Vancomycin-resistant Leuconostoc mesenteroides and Lactobacillus casei synthesize cytoplasmic peptidoglycan precursors that terminate in lactate

Abstract

The emergence of acquired high-level resistance among Enterococcus species has renewed interest in mechanisms of resistance to glycopeptide antibiotics in gram-positive bacteria. In Enterococcus faecalis and Enterococcus faecium, resistance is encoded by the van gene cluster and is due to the production of a peptidoglycan precursor terminating in D-alanyl-D-lactate, to which vancomycin does not bind. MostLeuconostoc and many Lactobacillus species are intrinsically resistant to high levels of glycopeptide antibiotics, but the mechanism of resistance has not been elucidated. To determine whether the mechanisms of resistance are similar in intrinsically resistant bacteria, cytoplasmic pepti4oglycan precursors were isolated from Leuconostoc mesenteroides and Lactobacilus casei and analyzed by mass spectrometry, revealing structuresGlycopeptide antibiotics such as vancomycin act by binding to the D-alanyl-D-alanine terminus of stem pentapeptides present in bacterial peptidoglycan (16,21). It had been widely assumed that this terminus was ubiquitous among eubacterial species producing peptidoglycan and that vancomycin resistance was therefore unlikely to emerge in the absence of a barrier to vancomycin binding such as the outer membrane of gram-negative bacteria. However, high-level transmissible resistance to glycopeptides has recently been recognized among several gram-positive species, particularly enterococci, in which the incidence of resistance has been rising among clinical isolates (2).Most Leuconostoc and Pediococcus and some Lactobacillus species have been described as being intrinsically resistant to glycopeptides (9,15,17,20), but the mechanism of resistance has not been systematically investigated, perhaps because these organisms were considered to be of little clinical significance. However, the widespread use of vancomycin has led to more frequent recognition of these species as opportunistic pathogens (9,20 depsipeptide (D-alanyl-D-hydroxy acid) which is added in place of D-alanyl-D-alanine to the UDP-muramyl-tripeptide precursor, resulting in a peptidoglycan precursor that is not bound by vancomycin (6). This hypothesis was supported by the observation that the MIC for resistant enterococci was diminished when tested with high concentrations of D-amino acids that can be incorporated into pentapeptide and that are bound by vancomycin to a greater extent than D-2-hydroxy acids (23). Subsequently, the cytoplasmic peptidoglycan precursor in resistant E. faecium and E. faecalis was identified as UDP-

D-Alanyl-D-Lactate and D-Alanyl-D-Alanine Synthesis by D-Alanyl-D-Alanine Ligase from Vancomycin-resistant Leuconostoc mesenteroides EFFECTS OF A PHENYLALANINE 261 TO TYROSINE MUTATION

“…This enzyme has the activity shown in Fig. 4B (20,21). These activities pinpoint this chromosomal D-Ala-D-Ala ligase as a key molecular determinant in the antibiotic resistance phenotype.…”

“…In these cases, PG intermediates terminating in DAla-D-Lac were also detected (20,21), suggesting a common evolutionary mechanism and a possible origin for at least the VanH and -A genes. Polymerase chain reaction (PCR) analysis has been used to identify and sequence fragments of the ddl genes in such organisms (22) Cloning, Subcloning, and Site-directed Mutagenesis-Cloning was carried out by amplification of a DNA fragment by using two rounds of PCR of genomic DNA purified from L. mesenteroides ATCC 8293.…”

“…A yield of 30 mg of enzyme from ϳ5 g (wet weight) of E. coli was obtained. (20,21), it may be that LmDdl2 functions in depsipeptide ligase mode in vivo.…”

“…Activity g/ml) (20,21) was subcloned into plasmid pET22b behind the T7 promoter as noted under "Experimental Procedures," expressed in E. coli BL21(DE3), and purified as summarized in Fig. 2.…”

Presence of UDP-N-acetylmuramyl-hexapeptides and -heptapeptides in enterococci and staphylococci after treatment with ramoplanin, tunicamycin, or vancomycin

“…In particular, the presence of unusual nucleotide precursors such as UDPMurNAc-pentadepsipeptide and UDP-MurNAc-tetrapeptide-D-Ser involved in the resistance of various gram-positive bacteria against glycopeptide antibiotics has been investigated by this method (1,4,5,12,13,21). More recently, qualitative and quantitative analyses of complex mixtures were greatly facilitated by coupling HPLC with mass spectrometry (MS).…”