Full text of DOI:10.1111/j.1348-0421.2002.tb02666.x

Microbiol. Immunol., 46(2), 109―113, 2002
Association of a Regulatory Gene, slyA with a
Mouse Virulence of Salmonella Serovar
Choleraesuis
,
1, 3
2
1, ‡1
3, ‡2
Aki Kaneko* , Masaharu Mita , Kachiko Sekiya , Hidenori Matsui
,
3
1
Kazuyoshi Kawahara , and Hirofumi Danbara
1
2
Department of Microbiology, Laboratory Animal Research Center, School of Pharmaceutical Science, Kitasato University,
3
Minato-ku, Tokyo 108
―8641, Japan, and Department of Bacteriology, The Kitasato Institute, Minato-ku, Tokyo 108―8642, Japan
Received August 16, 2001; in revised form, October 15, 2001. Accepted November 1, 2001
Abstract: The influence of slyA gene, originally found in Salmonella serovar Typhimurium as a regulatory
gene for the expression of virulence genes, on a mouse virulence of S. serovar Choleraesuis was investigated
by using an slyA-defective mutant. The defective mutant was constructed by the insertion of a kanamycin-
resistance gene (aph) into the cloned slyA gene, and the homologous recombination with the intact slyA gene
5
on the chromosome. The mutant strain showed the LD50 value for BALB/c mouse approximately 10
higher than that of the parent strain. The increase of the LD50 value was the same order as that shown by
the mutation of the slyA gene of S. serovar Typhimurium, although LD50 of the wild-type strain of S. serovar
Choleraesuis was 40-fold higher than that of S. serovar Typhimurium. The time course of infection
observed in the mice organs also proved the clear difference of the virulence between the parent and the
mutant strains. These results suggested that the slyA gene product functions as a virulence-associated reg-
ulator also in S. serovar Choleraesuis.
Key words: Virulence, slyA, Salmonella serovar Choleraesuis
For the full expression of the Salmonella virulence,
many classes of virulence genes such as inv genes (7),
pag genes (10), and plasmid-encoded spv genes (9, 11)
are required. Among them regulatory genes are of high-
est importance for the timely expression of the viru-
lence genes to adapt to the environmental changes during
the infection process. Besides phoP/phoQ (19) and
rpoS (27), a new regulatory gene slyA was cloned by
Libby et al. (15) from Salmonella serovar Typhimurium
as a cytolysin-encoding gene, but later it was proved to be
a gene encoding a transcriptional regulatory protein (5),
which belongs to a family of regulators including EmrR
enteric salmonellosis (29).
The slyA gene is commonly shared among major
pathogenic serovars of Salmonella (14, 15), and homol-
ogous genes have been found also in other members of
Enterobacteriaseae such as Escherichia coli (17, 22),
Shigella (17), and Yersinia (24). We have sequenced
ORF and upstream region of slyA of several serovars of
Salmonella, and found that all of the serovars so far
examined use the sequence TTG as initiation codon of
slyA, and the nucleotide sequences of this region were
highly conserved (14). In our study of Salmonella vir-
ulence, S. serovar Choleraesuis has been mainly used (1,
11, 13, 18), because this serovar shows a high tendency
to cause systemic salmonellosis to human and swine
compared with S. serovar Typhimurium (2, 25), and the
genes responsible for systemic salmonellosis are expect-
ed to be studied in simpler system by using this serovar.
In this context the function of slyA in S. serovar Choler-
aesuis has been investigated in relation to our previous
and ongoing studies on the pathogenesis of this serovar.
In the present study, slyA-defective mutants of S. serovar
(16), MarR (28), HprR (23), and PecS (21). This regu-
latory gene of S. serovar Typhimurium is required for
cytotoxicity to M-cells (4), survival in macrophages
(13), and resistance to oxidative stress (3). In contrast,
slyA is not involved in invasion or colonization in the
murine intestine, and therefore is not essential for the
*
Address correspondence to Dr. Aki Kaneko, Department of
Bacteriology, The Kitasato Institute, 5 1, Shirokane, Minato-
ku, Tokyo 108 8642, Japan. Fax:＋81 5791 6127. E-mail:
kaneko-a@kitasato.or.jp
―9―
―
―
―
Abbreviations: CFU, colony forming unit; LB broth, Luria-
Bertani broth; LPS, lipopolysaccharide; ORF, open reading
frame.
‡
1
Present addresses: Laboratory of Electron Microscopy.
Kitasato Institute for Life Science.
‡2
1
09

