Paradoxical response of Enterococcus faecalis to the bactericidal activity of penicillin is associated with reduced activity of one autolysin

Fontana, Roberta; Boaretti, Marzia; Grossato, A.; Tonin, Elena; Lleò, Maria M.; Satta, G.

doi:10.1128/aac.34.2.314

Cited by 35 publications

(21 citation statements)

References 20 publications

(12 reference statements)

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“…Wild-type cells harboring the pC3 cloning vector exhibited slightly faster autolysis than did untransformed cells, indicating that the presence of a plasmid per se can influence the autolysis kinetics. It has been reported that alterations in autolytic enzyme activity can influence the susceptibility to penicillin (6). We found that the MIC of penicillin for A9 was 6.25 g/ml and was identical to that for the wild type, in both N and BHI media.…”

Section: Vol 177 1995 Gene Controlling Muramidase-2 In E Hirae 5913supporting

confidence: 50%

Identification of a gene (arpU) controlling muramidase-2 export in Enterococcus hirae

1995

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Muramidase-2 of Enterococcus hirae is a 74-kDa peptidoglycan hydrolase that plays a role in cell wall growth and division. To study its regulation, we isolated a mutant defective in muramidase-2 release under certain growth conditions. This mutant had cell walls which apparently lacked 74-kDa muramidase-2 but which accumulated two proteolytic fragments of 32 and 43 kDa, which exhibited muramidase-2 activity in the membrane fraction. By complementation cloning, we identified a 2.6-kb fragment of the E. hirae chromosome containing a gene cluster coding for proteins of 58 to 137 amino acids. One of these genes (arpU), which encoded a 15.9-kDa protein, was shown to complement the defect of the A9 mutant in trans. We propose that this gene may be involved in the regulation of muramidase-2 export.Peptidoglycan hydrolases are enzymes responsible for a number of important biological functions such as cell wall growth, cell separation, peptidoglycan turnover, competence for genetic transformation, sporulation, and bacteriolysis induced by beta-lactam antibiotics (7,8,14,21,23,27). Regulation of autolysin activity is believed to occur mostly at the posttranslational level. Possible regulatory mechanisms include enzyme activation by changes in substrate structure, topological restriction of enzyme distribution in the cell wall, and control at the site of export (8).Two autolytic enzymes with muramidase activity have been described in Enterococcus hirae. Muramidase-1 is synthesized in a latent 130-kDa form, which is transported to specific sites in the cell wall, where it is converted to the active 87-kDa form (10). This enzyme can hydrolyze E. hirae cell walls but not Micrococcus luteus cell walls. The second enzyme, peptidoglycan hydrolase-2, or muramidase-2, is found in the culture medium during cell growth and is active on cell walls of M. luteus and purified peptidoglycan of E. hirae (5, 11). It has been postulated that muramidase-2 may operate in conjunction with muramidase-1 in peptidoglycan hydrolysis (2) and may facilitate cell separation (14,24). The E. hirae muramidase-2 gene has recently been cloned and sequenced (3,9).In an attempt to gain insight into the mechanisms that control muramidase-2 activity in E. hirae, we have characterized a mutant (A9) with medium-dependent impairment of muramidase-2 export. By functional complementation of the mutant with an E. hirae genomic bank, we cloned a gene (arpU) that would appear to be involved in muramidase-2 export. MATERIALS AND METHODSBacterial strains and growth conditions. Unless stated otherwise, E. hirae wild-type (ATCC 9790) and mutant cells were grown at 37ЊC in N medium (1% Bacto Peptone [Difco Laboratories, Detroit, Mich.], 0.5% yeast extract [Difco], 1% Na 2 HPO 4 ⅐ 2H 2 O). For some experiments, brain heart infusion (BHI; Difco) was used. To monitor growth, the optical density at 650 nm was measured in an LKB Ultrospec II.Selection of mutants. For the isolation of mutants deficient in muramidase-2, wild-type E. hirae was mutagenized as follows. Cells in mid...

