The Antibacterial Activity of N-Pentylpantothenamide is Due to Inhibition of CoA Synthesis

17 Pages·2010·0.53 MB·English

AAC Accepts, published online ahead of print on 4 January 2010
Antimicrob. Agents Chemother. doi:10.1128/AAC.01473-09
Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
2 The Antibacterial Activity of N-Pentylpantothenamide is Due
3 to Inhibition of CoA Synthesis
4
5
6
D
7 Jacob Thomas1,3 and John E. Cronan1,2 o
w
8 Departments of Biochemistry1 and Microbiology 2 University of Illinois at Urbana- nlo
a
9 Champaign, Urbana, IL 61801 d
e
10 d

f
11 ro
12 m

13 h
t
14 tp
:
15 /
/
a
16 Running Title: N-Pentylpantothenamide inhibits CoA synthesis a
17 c
.
a
18 s
m
19
.
20 o
r
g
21
/
22 3Present address: Department of Biology, Massachusetts Institute of Technology o
n
23 Cambridge, MA 02139
J
24 a
n
25 u
a
26 r
y
27
7
28 ,
2
29 0
1
30 Correspondence to: 9
31 John E. Cronan b
y
32 Department of Microbiology
g
33 University of Illinois u
e
34 B103 CLSL s
t
35 601 S Goodwin Ave
36 Urbana, IL 61801 U
37 Telephone: [phone], Fax: [phone].
38 E-mail: [email protected]
39
1
40 Abstract
41 Growth inhibition by the pantothenate analog, N-pentylpantothenamide (N5-Pan),
42 has been attributed to accumulation of acyl carrier protein carrying a prosthetic
43 group modified by incorporation of N5-Pan. This was attributed to an inability of
D
44 the AcpH acyl carrier protein phosphodiesterase to cleave the N5-Pan modified o
w
n
45 prosthetic group from the protein moiety. We report that AcpH readily removes lo
a
d
46 the N5-Pan modified prosthetic group both in vivo and in vitro and show that N5- e
d

f
47 Pan blocks CoA synthesis. ro
m

h
t
t
p
:
/
/
a
a
c
.
a
s
m
.
o
r
g
/

o
n

J
a
n
u
a
r
y

7
,

2
0
1
9

b
y

g
u
e
s
t
2
48 Introduction
49
50 The N-substituted pantothenamide antimetabolites are analogs of the CoA precursor,
51 pantothenic acid. These compounds have been utilized as antimicrobial agents (2, 18, 23),
52 as probes of the active site of pantothenate kinase (21) and to post-translationally modify
D
53 and visualize carrier proteins in vivo (14). o
w
n
54 N-Pentylpantothenamide (N5-Pan), the most studied of these compounds, has lo
a
d
55 been shown to participate in three of four CoA synthetic reactions (Fig 1) leading to the e
d

f
r
56 formation of the inactive CoA analog ethyldethia-CoA (18). Acyl carrier protein (ACP), o
m

h
57 the carrier of acyl intermediates of fatty acid synthesis (1), requires the posttranslational t
t
p
:
/
58 attachment of a prosthetic group 4’-phosphopantetheine derived from CoA for metabolic /a
a
c
59 function. Holo-ACP synthase (AcpS), the enzyme that catalyzes this reaction in .a
s
m
60 Escherichia coli (7, 15) also uses ethyldethia-CoA as a substrate in vivo leading to the .
o
r
g
61 accumulation of inactive ethyldethia-ACP and subsequent inhibition of fatty acid /
o
n

62 synthesis (23). Zhang and coworkers proposed that ACP phosphodiesterase (AcpH), the J
a
n
u
63 enzyme that hydrolyzes holo-ACP to apo-ACP and 4’-phosphopantetheine (19, 20), is a
r
y

64 unable to use ethyldethia-ACP as a substrate. This was postulated to lead to the observed 7
,

