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Infection and Immunity, May 2000, p. 2720-2727, Vol. 68, No. 5
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
The shdA Gene Is Restricted to Serotypes of
Salmonella enterica Subspecies I and Contributes to
Efficient and Prolonged Fecal Shedding
Robert A.
Kingsley,
Karin
van Amsterdam,
Naomi
Kramer, and
Andreas J.
Bäumler*
Department of Medical Microbiology and
Immunology, College of Medicine, Texas A&M University Health
Science Center, College Station, Texas 77843-1114
Received 12 November 1999/Returned for modification 4 January
2000/Accepted 10 February 2000
 |
ABSTRACT |
Little is known about factors which enable Salmonella
serotypes to circulate within populations of livestock and domestic fowl. We have identified a DNA region which is present in
Salmonella serotypes commonly isolated from livestock and
domestic fowl (S. enterica subspecies I) but absent from
reptile-associated Salmonella serotypes (S. bongori and S. enterica subspecies II to VII). This DNA region was cloned from Salmonella serotype Typhimurium
and sequence analysis revealed the presence of a 6,105-bp open reading frame, designated shdA, whose product's deduced amino acid
sequence displayed homology to that of AIDA-I from diarrheagenic
Escherichia coli, MisL of serotype Typhimurium, and IcsA of
Shigella flexneri. The shdA gene was located
adjacent to xseA at 52 min, in a 30-kb DNA region which is
not present in Escherichia coli K-12. A serotype Typhimurium shdA mutant was shed with the feces in reduced
numbers and for a shorter period of time compared to its isogenic
parent. A possible role for the shdA gene during the
expansion in host range of S. enterica subspecies I to
include warm-blooded vertebrates is discussed.
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INTRODUCTION |
Salmonella serotypes are
a frequent constituent of the intestinal flora of poikilothermic
animals. The percentage of apparently healthy, cold-blooded vertebrates
which harbor Salmonella serotypes ranges from 74 to 94%
(20, 28, 32, 34, 59), and these bacteria could thus be
considered part of the normal intestinal flora (23, 48).
Salmonella serotypes are also commonly isolated from a
fraction (usually <20%) of warm-blooded animal hosts (15, 31,
49, 54). Although chronic carriers, which appear healthy, are
observed within the human population and among warm-blooded animals
(22, 27, 35, 40), Salmonella serotypes are
commonly associated with illness in these hosts (55).
Consequently Salmonella serotypes are regarded as pathogens
rather than part of the normal intestinal flora of homeothermic animals.
On the basis of multilocus enzyme electrophoresis and comparative
sequence analysis of orthologous genes, two species, S. enterica and S. bongori, have been assigned to the
genus Salmonella (18, 46). S. enterica
is further subdivided into seven subspecies designated with roman
numerals (18, 44). While S. bongori and S. enterica subspecies II, IIIa, IIIb, IV, VI, and VII are mainly
associated with cold-blooded vertebrates, members of S. enterica subspecies I are frequently isolated from avian and
mammalian hosts (44). For instance, of the 90,201 Salmonella isolates collected between 1977 and 1992 by the
German National Reference Center for Enteric Pathogens from humans and
warm-blooded animals, 89,798 isolates (99.55%) belonged to S. enterica subspecies I (1). Currently it is not clear
which virulence mechanisms are responsible for the apparent adaptation
of S. enterica subspecies I to circulation within
populations of warm-blooded animals.
