FEMS Microbiology Letters 48 (1987) 229-233
Published by Elsevier
229
FEM 03002
Chromosomal-encoded siderophores are required
for mouse virulence of enteropathogenic Yersinia species
Jfirgen H e e s e m a n n
Institute for Medical Microbiology and Immunology, Universityof Hamburg, F.R.G.
Received 14 July 1987
Revision received 13 August 1987
Accepted 14 August 1987
Key words: Siderophore detection; Yersinia enterocolitica," Yersinia pseudotuberculosis; Mouse lethality;
Transposon mutagenesis
1. SUMMARY
Yersinia enterocolitica and Y. pseudotuberculosis
are enteropathogenic for humans. Essential virulence functions of these pathogens are determined
by a 40-mDa plasmid. Plasmid-bearing Y. pseudotuberculosis strains and Y. enterocolitica strains of
serotypes 0 : 8, 0 : 13, 0 : 20 and 0 : 40 are lethal for
mice. In contrast, human pathogenic Y. enterocolitica strains of serotype 0 : 3, 0 : 9 and 0 : 5.27 are
not mouse-lethal. Using a sensitive siderophore-indicator CAS-agar, we were able to detect siderophore production in all mouse-lethal Y. enterocolitica and Y. pseudotuberculosis strains mentioned
above. By Tn5-transposon insertions into the
chromosome of a serotype 0 : 8 strain we obtained
two siderophore-deficient mutants. Introduction
of the virulence plasmid did not render these
mutants mouse-lethal, indicating that siderophore
production is an essential virulence factor. The
h u m a n nonpathogenic, aerobactin-producing
strains of Y. intermedia, Y. kristensenii and Y.
Correspondence to: J. Heesemann, Institute for Medical Microbiology and Immunology, University of Hamburg, Hamburg,
F.R.G.
frederiksenii remained avirulent for mice after receiving the virulence plasmid of Y. enterocolitica.
Obviously the siderophore aerobactin does not
contribute to virulence in the genus Yersinia.
2. INTRODUCTION
Under iron-starvation many microorganisms release low-Mr compounds, termed siderophores,
which chelate iron. The resulting ferric-siderophore is trapped via siderophore receptors and is
subsequently transported into the cell. The importance of this kind of iron availability as a modulator of virulence of microorganisms has received
considerable experimental and speculative attention [1,2]. Recent studies on iron-assimilation and
virulence of the genus Yersinia have led to the
following results: (I) Siderophore activity could
not be detected with virulent Y. pestis, Y. pseudotuberculosis and Y. enterocolitica [3,4]. (II) Y. pseudotuberculosis and Y. enterocolitica can utilize
common siderophores like aerobactin, enterocholin and desferrioxamine [3,5,6]. (III) Y. pseudotuberculosis and the typical 'American' isolates of
Y. enterocolitica of serotypes 0 : 8, 0 : 13, 0 : 20 and
0378-1097/87/$03.50 © 1987 Federation of European Microbiological Societies
230
0:40 are lethal for mice [7,8]. (IV) The human
pathogenic Y. enterocolitica serotypes of 0 : 3, 0 : 9
and 0:5.27 (common for Europe) can become
lethal for mice, if the animals have been saturated
with iron or have been treated with desferrioxamine prior to challenge [6,9, Heesemann, in preparation]. (V) The mouse-lethal Y. enterocolitica
serotype 0 : 8 produces iron-regulated outer membrane proteins whereas the non-lethal serotypes of
0:3 and 0:9 do not [10]. (VI) Environmental
strains of Yersinia, namely Y. frederiksenii, Y.
intermedia and Y. kristensenii are able to produce
the siderophore aerobactin [4]. Paradoxically, these
strains are avirulent for humans and animals. From
these results, it was concluded that mouse-lethal
strains of Y. pseudotuberculosis and Y. enterocolitica may produce novel siderophores which have
not been recognized using common siderophore
assays [3,4]. Recently, a new universal method to
detect siderophores has been developed by using
the chrome azurol indicator agar [11]. Orange
halos around the colonies on blue (chrome azurol
iron complex) agar are indicative of siderophore
excretion by microorganisms. Using this method
we have screened various Yersinia-isolates for
siderophore production. Furthermore, these strains
have been assessed for lethal infections in mice.
As virulence of Yersinia is dependent on a 70-kb
plasmid, those strains of this study which were
originally plasmidless, received the virulene plasmid of Y. enterocolitica, serotype 0:8, via conjugation [12-15].
3. MATERIALS AND METHODS
3.1. Bacterial strains and plasmids
The Yersinia strains tested in this study are
listed in Table 1. For testing mouse-lethality the
strains Y-161M-Nal r, WA-1, WA-2, Y-NF-NaF,
Y. frederiksenii, Y. kristensenii and Y. intermedia
received the virulence plasmid of strain WA-314
via conjugational transfer [15]. All other strains
(with the exception of the plasmidless strain WAC-Nal r) harbored their original virulence plasmids. The Escherichia coli strains HB101 and
HB101 ::Tn5 have recently been described [17].