2
0
65 accumulation of this ACP species and cause the antimicrobial activity of N- 1
9

b
66 pentylpantothenamide. However, the gene encoding AcpH was unknown at that time y

g
u
67 precluding in vivo testing of this hypothesis. We report that AcpH readily hydrolyzes e
s
t
68 ethyldethia-ACP and that increasing or abolishing AcpH levels in vivo had only very
69 modest effects on sensitivity to N5-Pan.
70
3
71 Strain JT6 was constructed by transduction of E. coli K-12 strain UB1005 to tetracycline
72 resistance using strain CAG12184 (tolC::Tn10) (17) which lacks the outer membrane exit
73 duct of tripartite efflux pumps. An exponentially growing culture of strain JT6 in
74 minimal M9 medium containing 0.4% glycerol and 0.1% vitamin-free Casamino acids
75 was supplemented with 200 µg/ml N5-Pan synthesized by the method of Strauss and D
o
w
76 Begley (18). Samples of the culture were periodically removed, cell-free extracts n
lo
a
77 prepared and ACP pools were visualized by conformationally sensitive gel d
e
d
78 electrophoresis (5). As reported by other workers (23) a rapidly migrating ACP species f
r
o
m
79 corresponding to ethyldethia-ACP was observed (Fig 2, Panel A) that became the

h
t
t
80 dominant ACP species. p
:
/
/
a
81 a
c
.
a
s
82 In the model previously proposed (23), a strain lacking AcpH would be resistant to N5- m
.
o
83 Pan. Moreover, due to the increased turnover of holo-ACP, a strain overproducing AcpH r
g
/

o
84 should be more sensitive to N5-Pan. In order to test these predictions, strain JT13 n

J
a
85 lacking acpH was constructed by transduction of strain JT6 to chloramphenicol resistance n
u
a
86 using a P1 lysate made on the ∆acpH strain JT1 (19). Derivatives of strain JT6 ry

7
,
87 transformed with plasmid pJT5 which expresses AcpH under arabinose control (19) or 2
0
1
88 the empty vector pBAD322 (3) were also constructed. Contrary to our expectations, we 9

b
y
89 observed that the acpH null mutant strain JT13 was about 2-fold more sensitive to N5- g
u
e
90 Pan than the parent strain (Fig 3, Panel A) whereas the strain overexpressing AcpH was s
t
91 approximately 2-fold more resistant to the analog (Fig 3, Panel B).
92
4
93 To examine the ACP pools of these strains cultures were treated with 100 µg/ml N5-Pan,
94 crude extracts of the cells were made and the proteins were separated by
95 conformationally sensitive gel electrophoresis followed by electrophoretic transfer and
96 western blotting analysis with a specific anti-ACP antibody (Fig 2, Panel C). A
97 significant pool of holo-ACP was present in the AcpH overproducing strain 3 h after D
o
w
98 treatment with N5-Pan whereas in the empty vector control, the predominant species was n
lo
a
99 ethyldethia-ACP. The ACP pools of the acpH deletion strain JT13 did not differ d
e
d
100 markedly from the parental strain (data not shown). f
r
o
m
101

h
t
t
102 Purified ethyldethia-ACP was isolated as described previously (5) with the exception that p
:
/
/
a
103 400 µg/ml N5-Pan was added to the culture that overexpressed ACP. Both ethyldethia- a
c
.
a
s
104 ACP and holo-ACP were hydrolyzed by AcpH in vitro (Fig 2). Ethyldethia-ACP is m
.
o
105 therefore a substrate for AcpH both in vivo and in vitro and AcpH provides only a r
g
/

o
106 modestly increased resistance to N5-Pan. n

J
a
107 n
u
a
108 We then examined CoA synthesis in cultures treated with N5-Pan reasoning that, if CoA ry