S. bongori or S. enterica subspecies II to VII
are able to infect humans, colonize the intestine and cause disease
(1). Human infections with serotypes of S. bongori and S. enterica subspecies II to VII are rare
and are usually the result of contact with reptiles (21, 29, 42,
60). The symptoms of intestinal and extraintestinal infections
caused by reptile-associated Salmonella serotypes in humans
are, however, indistinguishable from those produced by nontyphoidal
serotypes of S. enterica subspecies I (1). These
data demonstrate that S. bongori or S. enterica subspecies II to VII are pathogenic for humans and suggest that the
scarcity of clinical cases of illness is a result of the absence of
these serotypes from animals from which we draw our food supply. This
raises the question as to which genetic traits enable serotypes of
S. enterica subspecies I to establish themselves in
populations of livestock or domestic fowl. Theoretical models to
describe the general principles concerning the ability of pathogens to invade, persist, and spread within a host population are well developed
(3). In order to become established in populations of
domesticated animals, a pathogen must generate on average more than one
secondary case of infection from a primary case. The average number of
animals in a susceptible host population which become infected from a
single case can be defined as the basic case reproductive number,
R0 (3). The basic case reproductive number of S. enterica subspecies I serotypes for higher
vertebrates must therefore be greater than one, since these pathogens
circulate in warm-blooded host populations. The absence of S. bongori and S. enterica subspecies II to VII serotypes
from populations of livestock or domestic fowl, on the other hand,
suggests that their basic case reproductive number for higher
vertebrates is less than one, a property apparently independent of
their ability to cause illness in these hosts (1). Thus, an
expansion in host range may have involved the acquisition of one or
more genetic determinants by a common ancestor of S. enterica subspecies I which increased the basic case reproductive
number (but not necessarily the lethality) of this organism for
warm-blooded vertebrates. To predict how acquisition of new genetic
material by a common ancestor of the S. enterica subspecies
I lineage may have contributed to its expansion in host range, it is
helpful to apply theoretical models which combine epidemiology with
population biology (3). In the case of direct transmission
(by the fecal-oral route or any other route), the basic case
reproductive number of a pathogen is directly proportional to the
duration, D, for which an infected host can transmit the
disease; the probability, 
, at which the disease is transmitted from
an infected animal to a susceptible host; and the density of
susceptible hosts, X (2):
|
(1)
|
An infected host can transmit the disease until it either dies of
natural causes (at the natural mortality rate, b), is killed by the pathogen (at the disease-induced mortality rate, 
), or is
able to clear the infection (at the clearance rate, 
)
(3). Thus the average lifespan of an infectious host,
D, can be described as follows:
|
(2)
|
After combining equations 1 and 2 it becomes clear that a
reduction of either the clearance rate, , or the disease induced mortality rate, , will result in an increase in the basic case reproductive number, R0, of a pathogen:
|
(3)
|
There is no evidence that members of S. enterica
subspecies I are less virulent or cause lower mortality rates in
warm-blooded hosts than serotypes of S. bongori or S. enterica II to VII. However, it is possible that a common ancestor
of the S. enterica subspecies I lineage may have increased
its basic case reproductive number for warm-blooded animals by reducing
the rate at which the infection is cleared from the feces. The genetic
determinants responsible for this phenotype are expected to be present
in S. enterica subspecies I but absent from serotypes of
S. bongori and S. enterica subspecies II to VII.
Here we describe the identification of a gene, termed shdA,
which is specific to S. enterica subspecies I serotypes and
study its role in fecal shedding during S. enterica serotype
Typhimurium infection of mice. This analysis is relevant for human
health, since little is known about virulence determinants required for circulation of enteric pathogens within animal reservoirs from which we
draw our food supply.
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MATERIALS AND METHODS |
Bacterial strains and culture conditions.
Strains CL1509
(aroA::Tn10), IR715 (virulent nalidixic
acid resistant derivative), AJB82
(aroA::Tn10
invA::TnphoA) and AJB75 (IR715
invA::TnphoA) are derivatives of
serotype Typhimurium strain ATCC 14028 (10, 56, 57). The
Salmonella reference B and SARC collections have been
published recently (17, 18). Escherichia coli
strains S17-1 
pir, and DH5 are described elsewhere
(26, 53). Strains were cultured aerobically at 37°C in
Luria-Bertani (LB) broth supplemented with the following antibiotics as
appropriate at the indicated concentrations: carbenicillin, 100 mg/liter (LB+Cb); chloramphenicol, 30 mg/liter (LB+Cm); tetracycline,
20 mg/liter (LB+Tc); kanamycin 60 mg/liter (LB+Km); or nalidixic acid,
50 mg/liter (LB+Nal).
Southern hybridization.
Isolation of genomic DNA and
Southern transfer of DNA onto a nylon membrane was performed as
recently described (5). Hybridization was performed at
65°C in solutions without formamide. Two 15-min washes were performed
under nonstringent conditions at room temperature in 2× SSC (1× SSC
is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% sodium dodecyl
sulfate. Labeling of DNA probes and detection of nucleotide sequences
were performed using the labeling and detection kit (nonradioactive)
from NEN.
Cloning of shdA.
