from patients with bronchiectasis, urinary tract, and middle-ear infections as well as from the equine guttural pouch of a mare having chronic mucopurulent nasal ...
Outer Membrane Protein LptD/OstA (PA0595) has a Novel Role in the Regulation of Alginate Synthesis in Pseudomonas aeruginosa 2, 3 Delgado ,
, Camila
Laura
2, Florez ,
Hansi
2 Kumari ,
and Kalai
2 Mathee
1Department
of Biological Sciences, College of Arts & Sciences, 2Department of Human & Molecular Genetics, Herbert Wertheim College of Medicine, 3Langone Medical Center, New York University School of Medicine, New York.
Abstract
Experiments and Results
Pseudomonas aeruginosa, a Gram-negative bacterium, is the leading cause of morbidity and mortality in cystic fibrosis (CF) patients. In the lungs of these patients, the presence of alginate-overproducing (Alg+) strains of P. aeruginosa is indicative of chronic infection and poor prognosis. Alginate-overproduction is generally due to a mutation in the gene mucA, encoding an anti-sigma factor, located in the algT/UmucABCD operon. MucA, an inner membrane protein, negatively regulates AlgT/U, a sigma factor and the master regulator of the alginate biosynthesis. To identify other key proteins in the alginate regulatory pathway, the constitutively alginate-overproducing strain PDO300 was used. This strain contains mucA22, a common allele identified in mucoid P. aeruginosa CF isolates. Our lab previously identified 34 nonmucoid variants of PDO300 called suppressors of alginate production (sap). Utilizing the P. aeruginosa cosmid library, cosmid pMO012217, harboring 18 open reading frames (ORFs), was found to restore the mucoid phenotype in sap strain sap27. Transposon mutagenesis of the cosmid revealed that an ORF in the lptD-operon was disrupted. The operon contains three genes: lptD-surA-pdxA. The lptD gene encodes an outer membrane protein (OMP) involved in lipopolysaccharide transport. SurA is the major chaperone for all OMPs while PdxA is responsible for vitamin B6 synthesis. We showed that expression of lptD alone restores the mucoid phenotype and overcomes algO (periplasmic protease protein similar to prc protein in Escherichia coli) mutation in sap27. Thus, we propose the novel role of LptD in alginate regulation in P. aeruginosa and it is the first report of an OMP component involved in alginate gene regulation.
Map of the cosmid pMO012217 Genomic Loca9on Cosmid pMO012217
655000
645000
635000
0
0
lptD operon
1
2
4
3
lptD
Transposon inser9on site
surA
Kb
pdxA
#
Image ID
Acquire Time
1
0000518_01
Aug 31, 2017 2:07:06 PM
Verification of lptD - operon
Image Display Values Channel
Lanes 8-13 900 bp
Chemi
surA
Sensitivity
Image Name
High
0000518_01
Comment
Localization of LptD
Color
Minimum
Maximum
K
Gray Scale (Black on White)
1.19
47.2
0
Frac3on W Induc3on - Lane 1
3
W + 2
S + 3
Lane 1, 1 Kb Ladder (M) Lanes 2-7 and Lanes 8-13 are the PCR products of the junction between lptD-surA and surA-pdxA, respectively. Lanes 2 and 8, products from the genomic DNA (G) used as positive controls; Lanes 3 and 9, products from the RNA before DNAse treatment and cDNA synthesis (bT); Lanes 5 and 11,DNA (cD) s: Lanes 4 and 10, RNA after the DNase treatment (aT); Lanes 6 and 12, without the reverse transcriptase (RT); and Lanes 7 and 13 absence of DNA polymerase (-P).
W V F
PAOΔalgO pLptD-SurA-LptE
Image Modifications
pAlgO
PDOΔalgO
IS + 4
MucB
C
2
PD
Z
Z
N
C ?
AlgO
Vector
PAOΔalgW pLptD-SurA-LptE PDOΔalgW pLptD-SurA-LptE
LptD
Detection of LptD protein by Western blot. His-tagged LptD was introduced into sap27. Whole cell fractions were extracted from cells that were uninduced (-) or induced (+) with 2 % arabinose for 3 hours at 25˚C. The
The presence of algO or lptD can restore algiante production in sap27.The presence of optP encoding another outermembrane protein failed to restore alginate production in sap27. Restoration of alginate production by lptD in PDOΔalgO (with muc22) requires algO and not in PA01 (with the wild-type mucA). Restoration of alginate production by lptD in PD0ΔalgO does not require algW. However, in PA01 (with the wild-type mucA), algW is required for LptD-dependent alginate production.
induced whole cell extract was further
Summary
separated into soluble (S) and insoluble (IS) fractions. The presence of LptD was shown using anti-His6 antibody.
sap27 (Vector)
PA01 (Vector)
sap27 (pLptD)
PDO300 (Vector)
sap27 (pLptD-Operon)
• • • • • • •
lptD-surA-pdxA genes form an operon sap27 harbors a mutation in algO Alginate production can be restored by algO AlgO is required for alginate production in PDO300 lptD can bypass algO mutation in sap27 lptD does not restore the mucoid phenotype when algO is deleted AlgW is not required for LptD-dependent alginate production in mucA22 background
sap27 (pVector)
sap27 (pMO012217)
Mechanism of MucA proteolysis
SspA
?
