Reaction Products and the X-ray Structure of AmpDh2, a Virulence Determinant of Pseudomonas aeruginosa

Martínez-Caballero, Siseth; Lee, Mijoon; Artola-Recolons, C.; Carrasco-López, César; Hesek, Dušan; Spink, Edward; Lastochkin, Elena; Zhang, Weilie; Hellman, Lance M.; Boggess, Bill; Mobashery, Shahriar; Hermoso, J.A.

doi:10.1021/ja405464b

Cited by 40 publications

(61 citation statements)

References 16 publications

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“…Very small amounts of product with GlcNAc-anhMurNAc attached to either 4-3, 4-4, or 3-3 cross-linked peptide stems were also observed (Ͻ0.25% of the total detector counts for each run). These are known products of partial amidase digestion (16). These results are consistent with a highly cross-linked sacculus, noted previously as being unusual among Gram-negatives (47), and a sacculus composed primarily of tetrapeptide stems (9).…”

Section: An Amic Q316k Mutation Has Only Intermediate Effects On Pg Fsupporting

confidence: 90%

Amidase Activity of AmiC Controls Cell Separation and Stem Peptide Release and Is Enhanced by NlpD in Neisseria gonorrhoeae

Lenz

Stohl

Robertson

et al. 2016

Journal of Biological Chemistry

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The human-restricted pathogen Neisseria gonorrhoeae encodes a single N-acetylmuramyl-L-alanine amidase involved in cell separation (AmiC), as compared with three largely redundant cell separation amidases found in Escherichia coli (AmiA, AmiB, and AmiC). Deletion of amiC from N. gonorrhoeae results in severely impaired cell separation and altered peptidoglycan (PG) fragment release, but little else is known about how AmiC functions in gonococci. Here, we demonstrated that gonococcal AmiC can act on macromolecular PG to liberate cross-linked and non-cross-linked peptides indicative of amidase activity, and we provided the first evidence that a cell separation amidase can utilize a small synthetic PG fragment as substrate (GlcNAc-MurNAc(pentapeptide)-GlcNAc-MurNAc (pentapeptide)). An investigation of two residues in the active site of AmiC revealed that Glu-229 is critical for both normal cell separation and the release of PG fragments by gonococci during growth. In contrast, Gln-316 has an autoinhibitory role, and its mutation to lysine resulted in an AmiC with increased enzymatic activity on macromolecular PG and on the synthetic PG derivative. Curiously, the same Q316K mutation that increased AmiC activity also resulted in cell separation and PG fragment release defects, indicating that activation state is not the only factor determining normal AmiC activity. In addition to displaying high basal activity on PG, gonococcal AmiC can utilize metal ions other than the zinc cofactor typically used by cell separation amidases, potentially protecting its ability to function in zinc-limiting environments. Thus gonococcal AmiC has distinct differences from related enzymes, and these studies revealed parameters for how AmiC functions in cell separation and PG fragment release. Peptidoglycan (PG)2 is a critical structural macromolecule that makes up the cell wall surrounding the cytoplasmic membrane of nearly all bacteria. This cage-like structure is made up of long polysaccharide strands that are composed of repeating units of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) with short peptide stems (3-5 amino acids) covalently attached to the MurNAc moiety (1). Depending on the organism, some proportion of peptide stems are crosslinked to peptides on adjacent strands, whereas others remain non-cross-linked. Cross-linking provides structural integrity for the macromolecule and contributes to the determination of bacterial shape.Neisseria gonorrhoeae (the gonococcus or GC) is a Gramnegative bacterium, obligate human pathogen, and etiologic agent of the sexually transmitted infection gonorrhea. Gonorrhea is the second most common reportable bacterial infection in the United States with an estimated 820,000 cases annually (2). A robust inflammatory response is the cause of much of the damage observed during GC infection and is triggered by the release of lipo-oligosaccharide, porin proteins, and fragments of PG (3-5). In particular, N. gonorrhoeae release a greater abundance of PG fragments than Escherichia co...

