Michel Arthur

QUANTITATIVE ANALYSIS OF THE METABOLISM OF SOLUBLE CYTOPLASMIC PEPTIDOGLYCAN PRECURSORS OF GLYCOPEPTIDE-RESISTANT ENTEROCOCCI

Transposon Tn1546 from Enterococcus faecium BM4147 mediates high‐level resistance to the glycopeptide antibiotics vancomycin and teicoplanin. Tn1546 encodes a dehydrogenase (VanH) and a ligase (VanA) that synthesize D‐alanyl‐D‐lactate (D‐Ala‐D‐Lac), a DD‐dipeptidase (VanX) that hydrolyses D‐Ala‐D‐Ala and a two‐component regulatory system (VanR–VanS) that controls transcription of the vanHAX operon. Strains of Enterococcus faecalis harbouring various copy numbers of the vanRSHAX cluster were tested to determine if there was a correlation between the levels of resistance to glycopeptides, the levels of expression of the corresponding resistance genes and the relative proportions of the different cytoplasmic peptidoglycan precursors. Increased transcription of the vanHAX operon was associated with increased incorporation of D‐Ala‐D‐Lac into peptidoglycan precursors to the detriment of D‐Ala‐D‐Ala, and with a gradual increase in the vancomycin‐resistance levels. More complete elimination of D‐Ala‐D‐Ala‐containing precursors was required for teicoplanin resistance. The VanY and VanZ proteins also encoded by Tn1546 were not effectors of the regulation of the vanHAX operon but contributed to vancomycin and teicoplanin resistance, respectively. Differences at the regulatory level accounted for phenotypic diversity in acquired glycopeptide resistance by production of D‐lac‐ending precursors.

The vanZ gene of Tn1546 from enterococcus faecium BM4147 confers resistance to teicoplanin

A five-gene cluster from Tn1546 confers resistance to the glycopeptide antibiotics vancomycin (Vm) and teicoplanin (Te) by synthesis of pentadepsipeptide peptidoglycan precursors terminating in D-lactate, which replaces D-alanine in the same position of precursors utilized by susceptible enterococci. Cloning and nucleotide sequencing indicated that Tn1546 contains an additional gene, designated vanZ, which confers low-level Te resistance, in the absence of the genes required for pentadepsipeptide synthesis. Analysis of cytoplasmic peptidoglycan precursors, accumulated in the presence of ramoplanin, showed that VanZ-mediated Te resistance does not involve incorporation of a substituent of D-alanine into the precursors.

The VANA glycopeptide resistance protein is related to D-alanyl-D-alanine ligase cell wall biosynthesis enzymes

SummaryInducible resistance to the glycopeptide antibiotics vancomycin and teicoplanin is mediated by plasmid pIP816 in Enterococcus faecium strain BM4147. Vancomycin induced the synthesis of a ca. 40 kDa membrane-associated protein designated VANA. The resistance protein was partially purified and its N-terminal sequence was determined. A 1761 by DNA restriction fragment of pIP816 was cloned into Escherichia coli and sequenced. When expressed in E. coli, this fragment encoded a ca. 40 kDa protein that comigrated with VANA from enterococcal membrane fractions. The ATG translation initiation codon for VANA specified the methionine present at the N-terminus of the protein indicating the absence of signal peptide processing. The amino acid sequence deduced from the sequence of the vanA gene consisted of 343 amino acids giving a protein with a calculated Mr of 37400. VANA was structurally related to the d-alanyl-d-alanine (d-ala-d-ala) ligases of Salmonella typhimurium (36% amino acid identity) and of E. coli (28%). The vanA gene was able to transcomplement an E. coli mutant with thermosensitive d-ala-d-ala ligase activity. Thus, the inducible resistance protein VANA was structurally and functionally related to cytoplasmic enzymes that synthesize the target of glycopeptide antibiotics. Based on these observations we discuss the possibility that resistance is due to modification of the glycopeptide target.

