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Infection and Immunity, November 2001, p. 7140-7145, Vol. 69, No. 11
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.11.7140-7145.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Identification of Genes Induced In Vivo during
Klebsiella pneumoniae CG43 Infection
Yi-Chyi
Lai,1
Hwei-Ling
Peng,2 and
Hwan-You
Chang1,*
Department of Life Science and Institute of
Biotechnology, National Tsing Hua University,1
and Department of Biological Science and Technology, National
Chiao Tung University,2 Hsin Chu, Taiwan,
Republic of China
Received 16 January 2001/Returned for modification 20 March
2001/Accepted 20 July 2001
 |
ABSTRACT |
A novel in vivo expression technology (IVET) was performed to
identify Klebsiella pneumoniae CG43 genes that are
specifically expressed during infection of BALB/c mice. The IVET
employed a UDP glucose pyrophosphorylase (galU)-deficient
mutant of K. pneumoniae which is incapable of utilizing
galactose and synthesizing capsular polysaccharide, as demonstrated by
its low virulence to BALB/c mice and a white nonmucoid colony
morphology on MacConkey-galactose agar. By using a functional
galU gene as the reporter, an IVE promoter could render the
galU mutant virulent while maintaining the white nonmucoid
colony phenotype. A total of 20 distinct sequences were obtained
through the in vivo selection. Five of them have been identified
previously as virulence-associated genes in other pathogens, while
another five with characterized functions are involved in regulation
and transportation of nutrient uptake, biosynthesis of isoprenoids, and
protein folding. No known functions have been attributed to the other
10 sequences. We have also demonstrated that 2 of the 20 IVE genes turn
on under iron deprivation, whereas the expression of another five genes
was found to be activated in the presence of paraquat, a superoxide generator.
 |
TEXT |
Klebsiella pneumoniae is
an important nosocomial pathogen that causes a wide range of
infections, including pneumonia, bacteremia, urinary tract infections,
and sometimes life-threatening septic shock. As an opportunistic
pathogen, it primarily attacks immunocompromised individuals who are
hospitalized and/or suffering from severe underlying diseases, such as
diabetes mellitus, chronic alcoholism, or pulmonary obstruction
(23). Many clinical strains of K. pneumoniae are highly resistant to antibiotics, indicating the relative
ineffectiveness of current therapy.
During infections, bacterial pathogens must adapt to various changes in
order to persist and proliferate in appropriate locations and to
circumvent host defenses. It is reasonable to assume that the
expression of many K. pneumoniae genes that participate in pathogenesis could be specifically induced within the host. Ideally, these in vivo-expressed (IVE) genes would serve as useful drug targets
and vaccine candidates. Several approaches, including in vivo
expression technology (IVET) (11, 15), comparative genomics (2), microarray DNA chips (7),
signature-tagged mutagenesis (18, 26), differential
display-PCR (1), and differential fluorescence detection
(31), have allowed the identification of genes that are
essential or specifically activated during infections. Nevertheless,
none of these approaches has been applied to K. pneumoniae,
primarily due to the limited number of mutants and genetic tools
available for the bacterium.
IVE technology (IVET) is a powerful technique that has been used
successfully for several important pathogens, including
Salmonella enterica serovar Typhimurium (15,
16), Yersinia enterocolitica (33),
Staphylococcus aureus (14), Pseudomonas
aeruginosa (32), Escherichia coli
(12), and Actinobacillus pleuropneumoniae
(9). The original IVET, designed to identify promoters
that turn on in vivo in S. enterica, used a tandem set of in
vivo and in vitro promoterless genes as the reporter (15).
There are now several different modifications of the IVET, such as the
use of auxotrophic markers and antibiotic resistance genes, and
induction of site-specific recombinase as the basis for the selection
systems (12, 14, 32). In view of the limited genetic tools
available for K. pneumoniae, we have designed a novel IVET
selection system for this heavily encapsulated bacterium.
Rationale for the galU-based IVET selection
system.
The rationale for the IVET developed for K. pneumoniae is shown in Fig. 1.