A cosmid bank of serotype
Typhimurium strain ATCC 14028 constructed in pLAFR2 and propagated in
E. coli LE392 has been described previously (38).
The bank was spread on LB+Tc plates, and 456 colonies were picked and
grown individually overnight. Cosmid DNA was prepared from 19 pools,
each containing 24 overnight cultures. Each pool was digested with
EcoRI, separated on an agarose gel and hybridized with probe
p5A8. The DNA of one pool hybridized with probe p5A8. The 24 strains
representing this pool were then grown individually, and cosmid DNA was
isolated, digested with EcoRI, and separated on an agarose
gel. Southern hybridization was performed to identify a cosmid
hybridizing with probe p5A8. Plasmid DNA for sequencing was isolated
using ion-exchange columns from Qiagen. Sequencing was performed by the
dideoxy chain termination method (50), using an AutoRead
Sequencing Kit (Pharmacia) and an ALF automatic sequenator. The
nucleotide sequences were analyzed using the MacVector 6.0.1 software
package (Oxford Molecular Group).
Construction of mutants.
Bacteriophage P22 HT105/1
int
was used for generalized transduction of
the aroA::Tn10 marker from serotype
Typhimurium strain CL1509 into RAK1 or AJB75 (10).
Transductants were routinely purified from contaminating phage by
streaking the strain twice for single colonies on Evans blue uridine
plates (16). Subsequently, strains were tested in a cross
streak for P22 sensitivity. Transductants were tested for growth on M9
minimal medium agar plates and on minimal medium agar plates
supplemented with aromatic amino acids (39). For
construction of a shdA mutant, a 3-kb fragment of cosmid
pRK824 was cloned into pBluescript KS(
) (52) to give rise
to plasmid pRA38. A chloramphenicol acetyltransferase (cat) gene was ligated into the BamHI site of the shdA
open reading frame cloned in pRA38 (see Fig. 3). The insert of the
resulting plasmid (pRA55) was excised with EcoRI and
SalI and cloned into the EcoRI- and
SalI-restricted suicide vector pGP704 (33) to give rise to plasmid pRA56. Exconjugants of a mating between serotype Typhimurium strain IR715 and E. coli strain S17-1
pir(pRA56) were selected on LB+Cm+Nal plates. An
exconjugant which was resistant to chloramphenicol
(shdA::cat allele) but sensitive to
carbenicillin (through loss of pGP704) was identified by patching
individual colonies on LB+Cb plates and was termed RAK1.
Animal experiments.
Six- to eight-week-old female BALB/c
(ByJ; Jackson Laboratory) mice were used throughout this study.
Bacteria were routinely cultured as standing overnight cultures prior
to infection. In all experiments the bacterial titer of the inoculum
was determined by spreading serial 10-fold dilutions on agar plates
containing appropriate antibiotics and determining the number of CFU.
The intestinal organ culture model has been described previously
(
7). The intestine was ligated at the distal end, filled
with 1 ml of a bacterial suspension containing 5 × 10
7 to 8 × 10
7 CFU of a mixture of the
two competing strains, and then ligated
at the proximal end and
incubated for 30 min at 37°C in 5% CO
2.
Nonadherent
bacteria were removed by five washes in phosphate-buffered
saline
(PBS), and sections of intestinal wall were homogenized
in 5 ml of PBS.
Dilutions were spread on LB plates containing
the appropriate
antibiotics. Experiments were repeated with organs
from three different
animals.
For competitive infection experiments, groups of four mice were
infected by oral gavage with an approximately 1:1 mixture
of mutant and
isogenic parent strains at a dose of approximately
10
9
CFU/mouse. Fecal pellets were collected daily and homogenized
in 1 ml
of PBS. The limit of detection was approximately 0.08
CFU/mg of feces.
Dilutions of fecal pellets were plated on LB
plates containing the
appropriate antibiotics. Data were normalized
by dividing the output
ratio (CFU of mutant/CFU of wild type)
by the input ratio (CFU of
mutant/CFU of wild type). In case only
one bacterial strain was
recovered from fecal pellets, the limit
of detection was determined for
the missing strain and used to
calculate of a minimum mutant/wild type
ratio. All data were converted
logarithmically prior to the calculation
of averages and statistical
analysis. Student's
t test was
used to determine whether the mutant/wild
type ratio in specimens
recovered from infected organs or fecal
pellets was significantly
different from the mutant/wild type
ratio present in the
inoculum.
For single infection experiments, groups of 12 BALB/c mice were
inoculated with 10
9 CFU of either CL1509
(
aroA::Tn
10-tet
r) or RAK7
(
aroA::Tn
10-tet
r
shdA::Cm
r). The presence of inoculum
strain in fecal pellets was determined
on 29 days during the first 79 days postinoculation (days 1 to
16, 18, 21, 24, 27, 31, 34, 37, 44, 51, 58, 65, 72, and 79 postinoculation).
