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Journal of Bacteriology, December 1998, p. 6764-6768, Vol. 180, No. 24
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
A Novel Serine/Threonine Protein Kinase Homologue
of Pseudomonas aeruginosa Is Specifically Inducible
within the Host Infection Site and Is Required for Full
Virulence in Neutropenic Mice
Jingyi
Wang,1
Caihe
Li,1
Hongjiang
Yang,1
Arcady
Mushegian,2 and
Shouguang
Jin1,*
Department of Microbiology and Immunology,
University of Arkansas for Medical Sciences, Little Rock, Arkansas
72205,1 and
Axys Pharmaceuticals,
Inc., La Jolla, California 920372
Received 21 July 1998/Accepted 8 October 1998
 |
ABSTRACT |
A genetic locus of Pseudomonas aeruginosa was
identified that is highly and specifically inducible during infection
of neutropenic mice. This locus, ppkA, encodes a protein
that is highly homologous to eukaryote-type serine/threonine protein
kinases. A ppkA null mutant strain shows reduced virulence
in neutropenic mice compared to the wild type. Overexpression of the
PpkA protein greatly inhibited the growth of Escherichia
coli or P. aeruginosa. However, a single amino acid
change at the catalytic site of the kinase domain eliminated the toxic
effect of PpkA on bacterial cells, suggesting that the kinase domain of
PpkA is functional within bacterial cells.
| |
TEXT |
We have previously reported a method
for the isolation of genes induced upon infection of neutropenic mice,
using Pseudomonas aeruginosa PAK. After five rounds of
selections, 22 different genetic loci were identified through
characterization of 45 randomly picked isolates (10). To
identify a locus that is the most highly inducible in vivo, two
additional rounds of selection were conducted with mice as described
earlier (10). A total of 48 colonies were picked and
analyzed by Southern hybridization followed by DNA sequencing, as
described previously (10). Fusion sites in 29 of them were
identical to the np6 locus and the remaining 19 were
identical to the np1 locus from our initial selection study (10). We focused our attention on the np1 locus
since it appears to encode a previously uncharacterized gene product.
The strains and plasmids used in this study are listed in Table
1.
The np1 locus is highly and specifically inducible in
host tissue.
To confirm the in vivo inducibility of the
np1 locus, we compared the in vivo (neutropenic mice) and in
vitro (minimal medium A [MinA]) (1a) replication rates between the
original isolate, NP1, and the parent purEK deletion strain,
PAK-AR2. Since purines are limited under either the in vivo or in vitro
assay conditions, the growth rate of the NP1 strain should be
proportional to the strength of the np1 promoter, which
controls purEK gene expression. Equal numbers of the two
bacterial strains were mixed and injected intraperitoneally into six
neutropenic mice (2 × 105 cells per mouse). Bacterial
cells were recovered from livers of the mice 24 and 48 h after
inoculation. The numbers of each bacterium were determined under
conditions that allowed the growth of both strains (L agar) or NP1 only
(L agar containing 150 µl of carbenicillin per ml). Assuming that the
two bacterial strains were cleared equivalently by the host defense
system, the ratios of the two bacterial strains in the animal tissue
reflect their relative replication rates in vivo, which is indicative
of the np1 promoter strength. As an in vitro control,
the same bacterial mixture was inoculated into 100 ml of MinA medium,
with a final concentration of 105/ml, and incubated at
37°C. As shown in Fig. 1, by 24 and
48 h of infection (in vivo), the NP1 strain had overgrown PAK-AR2
by 84- and >6 × 105-fold, respectively,
whereas in vitro the approximate 1:1 ratios were maintained at all
times. These data clearly indicate that the np1 locus is
specifically and highly expressed in the in vivo environment.
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FIG. 1.
Comparison of replication rates between NP1 and PAK-AR2
under in vivo and in vitro growth conditions. Numbers of each bacterial
strain recovered from livers of neutropenic mice (A) or from MinA
medium (B) after 24 and 48 h are shown. The in vivo data represent
an average from three mice at each time point. The in vitro data
represent an average of three independent mixed-culture tests.
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The np1 locus encodes a putative serine/threonine
protein kinase.
A cosmid clone containing the np1 locus
was identified from a cosmid clone bank of the PAK chromosomal DNA
(5) by colony hybridization, using a partial np1
gene fragment in pNP1 as a probe. DNA fragments surrounding the
original purEK fusion site were subcloned and sequenced. An
open reading frame (ORF) was identified with the predicted direction of
transcription of the np1 gene, encoding a 1,032-amino-acid
protein, which bears no obvious signal sequence in its N terminus.