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Section: Vol 177 1995 Gene Controlling Muramidase-2 In E Hirae 5913supporting

confidence: 50%

Identification of a gene (arpU) controlling muramidase-2 export in Enterococcus hirae

1995

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“…The paradoxical effect (Eagle phenomenon), which was first described by Eagle in 1948, is defined as a bactericidal activity that decreases when the concentration of the antibiotic increases. The mechanism of this phenomenon has not been clearly established; defective autolytic activity when exposed to high concentrations of antibiotics has been implicated for E. faecalis (37). However, the clinical relevance of this phenomenon is still unclear and needs further investigation.…”

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confidence: 97%

Activities of Fosfomycin and Rifampin on Planktonic and Adherent Enterococcus faecalis Strains in an Experimental Foreign-Body Infection Model

Oliva

Tafin

Maiolo

et al. 2014

Antimicrob Agents Chemother

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c Enterococcal implant-associated infections are difficult to treat because antibiotics generally lack activity against enterococcal biofilms. We investigated fosfomycin, rifampin, and their combinations against planktonic and adherent Enterococcus faecalis (ATCC 19433) in vitro and in a foreign-body infection model. The MIC/MBC log values were 32/>512 g/ml for fosfomycin, 4/>64 g/ml for rifampin, 1/2 g/ml for ampicillin, 2/>256 g/ml for linezolid, 16/32 g/ml for gentamicin, 1/>64 g/ml for vancomycin, and 1/5 g/ml for daptomycin. In time-kill studies, fosfomycin was bactericidal at 8؋ and 16؋ MIC, but regrowth of resistant strains occurred after 24 h. With the exception of gentamicin, no complete inhibition of growth-related heat production was observed with other antimicrobials on early (3 h) or mature (24 h) biofilms. In the animal model, fosfomycin alone or in combination with daptomycin reduced planktonic counts by Ϸ4 log 10 CFU/ml below the levels before treatment. Fosfomycin cleared planktonic bacteria from 74% of cage fluids (i.e., no growth in aspirated fluid) and eradicated biofilm bacteria from 43% of cages (i.e., no growth from removed cages). In combination with gentamicin, fosfomycin cleared 77% and cured 58% of cages; in combination with vancomycin, fosfomycin cleared 33% and cured 18% of cages; in combination with daptomycin, fosfomycin cleared 75% and cured 17% of cages. Rifampin showed no activity on planktonic or adherent E. faecalis, whereas in combination with daptomycin it cured 17% and with fosfomycin it cured 25% of cages. Emergence of fosfomycin resistance was not observed in vivo. In conclusion, fosfomycin showed activity against planktonic and adherent E. faecalis. Its role against enterococcal biofilms should be further investigated, especially in combination with rifampin and/or daptomycin treatment.

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“…In Enterococcus faecalis, two autolytic activities have been described (12). One of the corresponding proteins, designated AtlA in this report, has been identified (4,18), but its activity has not been characterized.…”

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confidence: 99%

Functional Analysis of AtlA, the MajorN-Acetylglucosaminidase ofEnterococcus faecalis

et al. 2006

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The major peptidoglycan hydrolase of Enterococcus faecalis, AtlA, has been identified, but its enzyme activity remains unknown. We have used tandem mass spectrometry analysis of peptidoglycan hydrolysis products obtained using the purified protein to show that AtlA is an N-acetylglucosaminidase. To gain insight into the regulation of its enzyme activity, the three domains of AtlA were purified alone or in combination following expression of truncated forms of the atlA gene in Escherichia coli or partial digestion of AtlA by proteinase K. The central domain of AtlA was catalytically active, but its activity was more than two orders of magnitude lower than that of the complete protein. Partial proteolysis of AtlA was detected in vivo: zymograms of E. faecalis extracts revealed two catalytically active protein bands of 62 and 72 kDa that were both absent in extracts from an atlA null mutant. Limited digestion of AtlA by proteinase K in vitro suggested that the proteolytic cleavage of AtlA in E. faecalis extracts corresponds to the truncation of the N-terminal domain, which is rich in threonine and glutamic acid residues. We show that the truncation of the N-terminal domain from recombinant AtlA has no impact on enzyme activity. The C-terminal domain of the protein, which contains six LysM modules bound to highly purified peptidoglycan, was required for optimal enzyme activity. These data indicate that AtlA is not produced as a proenzyme and that control of the AtlA glucosaminidase activity is likely to occur at the level of LysM-mediated binding to peptidoglycan.

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Paradoxical response of Enterococcus faecalis to the bactericidal activity of penicillin is associated with reduced activity of one autolysin

Cited by 35 publications

References 20 publications

Identification of a gene (arpU) controlling muramidase-2 export in Enterococcus hirae

Identification of a gene (arpU) controlling muramidase-2 export in Enterococcus hirae

Activities of Fosfomycin and Rifampin on Planktonic and Adherent Enterococcus faecalis Strains in an Experimental Foreign-Body Infection Model

Functional Analysis of AtlA, the MajorN-Acetylglucosaminidase ofEnterococcus faecalis

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