Approximately 20 mg of fresh fecal
pellets were resuspended in
PBS (pH 7.4), and bacteria were enumerated
on LB agar plates containing
tetracycline (20 µg/ml) or tetracycline
(20 µg/ml) and chloramphenicol
(50 µg/ml). The presence or absence
of the test strain in fecal
pellets was scored for each mouse (limit of
detection, 0.1 CFU/mg
of feces). The proportion of mice in each
inoculum group on each
day tested that contained serotype Typhimurium
in fecal pellets
was subjected to statistical analysis using the
Wilcoxon's signed-rank
test (
n = 29).
The 50% lethal morbidity dose (LD
50) of serotype
Typhimurium mutants was estimated by infecting groups of four mice
intragastrically
with serial 10-fold dilutions of bacterial cultures in
a 0.2-ml
volume. Lethal morbidity was recorded at 28 days
postinfection,
and the estimated LD
50 was calculated by the
method of Reed and
Muench (
45).
| |
RESULTS |
Identification of a DNA region restricted to S. enterica subspecies I.
We have recently screened a bank of
400 S. enterica serotype Gallinarum Mud-Cam transposon
mutants for virulence in day-of-hatch White Leghorn chicks. Virulence
data obtained for individual mutants during the initial screen were
inconsistent with experiments performed using this animal model to
confirm attenuation of individual mutants. We determined the
inconsistency of data to be the result of the antibiotic history from
battery-reared chicks. The fact that virulence defects could not be
confirmed for mutants identified in this screen prompted us to
discontinue the study. However, prior to this, the DNA flanking the
Mud-Cam insertion was cloned from one mutant, labeled, and used as a
probe to determine its phylogenetic distribution. Southern blot
analysis was performed with genomic DNA prepared from SARC
(18). This collection consists of 16 Salmonella
serotypes representing S. bongori (formerly S. enterica subspecies V) and S. enterica subspecies I to
VII. The 500-bp p5A8 DNA probe, derived from mutant G5A8, hybridized
with genomic DNA of strains from subspecies I, but no hybridization
signal was obtained with genomic DNA from isolates of S. bongori or S. enterica subspecies II to VII. Since the
host range of S. enterica subspecies I differs from that of
S. bongori or S. enterica subspecies II to VII we
decided to further characterize this DNA region.
A cosmid (pRK824) containing the
S. enterica subspecies I
specific DNA region was cloned from an
S. enterica serotype
Typhimurium
bank (
38) by hybridization with probe p5A8.
Restriction analysis
of cosmid pRK824 indicated that it carried an
insert of approximately
28 kb. To confirm that the cloned DNA region
was restricted to
S. enterica subspecies I, a 3-kb
ClaI restriction fragment hybridizing
with probe p5A8 was
cloned to give rise to plasmid pRA38. The
DNA probe derived from
plasmid pRA38 hybridized with genomic DNA
from
S. enterica
subspecies I but not genomic DNA from
S. bongori or
S. enterica subspecies II to VII (Fig.
1). To determine the
distribution of the
pRA38 DNA probe within
S. enterica subspecies
I, we used the
SARB collection, which includes 72 strains representing
37 serotypes of
S. enterica subspecies I (
17). A DNA probe
derived
from plasmid pRA38 hybridized with 69 of the 72 strains of SARB
collection. Southern blot analysis of 21 representative strains
from
SARB collection is shown in Fig.
1. No hybridization signal
was
obtained with genomic DNA from strains Dt1 (
S. enterica
serotype
Decatur), Ts3 (
S. enterica serotype Typhisuis) and
En2 (
S. enterica serotype Enteritidis). The multilocus
enzyme electrophoresis profile
of strain En2 is only distantly related
to that of other
S. enterica serotype Enteritidis clones
(En1, En3, and En7) which are present
in SARB and do hybridize with
pRA38 (
17).
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FIG. 1.
Phylogenetic distribution of the shdA gene
within the genus Salmonella. Southern blot analysis using
representative serotypes of S. enterica (subspecies are
indicated by roman numerals) and S. bongori is shown.
Genomic DNA prepared from the serotypes indicated on the left (strain
designations are indicated in parentheses) was hybridized with DNA
probes pRA58 (left panel) and pRA38 (right panel). The location of
these DNA probes (closed bars) relative to xseA and
shdA (arrows) is indicated on the map shown at the top.
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The shdA gene is located in the xseA-hisS
intergenic region.
