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

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.

Fig 1. Levels of secreted proteins are affected by MorA in P. aeruginosa.

(A) Top panel—Profiles of total extracellular protein (ECP) from P. aeruginosa PAO1 WT and morA KO culture supernatants. Samples were loaded based on protein secreted from equal number of cells. Protein bands were identified by MALDI-ToF-ToF. Bottom panel—Immunoblot of RNA polymerase (loading control) from cellular fractions of respective cultures. L-Protein ladder (Bio-Rad). (B) Percentage increase in levels of secreted proteins in morA KO compared to WT. Error bars represent mean +SE (n = 3). Student’s t-test, p-value<0.05.

Fig 2. 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.

Fig 3. Elastase activity, host cell morphological changes and invasion efficiency corroborate with differential protein secretion.

(A) Graph shows total active elastase per unit of total secreted extracellular proteins measured in P. aeruginosa PAO1 WT and morA KO. Activity was measured using elastin-congo red as substrate. Error bars represent mean +SE (n = 4). *Student’s t-test, p-value<0.05. ECP-extracellular protein. (B) Lung fibroblasts (MRC-5) infected with P. aeruginosa WT and mutant strains viewed by differential interference contrast microscopy. Images were captured 2 hours post-infection. MOI- Multiplicity of infection; 0 hr- No infection (control). (C) Graph shows difference in invasion efficiency of P. aeruginosa strains on lung fibroblasts (MRC-5) 2 hours post-infection. (D) Effect of MorA on invasion efficiency is consistent over a range of infection time and multiplicities of infection. Error bars represent mean +SE (n = 3). *Student’s t-test, p-value<0.05.

Fig 4

(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.