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Alternative glycopeptide resistance in Amycolatopsis balhimycina
Hans-Jörg Frasch1, Philip Steimle1, G. Gallo2, L. Kalan3, T. Schäberle1, A.-M. Puglia2, W. Wohlleben1, G. Wright3,E. Stegmann1
1 Interfakultäres
Institut für Mikrobiologie und Infektionsmedizin, Mikrobiologie/Biotechnologie, Eberhard-Karls-Universität Tübingen,
Auf der Morgenstelle 28, 72076 Tübingen, Germany
2 Dipartimento di Biologica Cellulare e dello Sviluppo, Università di Palermo, Viale delle Scienze, edificio 16, 90128 Palermo (Sicily)
3 Health Science Receiving, MacMaster University,1200 Main STW, L8N3Z5 Hamilton (Canada)
Introduction
sensitive cell wall
transglycosylation
The actinomycete Amycolatopsis balhimycina produces the vancomycin-type
glycopeptide balhimycin, which inhibits cell wall biosynthesis by binding to cell wall
precursors. Glycopeptide resistance is usually achieved by the synthesis of an alternative
cell wall. The endstanding D-alanine (D-Ala) in the pentapetide is replaced by a Dlactate (D-Lac), which reduces binding of the glycopeptide to its target (Fig. 1).
From resistant enterococci it is known that, this alteration of cell wall precursors requires
three enzymes: (I) VanH converts pyruvate to D-Lac, (II) VanA ligates D-Ala and D-Lac
and (III) VanX cleaves the ubiquitous D-Ala-D-Ala dipeptide. The expression of the
corresponding genes is controlled by the two component system VanRS. VanS senses
the presence of glycopeptides and phosphorylates VanR, which subsequently activates
the expression of the vanHAX-genes. An occassionally additional enzyme, VanY (IV),
cleaves the end standing D-Ala from pentapeptide precursors, thereby increasing the
resistance level (Fig 1).
Resistance to glycopeptides probably originated from producer strains which are immune
to their own product. Normally resistance genes are part of the biosynthetic gene cluster.
Surprisingly, the balhimycin gene cluster does not contain vanHAX-genes, but vanHAXhomologues were identified somewhere else in the chromosome.
To elucidate the complete resistance mechanism in A. balhimycina we have analysed the
function of the vanHAXb-genes by genetic, biochemical and analytical means.
Analysis of the vanHAXb-genes
A
transpeptidation
ADP
ATP
Pyruvate
P
D-Lac
D-Ala
D-Lac
(I)
(II)
promoter
activation
phosphorylation of VanR
orf1
IRL
transposase
orf2
vanR
resolvase
response
regulator
(IV)
vanS
histidine
kinase
vanH
vanA
dehydrogenase
ligase
vanX
vanY
dipeptidase
IRR
carboxy
peptidase
Fig 1 The biosynthesis of a glycopeptide resistant cell wall in enterococci
Analysis of the vanHAXb-mutant
GlcNac
UDP
PEP
Surprisingly, the vanHAXb deletion mutant produces balhimycin under
certain growth conditions, although the vanHAXb-genes are missing (data
not shown). Cell wall precursor analysis revealed the production of resistant
cell wall precusors under production conditions (Fig 5). Homology searches
in the genome of A. balhimycina delivered two genes encoding putative DAla-D-Ala-ligases, ddlAAb and ddlBAb. In vitro assays with purified protein
showed that DdlAAb exclusively forms D-Ala-D-Lac didepsipeptides (Fig 6).
DdlBAb showed no activity in the same assay (data not shown). A global
proteomic analysis showed that DdlAAb is not upregulated in response to
the loss of vanA, but is constitutively expressed, suggesting an innate lowlevel resistance to glycopeptides.
GlcNac - Enolpyruvate
MurB
NADP+
A
L-Ala
D-Glu
m-DAP
D-Ala
D-Ala
(III)
D-ala
1193
NADPH
UDP
UDP
A
MurNAc
MurC
D-lac
1194
D-ala
1193
D-Ala
D-Ala
P
MurA
Fig 2
A PCR Screeening of an A. balhimycina cosmid library using degenerated primers,
a 1,3 kB fragment was amplified which corresponds to vanA- homlogues.