The cosmid (pRK824) contained at least one
border of the subspecies I specific DNA region, since a DNA probe
(pRA58) generated from a 1.6-kb ClaI restriction fragment
hybridized with all serotypes of the SARC and SARB collections (Fig.
1). Nucleotide sequence analysis of pRA58 revealed that it contained
the 3' end of the serotype Typhimurium xseA gene, which
encodes the large subunit of exonuclease VII. Since this gene is shared
by both E. coli and serotype Typhimurium, it is likely to
account for the hybridization signal observed with the S. bongori or S. enterica subspecies II to VII serotypes.
Sequence homology between the E. coli and serotype
Typhimurium sequence ended 21 bp prior to the 3' end of
xseA, which defined the left end of the S. enterica subspecies I-specific DNA region. In the sequence of the
E. coli K-12 genome, the xseA gene is located
between the guaAB and hisS loci at 54 min
(14). However, genetic mapping data suggest that in serotype Typhimurium, guaAB and hisS are separated by a
30-kb DNA region which is absent from E. coli (Fig.
2) (47). Our data are
consistent with the presence of the shdA gene on a genetic
island present in the xseA-hisS intergenic region, which may
be as large as 30 kb.
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FIG. 2.
(Top) Comparison of the nucleotide sequences from
E. coli (E.c.) and S. enterica serotype
Typhimurium (S.t.) at the left boundary of the island. A putative
termination loop located downstream of the shdA gene is
indicated by arrows. (Bottom) Comparison of the genetic maps of
E. coli and S. enterica serotype Typhimurium
flanking the xseA gene. An approximately 30-kb DNA loop in
the guaAB-hisS intergenic region, which is present in
serotype Typhimurium but absent from E. coli, has been
described by Riley and Krawiec (47) and is shown as an open
bar.
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The nucleotide sequence analysis was extended to include a total of
6,831 bp (GenBank accession no.
AF091269). A single
open reading frame
of 6,105 bp transcribed in the opposite direction
to
xseA
was identified and designated
shdA. A putative termination
loop was identified downstream of the translational stop codon
of
shdA (stem, bp 6482 to 6495; loop, bp 6496 to 6499; stem, bp
6500 to 6514). The C-terminal domain (477 amino acids) of the
predicted
ShdA protein exhibited homology to the C-terminal 440
amino acids of
AIDA (34% identity) from diffuse adhering
E. coli (
11), the C-terminal 501 amino acids of MisL (36% identity)
from serotype Typhimurium (
13), and the C-terminal 353 amino
acids of IcsA (VirG) (30% identity) from
S. flexneri
(
12,
36).
The C terminus also contained five copies of a
12-amino-acid repeat,
which exhibited no homology to sequences in
available databases
(Fig.
3). Using the
SignalP program (41a), a putative signal peptide
was identified at the
amino terminus of ShdA. Similar to AIDA,
MisL, and IcsA, the signal
peptide was atypical, with the predicted
cleavage site (indicated by a
slash) following the alanine residue
at position 60 (LAMA/DNQV). The
N-terminal domain of ShdA (amino
acids 61 to 1558) did not exhibit
homology to sequences in GenBank
and contained nine copies of a
63-amino-acid repeat and three
copies of a 102-amino-acid repeat (Fig.
3).
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FIG. 3.
(Top) Pustell alignment of the ShdA amino acid sequence
against itself (window size, 10; minimum identity, 60%). Lines
parallel to the diagonal identified direct amino acid repeats. A
predicted signal peptide and the C-terminal domain, which has homology
to AIDA-I, MisL, and IcsA, are indicated in the ShdA primary structure
shown as an arrow below the Pustell alignment. The positions of nine
copies of a 63-amino-acid repeat (hatched bars numbered 1 to 9) and
three copies of a 102-amino-acid repeat (closed bars numbered I to III)
are indicated in the N-terminal domain of ShdA. The location of four
direct repeats of a 12-amino-acid sequence in the C-terminal domain of
ShdA are indicated (A-D). (Bottom) A CLUSTAL alignment of repeats I to
III, 1 to 9, and A to D is shown. Identical residues (shaded boxes) and
residues with similar biochemical properties (open boxes) are
indicated.
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Effect of a mutation in shdA on the disease-induced
mortality rate.
