Divergent Effects of Peptidoglycan Carboxypeptidase DacA on Intrinsic β-Lactam and Vancomycin Resistance

Park, Si Hyoung; Choi, Umji; Ryu, Su-Hyun; Lee, Han Byeol; Lee, Jinwon; Lee, Chang-Ro

doi:10.1128/spectrum.01734-22

Cited by 8 publications

(28 citation statements)

References 54 publications

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“…The role of the C-terminal membrane-anchoring domain of DacA was also analyzed. A previous report showed that PG carboxypeptidase activity of DacA(ΔC) was significantly higher than that of WT DacA (Park et al , 2022). DacA(ΔC) did not complement the alkaline and salt sensitivities of the dacA mutant or morphological defects in LB medium (Figure 3).…”

Section: Resultsmentioning

confidence: 83%

“…In a previous study, we demonstrated that DacA directly interacts with PBPs in a C-terminal domain-dependent manner (Park et al ., 2022). To determine whether these interactions are also detected between DacC and PBPs, we performed pull-down experiments using DacC-Flag and DacC(ΔC)-Flag strains expressing His-tagged or non-tagged PBPs.…”

Section: Resultsmentioning

confidence: 91%

“…The dacA mutant also exhibited salt sensitivity, which was fully complemented by the ectopic expression of DacA (Figure 3C). This phenotype was not detected in other PG carboxypeptidase mutants, including the dacC mutant (Park et al ., 2022), and additional deletions of DacC and DacD did not increase salt sensitivity of the dacA mutant (Figure 3–figure supplement 1), indicating that only DacA was associated with this phenotype. These data suggest that DacA is required to adapt to alkaline and salt stresses.…”

Section: Resultsmentioning

confidence: 93%

“…The C-terminal domain of DacC was dispensable for its enzymatic activity but was required for complementation of all phenotypes of the dacC mutant and physical interactions with PBPs, except for PBP2. The C-terminal domain of DacA is dispensable for its enzymatic activity (Park et al , 2022), but was required for complementation of all phenotypes of the dacA mutant, except for filamentous morphology under alkaline stress, and all physical interactions with PBPs. These results suggest that many functions of DacA and DacC are not linked to LD-transpeptidase, but are instead associated with DD-transpeptidases, PBPs.…”

Section: Discussionmentioning

confidence: 99%

“…DacD and DacC are expressed in mid-exponential growth and stationary phases, respectively (Buchanan and Sowell, 1982; Baquero et al , 1996). DacA, DacC, and DacD are suggested to be membrane-anchored via their C-terminal amphiphilic α-helix domains, and this domain is important for the morphological functioning of PBP5 (Nelson et al , 2002), its localization at the cell division site (Nelson et al , 2002; Potluri et al , 2010), and β-lactam resistance (Park et al , 2022).…”

Section: Introductionmentioning

confidence: 99%

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Coordinated and distinct LD-transpeptidase-independent roles of peptidoglycan carboxypeptidases DacC and DacA in stress adaptation and cell shape maintenance

Choi

Park

Lee

et al. 2022

Preprint

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Peptidoglycan (PG) is an essential bacterial architecture pivotal for shape maintenance and adaptation to osmotic stress. Although PG synthesis and modification are tightly regulated under harsh environmental stresses, few related mechanisms have been investigated. In this study, we aimed to investigate the coordinated and distinct roles of the PG carboxypeptidases DacC and DacA, in adaptation to alkaline and salt stresses and shape maintenance in Escherichia coli. We found that DacC is an alkaline PG carboxypeptidase, whose enzyme activity and protein stability are significantly enhanced under alkaline stress. Both DacC and DacA were required for bacterial growth under alkaline stress, whereas only DacA was required for the adaptation to salt stress. Under normal growth conditions, only DacA was necessary for cell shape maintenance, while under alkaline stress conditions, both DacA and DacC were necessary for cell shape maintenance, but their roles were distinct. Notably, all these roles of DacC and DacA were independent of LD-transpeptidases, which are necessary for the formation of PG 3-3 crosslinks and covalent bonds between PG and the outer membrane lipoprotein Lpp. Instead, DacC and DacA interacted with penicillin-binding proteins (PBPs), DD-transpeptidases, mostly in a C-terminal domain-dependent manner, and these interactions were necessary for most of their roles. Collectively, our results demonstrate the coordinated and distinct novel roles of PG carboxypeptidases in stress adaptation and shape maintenance and provide novel insights into the cellular functions of PG carboxypeptidases associated with PBPs.

