes in their levels were due to MorA expression.


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)

Effect on protein secretion by MorA in P. aeruginosa is c-di-GMP signaling dependent.(A) Top panel—Total extracellular protein (ECP) from culture supernatants of P. aeruginosa strains loaded based on protein secreted from equal number of cells. Black arrow indicates the position of elastase (LasB) band. Bottom panel—Immunoblot of RNA polymerase (loading control) on cellular fractions of respective cultures in top panel. L-Ladder (Bio-Rad). (B) Percentage increase in levels of secreted LasB based on protein band quantification by densitometry. Band intensity values of MorA insertion (morA KO) and deletion (ΔmorA) mutants are compared with wildtype (WT) while those of strains expressing MorA with mutations in GGDEF and EAL motifs (represented as ΔmorA–pUG* and ΔmorA–pUE* respectively) are compared with complementation strain (ΔmorA-pU) containing full length morA expressed in ΔmorA background. Error bars represent mean +SE (n = 5). Student’s t-test, p-value<0.05.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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pone.0123805.g002: Effect on protein secretion by MorA in P. aeruginosa is c-di-GMP signaling dependent.(A) Top panel—Total extracellular protein (ECP) from culture supernatants of P. aeruginosa strains loaded based on protein secreted from equal number of cells. Black arrow indicates the position of elastase (LasB) band. Bottom panel—Immunoblot of RNA polymerase (loading control) on cellular fractions of respective cultures in top panel. L-Ladder (Bio-Rad). (B) Percentage increase in levels of secreted LasB based on protein band quantification by densitometry. Band intensity values of MorA insertion (morA KO) and deletion (ΔmorA) mutants are compared with wildtype (WT) while those of strains expressing MorA with mutations in GGDEF and EAL motifs (represented as ΔmorA–pUG* and ΔmorA–pUE* respectively) are compared with complementation strain (ΔmorA-pU) containing full length morA expressed in ΔmorA background. Error bars represent mean +SE (n = 5). Student’s t-test, p-value<0.05.
Mentions: Based on our previous findings from gene expression profiling of MorA mutant [57] and other reports showing evidence that c-di-GMP regulating proteins control bacterial secretion systems [45, 46, 52–55], we tested the effect of MorA on the P. aeruginosa secretome. Profiles of extracellular proteins of P. aeruginosa PAO1 WT and morA KO were compared. RNA polymerase α-subunit levels in cellular fractions from the same samples were used as controls for biomass. Strains lacking MorA had higher overall levels of extracellular proteins than WT in planktonic culture supernatants (Fig 1A). In order to quantitate the levels of extracellular proteins, densitometry analysis of individual bands from the protein profiles was performed. As shown in Fig 1B, there was at least 50% increase in secretion due to morA mutation in six out of eight proteins observed. Decrease in levels of extracellular proteins upon morA complementation (Fig 2) further validated that changes in their levels were due to MorA expression.

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