A serotype Typhimurium strain in which the
shdA open reading frame was disrupted by insertion of a
cat gene was constructed by allelic exchange and was
designated RAK1. The insertion mutant was confirmed by Southern blot
analysis of chromosomal DNA prepared from RAK1 and hybridization with
the pRA38 DNA probe (data not shown). It is unlikely that insertional
inactivation of shdA resulted in a polar effect on the
expression of the adjacent xseA gene, since the
shdA and xseA genes are transcribed in opposite
orientations. The LD50 of strain RAK1 and that of its
isogenic parent (IR715) were determined and found to be identical.
These data show that mutational inactivation of shdA did not
alter the disease-induced mortality rate during serotype Typhimurium
infection of mice.
Effect of a mutation in shdA on fecal shedding.
We
determined the contribution of shdA to bacterial shedding in
the mouse typhoid model of serotype Typhimurium infection. Serotype
Typhimurium causes lethal signs of disease in mice starting at day 5 postinfection. Thus, in order to study fecal shedding beyond day 5 postinfection, strain RAK1 (shdA) was attenuated for mouse
virulence by introducing a mutation in aroA. Serotype Typhimurium aroA mutants are able to attach to and invade
the intestinal mucosa and colonize deeper tissues but are unable to multiply rapidly at these sites. Since bacterial shedding results from
bacterial colonization of an animal, we reasoned that inactivation of
aroA was unlikely to mask the effect of other genes on
shedding. To assess the effect of a mutation in shdA on
bacterial shedding, a group of four mice was infected with equal
numbers of CL1509 (aroA) and RAK7 (shdA aroA)
bacteria, and the bacteria were recovered from fecal pellets on
subsequent days. This analysis revealed that a mutation in
shdA significantly decreased the number of serotype
Typhimurium organisms shed in the feces (P < 0.01 at day 6 postinfection). The experiment was discontinued at day 6 postinfection, when RAK7 (shdA aroA) was not recovered from
the fecal pellets of three mice, while strain CL1509 (aroA)
was still shed with the feces of three animals.
The experiment was repeated with a group of six mice, and shedding was
monitored until day 35 postinfection. All mice shed
the inoculum on day
1 postinoculation, but on subsequent days
shedding was intermittent and
some animals cleared the inoculum.
Again, CL1509 (
aroA) was
recovered in significantly higher numbers
from fecal pellets than RAK7
(
shdA aroA). More importantly, CL1509
was shed for a longer
period than the
shdA mutant (RAK7). Figure
4 shows the combined results of both
shedding experiments. These
data show that a mutation in
shdA reduced the duration of bacterial
shedding, which is
indicative of an increased rate by which the
host could clear serotype
Typhimurium from intestinal contents
shed with the feces. The shedding
defect attributed to the
shdA mutation was confirmed using
single inoculation of groups of 12
mice with 10
9 CFU of
either RAK7 (
aroA shdA) or the CL1509 (
aroA)
parental
strain. Shedding in fecal pellets was scored for the presence
or absence of
Salmonella in each inoculum group on 29 occasions
over a 79-day period postinoculation. On 14 occasions during
this
period, a greater number of mice inoculated with the parental
strain (CL1509) were shedding serotype Typhimurium than were those
inoculated with the
shdA mutant (RAK7). The opposite was
true
on only three occasions. Statistical analysis of these data using
the Wilcoxon signed-rank test indicated that the
shdA mutant
was
cleared significantly earlier than the parental strain
(
P < 0.01).
Overall, these data suggest that a
mutation in
shdA increased
the clearance rate of serotype
Typhimurium from murine feces.
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FIG. 4.
Recovery of bacteria from fecal pellets collected after
inoculation of mice with an equal mixture of CL1509 (aroA)
and RAK7 (shdA aroA). Data for days 1 to 6 were from 10 mice, and data for subsequent days were from 6 mice. (A) For each
mouse, the output ratio (RAK7/CL1509) was determined daily. Data were
converted logarithmically and are given as means ± standard
errors (error bars). An asterisk below an error bar indicates that the
RAK7/CL1509 output ratio was significantly different (P < 0.05) from that present in the inoculum. (B) Total numbers of
CL1509 (open circles) and RAK7 (closed circles) recovered from fecal
pellets of mice. The limit of detection (1.2 × 101
CFU/mg of feces) is indicated by a broken line. Each circle represents
data for one strain from one animal. Animals for which no CFU of either
CL1509 or RAK7 were detectable are indicated below the broken line
along the x axis.
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Effect of a mutation in invA on fecal shedding.
