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Infection and Immunity, June 2000, p. 3772-3775, Vol. 68, No. 6
Department of Biochemistry, Microbiology, and Molecular
Genetics, University of Rhode Island, Kingston, Rhode Island
028811; Bacteriological and
Immunological Research and Development, Intervet International, 5830 AA
Boxmeer, The Netherlands2; Combined
Program in Pediatric Gastroenterology and Nutrition, Harvard Medical
School, Massachusetts General Hospital, Charlestown, Massachusetts
021203; Department of Gastrointestinal
Infections, Statens Seruminstitut, 2300 Copenhagen,4 and Department of
Microbiology, Technical University of Denmark, DK-2800
Lyngby,5 Denmark; and Scottish
Salmonella Reference Laboratory, Stobhill NHS Trust, Glasgow G21 3UW,
Scotland6
Received 16 December 1999/Returned for modification 17 February
2000/Accepted 6 March 2000
A minitransposon mutant of Salmonella enterica serovar
Typhimurium SR-11, SR-11 Fad Since Salmonella enterica
serovar Typhimurium is able to utilize phosphatidylserine as the sole
source of carbon, nitrogen, and phosphate for growth (11)
and since phosphatidylserine is a component of eucaryotic cell
membranes, it was of interest to determine whether mutants unable to
utilize either the glycerophosphate portion or the fatty acid portion
of the phospholipid for growth are attenuated. Several mutants unable
to utilize oleate as a sole carbon source were isolated after
mini-Tn10::d cam transposon mutagenesis (6), and one of them, designated serovar
Typhimurium SR-11 Fad SR-11 Fad
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
A Functional cra Gene Is Required for
Salmonella enterica Serovar Typhimurium Virulence in
BALB/c Mice
ABSTRACT
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Abstract
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References
, is unable to utilize
gluconeogenic substrates as carbon sources and is avirulent and
immunogenic when administered perorally to BALB/c mice (M. J. Utley et al., FEMS Microbiol. Lett., 163:129-134, 1998). Here,
evidence is presented that the mutation in SR-11 Fad that
renders the strain avirulent is in the cra gene, which
encodes the Cra protein, a regulator of central carbon metabolism.
TEXT
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Abstract
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References
(fatty acid), was totally avirulent
and immunogenic in BALB/c mice when administered perorally
(21). In addition, SR-11 Fad was found in the
liver and spleen in numbers 4 to 5 orders of magnitude lower than SR-11
(21). SR-11 Fad was also less virulent than
SR-11 when administered to mice intraperitoneally (21).
Furthermore, SR-11 Fad, in addition to being unable to
grow utilizing oleate as a sole carbon source, was also unable to grow
utilizing citrate, isocitrate, and acetate as sole carbon sources, all
of which were utilized well by SR-11 (21). SR-11
Fad grew slowly and to only one-tenth the final yield of
SR-11 utilizing pyruvate as the sole carbon source in liquid
culture (21). SR-11 Fad was, however,
able to utilize glucose, glycerol, and a number of sugars
sensitive to catabolite repression as the sole carbon sources
(14). In the present study, we identify the gene
made defective in SR-11 Fad by
mini-Tn10::d cam transposon
mutagenesis and show that the defect is responsible for SR-11
Fad avirulence in BALB/c mice.
is a cra (fruR)
mutant.
In order to identify the gene that is inactivated in SR-11
Fad, Southern hybridization was performed using a
digoxigenin (DIG)-labeled transposon-specific probe made from pJHA1
(Table 1). The transposon-specific probe
was prepared with a DIG High Prime DNA Labeling and Detection Starter
Kit II (Boehringer Mannheim, Indianapolis, Ind.) as described by the
manufacturer. The Tn10::d cam
minitransposon was located on a 4.5-kb PstI SR-11
Fad DNA fragment which was inserted into the unique
PstI site of pBluescript II SK(+) to create pJHA7 (Table 1).