Instead of using a set of tandem reporter genes, our IVET system
incorporates a copy of the promoterless galU gene as a
dual-function reporter. The galU gene encodes the enzyme UDP
glucose pyrophosphorylase, which regulates the supply of UDP galactose
and UDP glucose, two major precursors for the biosynthesis of capsule
polysaccharides (CPS) and lipopolysaccharides (LPS) in most enteric
bacteria. A GalU
mutant of K. pneumoniae produces defective forms of CPS and LPS and hence
loses virulence and the mucoid colony phenotype (5). In
addition, the K. pneumoniae GalU
strain is
incapable of fermenting galactose, a property that can be readily
distinguished by using MacConkey-0.4% galactose agar
(5). These unique properties make the galU gene
an ideal reporter system for IVE gene selection. The bacterial strains which are able to survive in vivo selection while exhibiting a white
nonmucoid colony phenotype on MacConkey-galactose agar would indicate
that the DNA fragment upstream of the galU reporter contains an IVE promoter.
Construction of galU-based reporter gene system.
Our IVET selection vector, designated pYC016 (Fig.
2), was constructed by using the
mobilizable plasmid pSUP102 (27) as the backbone. Plasmid
pYC016 was engineered sequentially in the order described as follows.
Initially, a PCR-amplified copy of the Pseudomonas
aeruginosa PAO1 galU gene coding sequence was inserted
into the unique ClaI site of pSUP102. Subsequently, a nonessential BamHI site in the plasmid was eliminated by
end-filling with Klenow fragment of DNA polymerase I, followed by
religation. Finally, a cassette from pKK232-8 (Amersham-Pharmacia,
Piscataway, N.J.) that contains a transcriptional terminator, multiple
cloning sites, and a set of three-way translational stop codons was
inserted into a ClaI site of the vector. The main features
of pYC016 include (i) a unique BamHI site which is
compatible with Sau3AI-digested chromosomal DNA fragments,
(ii) use of the galU gene of P. aeruginosa PAO1,
with the intention of minimizing the possibility of homologous recombination that could occur between the chromosomal and
plasmid-borne galU genes in K. pneumoniae, (iii)
the presence of translational stop codons in all three reading frames
preceding the ribosome-binding site of the galU gene to
ensure transcriptional fusions between inserted DNA fragments and the
galU gene, and (iv) a transcriptional terminator,
rrnB T1 (4), upstream of the cloned
chromosomal fragment to prevent transcription of the galU
gene from a fortuitous plasmid promoter.

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FIG. 2.
Diagram of K. pneumoniae IVET vector pYC016,
showing the locations of the transcriptional terminator
(rrnB T1), multiple cloning sites (MCS),
three-way translational stop codons, and promoterless galU
gene. Only the BamHI site in the multiple cloning site is
unique in the vector.
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|
Construction of K. pneumoniae IVET library.
Two
isogenic K. pneumoniae strains were used in this study.
K. pneumoniae CG43 is a clinical isolate which is highly
virulent to laboratory mice, with a 50% lethal dose (LD50)
of 10 CFU (22). K. pneumoniae CG43-17 is a
GalU
derivative of CG43, generated previously by
Tn5 insertion mutagenesis. The virulence of K. pneumoniae CG43-17 was found to be significantly attenuated
(LD50, ~106 CFU) (5).
High-molecular-weight chromosomal DNA of K. pneumoniae CG43
was purified and partially digested with Sau3AI. Fragments ranging from 0.5 to 1 kb in size were purified from agarose gels and
ligated into the unique BamHI site of pYC016. The ligation mixture was transformed into E. coli S17-1 (27)
(hsdR recA pro RP4-2 [Tc::Mu;
Km::Tn7]) and selected on Luria-Bertani (LB) agar supplemented with chloramphenicol (30 µg/ml). Approximately 5 × 105 of the transformants, 90% of which contain a plasmid
with an insert, were obtained. These plasmids were then mobilized into K. pneumoniae CG43-17 by conjugation. The transconjugants
were selected on MacConkey-0.4% galactose plates supplemented with kanamycin (75 µg/ml), chloramphenicol (30 µg/ml), and ampicillin (50 µg/ml). After an 8-h incubation at 37°C, red mucoid colonies were scored as Gal+, whereas white nonmucoid colonies were
scored as Gal
. The red-white
(Gal+:Gal
) ratio of the transconjugants was
about 1:3.
Screening of K. pneumoniae IVE genes.
A pilot
study was performed with about 103 CFU of the IVE library.