7
,
109 synthesis was inhibited by the analog or its products in vivo, ethyldethia-CoA would 2
0
1
110 constitute the sole source of cellular ACP prosthetic groups thereby leading to the 9

b
y
111 observed preferential accumulation of ethyldethia-ACP over holo-ACP. The β-alanine g
u
e
112 auxotrophic strain JT7 carrying a panD deletion (4, 22), was constructed by transducing s
t
113 NRD45 (UB1005 ∆panD) to tetracycline resistance using the strain CAG12184 lysate as
114 above. The cellular CoA and ACP pools were specifically labeled using tritiated β-
115 alanine (12, 16). A culture of strain JT7 was grown to an optical density of 0.2 in
5
116 minimal M9 medium containing 0.4% glycerol, 0.1% Casamino acids and 4 µM β-[3-
117 3H]alanine (0.25 Ci/mol), the culture divided in half and 100 µg/ml N5-Pan was added to
118 one culture. After incubation for a further 90 min (approximately 2 doublings), the CoA
119 pools were extracted by the method of Iram and Cronan (9) and analyzed by reverse
120 phase HPLC on a C18 column. After treatment with the analog, CoA synthesis was D
o
w
121 severely inhibited, leading to over 10-fold decreased levels of the key metabolic n
lo
a
122 intermediate, acetyl-CoA (Fig 4). d
e
d
123 f
r
o
m
124 Zhang and coworkers argued that N5-Pan did not inhibit CoA synthesis based on the

h
t
t
125 finding that the major effect of depletion of intracellular CoA, inhibition of protein p
:
/
/
a
126 synthesis (11, 13), was not observed (23). However, their studies were done in the a
c
.
a
s
127 presence of exogenous amino acids which relieve the amino acid starvation caused by m
.
o
128 CoA depletion (13). r
g
/

o
129 We attempted to determine if a specific step of CoA synthesis was inhibited by N5-Pan n

J
a
130 by separately overproducing each of the four CoA synthetic enzymes CoaA, CoaD, CoaE n
u
a
131 and Dfp in the expression vector pBAD24 (8). Overproduction of these enzymes failed ry

7
,
132 to impart N5-Pan resistance and instead caused increased sensitivity to N5-Pan (Fig 3, 2
0
1
133 Panel C), likely due to increased flux of the analog through the synthetic pathway. 9

b
y
134 Identification of the specific enzyme(s) inhibited will require further in vitro analysis. g
u
e
135 s
t
136 Inhibition by pantothenamides of the CoA synthetic enzyme pantothenate kinase, CoaA,
137 was reported previously (2, 10, 18, 21), but because neither overexpression of CoaA (this
138 work) nor supplementation of the medium with pantothenate (23) relieved growth
6
139 inhibition, it is uncertain whether inhibition of this enzyme is sufficient to account for the
140 toxicity of N5-Pan. Inhibition of the fatty acid synthetic enzymes by ethyldethia-ACP
141 may also be responsible for part of the toxic effects of the analog. However,
142 supplementation of Streptococcus pneumoniae with exogenous fatty acids was
143 insufficient to restore growth (23). Thus, the most likely scenario seems that the mode of D
o
w
144 action of the pantothenamide analogs is through combined inhibition of CoA and fatty n
lo
a
145 acid synthetic enzymes and perhaps of enzymes that utilize CoA or acetyl-CoA. d
e
d

f
r
o
m

h
t
t
p
:
/
/
a
a
c
.
a
s
m
.
o
r
g
/

o
n

J
a
n
u
a
r
y

7
,

2
0
1
9

b
y

g
u
e
s
t
7
146 ACKNOWLEDGMENTS
147 This work was supported by NIH research grant AI15650 from the National Institute of
148 Allergy and Infectious Diseases.
149
D
o
w
n
lo
a
d
e
d

f
r
o
m

h
t
t
p
:
/
/
a
a
c
.
a
s
m
.
o
r
g
/

o
n

J
a
n
u
a
r
y

7
,

2
0
1
9

b
y

g
u
e
s
t
8
150 References
151 1. Byers, D. M., and H. Gong. 2007. Acyl carrier protein: structure-function
152 relationships in a conserved multifunctional protein family. Biochem. Cell. Biol. 85:649-
153 662.
D
154 2. Clifton, G., S. R. Bryant, and C. G. Skinner. 1970. N'-(substituted) o
w
n
155 pantothenamides, antimetabolites of pantothenic acid. Arch. Biochem. Biophys. 137:523- lo
a
d
156 528. e
d

f
157 3. Cronan, J. E. 2006. A family of arabinose-inducible Escherichia coli expression ro
m

