Low-Level Kanamycin Resistance in Mycobacterium tuberculosis: Molecular Mechanisms and Clinical Implications Pubblico
Zaunbrecher, Mary Analise (2010)
Abstract
Low-Level Kanamycin Resistance in Mycobacterium
tuberculosis:
Molecular Mechanisms and Clinical Implications
Mary Analise Zaunbrecher
The aminoglycosides kanamycin (KAN) and amikacin (AMK) are required
for the
treatment of multidrug-resistant tuberculosis (MDR-TB) and
resistance to these drugs is a
growing concern. Knowledge of the mechanism(s) responsible for KAN
resistance is
limited. High-level resistance has been attributed to mutations in
the 16S rRNA gene,
rrs. However, in 30-80% of KAN resistant clinical isolates,
low-level resistance is
observed which cannot be ascribed to any known mechanism. This work
investigates the
molecular basis of low-level KAN resistance through either the
utilization of a cosmid
library or whole genome sequencing analysis of spontaneous
low-level KAN resistant
mutants. Mutations in the -10 and -35 regions promoter region of
eis (Rv2416c), a gene
encoding a previously uncharacterized aminoglycoside
acetyltransferase, were found to
confer low-level KAN resistance. These mutations led to a 20- to
180-fold increase in
the amount of eis leaderless mRNA transcript with a
corresponding increase in protein
expression. In vitro acetyltransferase assays confirmed that
the Eis protein acetylates and
inactivates KAN and AMK. 80% of clinical isolates that exhibit
low-level mono-KAN
resistance harbor eis promoter mutations. Experiments
presented here demonstrate that
low-level KAN resistance is also conferred by mutations in the
promoter region of the
transcriptional activator, whiB7 that cause a 23- to
145-fold increase in whiB7 transcripts.
qRT-PCR assays demonstrate that increased whiB7 expression
enhances expression of
genes in the WhiB7 regulon including eis and the tap
(Rv1258c) efflux pump. The
increased expression of eis confers KAN resistance, and
increased expression of tap
confers cross-resistance
to streptomycin (STR), presumably by enhanced efflux of STR.
Overall, this study identifies the molecular basis for the
involvement of eis in KAN
resistance, tap in STR resistance, and whiB7 in KAN
and STR cross-resistance.
Together, these data provide a means to develop rapid diagnostics
of drug resistant strains
and ultimately may affect how treatment is designed for MDR- and
XDR-TB cases.
Table of Contents
Table of Contents
Chapter 1.
Background……………………………………………………….....
1
Background
References………………………………………….......
38
Background
Figures…………………………………………………
54
Chapter 2.
Overexpression of the chromosomally encoded aminoglycoside
acetyltransferase eis confers kanamycin resistance in
Mycobacterium
tuberculosis……………..………………………….
57
Chapter 2
References………………………………………………..
79
Chapter 2
Tables/Figures……………………………………………
83
Chapter 2 Supplemental
Tables/Figures…………………………….
90
Chapter 3.
Mutations in the whiB7 promoter lead to cross resistance
to
kanamycin and streptomycin in Mycobacterium
tuberculosis……… 101
Chapter 3
References………………………………………………..
122
Chapter 3
Tables/Figures……………………………………………
127
Chapter 3 Supplemental
Tables/Figures…………………………….
135
Chapter 4.
Kanamycin resistant Mycobacterium tuberculosis
harboring
mutations in the eis promoter display enhanced growth
in
macrophages…………………………………………………………
144
Chapter 4
References………………………………………………..
158
Chapter 4
Tables/Figures……………………………………………
161
Chapter 5.
Conclusion…………………………………………………………...
167
Conclusion
References………………………………………………
173
Chapter 2.
Table 1
eis mutations and aminoglycoside resistance
levels………..……….
85
Table 2
Distribution of eis mutations in clinical
isolates…………….…....…
86
Table S1
Strains used in this
study…………………………………………….
94
Table S2
Plasmids and phage used in this
study………………………………
95
Table S3
Primer
sequences……………………………………………………
96
Chapter 3
Table 1
Spontaneous mutant survey and
derivatives………………………
129
Table 2
Effect of the whiB7 ∆C-125 mutation on the emergence
of high
level streptomycin resistant
mutants…………………………….......
130
Table S1
Strains used in this
study…………………………………………….
139
Table S2
Plasmids and phage used in this
study………………………………
140
Table S3
Primer
sequences……………………………………………………
141
Chapter 4
Table 1
Strains and phage used in this
study…………………………………
162
Chapter 1.
Figure 1
Structure of kanamycin and
amikacin……………………………….
55
Figure 2
Structure of
streptomcyin……………………………………………
56
Chapter 2
Figure 1
Characterization of eis promoter and
expression……………………
87
Figure 2
Analysis of eis expression and acetyltransferase
activity…………...
88
Figure 3
Eis acetylates kanamycin and amikacin, kinetic
analysis…………...
89
Figure S1
sigA transcript levels in H37Rv and
K204…………………………..
97
Figure S2
Immunoblot analysis of cell filtrate proteins from wild type
strains
and eis
mutants………………………………………………………
98
Figure S3
Promoter truncation
constructs………………………………………
99
Figure S4
Disk diffusion assays for kanamycin, amikacin, and
streptomycin… 100
Chapter 3.
Figure 1
whiB7 promoter and
mutations……………………………………...
131
Figure 2
whiB7 mutations confer increased expression of whiB7,
eis,
and
tap……………………………………………………………….
132
Figure 3
K301 derivatives on kanamycin and
streptomycin………………….
133
Figure 4
whiB7 is necessary for basal level eis and tap
expression…………..
134
Figure S1
qRT-PCR analysis of eis in K204 and K204∆
whiB7……………….
142
Figure S2
Acetyltransferase activity of clinical isolates with
unexplained
KAN
resistance……………………………………………………..
143
Chapter 4
Figure 1
Analysis of eis
expression…………………………………………..
163
Figure 2
Comparison of intracellular
growth…………………………………
164
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