Role of smeU1VWU2X Operon in the Alleviation of Oxidative Stresses and the Occurrence of

42 Pages·2017·2.08 MB·English

AAC Accepted Manuscript Posted Online 4 December 2017
Antimicrob. Agents Chemother. doi:10.1128/AAC.02114-17
Copyright © 2017 American Society for Microbiology. All Rights Reserved.
1 Role of smeU1VWU2X Operon in the Alleviation of Oxidative Stresses and the
2 Occurrence of Sulfamethoxazole/trimethoprim-resistant Mutants in
3 Stenotrophomonas maltophilia
4
5 Chao-Jung Wu,a Tsu-Ting Chiu,a Yi-Tsung Lin,b,c Yi-Wei Huang,a Li-Hua Li,d,e and D
o
w
n
6 Tsuey-Ching Yanga* lo
a
d
e
d
7 Department of Biotechnology and Laboratory Science in Medicine, National

f
r
o
m
8 Yang-Ming University, Taipei, Taiwana; Division of Infectious Diseases, Department
h
t
t
p
:
9 of Medicine, Taipei Veterans General Hospital, Taipei, Taiwanb; School of Medicine, //a
a
c
.
a
10 National Yang-Ming University, Taipei, Taiwanc; Department of Pathology and s
m
.
o
11 Laboratory Medicine, Taipei Veterans General Hospital, Taipei, Taiwand; School of rg
/

o
n

12 Medical Laboratory Science and Biotechnology, College of Medical Science and A
p
r
il
13 Technology, Taipei Medical University, Taipei, Taiwane 2,

2
0
1
14 9

b
y

g
15 u
e
s
t
16 Running title: SmeU1VWU2X operon and oxidative stress
17
18 For correspondence
19 Tsuey-Ching Yang
1
20 E-mail: [email protected]
21 Mailing address: 155 Section 2, Lie-Nong Street, Taipei, 112, Taiwan, R.O.C.
22 Tel: [phone]
23 Fax: [phone]
24 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

A
p
r
il
2
,

2
0
1
9

b
y

g
u
e
s
t
2
25 Abstract
26 Overexpression of resistance-nodulation-division (RND)-type efflux pumps is an
27 important mechanism for bacteria to combat antimicrobials. RND efflux pumps are
28 also critical for bacterial physiology such as oxidative stress tolerance.
29 Stenotrophomonas maltophilia, a multi-drug resistant opportunistic pathogen, harbors D
o
w
n
30 eight RND-type efflux pump operons. Of them, smeU1VWU2X operon is unique for lo
a
d
e
d
31 its possession of two additional genes, smeU1 and smeU2, which encode proteins of

f
r
o
m
32 short-chain dehydrogenase/reductase (SDR) family. Overexpression of the SmeVWX
h
t
t
p
:
/
33 pump is known to contribute to the acquired resistance to chloramphenicol, quinolone, /a
a
c
.
a
34 and tetracycline; however, SmeU1 and SmeU2 are little involved in this phenotype. In s
m
.
o
r
35 this article, we further linked smeU1VWU2X operon to oxidative stress alleviation and g
/

o
n

36 sulfamethoxazole/trimethoprim (SXT)-resistant mutants occurrence. SmeU1VWU2X A
p
r
il
2
37 operon was inducibly expressed upon the challenge of menadione (MD), plumbagin ,

2
0
1
38 (PL), and hydrogen peroxide (H O ), verified by chromosomal smeU1VWU2X-xylE 9
2 2
b
y

g
39 transcriptional fusion construct and quantitative real-time PCR (qRT-PCR). The u
e
s
t
40 MD-mediated smeU1VWU2X upexpression was totally dependent on SoxR, partially
41 relied on SmeRv, but was less relevant to OxyR. SmeRv, but not SoxR and OxyR,
42 played a regulatory role in the H O -mediated smeU1VWU2X upexpression. The
2 2
43 significance of smeU1VWU2X upexpression was investigated in respect of oxidative
3
44 stress alleviation and SXT-resistant mutants occurrence. Overexpression of
45 smeU1VWU2X operon contributed to the alleviation of MD-mediated oxidative stress.
46 Of the encoded proteins, SmeVWX pump and SmeU2, rather than SmeU1,
47 participated in the MD tolerance. Furthermore, we also demonstrated that
48 MD-mediated expression of smeU1VWU2X operon decreased SXT resistance D
o
w
n
49 frequency when S. maltophilia was grown in the reactive oxygen species (ROS)-rich lo
a
d
e
d
50 environment.