The study demonstrated that after infections in BALB/c mice and
subsequent recovery from spleen, the ratio of red to white colonies
shifted from the original 1:3 to 60:1, indicating that most of the
Gal
clones were eliminated effectively in vivo. To reduce
the number of mice used in this study as well as to minimize undesired
bacteria of the Gal+ background, approximately 5 × 104 mostly white nonmucoid colonies were collected manually
and separated into three batches for the experiments that followed.
Male BALB/c mice (6 to 8 weeks old) with an average weight of 25 g were
obtained from the animal center of National Taiwan University and
acclimatized in an animal house of our institute for 3 days.
Exponential-phase K. pneumoniae was obtained by
diluting an overnight broth culture 100-fold into warmed LB broth and
shaking at 37°C until the optical density at 600 nm
(OD600) reached 0.3 to 0.4.
Typically 104 CFU were used to infect a BALB/c mouse. Prior
to infection, bacteria were washed twice and resuspended in 200 µl of
1× phosphate-buffered saline (PBS), and the suspension was then
injected intraperitoneally into a BALB/c mouse. The infected mice were
sacrificed after 24 to 48 h, and the spleens were dissected, homogenized, serially diluted, and plated on MacConkey-galactose agar
supplemented with appropriate antibiotics. The surviving white
nonmucoid colonies were then collected for an additional two rounds of
in vivo selection. In three independent experiments, each utilizing a
different pool of white nonmucoid bacteria and three mice per round of
selection, we arbitrarily picked 30 to 60 white nonmucoid colonies from
the postselection pool of each infected mouse for plasmid DNA
preparation and restriction endonuclease digestion analysis. A total of
20 distinct eletrophoretic patterns were observed, and these IVE clones
were subjected to further characterization.
Verification of inducibility of galU fusion constructs
in vivo and in vitro.
The plasmid DNA was extracted from the 20 IVE clones and retransformed into K. pneumoniae CG43-17, and
the transformants were tested individually for virulence in BALB/c.
Approximately 1 × 106 to 5 × 107
CFU could be recovered from 1 g of spleen from the sick mice. The
number is comparable to that of the wild-type K. pneumoniae CG43. In contrast, less than 10 CFU of K. pneumoniae
CG43-17(pYC016) was observed under these conditions. Moreover, the use
of a standard assay method (5) indicated that all these
clones did not exhibit detectable UDP glucose pyrophosphorylase
activity, confirming that the promoters were indeed turned off under in
vitro growth conditions.
A serum susceptibility assay, which correlates LPS quantity in the
gram-negative bacteria, was also performed. Less than 1% human serum
was sufficient to achieve 50% killing of K. pneumoniae CG43-17, a mutant known to be incapable of synthesizing intact LPS
(5). The K. pneumoniae CG43-17 IVE clones
exhibited a similar behavior towards 1% human serum and were also
killed efficiently. However, the concentration of human serum required
to effectively kill the wild-type K. pneumoniae CG43 must
exceed 50%. Together, these results indicated that the galU
fusion clones were not expressed when grown in enriched medium but
could be induced preferentially during infection of BALB/c mice.
Nucleotide sequence determination of IVE genes.
DNA sequence
determination was carried out by the PCR-mediated Taq
DyeDeoxy Terminator Cycle sequencing kit on an Applied Biosystems model
373A DNA sequencer. The homology search of the GenBank/EMBL and
SwissProt databases was performed using the BLAST programs provided by
the National Center of Biotechnology Information through the Internet.
The result of the sequence analysis is shown in Table
1.
Among the 20 IVE sequences, 5 have been shown to be genes essential for
in vivo growth identified previously in other pathogens, demonstrating
the effectiveness of the galU-based selection strategy. iucA and fepA are both involved in iron
acquisition, and expression of these genes is known to be activated
during infection (3, 8, 11, 17, 20, 31). The
ptfA gene encodes a phosphotransfer system for fructose
uptake. By using signature-tagged mutagenesis, it has been demonstrated
that this gene is crucial for Vibrio cholerae to survive in
the host (6). The gene product of rbsR is a
repressor responsible for regulating the expression of rbsC, the ribose permease-encoding gene. The expression of rbsR
was found to be essential for Brucella melitensis to survive
in the host (13). lysA encodes diaminopimelate
decarboxylase for lysine biosynthesis (28). It has been
shown that in Staphylococcus aureus, lysA is
preferentially expressed during infection, presumably due to limited
supply of lysine in the host (18).