The first 489 bp in the 4.5-kb SR-11 Fad insert in pJHA7
at the T3 promoter end were sequenced. The sequence was essentially
identical to the last 180 bp of the serovar Typhimurium LT2
ilvI gene, the first 306 bp of the serovar Typhimurium LT2 ilvH gene, and the three intervening base pairs (GenBank
accession number X55456). The first 470 bp at the opposite end of the 4.5-kb SR-11 Fad insert in pJHA7, sequenced from the T7
promoter, were 87% identical to yabB, a gene of unknown
function in Escherichia coli (GenBank accession number
AE000118 U00096). In both serovar Typhimurium LT2 and E. coli, ilvI and ilvH are immediately upstream
of the fruR gene, which is 601 bp upstream of
yabB in E. coli (GenBank accession number
AE000118 U00096). Sequencing from the chloramphenicol resistance gene
in pJHA7 determined that the mini-Tn10::d
cam transposon was indeed inserted 45 bp upstream of the 3'
end of the LT2 fruR gene (reference 9 and
GenBank accession number X55456). The fruR gene has recently
been renamed cra (catabolite repressor/activator)
(18). A diagram of the genes present in the 4.5-kb
PstI SR-11 Fad DNA fragment in pJHA7 and the
portions sequenced is presented in Fig.
1.
TABLE 1.
Bacterial strains and plasmids
View larger version (9K):
[in a new window]
FIG. 1.
Genes present in the SR-11 Fad 4.5-kb
PstI DNA fragment that contains the chloramphenicol
resistance gene (cam). Heavy lines below the genes denote
regions in the fragment that were sequenced. Sequencing reactions were
performed with the BigDye Terminator Cycle Sequencing Kit, and
reactions were analyzed on the ABI PRISM 310 (PE Applied Biosystems,
Forest City, Calif.). Nucleotide sequences were analyzed using Clone
Manager and Align Plus 3 programs (Sci-Ed Software, Durham, N.C.).
Arrows above the genes denote the direction of transcription. Arrows
below the genes denote the positions of the forward and reverse primers
used for PCR amplification of the wild-type and defective
cra genes. P, PstI.
. Both mutants were essentially
identical with respect to growth on M9 minimal agar plates containing
either sodium oleate (5 mM), sodium citrate (0.2% [wt/wt]),
potassium acetate (0.4% [wt/wt]), sodium pyruvate (0.4% [wt/wt]),
sodium succinate (0.6% [wt/wt]), potassium fumarate (0.4%
[wt/wt]), glucose (0.2% [wt/wt]), or glycerol (0.2% [wt/wt]),
i.e., they failed to grow on the gluconeogenic substrates as sole
carbon sources in 48 h at 37°C but grew as well as their
wild-type parents on glucose and glycerol. The wild-type SR-11
cra gene was cloned into pBR322 to create pJHA8 (Table 1). Both SR-11 Fad and LJ2443, when complemented with pJHA8,
regained the ability to grow on M9 agar plates containing the
aforementioned gluconeogenic substrates as sole carbon sources. pBR322
did not functionally complement the cra mutation in either
SR-11 or LJ2443. These experiments established conclusively that
serovar Typhimurium SR-11 Fad is a cra mutant.
SR-11 Fad (pJHA8) is virulent.
It was possible
that inactivation of the cra gene with the
mini-Tn10::d cam insertion caused a
downstream effect that resulted in avirulence. We therefore
complemented SR-11 Fad with pJHA8, which contains the
wild-type SR-11 cra gene as the only nonvector gene in
pBR322, and tested its virulence.
(pBR322) or
4.3 × 108 CFU per mouse of SR-11 Fad
(pJHA8). By 8 days postinfection, three of the four mice infected with
SR-11 Fad (pJHA8) had died. The fourth mouse infected
with SR-11 Fad (pJHA8) appeared to be very sick for
several days (ruffled fur, loss of appetite, huddled) but recovered. In
contrast, the four mice infected with SR-11 Fad (pBR322)
remained alive and healthy. Since complementing SR-11 Fad
with the wild-type cra gene results in renewed virulence, it is the defect in the cra gene caused by the
mini-Tn10::d cam insertion that
renders the strain avirulent.
A serovar Typhimurium UK-1 cra mutant constructed by
allelic exchange is avirulent.
It was of interest to determine
whether a cra mutant of a second serovar Typhimurium strain
was also avirulent. Therefore, a cra mutant of the virulent
strain serovar Typhimurium UK-1 (23), designated UK-1
Fad AX-1 (allelic exchange), was constructed by allelic
exchange of the wild-type cra gene with the mutant
cra gene using pMJN10 (see Table 1). pMJN10 contains the
mutant cra gene and flanking sequences as a 4.5-kb
PstI DNA fragment (Fig. 1) in pLD55, an allelic exchange
vector (15). That the only cra gene that UK-1 Fad AX-1 contained was the defective cra gene
was shown by four lines of evidence. First, UK-1 Fad AX-1
was unable to grow on any of the gluconeogenic substrates. Second,
complementing UK-1 Fad AX-1 with the wild-type
cra gene returned its ability to grow on the gluconeogenic
substrates. Third, when PstI-cut UK-1 Fad AX-1
DNA was probed with either the wild-type cra gene or the chloramphenicol resistance gene in Southern hybridization experiments, a single 4.5-kb band was detected, as expected if the 3.1-kb
PstI DNA fragment containing the wild-type cra
gene was replaced with the defective cra gene (not shown).
