Changes to its peptidoglycan-remodeling enzyme repertoire modulate ß-lactam resistance in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a leading cause of hospital-acquired infections and is resistant to many antibiotics. Among its primary mechanisms of resistance is expression of a chromosomally encoded AmpC ß-lactamase that inactivates ß-lactams. The mechanisms leading to AmpC expression in P. aeruginosa remain incompletely understood but are intricately linked to cell wall metabolism. To better understand the roles of peptidoglycan-active enzymes in AmpC expression-and consequent ß-lactam resistance-a phenotypic screen of P. aeruginosa mutants lacking such enzymes was performed. Mutants lacking one of four lytic transglycosylases (LTs) or the nonessential penicillin-binding protein PBP4 (dacB) had altered ß-lactam resistance. mltF and slt mutants with reduced ß-lactam resistance were designated WIMPs (wall-impaired mutant phenotypes), while highly resistant dacB, sltB1, and mltB mutants were designated HARMs (high-level AmpC resistant mutants). Double mutants lacking dacB and sltB1 had extreme piperacillin resistance (>256 µg/ml) compared to either of the single knockouts (64 µg/ml for a dacB mutant and 12 µg/ml for an sltB1 mutant). Inactivation of ampC reverted these mutants to wild-type susceptibility, confirming that AmpC expression underlies resistance. dacB mutants had constitutively elevated AmpC expression, but the LT mutants had wild-type levels of AmpC in the absence of antibiotic exposure. These data suggest that there are at least two different pathways leading to AmpC expression in P. aeruginosa and that their simultaneous activation leads to extreme ß-lactam resistance.

Maintaining network security: how macromolecular structures cross the peptidoglycan layer.

Peptidoglycan plays a vital role in bacterial physiology, maintaining cell shape and resisting cellular lysis from high internal turgor pressures. Its integrity is carefully maintained by controlled remodeling during growth and division by the coordinated activities of penicillin-binding proteins, lytic transglycosylases, and N-acetylmuramyl-l-alanine amidases. However, its small pore size (∼2 nm) and covalently closed structure make it a formidable barrier to the assembly of large macromolecular cell-envelope-spanning complexes involved in motility and secretion. Here, we review the strategies used by Gram-negative bacteria to assemble such macromolecular complexes across the peptidoglycan layer, while preserving its essential structural role. In addition, we discuss evidence that suggests that peptidoglycan can be integrated into cell-envelope-spanning complexes as a structural and functional extension of their architecture.

The peptidoglycan-binding protein FimV promotes assembly of the Pseudomonas aeruginosa type IV pilus secretin.

The Pseudomonas aeruginosa inner membrane protein FimV is among several proteins of unknown function required for type IV pilus-mediated twitching motility, arising from extension and retraction of pili from their site of assembly in the inner membrane. The pili transit the periplasm and peptidoglycan (PG) layer, ultimately exiting the cell through the PilQ secretin. Although fimV mutants are nonmotile, they are susceptible to killing by pilus-specific bacteriophage, a hallmark of retractable surface pili. Here we show that levels of recoverable surface pili were markedly decreased in fimV pilT retraction-deficient mutants compared with levels in the pilT control, demonstrating that FimV acts at the level of pilus assembly. Levels of inner membrane assembly subcomplex proteins PilM/N/O/P were decreased in fimV mutants, but supplementation of these components in trans did not restore pilus assembly or motility. Loss of FimV dramatically reduced the levels of the PilQ secretin multimer through which pili exit the cell, in part due to decreased levels of PilQ monomers, while PilF pilotin levels were unchanged. Expression of pilQ in trans in the wild type or fimV mutants increased total PilQ monomer levels but did not alter secretin multimer levels or motility. PG pulldown assays showed that the N terminus of FimV bound PG in a LysM motif-dependent manner, and a mutant with an in-frame chromosomal deletion of the LysM motif had reduced motility, secretin levels, and surface piliation. Together, our data show that FimV's role in pilus assembly is to promote secretin formation and that this function depends upon its PG-binding domain.