The other five K. pneumoniae IVE genes found in this study
that have been characterized in other organisms include uup
(25), an ATP-binding cassette type transporter encoding
gene; yaeM (29), which encodes
1-deoxy-D-xylulose 5-phosphate reductoisomerase, which is
responsible for terpenoid synthesis; gyrA (19),
DNA gyrase subunit A; tdcA (10), the product of
which is a transcription activator for the tdc operon, which
encodes a system involved in threonine and serine metabolism and
transport during anaerobic growth; and ppiA
(30), peptidylprolyl cis-trans isomerase A. Among the remaining 10 sequences of unknown function, 7 matched the
Escherichia coli hypothetical protein-encoding genes. Two sequences were found in the genome database of K. pneumoniae
MGH 78578, established in the Genomic Sequencing Center at Washington University, St. Louis, Mo., and one was a novel sequence.
Inducibility of IVE promoters under iron deprivation and oxidative
stress.
On entering the host, bacterial pathogens must circumvent
iron deprivation and the attack of reactive oxygen species produced by
the immune cells. Therefore, it is reasonable to assume that some of
the IVE promoters identified in this study may encode a product that
assists the bacteria in countering these stresses. To verify the
possibility, the IVE clones were spotted individually on
MacConkey-galactose plate containing either 200 µM 2',2'-dipyridyl, an iron chelator, or 10 µM paraquat, a superoxide generator. The phenotype of the colonies after overnight incubation at 37°C was then
examined. Two IVE clones, AJ277397 and AJ292298, representing the promoters of two iron acquisition genes, iucA and
fepA, respectively, were found to display the red mucoid
colony phenotype in the presence of 2',2'-dipyridyl that reflects the
activation of the promoters under iron deprivation conditions (Fig.
3).

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FIG. 3.
Phenotypes of IVE clones on MacConkey-galactose agar
containing 10 µM paraquat (A) or 100 µM 2',2'-dipyridyl (B). The
bright red color of the colonies indicates expression of the
galU reporter gene.
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|
Paraquat was found to activate five IVE clones, including
lysA (AJ277396) and four carrying genes of no known function (AJ292306, AJ292315, AJ292301, and AJ292302). Our result indicates that
many genes are likely to be turned on to counter oxidative stress
during infection in the host. The number may be more, since it has been
shown that E. coli responds to the redox stress imposed by
paraquat by activating the synthesis of as many as 80 polypeptides
(21). The identification of lysA as a
superoxide-inducible gene is intriguing, although we do not have a good
explanation for this finding yet.
In summary, we have constructed a novel IVET for identification of
virulence-associated genes in gram-negative bacteria. By using this
system, we have successfully identified 20 IVE genes in K. pneumoniae. In addition to being used in IVE gene identification, the convenient red-white selection on MacConkey-galactose plates provided by the galU reporter system might be applied in
identifying genes specifically expressed under certain growth
conditions, such as iron deprivation and oxidative stress. Like many
other IVET, the procedure demands that the reporter gene be expressed throughout the course of an infection. Therefore, this strategy may be
unable to identify certain IVE genes that are expressed only during a
specific stage of infection. However, this drawback can be complemented
by screening the IVE library in multiple animal infection models, as
demonstrated in Streptococcus pneumoniae with
signature-tagged mutagenesis (24).
 |
ACKNOWLEDGMENTS |
This work was supported in part by the National Science Council of
the Republic of China (NSC89-2320-B-009-001 to H.L.P. and 89-2320-B-007-002 to H.Y.C.) and VTY Joint Research Program, Tsou's Foundation (VTY89-p4-28 to H.Y.C.).
We are grateful to J. Vatsyayan for critical reading of the manuscript.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Life Science, National Tsing Hua University, 101 Kuan-Fu Road, 2nd
Sec., Hsin Chu, Taiwan, Republic of China. Phone: 886-3-5742910. E-mail: hychang{at}life.nthu.edu.tw.
Editor:
V. J. DiRita
 |
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Infection and Immunity, November 2001, p. 7140-7145, Vol. 69, No. 11
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.11.7140-7145.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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