ClpP
ClpX ClpP2
RNAP α2ββʹ ω
AlgT/U
7
AlgT/U
RIP cascade: C-terminal WVF motif of MucE primes the AlgW protease (1). AlgW cleaves MucA (2), followed by MucP (3). SspA binds to the N-terminus portion of MucA (4) bringing the fragment close enough to the ClpXP complex for degradation (6). The free AlgT/U binds to RNA polymerase to initiate transcription of genes in the alg regulon (7) leading to alginate production. The roles of MucD and AlgO have not been established. LptD may be involved in initiating MucA proteolysis via AlgO pathway and bypass AlgW or by displacing MucB depending on strains used.
PA01 (mucA)
sap27 has algO mutation
AlgT/U
Strain 106 PA01
sap27
107
318
319
320
GCC
GAG
. . CTG
GAT
GAA
Ala
Glu
. . Leu
ASP
Glu
GCC
GAT
. . CCT
GGA
TGA
Gla
Asp
. . Pro
Gly
*
There was no mutation in lptD operon in sap27. However, sequencing of algO revealed an insertion of a single nucleotide, thymine (T), at the position 3,643,250 on the genome. This corresponds to the codon 107 that results in a frame-shift mutation leading to premature termination of algO at codon 320, which is converted from glutamic acid to UGA, an opal stop codon.
Z
PDZ
AlgW
PD
The cosmid pMO012217 introduced into sap27 restoring the mucoid phenotype. PAO1 and PDO300 were used as negative and positive controls, respectively. The plasmid pLAFR3 was used as a control vector. (b) The plasmids pLptD and pLptD-operon which harbor lptD and lptD-surA-pdxA were separately introduced into sap27 restoring the mucoid phenotype The strains were plated on LB containing 2% arabinose (Ara) where indicated and incubated at 37 C for 36 hours.
6
(b) PD
Z
PDO300 (mucA22)
AlgW
MucB
MucB
MucA22
3
AlgT/U
4
5
* *
Vector
PDO300
MucA
MucP
MucA
AlgW
N
N
AlgT/U
*
pLptD-SurA-LptE
(a)
Inner Membrane
*
Project
Vector
?
PDZ
Vector
Vector
(b) + Ara PA01 (pMO012217)
*
Z
? PD
PD Z
C
PDZ
1
PDZ Z PD
pLptD Page 1
PD
Periplasm
(a) + Ara
*
Vector
sap22
1 1.5 0.517
1 0.5
*
pOptP
pdxA
Ladder G bT aT cD -RT -P G bT aT cD -RT -P Ladder 1 2 3 4 5 6 7 8 9 10 11 12 13 14 kb
*
pAlgO
653,902
Acquisition Information
kb
120
*
pLptD-SurA-LptE
Image ID: 0000518_01 Acquire Time: Aug 31, 2017 2:07:06 PM
Lanes 2-7 1000 bp
100
*
pLptD-operon
sap27
GTGGACGATGACGCCTTCCTCG
PDO300 (pMO012217)
?
80
pLptD-SurA
5
The cosmid was identified in the en masse complementation with the MTP library with sap27 that restored the mucoid phenotype. The cosmid contains 18 open reading frames (ORF). Transposon mutagenesis of this cosmid resulted in the loss of phenotype. The transposon was localized to the 3’end of lptD/ostA encoding an outer membrane protein involved in LPS biosynthesis.
LptD
MucD
60
Vector
lptD restores alginate production
MucE
40
GTGAAGATCAAGCTATGTAA CAGTGAGCC
Introduction
Outer Membrane
20
Alginate (μg/ml)
pLptD
lptD/ostA
Pseudomonas aeruginosa is a Gram-negative aerobic bacterium with polar monotrichous flagella and surface pili that facilitate adherence to respiratory epithelium. Alginate-producing P. aeruginosa is primarily associated with CF individuals but occasionally has been isolated from patients with bronchiectasis, urinary tract, and middle-ear infections as well as from the equine guttural pouch of a mare having chronic mucopurulent nasal discharge. The master regulator of alginate production in P. aeruginosa is the extracytoplasmic sigma factor AlgT/U (σ22) that has high homology to stress response sigma factor SigE (σE) found in E. coli, Streptomyces coelicolor, Bacillus subtilis and Salmonella typhimurium. The AlgT/U is encoded in an algT/U-mucA-mucB-mucC-mucD operon that plays an essential role in turning a nonmucoid P. aerguinosa into a mucoid phenotype. MucA is an anti sigma factor that sequesters AlgT/U and prevents its activity. Release of AlgT/U requires a sequential degradation of MucA through a process known as regulated inner-membrane proteolysis (RIP) cascade as shown in fig , the process that is conserved from bacteria to humans. All the proteins involved in the process are shown in table below.
665000
Alginate quantification
LptD
Mucoid
AlgT/U
AlgO
Mucoid
We propose that (a) in PAO1, increased expression of lptD displaces MucB and induces AlgW to cleave MucA resulting in proteolysis of MucA as described previously in Figure. 1. There might not be any role for AlgO in PAO1 under the tested conditions; (b) in the mucA22 background, MucB cannot bind to MucA, thus the cleavage site of MucA22 is exposed. The overexpression of lptD might activate the truncated AlgO leading to MucA22 proteolysis via an unknown mechanism
Acknowledgments This research was supported by Florida International University Presidential Fellowship and Teaching Assistantship (SP), NIH/NIGMS R25 GM061347 (CD, LF). We thank all Mathee lab members for their support.