The vertebrate lysozyme inhibitor Ivy functions to inhibit the activity of lytic transglycosylase.

The proteinaceous inhibitor of vertebrate lysozymes (Ivy) is produced by a collection of Gram-negative bacteria as a stress response to damage to their essential cell wall component peptidoglycan. A paralog of Ivy, Ivyp2 is produced exclusively by a number of pseudomonads, including Pseudomonas aeruginosa, but this protein does not inhibit the lysozymes, and its function was unknown. In this study, we demonstrate that the production of Ivy (homologs of both Ivyp1 and Ivyp2) correlates with bacteria that do not O-acetylate their peptidoglycan, a modification that controls the activity of the lytic transglycosylases. Furthermore, we show that both Ivy proteins are potent inhibitors of the lytic transglycoslyases, enzymes involved in the biosynthesis and maintenance of peptidoglycan. These data suggest that the true physiological function of the Ivy proteins is to control the autolytic activity of lytic transglycosylases within the periplasm of Gram-negative bacteria that do not produce O-acetylated peptidoglycan and that the inhibition of exogenous lysozyme by Ivy is simply a fortuitous coincidence.

The C-terminal domain of Escherichia coli YfhD functions as a lytic transglycosylase.

The hypothetical Escherichia coli protein YfhD has been identified as the archetype for the family 1B lytic transglycosylases despite a complete lack of experimental characterization. The yfhD gene was amplified from the genomic DNA of E. coli W3110 and cloned to encode a fusion protein with a C-terminal His(6) sequence. The enzyme was found to be localized to the outer membrane of E. coli, as would be expected for a lytic transglycosylase. Its gene was engineered for the production of a truncated soluble enzyme derivative lacking an N-terminal signal sequence and membrane anchor. The soluble YfhD derivative was purified to apparent homogeneity, and three separate in vitro assays involving high pressure liquid chromatography and matrix-assisted laser desorption ionization time-of-flight mass spectrometry were used to demonstrate the YfhD-catalyzed release of 1,6-anhydromuro-peptides from insoluble peptidoglycan. In addition, an in vivo bioassay developed using the bacteriophage lambda lysis system confirmed that the enzyme functions as an autolysin. Based on these data, the enzyme was renamed membrane-bound lytic transglycosylase F. The modular structure of MltF was investigated through genetic engineering for the separate production of identified N-terminal and C-terminal domains. The ability to bind peptidoglycan and lytic activity were only associated with the isolated C-terminal domain. The enzymatic properties of this lytic transglycosylase domain were found to be very similar to those of the wild-type enzyme. The one notable exception was that the N-terminal domain appears to modulate the lytic behavior of the C-terminal domain to permit continued lysis of insoluble peptidoglycan, a unique feature of MltF compared with other characterized lytic transglycosylases.

Production and purification of the bacterial autolysin N-acetylmuramoyl-L-alanine amidase B from Pseudomonas aeruginosa.

The bacterial cell wall heteropolymer peptidoglycan is not a static structure as it is constantly being made and recycled throughout the bacterium's life cycle. This turnover of peptidoglycan is a highly coordinated event involving a complement of autolytic enzymes that include those with specificity for either the carbohydrate or the peptide linkages of peptidoglycan. One major class of these autolysins are the N-acetylmuramoyl-L-alanine amidases which cleave the amide linkage between the stem peptides and the lactyl moiety of muramoyl residues. They are required in the periplasm for cell separation during division and in both the periplasm and cytoplasm to trim soluble released PG fragments during turnover for recycling. The gene encoding N-acetylmuramoyl-L-alanine amidase B in Pseudomonas aeruginosa was cloned and over-expressed in Escherichia coli. The recombinant protein with a C-terminal His-tag was purified to apparent homogeneity by a combination of affinity and cation-exchange chromatographies using Ni(2+)NTA-agarose and Source S, respectively. Four separate assays involving zymography, light scattering, HPLC and MALDI-TOF mass spectrometry were used to confirm the activity of the protein as an N-acetylmuramoyl-L-alanine amidase.