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Section: Resultsmentioning

confidence: 83%

Section: Resultsmentioning

confidence: 91%

Section: Resultsmentioning

confidence: 93%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Coordinated and distinct LD-transpeptidase-independent roles of peptidoglycan carboxypeptidases DacC and DacA in stress adaptation and cell shape maintenance

Choi

Park

Lee

et al. 2022

Preprint

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Adapting antibacterial display to identify serum-active macrocyclic peptide antibiotics

Randall,

Groover,

O'Donnell

et al. 2023

PNAS Nexus

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The lack of available treatments for many antimicrobial resistant infections highlights the critical need for antibiotic discovery innovation. Peptides are an underappreciated antibiotic scaffold because they often suffer from proteolytic instability and toxicity towards human cells, making in vivo use challenging. To investigate sequence factors related to serum activity, we adapt an antibacterial display technology to screen a library of peptide macrocycles for antibacterial potential directly in human serum. We identify dozens of new macrocyclic peptide antibiotic sequences and find that serum activity within our library is influenced by peptide length, cationic charge, and the number of disulfide bonds present. Interestingly, an optimized version of our most active lead peptide permeates the outer membrane of gram-negative bacteria without strong inner membrane disruption and kills bacteria slowly while causing cell elongation. This contrasts with traditional cationic antimicrobial peptides, which kill rapidly via lysis of both bacterial membranes. Notably, this optimized variant is not toxic to mammalian cells and retains its function in vivo, suggesting therapeutic promise. Our results support the use of more physiologically relevant conditions when screening peptides for antimicrobial activity which retain in vivo functionality.

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O antigen biogenesis sensitisesEscherichia coliK-12 to bile salts, a likely cause for how it lost its O antigen

Qin

Hong

Morona

et al. 2023

Preprint

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Escherichia coliK-12 is a model organism for bacteriology and has served as a workhorse for molecular biology and biochemistry for over a century since its first isolation in 1922. However,Escherichia coliK-12 strains are phenotypically devoid of an O antigen (OAg) since early reports in the scientific literature. Recent studies reported the presence of independent mutations that abolish OAg biogenesis inE. coliK-12 strains from the same original source, suggesting unknown evolutionary forces have selected for loss of OAg during the early propagation of K-12. Here, we show for the first time that restoration of OAg inE. coliK-12 strain MG1655 synergistically sensitises bacteria to vancomycin with bile salts (VBS). Suppressor mutants surviving lethal doses of VBS mostly contained disruptions in OAg biogenesis. We present data supporting a model where the transient presence and accumulation of lipid-carried OAg intermediates in the bacterial periplasm interfere with peptidoglycan synthesis, causing growth defects that are synergistically enhanced by bile salts. Lastly, we demonstrate that continuous bile salt exposure of OAg-producing MG1655 in the laboratory, can recreate a scenario where OAg disruption is selected for. Hence our work provides a likely explanation for the long-held mystery of howE. coliK-12 lost its OAg production and opens new avenues for exploring long-standing questions on the intricate network coordinating the synthesis of different cell envelope components in Gram-negative bacteria.Significance statementEscherichia coliK-12 is the most studied microorganism, widely used in laboratories for studying bacteriology and as a tool for molecular biology. The reason why it is devoid of O antigen remains a long-standing question. Our work has uncovered a previously unknown selection pressure of bile salts on bacterial O antigen biogenesis, which provides a plausible scenario for how the early propagation ofE. coliK-12 strains in bile salt containing media could have led to loss of O antigen in K-12. Our results also suggest that the accumulation of O antigen intermediates in the bacterial periplasm may interfere with bacterial cell wall synthesis, which paves a new research direction into the interplay of different cell envelope component synthesis pathways.

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Divergent Effects of Peptidoglycan Carboxypeptidase DacA on Intrinsic β-Lactam and Vancomycin Resistance

Cited by 8 publications

References 54 publications

Coordinated and distinct LD-transpeptidase-independent roles of peptidoglycan carboxypeptidases DacC and DacA in stress adaptation and cell shape maintenance

Coordinated and distinct LD-transpeptidase-independent roles of peptidoglycan carboxypeptidases DacC and DacA in stress adaptation and cell shape maintenance

Adapting antibacterial display to identify serum-active macrocyclic peptide antibiotics

O antigen biogenesis sensitisesEscherichia coliK-12 to bile salts, a likely cause for how it lost its O antigen

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