The N-terminal third of the protein is similar to serine/threonine
protein kinases found in bacteria and eukaryotes. The greatest
similarity observed was to the putative kinases from
Myxococcus xanthus (
7),
Mycobacterium leprae
(
2),
Mycobacterium tuberculosis (
1),
and
Streptomyces coelicolor (
9). Multiple
sequence
alignment of the putative bacterial kinases with their
better-studied
eukaryotic counterparts revealed pronounced conservation
of at
least 10 of the known 12 motifs that define the Ser/Thr protein
kinase superfamily in eukaryotes (
3,
4), including an
ATP-binding
glycine loop in subdomain I, an invariant lysine residue
involved
in interaction with
- and
- phosphates in subdomain II,
a "kinase
loop" motif with an invariant catalytic aspartate in
subdomain
Vib (amino acid 129), and a threonine residue in motif VIII
that
is frequently autophosphorylated (Fig.
2). The C-terminal two-thirds
of the
np1 ORF, rich in proline, shares no similarity to any known
sequences. Prediction of globular and nonglobular regions, using
local
sequence complexity measures (
11), detects a nonglobular,
elongated protein segment in the area spanning amino acids 280
to 660 and a globular structure in the remaining C-terminal portion
of the
protein.
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FIG. 2.
Alignment of PpkA with related bacterial and eukaryotic
Ser/Thr-like protein kinases. Unique identifiers in SWISSPROT or PDB
databases are shown. Distances between the ungapped blocks of the
highest similarity and the protein termini are indicated by numbers.
Invariant residues are shown in boldface. Highly conserved bulky
hydrophobic residues (I, L, M, V, F, Y, and W) and small-side-chain
residues (A, G, and S) are also highlighted. Functionally important
residues in motifs I, II, VIb, and VIII (see the text) are underlined.
In the motif line, the conserved motifs in Ser/Thr kinases, as defined
in Hanks and Hunter (3, 4), are indicated. In the secondary
structure line, -helices and -strands in the known
three-dimensional structure of twitchin (1KOA) are indicated.
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The
purEK gene fusion in the original isolate, NP1, occurred
at amino acid 561. Further analysis of the other isolate, NP6,
indicated that it had a
purEK fusion to the same gene, but
the
fusion site resided in the N-terminal end, at amino acid position
198. These results indicate that we had actually isolated a single
locus that is the most highly inducible in vivo, having the
purEK gene fused at two different sites. This locus is
designated
ppkA (
Pseudomonas protein
kinase).
The ppkA locus is required for full bacterial virulence
in neutropenic mice.
To investigate the role of the
ppkA gene in bacterial virulence, a ppkA
insertional null mutant was generated. An 
fragment (8),
coding for resistance to spectinomycin and streptomycin, was inserted
into the 5' structural ppkA gene on pPKN-S, resulting in
pSJ9711. The mutant ppkA gene was then introduced into the chromosome of the wild-type PAK strain by electroporation
(6). The ppkA null mutant strain, designated
PKN-A, was confirmed by Southern hybridization of the chromosomal DNA
(Fig. 3).
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FIG. 3.
Southern hybridization of chromosomal DNA from strains
PAK, PKN-A, and PKN-AC. (Top) Restriction map of the region containing
the ppkA gene in PKN-A and PKN-AC. B, BamHI; P,
PstI; R, EcoRI. (Bottom) Chromosomal DNA from
strains PAK (lanes 1, 3, and 5), PKN-A (lanes 2, 4, and 6), and PKN-AC
(lane 7) were digested with EcoRI (lanes 1 and 2),
BamHI (lanes 3 and 4), or PstI (lanes 5, 6 and
7). A 4-kb SalI fragment containing the 5' ppkA
gene was used as a probe.
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The PKN-A strain did not show any traits distinguishable from those of
wild-type PAK when grown on either rich or minimal
medium; however, a
clear difference in virulence in neutropenic
mice was observed. Tests
of virulence in neutropenic mice were
conducted as described earlier
(
10), and the number of animal
deaths was observed at 6-h
intervals for a total of 72 h. As shown
in Fig.
4, about 10-fold-more PKN-A cells were
needed to cause
a similar lethal effect in neutropenic mice compared to
the wild-type
PAK strain. Furthermore, the
ppkA mutant
caused on average 8 to
12 h of delay in the times of the animal
deaths compared to the
wild type.
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FIG. 4.
Survival rates of neutropenic mice infected with strain
PAK, PKN-A, or PKN-AC. Bacteria were injected intraperitoneally at
doses of 103, 104, and 105 cells,
and animal death was observed at 6-h intervals for a total of 72 h. The numbers of dead animals by 72 h over the total numbers of
animals tested are shown next to the strains used.
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To see whether the delay in the times of animal deaths caused by PKN-A
is a direct result of the
ppkA mutation or of a polar
effect
on downstream genes, the pPKN-S
plasmid, containing a
3'-end-truncated version of the
ppkA gene, was
electroporated
into PKN-A cells and a single crossover through the left
arm (5'
to the "
" insertion site of the
ppkA gene in
PKN-A) was selected
for, as depicted in Fig.
3. The resulting strain,
PKN-AC, has
a single-copy, stably maintained
ppkA gene (Fig.