f
r
o
m
51
h
t
t
p
:
/
52 /a
a
c
.
a
53 s
m
.
o
r
54 g
/

o
n

55 A
p
r
il
2
56 ,

2
0
1
57 9

b
y

g
58 u
e
s
t
59
60
61
62
4
63 Introduction
64 Aerobic metabolism is the main mechanism for energy acquisition for normal growth
65 of aerobic bacteria. However, reactive oxygen species (ROSs), the toxic by-products
66 of aerobic metabolism, can cause lethal damage to cellular proteins, membrane, and
67 nucleic acids (1). Bacteria also encounter extreme oxidative stress from the D
o
w
n
68 environment, such as ionizing radiation, near-UV radiation, or numerous superoxide lo
a
d
e
69 (O -)-generating compounds such as menadione (MD) and plumbagin (PL) (2). In d
2 f
r
o
m
70 addition, some immune cells exploit oxidative stress as a weapon to kill invading
h
t
t
p
:
/
71 pathogenic bacteria (3). For their survival, bacteria have developed an array of /a
a
c
.
a
72 defence systems that can efficiently destroy or extrude ROSs to alleviate ROS stress, s
m
.
o
r
73 including non-enzymatic antioxidants (such as -carotene, ascorbic acid, α-tocopherol, g
/

o
n

74 and glutathione), specific enzymatic systems (such as superoxide dismutases, A
p
r
il
2
75 catalases, hydroperoxidase, and glutathione peroxidase), and efflux pump systems (4). ,

2
0
1
76 Efflux pumps are integral membrane proteins capable of extruding a broad range 9

b
y

g
77 of structurally different compounds from the bacterial cells in an energy-dependent u
e
s
t
78 manner. Bacterial efflux pumps are categorized into five families: ABC superfamily,
79 the major facilitator superfamily (MFS), the small multidrug resistance (SMR) family,
80 the multidrug and toxic compound extrusion (MATE) family, and the
81 resistance-nodulation division (RND) superfamily (5). Among these, the tripartite
5
82 RND-type efflux pump, consisting of cytoplasmic, periplasmic, and outer-membrane
83 proteins, is found uniquely in gram-negative bacteria. Most of the genes coding for
84 RND-type efflux pumps are located in one operon. RND-type efflux pump was first
85 highlighted for its contribution to multidrug resistance. It has later attracted attention
86 for its physiological functions. There is accumulated evidence supporting the roles D
o
w
n
87 played by efflux pumps in detoxification of intracellular metabolites, intercellular lo
a
d
e
d
88 signal trafficking, bacterial virulence, biofilm formation, motility, envelope stress

f
r
o
m
89 response, and oxidative stress alleviation (6-9). RND-type efflux pumps are often
h
t
t
p
:
/
90 expressed under precise and elaborate control at the level of transcription. It is /a
a
c
.
a
91 believed that transcription of RND-type pumps can be activated by certain kinds of s
m
.
o
r
92 stress and (or) their natural substrates, and their overexpression should be relevant to g
/

o
n

93 the alleviation of stress. Therefore, efflux pumps should provide a benefit to bacterial A
p
r
il
2
94 survival in different environments by responding to assorted signals. ,

2
0
1
95 Stenotrophomonas maltophilia, an aerobic, non-fermentative, gram-negative 9

b
y

g
96 bacterium, is ubiquitous in aqueous environments, soil, and plants. It is naturally u
e
s
t
97 resistant to many broad-spectrum antibiotics, which makes treatment extremely
98 difficult (10). S. maltophilia encodes eight RND-type efflux pumps, SmeABC,
99 SmeDEF, SmeGH, SmeIJK, SmeMN, SmeOP, SmeVWX, and SmeYZ (11). We were
100 interested in the SmeVWX pump because the components of this pump are encoded
6
101 by a five-gene operon, smeU1-V-W-U2-X, in which smeU1 and smeU2 encode
102 proteins of short-chain dehydrogenase/reductase (SDR) family (12-13). Our earlier
103 work had led to the observation that the expression of smeU1-V-W-U2-X operon was
104 positively regulated by the divergently transcribed LysR-type transcriptional regulator
105 gene smeRv. Overexpression of SmeVWX pump contributes to acquisition of D
o
w
n
106 resistance to chloramphenicol, quinolone, and tetracyclines, but enhances the lo
a
d
e
d
107 susceptibility to aminoglycosides (12). In our on-going study concerning the hydrogen

f
r
o
m
108 peroxide-mediated oxidative stress response, we performed genome-wide analysis of
h
t
t
p
:
/
109 the transcriptome profile of logarithmic-phase S. maltophilia KJ cells upon the /a
a
c
.
a
110 challenge with hydrogen peroxide. We noticed that the transcripts of s
m
.
o
r
111 smeU1-V-W-U2-X operon were noticeably increased upon treatment with hydrogen g
/

o
n

112 peroxide (Table S1). A recent report also indicated that Vitamin K3 (MD) induces the A
p
r
il
113 expression of SmeVWX pump (14). These evidences link smeU1VWU2X operon to 2,