The mobilizable cloning vector pRK290B, the
Table 1
Association of mouse lethality with siderophore production
Yersinia, harboring
Strain
for
virulence plasmids
Serotype
Lethality
Siderophore
A 2628 a
0 :8
+
Y7P
0:8
+
+
WA a
WA-314 b
0:8
0 :8
+
+
+
+
W A - C - N a l r b.g
Y-161M-Nal rb
0 :8
0:8
--
+
-
]I. e n t e r o c o l i t i c a
a
+
WA-1 c
0:8
-
-
WA-2 c
0 :8
-
-
1209-79 ~
0 : 13
+
1223-75-1 ~
3973-76 a
0 : 20
0 : 40
+
+
Y-NF-Nal rb
0:5
-
Y-108 Nal rb
0:3
-
-
Y-96 Nal r b
0 :9
-
-
Y-5,27 d
0 : 5.27
--
--
Y. p s e u d o t u b e r c u l o s i s
Y-P-I d
Y-P-III *
Y. f r e d e r i k s e n i i
I
+
lII
+
d
+
+
(+ ) f
-
+
(+ ) f
_
+
Y. k r i s t e n s e n i i d
_
+
Y. i n t e r m e d i a
_
+
d
a,b S t r a i n s w h i c h h a v e b e e n d e s c r i b e d i n [8] a n d [16], r e s p e c tively.
c
Strains described in this study.
d
Strains, obtained
from the Hygiene Institute of Hamburg,
F.R.G.
e
Strain,
obtained
from
the Institute
of Medical
Microbi-
ology, Turku, Finland
f
Small halo, because of slowly growing culture.
g
Plasmidless derivative of strain WA-314.
helper plasmid pRK2073 and the mobilizable
cointegrate pRK290B8-5 :: p0 : 8 consisting of the
vector pRK290B and the virulence plasmid of
strain WA-314 (Table 1) have been characterized
previously [15,17].
3.2. Siderophore detection
For siderophore detection a loop of a bacterial
suspension in water was streaked upon chrome
azurol S agar (CAS-agar) and incubated for 2 days
at 28 °C. The red halos were then photographed
using a red filter system. CAS-agar has been prepared according to the instructions for E. coli
strains [11].
231
3.3. Virulence assay (15)
The lethal response in laboratory mice
(NMRI-strain) was determined by injecting in-
traperitoneally 0.5 ml of 10 7 bacteria in 0.9%
NaCI. Mice were examined daily for death over 4
weeks.
Fig. 1. CAS-agar plates showing siderophore-producing (with haloes) and non-producing stratus (without haloes). Strains of Table 1
are denoted as follows: 13: 1209-79; 3:Y-108 Nal~; 9:Y-96 Nalr; 20: 1223-75-1; 8R: Y-161M-Nalr; 8: WA-C-Nalr; YP: Y-P-I (Y.
pseudotuberculosis); V: Y-NF-Nalr; W: WA-314; Wl: WA-1 (insertion mutant); W 2 : W A - 2 (insertion mutant); Yi: Yersinia
intermedia.
232
3. 4. Transposon mutagenesis
The cointegrate pRK290B8-5 :: pO : 8 was
mobilized from E. coli HB101 :: Tn5 into Y. enterocolitica strain WA-C-Nal r under kanamycin and
nalidixic acid selection [18]. A transconjugant
harboring a virulence plasmid with a Tn5-transposon insertion was then incubated at 37 ° C for 20 h
under kanamycin selection to eliminate the plasmid (the virulence plasmid has a temperature-sensitive replicon, [12,13]). Then the culture was
streaked on calcium-deficient solid medium
(Mox-agar, [15]) containing 5 0 / , g / m l kanamycin.
Under these conditions, the plasmidless derivatives grew much faster than the plasmid-bearing
strains and thus mutants with a T n 5 insertion in
the chromosome could be isolated. About one
thousand insertion mutants were screened for altered siderophore production using CAS-agar
plates.
recent study which clearly demonstrated that the
antigen reference strain of serotype 0 : 8, Y-161MNal r, remained avirulent after introduction of the
virulence plasmid of strain WA-314 [16]. Obviously, this strain is a spontaneous siderophore-deficient mutant and thus has lost a relevant virulence function. On the other hand, siderophore
production per se did not render Yersinia strains
mouse-virulent. As can be demonstrated by the
environmental strains of Y. intermedia, Y. kristensenii and Y. frederiksenii which are strong
siderophore (aerobactin) producers [4], mouse
lethality could not be established in these strains
after introduction of a Yersinia virulence plasmid.
F r o m this we may suggest that aerobactin is not a
virulence factor in Yersinia.
Further genetical and biochemical studies are
necessary to characterize these novel siderophores
of virulent Yersinia in order to elucidate their role
in the pathogenesis of Yersinia infection in mice.
4. RESULTS A N D D I S C U S S I O N
ACKNOWLEDGEMENTS
Yersinia strains of various origin were screened
for orange halo production on CAS-agar plates
(Table 1). As seen in Fig. 1 well-focused orange
zones on blue agar could be observed with all
mouse-lethal Y. enterocolitica and Y. pseudotuberculosis strains. The non-lethal Y. enterocolitica
strains were also able to grow well on CAS-agar
plates but the color did not turn from blue to
orange. Probably these strains obtained iron via a
low affinity pathway, whereas mouse lethal strains
excreted siderophores which remove the iron from
the indicator dye. Plasmid-cured strains e.g. WAC-Nal r (Table 1) produced similar halos as their
parent strains, indicating that siderophore production is chromosomally encoded. This is supported
by the defect of siderophore release of the two
Tn5-insertion mutants WA-1 and WA-2 (Table 1,
Fig. 1). In order to test the contribution of siderophore production for animal virulence, the serotype 0 : 8 virulence plasmid was reintroduced into
the mutants of WA-1 and WA-2. The obtained
transconjugants were non-lethal for mice, indicating that siderophore production enhances the virulence potential of Y. enterocolitica. Further support for this conclusion could be obtained from a
I am grateful to A. Grote for screening the
transposon mutants. I thank Dr. Wachsmuth,
CDC, Atlanta and Dr. Aleksid, Hygiene Institute,
Hamburg, for providing us with various Yersinia
species.
This study was supported by the Deutsche Forschungsgemeinschaft (grant No. He 1297/1-4).
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