1
10
A. KANEKO ET AL
Table 1. Bacterial strains and plasmids
Strain and plasmid
Relevant characteristics
Reference
Strain
－
E. coli DH5 α
E. coli S17-1 λpir
F Φ80dlacZ∆M15 ∆(lacZYA-argF)U169 endA1 recA1 hsdR17
RP4-2-Tc::Mu-Km::Tn7 λpir
Gibco/BRL
26
S. serovar Choleraesuis
r
RF-1
KAD001
wild type, Rf , derived from a swine isolate
12
r
RF-1 slyASC::aph, Km
This study
S. serovar Typhimurium
r
SR-11
wild type, Nr
8
r
KAD101
SR-11 slyAST::aph, Km
This study
Plasmid
pUC119
pUC4K
r
cloning vector, Ap
Toyobo
Toyobo
26
r
r
Km cassette (aph), Ap ,Km
suicide vector for homologous recombination, Ap
r
pCVD442
pKAD301
pKAD302
pKAD306
pUC119, slyASC
pUC119, slyASC::aph
pCVD442, slyASC::aph
14
This study
This study
pKAD308
pKAD309
pKAD313
pUC119, slyAST
pUC119, slyAST::aph
pCVD442, slyAST::aph
14
This study
This study
Choleraesuis and S. serovar Typhimurium were con-
structed and the decrease of virulence from these muta-
tions were compared.
The slyA-defective mutants were constructed by the
method of homologous recombination. The slyA genes
of S. serovar Choleraesuis RF-1 and S. serovar
Typhimurium SR-11 were amplified by PCR and ligated
into pUC119 as described in a previous paper (14). The
kanamycin-resistance cassette gene (aph) was prepared
from pUC4K by BamHI digestion, blunted, and inserted
into RsaI site of slyA gene to form pKAD302 and
pKAD309. The mutated slyA genes (slyA::aph) were
prepared from pKAD302 and pKAD309 by BamHI and
EcoRI digestions, blunted, and ligated into SmaI site of
the suicide vector pCVD442 to form pKAD306 and
pKAD313.
Conjugation and allelic exchange was performed
using pKAD306 and pKAD313 by the conventional
method (26), and putative transconjugants were desig-
nated KAD001 and KAD101. The bacterial strains and
plasmids used or prepared in this study are summarized
in Table 1. The chromosomal DNA preparations of these
transconjugants were digested, and the insertion of the
aph gene was examined by Southern hybridization.
When the 40 bp (from 318 to 357) of slyA gene from S.
serovar Typhimurium (15) was used as a DNA probe,
increase of molecular size of the slyA-containing frag-
ments was observed for both transconjugant strains,
Fig. 1. Southern hybridization of wild-type and slyA mutant
strains by the 40 bp DNA probe from slyA gene. Chromosomal
DNA prepared from each strain was digested with NotI. Lanes 1,
RF-1; 2, KAD001; 3, SR-11; 4, KAD101.
KAD001 and KAD101 as shown in Fig. 1. These results
indicated that slyA genes of the parent strains were
knocked out by the aph gene insertion. By the slyA
mutation, colony morphology, polysaccharide length of

NOTES
111
LPS, O and H antigenicity, and growth rate in LB broth
were found to be unchanged.
Table 2. Effect of the slyA mutation on Salmonella virulence
Strain
Genotype
LD50(CFU/mouse)
The parent (wild-type) strains and the slyA-defective
strains were grown in LB broth and inoculated to
BALB/c mice (female, 5 weeks old, CLEA Japan,
Tokyo) intraperitoneally (5 mice/dose) according to the
method described previously (20). The LD50s of the
strains were determined by the method of Beherns-Kar-
ber (6). All animal experiments were done in accordance
with the Kitasato University Guideline for Animal Care
and Experimentation. As shown in Table 2, LD50 of S.
serovar Typhimurium was much lower than that of S.
serovar Choleraesuis as reported previously (20). The
LD50 values in this study are generally higher than those
reported in the previous paper (20), but these differ-
S. serovar Choleraesuis
2
RF-1
KAD001
wild type
slyASC::aph
7.6×10
7
5.7×10
S. serovar Typhimurium
1
SR-11
wild type
1.9×10
6
KAD101
slyAST::aph
1.1×10
Salmonella strains grown overnight in LB broth were recov-
ered by centrifugation and resuspended in sterile PBS ( ) to
―
1
8
approximately 5×10 to10 CFU/ml. The actual number of
bacteria present was determined by plating on agar medium.
BALB/c mice were challenged intraperitoneally (0.2 ml of
bacterial suspension), and observed for 14 days.
Fig. 2. Course of infection of wild-type and slyA mutant strains. A: S. serovar Choleraesuis KAD001 (dotted line) and RF-1(solid line).
4
B: S. serovar Typhimurium KAD101 (dotted line) and SR-11(solid line). Mice were inoculated intraperitoneally with 1×10
CFU/mouse, and sacrificed on the days indicated. Livers (□, ■), spleens (△, ▲) and intestinal lymph nodes (○, ●) were dissected
and homogenized, and the number of bacteria in each organ was determined by plating homogenates onto LB agar. Asterisks indicate
the death of mice.

1
12
A. KANEKO ET AL
ences might arise from the difference of mice age used in
the experiments. By the mutation in slyA, LD50 of S.
serovar Choleraesuis was increased in the order of 10 ,
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