Resistance Phenotypes Mediated by Aminoacyl-Phosphatidylglycerol Synthases

Abstract

The specific aminoacylation of the phospholipid phosphatidylglycerol (PG) with alanine or with lysine catalyzed by aminoacylphosphatidylglycerol synthases (aaPGS) was shown to render various organisms less susceptible to antibacterial agents. This study makes use of Pseudomonas aeruginosa chimeric mutant strains producing lysyl-phosphatidylglycerol (L-PG) instead of the naturally occurring alanyl-phosphatidylglycerol (A-PG) to study the resulting impact on bacterial resistance. Consequences of such artificial phospholipid composition were studied in the presence of an overall of seven antimicrobials (␤-lactams, a lipopeptide antibiotic, cationic antimicrobial peptides [CAMPs]) to quantitatively assess the effect of A-PG substitution (with L-PG, L-PG and A-PG, increased A-PG levels). For the employed Gram-negative P. aeruginosa model system, an exclusive charge repulsion mechanism does not explain the attenuated antimicrobial susceptibility due to PG modification. Additionally, the specificity of nine orthologous aaPGS enzymes was experimentally determined. The newly characterized protein sequences allowed for the establishment of a significant group of A-PG synthase sequences which were bioinformatically compared to the related group of L-PG synthesizing enzymes. The analysis revealed a diverse origin for the evolution of A-PG and L-PG synthases, as the specificity of an individual enzyme is not reflected in terms of a characteristic sequence motif. This finding is relevant for future development of potential aaPGS inhibitors.

Deciphering the tRNA-dependent lipid aminoacylation systems in bacteria: Novel components and structural advances

“…38 Several studies have tried to determine the phenotypical changes correlated with synthesis of Lys-PG compared with Ala-PG in S. aureus and P. aeruginosa . 24,30 These investigations showed that substitution of Lys-PG (which confers a net charge of +1) with Ala-PG (which bears a neutral net charge) did not affect bacterial susceptibility to CAMPs such as nisin and gallidermin, or to the CAMP-like antibiotic daptomycin. These findings suggest that the α amino group of the aa moiety of aa-PG alone is able to enhance antimicrobial resistance in bacteria.…”

“…23 In more recent years, it was shown that LysPGS is only one member of a large family of aminoacyl-PG synthase (aaPGS) homologs spanning nearly all known bacterial phyla. Some homologs are also present in archaeal methanogens, 24 but no examples have been identified in eukaryotic genomes. 1 Aminoacyl-phosphatidylglycerol synthases (aaPGSs), as these enzymes are collectively called, are bifunctional enzymes.…”

“…The effects of aa-PG on antibiotic resistance have been investigated in several important human pathogens such as S. aureus (for review see [4]), L. monocytogenes , 53 B. anthracis , 58 P. aeruginosa , 24,59 M. tuberculosis , 29,60 Enterococcus species, 35,36,61 and B. subtilis 62,63 (for review see [1]). aa-PGs in these organisms primarily enhance bacterial resistance to positively charged compounds targeting the membrane (such as CAMPs) and last resort lipopetides like daptomycin.…”

Structures of two bacterial resistance factors mediating tRNA-dependent aminoacylation of phosphatidylglycerol with lysine or alanine

“…Accordingly, aa-PGS enzymes can be considered virulence factors and thus have been termed "multiple peptide resistance factors." In Pseudomonas aeruginosa, aminoacylation of PG also was found in response to acidic environmental conditions (2,5), inter alia. P. aeruginosa is the dominant pathogen that colonizes the lung of patients suffering from cystic fibrosis.…”

“…1) reduce the overall net negative charge of the membrane, making it less susceptible to cationic antimicrobial peptides (CAMPs). Such CAMPs often are synthesized as innate immunity host-defense peptides in response to bacterial infections (2)(3)(4)(5). Fundamental work with the Gram-positive pathogen Staphylococcus aureus indicated aa-PGS-mediated nonsusceptibility to vancomycin (an antibiotic of last resort), resistance to host antimicrobial peptides (defensins), and protection against neutrophil killing (6)(7)(8)(9).…”