B RT-PCR with primers overlapping vanHb and vanAb , RNA extraction after 15, 39 and
54 h hours of growth
L-Ala
D-Glu
m-DAP
D-Ala
D-Lac
D-Ala
UDP
B
glycopeptide
glycopeptide
binding
Interplay of cell wall
biosynthesis pathways
Homologues to vanHAXb-genes were identified in an A. balhimyina
cosmid library by a PCR-screening using degenerated primers (Fig 2A).
RT-PCR analysis revealed that these genes are expressed as an operon
prior to antibiotic biosynthesis (Fig 2B). LC-MS analysis of extracted cell
wall precursors (cwp) showed that resistant cell wall precursors ending
on D-Lac are predominant under different growth conditions (Fig. 3) and
deletion of the vanHAXb-operon resulted in sensitivity to 50 µg/ml
balhimycin (Fig 4) showing that the vanHAXb– genes are functional.
resistant cell wall
L-Ala
D-lac
1194
D-ala
1193
B
Fig 5 Cell wall precursor pattern in LC-MS of A. balhimycina ΔvanHAX
A In non production medium
B In production medium
Sensitive cwp ending on D-alanine elute at retention time of 10-11 min.
Resistant cwp ending on D-lactate elute at retention time of 15-16 min.
MurNAc
D-lac
1194
B
MurD
Fig 3 Cell wall precursor pattern in LC-MS of A. balhimycina WT
A In non production medium
B In production medium
Sensitive cwp ending on D-alanine elute at retention time of 10 -11 min
Resistant cwp ending on D-lactate elute at retention time of 15 -16 min
UDP
A. balhimycina
vanHAXb
WT
m-Dap
Fig 6 Thin-layer-chromatography of DdlA product formation
A) 2 mM D-Lac B) 2 mM D-Lac + 2 mM D- Ala C) 2 mM D-Ala + 1 mM D-Ala
VanA forms D-Ala-D-Lac-didepsipeptide as reference
DdlBEC forms D-Ala-D-Ala dipeptide as reference
Wildtyp
ΔvanHAXb
MurNAc
Fig 7 Comparison of DdlA-spots (outlined) in a 2D-DIGE analysis in A. balhimycina WT
and ΔvanHAXb. In both strains DdlAAb is expressed at the same level.
Fig 4 Resistance assay of A. balhimycina WT and ΔvanHAXb on YM-Agar 50 µg/ml
balhimycin
MurF
MurF
D-Ala-D-Ala
Ligase
VanA
Pyr
UDP
Alanine
racemase
L-Ala
MurNAc
MurE
UDP
VanH
D-Glu
D-Lac
D-Ala
MurNAc
UDP
MurNAc
D-Ala-D-Ala
D-Ala-D-Lac
D-Ala-D-Ala
Ligase
D-Ala-D-Ala
VanX
Canonical
Resistance
mechanism
Alanine
racemase
Non-canonical
Resistance
mechanism
D-Ala
DdlAAb
D-Ala-D-Lac
L-Ala
?
D-Lac
Amycolatopsis balhimycina possesses an alternative glycopeptide resistance mechanism
Stegmann et al, 2010, Glycopeptide biosynthesis in the context of basic cellular functions, Current opinion in microbiology, submitted
Wohlleben et al, 2009, Chapter 18: Molecular genetic approaches to analyze glycopeptide biosynthesis, Methods Enzymol, 458: 459-489
Gallo et al., 2010, Differentiell proteomic analysis reveals novel links between primary metabolism and antibiotic production in Amycolatopsis balhimycina, Proteomics , [Epub head of print]
Pelzer et al., 1999, Identification and analysis of the balhimycin biosynthetic gene cluster and its use for manipulating glycopeptide biosynthesis in Amycolatopsis mediteranei DSM5908; Antimicrob. Agents Chemother., 43: 1565-1573.
Pyr