Requirement of the VanY and VanX D,D-peptidases for glycopeptide resistance in enterococci

Transposon Tn1546 confers resistance to glycopeptide antibiotics in enterococci and encodes two D,D‐peptidases (VanX and VanY) in addition to the enzymes for the synthesis of D‐alanyl‐D‐lactate (D‐Ala‐D‐Lac). VanY was produced in the baculovirus expression system and purified as a proteolytic fragment that lacked the putative N‐terminal membrane anchor of the protein. The enzyme was a Zn2+‐dependent D,D‐carboxypeptidase that cleaved the C‐terminal residue of peptidoglycan precursors ending in R‐D‐Ala‐D‐Ala or R‐D‐Ala‐D‐Lac but not the dipeptide D‐Ala‐D‐Ala. The specificity constants kcat/Km were 17‐ to 67‐fold higher for substrates ending in the R‐D‐Ala‐D‐Ala target of glycopeptides. In Enterococcus faecalis, VanY was present in membrane and cytoplasmic fractions, produced UDP‐MurNAc‐tetrapeptide from cytoplasmic peptidoglycan precursors and was required for high‐level glycopeptide resistance in a medium supplemented with D‐Ala. The enzyme could not replace the VanX D,D‐dipeptidase for the expression of glycopeptide resistance but a G237D substitution in the host D‐Ala:D‐Ala ligase restored resistance in a vanX null mutant. Deletion of the membrane anchor of VanY led to an active D,D‐carboxypeptidase exclusively located in the cytoplasmic fraction that did not contribute to glycopeptide resistance in a D‐Ala‐containing medium. Thus, VanX and VanY had non‐overlapping functions involving the hydrolysis of D‐Ala‐D‐Ala and the removal of D‐Ala from membrane‐bound lipid intermediates respectively.

Mutations leading to increased levels of resistance to glycopeptide antibiotics in VanB-type enterococci.

The vanB gene cluster mediates glycopeptide resistance by production of peptidoglycan precursors ending in the depsipeptide D‐alanyl‐D‐lactate (D‐Ala‐D‐Lac) instead of D‐Ala‐D‐Ala found in susceptible enterococci. Synthesis of D‐Ala‐D‐Lac and hydrolysis of D‐Ala‐D‐Ala is controlled by the VanRBSB two‐component regulatory system that activates transcription of the resistance genes in response to vancomycin but not to teicoplanin. Two substitutions (A30→G or D168→Y) in the VanSB sensor kinase resulted in induction by teicoplanin, indicating that the N‐terminal domain of the protein was involved in glycopeptide sensing. A substitution (T237→K) located in the vicinity of the putative autophosphorylation site of VanSB (H233) was associated with a constitutive phenotype and affected a conserved residue known to be critical for the phosphatase activity of related kinases. A mutant producing an impaired host D‐Ala:D‐Ala ligase required vancomycin for growth, since D‐Ala‐D‐Lac was only produced under inducing conditions. The ddl and vanSB mutations, alone or in combination, resulted in various resistance phenotypes that were determined by the amount of D‐Ala‐D‐Ala and D‐Ala‐D‐Lac incorporated into peptidoglycan precursors under different inducing conditions.

Regulated interactions between partner and non-partner sensors and response regulators that control glycopeptide resistance gene expression in enterococci.

Transcription of the vanA and vanB glycopeptide resistance gene clusters is regulated by the VanRS and VanRBSB two-component regulatory systems, respectively. Histidine to glutamine substitutions were introduced at positions 164 of VanS and 233 of VanSB to prevent autophosphorylation of the sensor kinases and transfer of the phosphate groups to the VanR and VanRB response regulators. VanSH164Q and VanSBH233Q abolished activation of VanR and VanRB by host kinases. The phosphatase activity of VanSBH233Q was negatively modulated by vancomycin whereas VanSH164Q prevented transcription of the resistance genes under all growth conditions. Cross-talk was detected between VanRB and VanS in a vanSB null mutant. VanR is required for activation of promoters PR and PH allowing transcription of the regulatory (vanRS) and resistance (vanHAXYZ) genes, respectively. Under non-inducing conditions, activation of VanR by cross-talk was blocked by the presence of a multicopy plasmid carrying PH. Presence of the high-affinity VanR-binding sites of the regulatory region of PH on the multicopy vector probably sequestered VanR, thereby preventing autoactivation of the PR promoter. Under such circumstances, stimulation of the host kinase by glycopeptides or moenomycin was required for expression of the resistance genes.

