Enterococcal Cell Wall

Coyette, Jacques; Hancock, Lynn E.

doi:10.1128/9781555817923.ch5

Cited by 10 publications

(4 citation statements)

References 165 publications

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“…The molecular structure of this m/z ϭ 817.3938 peak material was deduced to be N-acetylmuramoyl-Lalanine-D-glutamic acid-L-lysine-(L-alanine)-D-alanine-D-alanine ( Fig. 9B), a composition in agreement with that determined for the E. faecalis peptidoglycan (see reviews by Coyette and Hancock [30], Bouhss et al [31], and Hancock et al [32]). This interpretation is supported by the results of a high-resolution targeted tandem MS (MS/MS) analysis of the major digestion product seen at m/z ϭ 817.40 in the initial MS spectrum ( Fig.…”

Section: Figsupporting

confidence: 79%

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Bacteriophage φEf11 ORF28 Endolysin, a Multifunctional Lytic Enzyme with Properties Distinct from All Other Identified Enterococcus faecalis Phage Endolysins

Zhang

Buttaro

Fouts

et al. 2019

Appl Environ Microbiol

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Ef11 is a temperate Siphoviridae bacteriophage that infects strains of Enterococcus faecalis. The Ef11 genome, encompassing 65 open reading frames (ORFs), is contained within 42,822 bp of DNA. Within this genome, a module of six lysis-related genes was identified. Based upon sequence homology, one of these six genes, ORF28, was predicted to code for an N-acetylmuramoyl-Lalanine amidase endolysin of 46.133 kDa, composed of 421 amino acids. The PCRamplified ORF28 was cloned and expressed, and the resulting gene product was affinity purified to homogeneity. The purified protein was obtained from a fusion protein that exhibited a molecular mass of 72.5 kDa, consistent with a 46.1-kDa protein combined with a fused 26.5-kDa glutathione S-transferase tag. It produced rapid, profound lysis in E. faecalis populations and was active against 73 of 103 (71%) E. faecalis strains tested. In addition, it caused substantial destruction of E. faecalis biofilms. The lysin was quite stable, retaining its activity for three years in refrigerated storage, was stable over a wide range of pHs, and was unaffected by the presence of a reducing agent; however, it was inhibited by increasing concentrations of Ca 2ϩ . Liquid chromatography-mass spectrometry analysis of E. faecalis cell wall digestion products produced by the ORF28 endolysin indicated that the lysin acted as an N-acetylmuramidase, an endo-␤-N-acetylglucosaminidase, and an endopeptidase, rather than an N-acetylmuramoyl-L-alanine amidase. The Ef11 ORF28 lysin shared 10% to 37% amino acid identity with the lytic enzymes of all other characterized E. faecalis bacteriophages. IMPORTANCE The emergence of multidrug-resistant pathogenic microorganisms has brought increasing attention to the urgent need for the development of alternative antimicrobial strategies. One such alternative to conventional antibiotics employs lytic enzymes (endolysins) that are produced by bacteriophages in the course of lytic infection. During lytic infection by a bacteriophage, these enzymes hydrolyze the cell wall peptidoglycan, resulting in the lysis of the host cell. However, external endolysin application can result in lysis from without. In this study, we have cloned, expressed, purified, and characterized an endolysin produced by a bacteriophage infecting strains of Enterococcus faecalis. The lysin is broadly active against most of the tested E. faecalis strains and exhibits multifunctional enzymatic specificities that differ from all other characterized endolysins produced by E. faecalis bacteriophages.

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Section: Figsupporting

confidence: 79%

“…Furthermore, antiserum raised to the E. faecium cell wall polysaccharide failed to recognize the polysaccharide purified from the cell wall of an E. faecalis strain. Any or all of these differences could explain the differences in sensitivity to the lysin between strains of E. faecalis and E. faecium (30)(31)(32).…”

Section: Discussionmentioning

confidence: 99%

Bacteriophage φEf11 ORF28 Endolysin, a Multifunctional Lytic Enzyme with Properties Distinct from All Other Identified Enterococcus faecalis Phage Endolysins

Zhang

Buttaro

Fouts

et al. 2019

Appl Environ Microbiol

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“…The enterococcal cell wall is a multi-layered network and is characterized by the presence of peptidoglycan, teichoic acid, and polysaccharides [ 31 , 32 ]. The main component of the cell wall is peptidoglycan, which is a mesh-like structure consisting of parallel glycan chains cross-linked by amino acids [ 31 ].…”

Section: Resultsmentioning

confidence: 99%

ddcP, pstB, and excess D-lactate impact synergism between vancomycin and chlorhexidine against Enterococcus faecium 1,231,410