Fourth, when UK-1 and UK-1 AX-1 DNA were amplified by PCR using the
forward and reverse primers used to amplify the SR-11 wild-type
cra gene (see Fig. 1 and the legend to Fig.
2), the amplified fragments were as
expected (Fig. 2), i.e., in UK-1 a 1.4-kb fragment containing the
wild-type cra gene and in UK-1 Fad AX-1 a
2.8-kb fragment (the 1.4-kb cra fragment containing the inserted 1.4-kb chloramphenicol resistance gene).
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AX-1 is avirulent, four
BALB/c mice were infected perorally with UK-1 (2.6 × 108 CFU per mouse), and four mice were identically infected
with UK-1 Fad AX-2 (1.8 × 108 CFU per
mouse). By day 8 postinfection, all four UK-1 infected mice had died,
whereas the four mice infected with UK-1 AX-1 were healthy and active
and had never appeared to be sick. The UK-1 Fad
AX-1-infected mice were still healthy and active when the experiment was terminated at 3 weeks postinfection. Therefore, avirulence caused
by a defect in cra is not restricted to SR-11.
SR-11 Fad AX-2 was also constructed by allelic exchange
of the wild-type cra gene with the mutant cra
gene using pMJN10 (see Table 1). SR-11 Fad AX-2 had the
phenotype of a cra mutant with respect to utilization of
carbon sources. Moreover, SR-11 Fad AX-2 regained the
wild-type phenotype, when complemented with the wild-type
cra gene, and contained the chloramphenicol resistance gene
on a 4.5-kb PstI fragment, and the expected 2.8-kb PCR
product was amplified from SR-11 Fad AX-2 using the
forward and reverse primers specific to the SR-11 wild-type
cra gene (Fig. 2). SR-11 Fad AX-2 was also
found to be avirulent. Four BALB/c mice were infected perorally with
SR-11 (2.1 × 108 CFU per mouse), and five mice were
identically infected with SR-11 Fad AX-2 (2.8 × 108 CFU per mouse). By day 8 postinfection, all four SR-11
infected mice had died, whereas the five mice infected with SR-11
Fad AX-2 were healthy and active and had never appeared
to be sick. The SR-11 Fad AX-2-infected mice were still
healthy and active when the experiment was terminated at 3 weeks postinfection.
Behavior of SR-11 and SR-11 Fad in infection
models.
Serovar Typhimurium utilizes nutrients present in
intestinal mucus for growth in the intestine (12, 14).
Mammalian intestinal mucus contains several sugars that SR-11 and SR-11
Fad could metabolize via glycolytic pathways, including
N-acetylglucosamine, N-acetylgalactosamine,
galactose, fucose, sialic acid, ribose, arabinose, mannose, gluconate,
and the hexuronates glucoronate and galacturonate (1, 17,
20). Since serovar Typhimurium SR-11 Fad is not
defective in its ability to utilize sugars for growth (21),
it is unlikely that it is defective in the ability to grow in the mouse
intestine. In support of this view, in a previous study (8),
when SR-11 and SR-11 Fad were inoculated (4.0 × 104 CFU/ml) into mouse intestinal mucus in vitro, they grew
equally well, reaching levels of about 6.0 × 108
CFU/ml in 24 h at 37°C. For this reason, we turned our attention to the ability of SR-11 Fad to adhere to and invade
intestinal epithelial cells and to survive within macrophages.
invaded
BALB/c mouse M cells equally well after infection of ileal loops, made
as described previously (10), and both were found in
enclosed vacuoles within the M cells. In addition, differences were not
found in the ability of SR-11 and SR-11 Fad to adhere to
and invade human T-84 intestinal epithelial monolayers using procedures
described previously (13), i.e., 2.41 ± 0.74% (mean ± the standard deviation [SD] of triplicate samples) of the SR-11 inoculum (20 CFU per T84 cell) and 2.06 ± 0.50% of the SR-11 Fad inoculum (20 CFU per T84 cell) became cell
associated in 60 min. In the same time, 0.094 ± 0.009% of the
SR-11 inoculum and 0.089 ± 0.016% of the SR-11 Fad
inoculum were internalized. Collectively, these results make it
unlikely that SR-11 Fad is avirulent because it is
defective in its ability to adhere to and invade M cells and/or
intestinal epithelial cells.