Single-cell analysis of glycopeptide resistance gene expression in teicoplanin-resistant mutants of a VanB-type Enterococcus faecalis.

The vanB gene cluster confers resistance to vancomycin but not to the related antibiotic teicoplanin, as the VanRBSB two‐component regulatory system triggers expression of the glycopeptide resistance genes only in response to vancomycin. The VanRB regulator activates promoters PRB and PYB for transcription of the regulatory (vanRBSB) and resistance (vanYBWHBBXB) genes respectively. The gfpmut1 gene encoding a green fluorescent protein was fused to PYB to analyse promoter activation in single cells by fluorescence microscopy and flow cytometry. Characterization of 17 teicoplanin‐resistant mutants indicated that amino acid substitutions on either side of the VanSB autophosphorylation site led to a constitutive phenotype. Substitutions in the membrane‐associated domain resulted in a gain of function, as they allowed induction by teicoplanin. A vanSB null mutant expressed gfpmut1 at various levels under non‐inducing conditions, and the majority of the bacteria were not fluorescent. Bacteria grown in the presence of vancomycin or teicoplanin were homogeneously fluorescent. The increase in the number of fluorescent bacteria resulted from induction in negative cells rather than from selection of a resistant subpopulation, indicating that VanRB was activated by cross‐talk. Transglycosylase inhibition was probably the stimulus for the heterologous kinase, as moenomycin was also an inducer.

Rapid Cytolysis of Mycobacterium tuberculosis by Faropenem, an Orally Bioavailable β-Lactam Antibiotic

ABSTRACT Recent clinical studies indicate that meropenem, a β-lactam antibiotic, is a promising candidate for therapy of drug-resistant tuberculosis. However, meropenem is chemically unstable, requires frequent intravenous injection, and must be combined with a β-lactamase inhibitor (clavulanate) for optimal activity. Here, we report that faropenem, a stable and orally bioavailable β-lactam, efficiently kills Mycobacterium tuberculosis even in the absence of clavulanate. The target enzymes, l,d-transpeptidases, were inactivated 6- to 22-fold more efficiently by faropenem than by meropenem. Using a real-time assay based on quantitative time-lapse microscopy and microfluidics, we demonstrate the superiority of faropenem to the frontline antituberculosis drug isoniazid in its ability to induce the rapid cytolysis of single cells. Faropenem also showed superior activity against a cryptic subpopulation of nongrowing but metabolically active cells, which may correspond to the viable but nonculturable forms believed to be responsible for relapses following prolonged chemotherapy. These results identify faropenem to be a potential candidate for alternative therapy of drug-resistant tuberculosis.

Combinations of β-Lactam Antibiotics Currently in Clinical Trials Are Efficacious in a DHP-I-Deficient Mouse Model of Tuberculosis Infection

ABSTRACT We report here a dehydropeptidase-deficient murine model of tuberculosis (TB) infection that is able to partially uncover the efficacy of marketed broad-spectrum β-lactam antibiotics alone and in combination. Reductions of up to 2 log CFU in the lungs of TB-infected mice after 8 days of treatment compared to untreated controls were obtained at blood drug concentrations and time above the MIC (T>MIC) below clinically achievable levels in humans. These findings provide evidence supporting the potential of β-lactams as safe and mycobactericidal components of new combination regimens against TB with or without resistance to currently used drugs.

Bactericidal Activity of Gentamicin against Enterococcus faecalis In Vitro and In Vivo

ABSTRACT The activity of gentamicin at various concentrations against two strains of Enterococcus faecalis was investigated in vitro and in a rabbit model of aortic endocarditis. In vitro, gentamicin at 0.5 to 4 times the MIC failed to reduce the number of bacteria at 24 h. Rabbit or human serum dramatically increased gentamicin activity, leading to a ≥3-log10 CFU/ml decrease in bacterial counts when the drug concentration exceeded the MIC. Susceptibility testing in the presence of serum was predictive of in vivo activity, since gentamicin alone significantly reduced the number of surviving bacteria in the vegetations if the peak-to-MIC ratio was greater than 1. However, gentamicin selected resistant mutants in rabbits. The intrinsic activity of gentamicin should be taken into account in evaluation of combinations of gentamicin and cell wall-active agents against enterococci.