Bhardwaj¹,

Islam²,

Kim³

et al. 2021

PLoS ONE

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Vancomycin-resistant enterococci (VRE) are important nosocomial pathogens that cause life-threatening infections. To control hospital-associated infections, skin antisepsis and bathing utilizing chlorhexidine is recommended for VRE patients in acute care hospitals. Previously, we reported that exposure to inhibitory chlorhexidine levels induced the expression of vancomycin resistance genes in VanA-type Enterococcus faecium. However, vancomycin susceptibility actually increased for VanA-type E. faecium in the presence of chlorhexidine. Hence, a synergistic effect of the two antimicrobials was observed. In this study, we used multiple approaches to investigate the mechanism of synergism between chlorhexidine and vancomycin in the VanA-type VRE strain E. faecium 1,231,410. We generated clean deletions of 7 of 11 pbp, transpeptidase, and carboxypeptidase genes in this strain (ponA, pbpF, pbpZ, pbpA, ddcP, ldtfm, and vanY). Deletion of ddcP, encoding a membrane-bound carboxypeptidase, altered the synergism phenotype. Furthermore, using in vitro evolution, we isolated a spontaneous synergy escaper mutant and utilized whole genome sequencing to determine that a mutation in pstB, encoding an ATPase of phosphate-specific transporters, also altered synergism. Finally, addition of excess D-lactate, but not D-alanine, enhanced synergism to reduce vancomycin MIC levels. Overall, our work identified factors that alter chlorhexidine and vancomycin synergism in a model VanA-type VRE strain.

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“…9 μg/ml H-CHG is the MIC for E. faecium 1,231,410; 1.2 μg/ml H-CHG and 2.4 μg/ml H-CHG are 1/4X MIC and 1/2X MIC, respectively.Deletion of ddcP alters the synergism phenotype. The enterococcal cell wall is a multilayered network and is characterized by the presence of peptidoglycan, teichoic acid, and polysaccharides(27,28). The main component of the cell wall is peptidoglycan, which is a mesh-like structure consisting of parallel glycan chains cross-linked by amino acids(27).…”

mentioning

confidence: 99%

ddcP, pstB, and excess D-lactate impact synergism between vancomycin and chlorhexidine against Enterococcus faecium 1,231,410

Bhardwaj¹,

Islam²,

Kim³

et al. 2018

Preprint

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28Enterococci are Gram-positive gastrointestinal tract colonizers of humans and animals. 29Vancomycin-resistant enterococci (VRE) are important nosocomial pathogens and can cause 30 life-threatening infections. To control hospital-associated infections, skin antisepsis and bathing 31 utilizing chlorhexidine is recommended for VRE patients in acute care hospitals. Previously, we 32 reported that exposure to inhibitory chlorhexidine levels induced the expression of vancomycin 33 resistance genes in VanA-type Enterococcus faecium. However, vancomycin susceptibility 34 actually increased for VanA-type E. faecium in the presence of chlorhexidine. Hence, a 35 synergistic effect of the two antimicrobials was observed. In this study, we tested various 36 models to elucidate the mechanism(s) of synergism between chlorhexidine and vancomycin. 37We deleted each of the pbp genes from a model VanA-type VRE E. faecium strain. We found 38 that deletion of ddcP, a membrane-bound carboxypeptidase, resulted in partial loss of 39 synergism. Interestingly, addition of excess D-lactate, but not D-alanine, enhanced synergism. 40Furthermore, we isolated a synergy escaper mutant in E. faecium and utilized whole genome 41 sequencing to determine that a mutation in a gene encoding an ATPase of phosphate-specific 42 transporters (pstB) also resulted in loss of synergism. Our study is significant because 43 understanding the mechanisms for chlorhexidine-induced vancomycin resensitization in VRE 44 could lead to new combinatorial therapeutics to treat VRE infections. 45 46 47 48 49 50 51 52 Enterococcus faecium and E. faecalis are Gram-positive commensal bacteria inhabiting the 55 gastrointestinal tracts of humans and animals (1). A recently published evolutionary history of 56 the enterococci elucidated how these bacteria became the leading causes of hospital-57 associated infections (2). The ability to survive in harsh environmental conditions including 58 starvation and desiccation facilitated the emergence of hospital-adapted strains which are 59 resistant to the action of antibiotics and disinfectants. Hospital-adapted enterococcal strains 60 have limited treatment options and are typically characterized by high-level resistance to 61 vancomycin, a glycopeptide antibiotic which inhibits the process of peptidoglycan synthesis (3, 62 4). Vancomycin-resistant enterococci (VRE) synthesize peptidoglycan precursors for which 63 vancomycin has low affinity (5-8). Vancomycin resistance in hospital-adapted enterococcal 64isolates occurs through the horizontal acquisition of resistance genes (9, 10). For VanA-type 65 VRE, vancomycin resistance is conferred and controlled by the activities encoded by the 66 vanRS, vanHAX, and vanYZ genes. 68Patients in critical care units are frequently bathed or cleansed with chlorhexidine, a cationic cell 69 membrane-targeting antimicrobial, to reduce the occurrence of hospital-associated infections (11)(12)(13). Chlorhexidine interacts with the negatively charged phospholipids and proteins on the 71 cell membra...

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Enterococcal Cell Wall

Cited by 10 publications

References 165 publications

Bacteriophage φEf11 ORF28 Endolysin, a Multifunctional Lytic Enzyme with Properties Distinct from All Other Identified Enterococcus faecalis Phage Endolysins

Bacteriophage φEf11 ORF28 Endolysin, a Multifunctional Lytic Enzyme with Properties Distinct from All Other Identified Enterococcus faecalis Phage Endolysins

ddcP, pstB, and excess D-lactate impact synergism between vancomycin and chlorhexidine against Enterococcus faecium 1,231,410

ddcP, pstB, and excess D-lactate impact synergism between vancomycin and chlorhexidine against Enterococcus faecium 1,231,410

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