During competitive infection experiments, serotype Typhimurium strains
carrying a mutation in invA are absent from feces more frequently than the wild type on days 3 and 5 postinoculation (10). However, the shedding defect previously reported for
invA mutants was based on observation restricted to two
occasions postinoculation. Observations from shedding experiments
described above indicated that shedding is highly variable from day to
day. In order to investigate whether a serotype Typhimurium strain
containing a mutation in invA has a similar shedding defect
to that observed for strains containing a mutation in shdA,
eight mice were inoculated with an equal mixture of strain AJB82
(invA aroA) and its isogenic parental strain (CL1509). Fewer
mice shed the invA mutant and at lower numbers at earlier
time points. However, the opposite was observed at later time points
(Fig. 5). Although a mutation in
invA reduced bacterial shedding at early times
postinfection, there was no evidence for an effect on bacterial
clearance from the feces at the end of the experiment.
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FIG. 5.
Recovery of bacteria from fecal pellets collected after
inoculation of mice with an equal mixture of CL1509 (aroA)
and AJB82 (invA aroA). (A) For each mouse, the output ratio
(RAK82/CL1509) was determined daily. Data were converted
logarithmically and are given as means ± standard errors (error
bars). An asterisk below an error bar indicates that the RAK82/CL1509
output ratio was significantly different (P < 0.05)
from that present in the inoculum. (B) Total numbers of CL1509 (open
circles) and RAK82 (closed circles) recovered from fecal pellets of
mice. The limit of detection (1.2 × 101 CFU/mg of
feces) is indicated by a broken line. Each circle represents data for
one strain from one animal. Animals for which no CFU of either CL1509
or RAK82 were detectable are indicated below the broken line along the
x axis.
|
|
A mutation in shdA does not affect colonization of the
villous intestine or Peyer's patches in an intestinal organ culture
model.
It is known that invA is required for invasion
of the mucosal epithelium, particularly at the Peyer's patches
(25). To compare the contributions of shdA and
invA to colonization of the small intestine, we used the
intestinal organ culture model (7). Equal numbers of RAK1
(shdA) and its parent (IR715) were injected into loops
formed from fresh mouse ileum, and following a 30-min incubation
period, CFU of each strain were enumerated in the villous intestine and
Peyer's patch regions. No significant difference in colonization of
these tissues by the shdA strain and parental strain was
observed. In a second experiment, an equal mixture of a serotype
Typhimurium invA mutant (AJB75) and its isogenic parent
(IR715) was inoculated into ligated intestinal loops. The invA mutant (AJB75) was recovered in significantly lower
numbers from Peyer's patches than the parental strain, thus confirming the role of SPI1 in colonizing this organ.
| |
DISCUSSION |
A primary pathogen can be defined as an organism which is capable
of entering a host, finding a unique niche in which to multiply, avoiding or subverting the host defenses, and being transmitted to
a susceptible host (24). All members of the genus
Salmonella fit this description, as they are pathogenic for
humans (1). However, serotypes of S. enterica
subspecies I differ from S. bongori and S. enterica subspecies II to VII serotypes with regard to animal
reservoir. While human infections with S. bongori and S. enterica subspecies II to VII are rare and result from
contact with reptiles (21, 29, 42, 60), serotypes of
S. enterica subspecies I are frequently associated with
disease, and most cases can be traced back to livestock or domestic
fowl (4, 41). Thus, it could be speculated that serotypes of
S. enterica subspecies I possess one or more genes which
enable these pathogens to invade, persist, and spread within
warm-blooded host populations, thereby resulting in their introduction
into food items originating from domesticated animals. S. bongori and S. enterica subspecies II to VII, on the
other hand, lack these genes and are unable to circulate in populations
of livestock and domestic fowl.
We characterized shdA, a gene encoded on a genetic island
which is present in serotypes of S. enterica subspecies I. Unlike previously identified virulence gene clusters, such as SPI1,
SPI2, SPI3, agf, fim, lpf, and
spv, the shdA gene was absent from lineages other
than subspecies I (6, 13, 19, 30, 37). Although virulence
determinants, which are restricted to subspecies I have been identified
previously, these are present in only a small number of serotypes. For
instance, the SARB collection which consists of 72 strains from
S. enterica subspecies I, contains 3 isolates carrying the
viaB region, 10 isolates possessing the sef
operon, and 9 isolates hybridizing with the pef operon
(6, 51). In contrast, shdA was present in 69 of
the 72 strains of the SARB collection, suggesting that it was acquired
early in the divergence of the S. enterica subspecies I
lineage (Fig. 1). It has been postulated that the ability of S. enterica subspecies I serotypes to circulate in populations of
warm-blooded animals is a new trait, since extant serotypes of all
other phylogenetic lineages within the genus Salmonella are
associated with cold-blooded vertebrates (8, 43). Our data
suggest that this expansion in host range to include warm-blooded
vertebrates was accompanied by acquisition of the shdA gene
by a common ancestor of S. enterica subspecies I.