The ability to survive in macrophages in vitro has been correlated with
S. enterica pathogenicity in mice (7, 16).
Resident peritoneal macrophages were isolated from BALB/c mice
(7), and survival of SR-11 and SR-11 Fad in
macrophages was determined as described previously (7). Both
SR-11 and SR-11 Fad survived equally well in resident
peritoneal macrophages. That is, after 60 min 0.057 ± 0.0075%
(mean ± the SD of triplicate samples) of the SR-11 inoculum and
0.056 ± 0.018% of the SR-11 Fad inoculum survived
and, in each case, both strains remained at the 1-h survival level for
the next 23 h. While these data rule out the possibility that
SR-11 Fad is inherently inferior to SR-11 in surviving in
BALB/c peritoneal macrophages, they do not rule out the possibility
that SR-11 Fad is avirulent because its ability to
survive and grow in macrophages in vivo is decreased relative to that
of SR-11. Serovar Typhimurium may, in fact, utilize gluconeogenic
substrates for survival and growth in macrophages in vivo (e.g.,
succinate, pyruvate, etc.). If so, SR-11 Fad would be at
a distinct disadvantage. In the present experiments, the BALB/c
resident peritoneal macrophages were continuously bathed in RPMI 1640 medium. RPMI 1640 medium contains 2 g of glucose per liter which
SR-11 and SR-11 Fad metabolize equally well.
In summary, a functional cra gene is necessary for serovar
Typhimurium virulence in BALB/c mice. The Cra protein is a regulator of
central carbon metabolism (18). When interacting with the genes it regulates, i.e., in the presence of gluconeogenic substrates (18), the Cra protein positively regulates transcription of those genes encoding biosynthetic and oxidative enzymes (e.g., key
enzymes in the tricarboxylic acid cycle, the glyoxylate bypass, the
gluconeogenic pathway, and electron transport) and negatively regulates
transcription of genes encoding glycolytic enzymes, e.g., key enzymes
in the Embden-Meyerhof and Entner-Doudoroff pathways (18).
Since in a cra mutant the genes involved in glycolytic pathways are highly expressed, whereas the expression of genes involved
in the gluconeogenic pathway and the glyoxylate bypass is reduced, it
is possible that gluconeogenesis is required for serovar Typhimurium
virulence. In support of this view, it has recently been reported that
a MudJ insertion in a putative malate oxidoreductase gene,
involved in gluconeogenesis, renders serovar Typhimurium avirulent and
immunogenic (22). Since SR-11 Fad grows as
well as SR-11 in intestinal mucus (8), enters M cells as
well as SR-11 in vivo, and adheres to epithelial cells as well as SR-11
in vitro, it is unlikely that SR-11 Fad is avirulent
because it is either defective in its ability to grow in the intestine
or defective in its ability to adhere to and enter intestinal
epithelial cells and M cells in vivo. If, however, the only available
carbon sources for S. enterica serovar Typhimurium survival
and growth in M cells and/or in macrophages in vivo are the
gluconeogenic substrates, since SR-11 Fad would be unable
to grow under these conditions, it would not be surprising if this were
the reason for its avirulence in mice. In situ experiments will be
required to test this hypothesis.
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ACKNOWLEDGMENTS |
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This study was supported by a grant to P.S.C. from Intervet International, Boxmeer, The Netherlands, and from the University of Rhode Island Foundation and by NIH grant DK50989 to B.A.M.
We thank Roy R. Curtiss III, Washington University, St. Louis, Mo; V. de Lorenzo, Centro de Investigaciones Biologicas, Consejo Superior de Investigaciones, Cientificas, Madrid, Spain; and Milton Saier, University of California at San Diego, La Jolla, for their kind gifts of bacterial strains. We also thank Nafisa Ghori, Stanford University School of Medicine, Stanford, Calif., for excellent technical assistance with the transmission electron microscopy.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Biochemistry, Microbiology, and Molecular Genetics, Morrill Science Bldg., 45 Lower College Rd., University of Rhode Island, Kingston, RI 02881. Phone: (401) 874-5920. Fax: (401) 874-2202. E-mail: pco1697u{at}postoffice.uri.edu.
Editor: J. T. Barbieri
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Infection and Immunity, June 2000, p. 3772-3775, Vol. 68, No. 6
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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