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Section: An Amic Q316k Mutation Has Only Intermediate Effects On Pg Fsupporting

confidence: 90%

Amidase Activity of AmiC Controls Cell Separation and Stem Peptide Release and Is Enhanced by NlpD in Neisseria gonorrhoeae

Lenz

Stohl

Robertson

et al. 2016

Journal of Biological Chemistry

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“…AmpDh2 and AmpDh3 are periplasmic enzymes mainly acting directly on the insoluble polymeric sacculus of the peptidoglycan for cell wall remodeling (36, 37). However, they can also process soluble 1,6-anhydromuropeptides but with a lower efficiency than that for AmpD (36–38). Thus, candidates for triggers of the fitness and virulence impairment in the amidase triple mutant would be the accumulation of soluble 1,6-anhydromuropeptides and/or the alteration of cell wall remodeling itself.…”

Section: Discussionmentioning

confidence: 99%

Impact of AmpC Derepression on Fitness and Virulence: the Mechanism or the Pathway?

Pérez-Gallego

Torrens

Castillo-Vera

et al. 2016

mBio

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Understanding the interplay between antibiotic resistance and bacterial fitness and virulence is essential to guide individual treatments and improve global antibiotic policies. A paradigmatic example of a resistance mechanism is the intrinsic inducible chromosomal β-lactamase AmpC from multiple Gram-negative bacteria, including Pseudomonas aeruginosa, a major nosocomial pathogen. The regulation of ampC expression is intimately linked to peptidoglycan recycling, and AmpC-mediated β-lactam resistance is frequently mediated by inactivating mutations in ampD, encoding an N-acetyl-anhydromuramyl-l-alanine amidase, affecting the levels of ampC-activating muropeptides. Here we dissect the impact of the multiple pathways causing AmpC hyperproduction on P. aeruginosa fitness and virulence. Through a detailed analysis, we demonstrate that the lack of all three P. aeruginosa AmpD amidases causes a dramatic effect in fitness and pathogenicity, severely compromising growth rates, motility, and cytotoxicity; the latter effect is likely achieved by repressing key virulence factors, such as protease LasA, phospholipase C, or type III secretion system components. We also show that ampC overexpression is required but not sufficient to confer the growth-motility-cytotoxicity impaired phenotype and that alternative pathways leading to similar levels of ampC hyperexpression and resistance, such as those involving PBP4, had no fitness-virulence cost. Further analysis indicated that fitness-virulence impairment is caused by overexpressing ampC in the absence of cell wall recycling, as reproduced by expressing ampC from a plasmid in an AmpG (muropeptide permease)-deficient background. Thus, our findings represent a major step in the understanding of β-lactam resistance biology and its interplay with fitness and pathogenesis.

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“…The number of glycines may vary from four to six, or one glycine may be substituted by a serine or an alanine (43,44). Although structures of several bacterial amidases have been determined (19,(45)(46)(47)(48)(49)(50)(51)(52), the amidase AmiD from E. coli is the only catalytically active amidase for which structural data of an uncleaved ligand-enzyme complex have been available prior to this work (21).…”

Section: Discussionmentioning

confidence: 99%

Structure-Function Analysis of Staphylococcus aureus Amidase Reveals the Determinants of Peptidoglycan Recognition and Cleavage

Büttner

Zoll

Nega

et al. 2014

Journal of Biological Chemistry

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Background: Autolysins ensure the plasticity of bacterial cell walls, and deletion leads to impaired cell clusters. Results: High resolution structures of Staphylococcus aureus amidase AmiA shed light on peptidoglycan binding and cleavage. Conclusion: AmiA distinguishes peptidoglycan mostly by the peptide, and cleavage is facilitated by a zinc-activated water molecule. Significance: These structures will inform strategies to develop new therapeutics against MRSA.

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Reaction Products and the X-ray Structure of AmpDh2, a Virulence Determinant of Pseudomonas aeruginosa

Cited by 40 publications

References 16 publications

Amidase Activity of AmiC Controls Cell Separation and Stem Peptide Release and Is Enhanced by NlpD in Neisseria gonorrhoeae

Amidase Activity of AmiC Controls Cell Separation and Stem Peptide Release and Is Enhanced by NlpD in Neisseria gonorrhoeae

Impact of AmpC Derepression on Fitness and Virulence: the Mechanism or the Pathway?

Structure-Function Analysis of Staphylococcus aureus Amidase Reveals the Determinants of Peptidoglycan Recognition and Cleavage

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