158 vectors having pBR322 copy control. Plasmid. 55:152-157. h
t
t
p
:
159 4. Cronan, J. E., Jr. 1980. β-alanine synthesis in Escherichia coli. J. Bacteriol. //
a
a
c
160 141:1291-1297. .
a
s
m
161 5. Cronan, J. E., and J. Thomas. 2009. Bacterial fatty acid synthesis and its
.
o
r
g
162 relationships with polyketide synthetic pathways. Methods Enzymol. 459:395-433. /

o
n
163 6. De Lay, N. R., and J. E. Cronan. 2006. Gene-specific random mutagenesis of J
a
n
164 Escherichia coli in vivo: isolation of temperature-sensitive mutations in the acyl carrier u
a
r
y
165 protein of fatty acid synthesis. J. Bacteriol. 188:287-296. 7
,

2
166 7. Flugel, R. S., Y. Hwangbo, R. H. Lambalot, J. E. Cronan, Jr., and C. T. Walsh. 0
1
9

167 2000. Holo-(acyl carrier protein) synthase and phosphopantetheinyl transfer in b
y

g
168 Escherichia coli. J. Biol. Chem. 275:959-968. u
e
s
t
169 8. Guzman, L. M., D. Belin, M. J. Carson, and J. Beckwith. 1995. Tight regulation,
170 modulation, and high-level expression by vectors containing the arabinose PBAD
171 promoter. J. Bacteriol. 177:4121-4130.
9
172 9. Iram, S. H., and J. E. Cronan. 2006. The β-oxidation systems of Escherichia coli
173 and Salmonella enterica are not functionally equivalent. J. Bacteriol. 188:599-608.
174 10. Ivey, R. A., Y. M. Zhang, K. G. Virga, K. Hevener, R. E. Lee, C. O. Rock, S.
175 Jackowski, and H. W. Park. 2004. The structure of the pantothenate kinase-ADP-
176 pantothenate ternary complex reveals the relationship between the binding sites for D
o
w
177 substrate, allosteric regulator, and antimetabolites. J. Biol. Chem. 279:35622-35629. n
lo
a
178 11. Jackowski, S., and C. O. Rock. 1986. Consequences of reduced intracellular d
e
d
179 coenzyme A content in Escherichia coli. J. Bacteriol. 166:866-871. f
r
o
m
180 12. Jackowski, S., and C. O. Rock. 1981. Regulation of coenzyme A biosynthesis. J.
h
t
t
p
181 Bacteriol. 148:926-932. :
/
/
a
a
182 13. Keating, D. H., Y. Zhang, and J. E. Cronan, Jr. 1996. The apparent coupling c
.
a
s
183 between synthesis and posttranslational modification of Escherichia coli acyl carrier m
.
o
184 protein is due to inhibition of amino acid biosynthesis. J. Bacteriol. 178:2662-2667. rg
/

o
185 14. Meier, J. L., A. C. Mercer, H. Rivera, Jr., and M. D. Burkart. 2006. Synthesis and n

J
a
186 evaluation of bioorthogonal pantetheine analogues for in vivo protein modification. J. n
u
a
r
187 Am. Chem. Soc. 128:12174-12184. y

7
,

188 15. Polacco, M. L., and J. E. Cronan, Jr. 1981. A mutant of Escherichia coli 2
0
1
9
189 conditionally defective in the synthesis of holo-[acyl carrier protein]. J. Biol. Chem.
b
y

190 256:5750-5754. g
u
e
s
191 16. Rock, C. O., and J. E. Cronan, Jr. 1981. Acyl carrier protein from Escherichia coli. t
192 Methods Enzymol. 71 Pt C:341-351.
193 17. Singer, M., T. A. Baker, G. Schnitzler, S. M. Deischel, M. Goel, W. Dove, K. J.
194 Jaacks, A. D. Grossman, J. W. Erickson, and C. A. Gross. 1989. A collection of strains
10