A Proteolytic Complex Targets Multiple Cell Wall Hydrolases in Pseudomonas aeruginosa

Abstract

Carboxy-terminal processing proteases (CTPs) occur in all three domains of life. In bacteria, some of them have been associated with virulence. However, the precise roles of bacterial CTPs are poorly understood, and few direct proteolytic substrates have been identified. One bacterial CTP is the CtpA protease of Pseudomonas aeruginosa, which is required for type III secretion system (T3SS) function and for virulence in a mouse model of acute pneumonia. Here, we have investigated the function of CtpA in P. aeruginosa and identified some of the proteins it cleaves. We discovered that CtpA forms a complex with a previously uncharacterized protein, which we have named LbcA (lipoprotein binding partner of CtpA). LbcA is required for CtpA activity in vivo and promotes its activity in vitro. We have also identified four proteolytic substrates of CtpA, all of which are uncharacterized proteins predicted to cleave the peptide cross-links within peptidoglycan. Consistent with this, a ctpA null mutant was found to have fewer peptidoglycan cross-links than the wild type and grew slowly in salt-free medium. Intriguingly, the accumulation of just one of the CtpA substrates was required for some ΔctpA mutant phenotypes, including the defective T3SS. We propose that LbcA-CtpA is a proteolytic complex in the P. aeruginosa cell envelope, which controls the activity of several peptidoglycan cross-link hydrolases by degrading them. Furthermore, based on these and other findings, we suggest that many bacterial CTPs might be similarly controlled by partner proteins as part of a widespread mechanism to control peptidoglycan hydrolase activity.

Identification of Drug Resistance Determinants in a Clinical Isolate of Pseudomonas aeruginosa by High-Density Transposon Mutagenesis

“…MepM1 (YebA, PA0667) belongs to a group of murein endopeptidases (EPs) which putatively modulate PG cross-linking (43). A study revealed that the protease CtpA (PA5134) inactivates various EPs, namely, those encoded by PA0667 (TUEID40_04290 or mepM1), PA4404 (TUEID40_02316), PA1198 (TUEID40_01415), and PA1199 (TUEID40_ 01414), and thereby controls the level of PG cross-linking (43). TUEID40_01415 also showed reduced read counts upon treatment with MEM and/or FEP, but to a much lesser extent than MepM1 did (Data Set S3).…”

Regulated, sequential processing by multiple proteases is required for proper maturation and release of Bordetella filamentous hemagglutinin

“…Our data are consistent with direct interaction between CtpA and the prodomain. However, because it is likely that CtpA has multiple degradation targets, some of which may include homologs of identified target peptidoglycan hydrolases, it is possible that CtpA is necessary for prodomain degradation due to indirect effects, such as cleavage of another protein or changes in the peptidoglycan (Singh et al, 2015;Srivastava et al, 2018).…”

“…Size of 250 kDa protein standard is indicated at right. [Colour figure can be viewed at wileyonlinelibrary.com] as the CtpA substrate (Srivastava et al, 2018). Multiple observations suggest that a third, unidentified protease (P3) is responsible for removing C-terminal residues to convert FhaB into a CtpA substrate.…”

Altered immunity of laboratory mice in the natural environment is associated with fungal colonization

“…We recently described a mesocosm system in which SPF mice are 'rewilded' through 70 controlled release into an outdoor enclosure facility (7). A key feature of the enclosure is that a 71 zinced iron wall excludes predators and rodents harboring disease-causing infectious agents, 72 while allowing exposure to natural soil, vegetation, and weather.…”

“…A key feature of the enclosure is that a 71 zinced iron wall excludes predators and rodents harboring disease-causing infectious agents, 72 while allowing exposure to natural soil, vegetation, and weather. Mice rewilded through transient 73 release into the enclosure acquire a bacterial microbiota characterized by increased diversity, and 74 display heightened susceptibility to helminth infection (7). To determine the consequence of 75 microbial colonization in the natural environment on the steady-state immune system, we applied 76 multicolor flow cytometry to analyze the immune cell composition of blood and mesenteric 77 lymph nodes (MLNs) from SPF mice aged 6-8 weeks old at the time of release into the enclosure 78 and captured 6-7 weeks later, herein referred to as rewilded mice.…”