3 and data not
shown). As shown by the virulence test results in Fig.
4, PKN-AC
caused an animal death rate similar to that caused by the
wild-type
PAK strain. These results indicated that the N-terminal
two-thirds
of the PpkA protein is sufficient to complement the PKN-A
mutant
and that the
ppkA gene is solely responsible for the
reduced bacterial
virulence of the mutant strain PKN-A. Furthermore,
searching the
database of the unfinished contigs from the
Pseudomonas genome
projects (accessible at
http://www.ncbi.nlm.nih.gov/BLAST/pseudoabl.html)
revealed
that, in addition to PpkA itself, there are at least
three other
PpkA-related sequences, which may account for the
moderate reduction of
virulence of PKN-A.
The kinase domain of PpkA affects growth of E. coli and
P. aeruginosa.
To confirm the size of the PpkA ORF as well
as to study the biochemical properties of PpkA, we attempted to
overproduce the PpkA protein in E. coli. The first ATG codon
of PpkA was fused in frame behind the glutathione
S-transferase (GST) gene in pGEX5x-1, resulting in pHJY9.
This construct was sequenced to confirm the in-frame gene fusion.
E. coli harboring the fusion construct, pHJY9, grows slowly
and forms small colonies on media even in the absence of
isopropyl--D-thiogalactopyranoside (IPTG), compared to
E. coli harboring the fusion vector only. In the presence of IPTG (>0.1 mM), E. coli containing pHJY9 hardly grows on
minimal or rich medium and prolonged incubation in liquid medium leads to bacterial lysis, indicating that the PpkA portion of the fusion protein is toxic to E. coli.
We next asked if the kinase activity of PpkA plays any role in
toxicity. Since the conserved aspartic acid residue (amino
acid 129),
residing within the catalytic loop of the kinase domain,
is required
for the catalytic activity of the enzyme in all characterized
Ser/Thr
protein kinases (
3,
4), it was mutated to an asparagine
by
site-directed mutagenesis. The mutant gene,
ppkA(D129N), was
then fused behind the
gst gene as in pHJY9, resulting in
pHJY10.
In contrast to
E. coli harboring pHJY9,
E. coli harboring pHJY10
grows normally on media in the presence or
absence of IPTG. Furthermore,
a 150-kDa GST-PpkA(D129N) fusion protein
was highly and specifically
produced in the presence of IPTG (Fig.
5), demonstrating that
the toxic effect
of PpkA is due to its kinase domain. Excluding
the 25-kDa GST portion,
the size of the PpkA protein in the above
fusion construct is in good
agreement with the molecular mass
predicted from the DNA sequence. By
using antibody against GST,
an IPTG induction-specific 150-kDa GST-PpkA
fusion protein was
also detected in
E. coli containing pHJY9
(data not shown), including
a series of smaller bands, mainly the
visible 40-kDa protein band
(Fig.
4, lanes 3 and 4), representing
breakdown products.
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FIG. 5.
Coomassie blue-stained sodium dodecyl
sulfate-polyacrylamide gel of total bacterial cell extracts. E. coli DH5 harboring GST fusion vector pGEX5x-1 (lanes 1 and 2),
GST-PpkA fusion construct pHJY9 (lanes 3 and 4), or GST-PpkA(D129N)
fusion construct pHJY10 (lanes 5 and 6) was grown in the presence
(lanes 2, 4, and 6) or absence (lanes 1, 3, and 5) of 0.1 mM IPTG. Cell
extracts from the same number of bacterial cells were loaded in each
lane.
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Although the
tac promoter that drives the expression of the
gst gene is not as strong a promoter in
P. aeruginosa as it is
in
E. coli, the same toxic effect
of GST-PpkA on
P. aeruginosa was observed when pHJY9 was
introduced into the chromosome of
PAK by single crossover (PAK×pHJY9),
whereas pHJY10 had no toxic
effect. Taken together, the above
observations clearly indicate
that the kinase domain of PpkA has an
enzymatic function within
bacterial cells and high-level substrate
phosphorylation might
have led to the inhibitory effect on bacterial
growth.
Nucleotide sequence accession number.
The nucleotide sequence
of ppkA has been submitted to the GenBank databases under
accession number AF035395.
| |
ACKNOWLEDGMENTS |
We thank Marie Chow for many suggestions and stimulating
discussions, Linda Thompson for statistical analysis of the data, and
Allen Gies for running the automated DNA sequencer.
This work was supported by NIH grants R29AI39524 and 5P20RR11815.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Immunology, Mail Slot 511, 4301 W. Markham, Little
Rock, AR 72205. Phone: (501) 296-1396. Fax: (501) 686-5359. E-mail: JINSHOUGUANG{at}EXCHANGE.UAMS.EDU.
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Journal of Bacteriology, December 1998, p. 6764-6768, Vol. 180, No. 24
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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