Our results show that mutational inactivation of shdA
resulted in recovery of serotype Typhimurium at lower numbers and for a
shorter period of time from murine fecal pellets than its isogenic parent (Fig. 4). It is unlikely that shdA is the only factor
involved in prolonged fecal shedding of serotype Typhimurium from mice. Indeed, previous studies have shown that Typhimurium strains containing a mutation in invA are less likely to be recovered from
fecal pellets of mice at days 3 and 5 postinfection than their isogenic parent (10). However, comparison of the shedding defects of strains AJB82 (invA) and RAK7 (shdA) demonstrated
that inactivation of shdA reduced bacterial shedding at
later time points (day 11 postinfection or subsequent days) to a
greater extent than a mutation in invA (Fig. 4 and 5).
Another group of virulence determinants previously implicated in
bacterial shedding are fimbrial adhesins of serotype Typhimurium. A
serotype Typhimurium agf pef fim lpf mutant is recovered in
significantly lower numbers from fecal pellets at 5 days postinfection
than the isogenic wild type during a competitive infection experiment
(58). Thus, inactivation of genes required for attachment to
or invasion of the intestinal mucosa may result in reduced bacterial
shedding. While attachment of serotype Typhimurium to the murine small
intestine can be detected using ligated ileal loops (7, 9),
mutational inactivation of shdA did not reduce bacterial
numbers recovered from this model (Fig.
6A). In contrast, a serotype Typhimurium
invA mutant colonized Peyer's patches at reduced levels in
the organ culture model, suggesting that bacterial invasion can be
detected using this assay (Fig. 6B). These data suggest that unlike
mutations in fimbrial biosynthesis or invasion genes, the shedding
defect of a shdA mutant was not caused by decreased
bacterial attachment to or invasion of the mucosa of the murine small
intestine but may be due to a different mechanism.
View larger version (11K):
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|
FIG. 6.
Recovery of bacteria from the intestinal organ culture
model. Intestinal loops were infected with an equal mixture of RAK1
(shdA) and IR715 (wild type) (A) or AJB75 (invA)
and IR715 (wild type) (B). The output ratios were determined for
Peyer's patches (PP) and villous intestine (VI). Data were converted
logarithmically and are given as means ± standard errors (error
bars).
|
|
A mutation in invA or the simultaneous inactivation of the
agf, pef, fim, and lpf
operons results in a 50- and 26-fold attenuation of serotype
Typhimurium for mouse virulence, respectively (25, 58). The
attenuating effect of these mutations suggests that the corresponding
attachment or invasion genes increase the disease-induced mortality
rate, , which is expected to result in a reduction of the basic case
reproductive number, R0 of serotype Typhimurium (equation 3). At the same time, however, invA,
agf, pef, fim, and lpf may
reduce the clearance rate, , which would be predicted to increase
the basic case reproductive number. It is therefore difficult to
predict whether the net result of expressing fimbriae or invasion genes
is an increase or a decrease in the basic case reproductive number of
serotype Typhimurium. In contrast, mutational inactivation of
shdA did not reduce the disease-induced mortality rate, ,
but decreased the duration of shedding (Fig. 4). This phenotype is
consistent with a role of shdA in decreasing the clearance
rate, , thereby resulting in an increase in the basic case
reproductive number, R0, of serotype Typhimurium
(equation 3). The phylogenetic distribution of shdA and its
predicted effect on the basic case reproductive number are consistent
with the idea that acquisition of this gene may have contributed to the expansion in host range of S. enterica subspecies I to
include warm-blooded animals.
| |
ACKNOWLEDGMENTS |
We are grateful to Renée Tsolis for helpful suggestions on
the manuscript and Kenneth Sanderson for providing strains from the
SARB and SARC collections.
Work in A.B.'s laboratory is supported by Public Health Service grants
AI40124 and AI44170 and grant 9802610 from the U.S. Department of Agriculture.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Medical Microbiology and Immunology, 407 Reynolds Medical Building,
College of Medicine, Texas A&M University Health Science Center,
College Station, TX 77843-1114. Phone: (409) 862-7756. Fax: (409)
845-3479. E-mail: abaumler{at}tamu.edu.
Editor:
A. D. O'Brien
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Abstract
Introduction