2
0
1
114 oxidative stress response and alleviation. In this report, we have documented that the 9

b
y

g
115 smeU1VWU2X operon is expressed in response to oxidative stress and this expression u
e
s
t
116 contributes to alleviation of oxidative stress. We also elucidated the participation of
117 SoxR, OxyR, and SmeRv in MD-mediated smeU1VWU2X operon up-expression.
118 Finally, we also investigated the impact of smeU1VWU2X operon and oxidative stress
119 on the occurrence of sulfamethoxazole/trimethoprim (SXT)-resistant mutants.
7
120 Materials and Methods
121 Bacterial strains, primers, and growth condition
122 The strains, plasmids, and primers used in this study were listed in Table S2. All
123 primers used in this study were designed based on the genome sequence of S.
124 maltophilia K279a. Bacteria were cultured aerobically at 37C with agitation in the D
o
w
n
125 Lauria-Bertani (LB) broth unless special description. The bacterial cell density was lo
a
d
e
d
126 determined by measuring the optical density at 450 nm, since the bacterial suspension

f
r
o
m
127 of S. maltophilia displays a more sensitive absorbance against 450nm than against 600
h
t
t
p
:
/
128 nm (15). Two different oxidative stresses, H2O2 and MD, were used in this study. The /a
a
c
.
a
129 H O challenge was 0.01% for 10 min against 6-h grown logarithmic-phase cells, the s
2 2 m
.
o
r
130 same treatment as our previous transcriptome analysis (Table S1). The MD stress was g
/

o
n

131 designed as 16 μg/ml for 6 h, since MD of 16 g/ml made little impact on bacterial A
p
r
il
2
132 growth and 6 h is an adequate time for the obtainment of logarithmic-phase cells. ,

2
0
1
133 9

b
y

g
134 Construction of the chromosomal smeU1VWU2X-xylE single-copy u
e
s
t
135 transcriptional fusion strain, KJVWX23 and KJ09CVWX23
136 In order to assess the expression of smeU1VWU2X operon, we constructed a
137 smeU1VWU2X-xylE transcriptional fusion in the chromosome. Three notes were
138 consideration for the xylE trasnscriptional fusion made with the last gene of the
8
139 operon: (i) avoidance of the xylE insertion-mediated polar effect, which may cause
140 unexpected effect, (ii) assurance of the presence of stable smeU1VWU2X transcript,
141 which is importance for the following functional evaluation of smeU1VWU2X operon,
142 and (iii) no individual promoter for the smeX gene, which has been verified in our
143 previous study (12). The KJVWX23 and KJ09CVWX23 were constructed by double D
o
w
n
144 crossover homologous recombination between the bacterial chromosome and the lo
a
d
e
d
145 constructed recombinant plasmid pVWX23. Plasmid pVWX23 was constructed as

f
r
o
m
146 follows: an 840-bp DNA fragment containing the partial C-terminus of smeX gene
h
t
t
p
:
/
147 and downstream of the smeX gene was obtained by PCR using the primer sets of /a
a
c
.
a
148 X1-F/X1-R (Table S2) and cloned into pEX18Tc, generating plasmid pX1. A xylE s
m
.
o
149 cassette retrieved from pTxylE (15) was inserted into the SphI site of pX1, yielding rg
/

o
n

150 pVWX23. The orientation of xylE, confirmed by sequencing, was the same as that of A
p
r
il
2
151 the smeX gene. The plasmid mobilization and transcription fusion construct selection ,

2
0
1
152 were performed as descried previously (16). The xylE gene in pVWX23 was inserted 9

b
y

g
153 onto the intergenic regions (IG) downstream the smeX gene of strains KJ and KJ09C, u
e
s
t
154 without disrupting any gene, yielding KJVWX23 and KJ09CVWX23 respectively.
155
156 Construction of deletion mutants KJOxyR and KJSoxR
157 Plasmids pOxyR, and pSoxR were prepared for the construction of KJOxyR and
9
158 KJSoxR respectively. Two PCR amplicons, corresponding to upstream and
159 downstream of oxyR gene, were amplified using primer sets of OxyR5-F/OxyR5-R
160 and OxyR3-F/OxyR3-R (Table S2), and subsequently cloned into pEX18Tc to yield
161 the recombinant plasmid pOxyR, in which the cloned oxyR gene was partially
162 deleted. Similar strategy for pSoxR construction was done. Primer sets D
o
w
n
163 SoxR5-F/SoxR5-R and SoxR3-F/SoxR3-R (Table S2) were used for the construction lo
a
d
e
d
164 of pSoxR. The plasmid mobilization and transconjugants selection were performed

f
r
o
m
165 as descried previously (16). The correctness of mutants was confirmed by PCR and
h
t
t
p
:
/
166 DNA sequencing. The growths of oxyR and soxR mutants were assessed and showed /a
a
c
.
a
167 no difference compared to the parental strain KJ (Data not shown). s
m
.
o
r
168 g
/

o
n

169 Construction of complementation plasmids, pSoxR, and pSmeRv A
p
r
il
2
170 The intact soxR and smeRv genes were amplified by PCR using the primer sets of ,

2
0
1
171 SoxR-F/SoxR-R, and SmeRv-F/SmeRv-R, and then cloned into pRK415, yielding 9

b
y

g
172 plasmids pSoxR and pSmeRv respectively. u
e
s
t
173
174 C23O activity determination
175 The product of xylE gene, catechol 2, 3-dioxygenase (C23O), catalyzes the hydrolysis
176 of catechol into the yellow 2-hydroxymuconate semialdehyde. The C23O activity was
10