Veterinary Microbiology 144 (2010) 246–249

Short communication

The cation-uptake regulators AdcR and Fur are necessary for full virulenceof Streptococcus suis

Jesus Aranda a, Maria Elena Garrido a, Nahuel Fittipaldi b, Pilar Cortes a, Montserrat Llagostera a,Marcelo Gottschalk b, Jordi Barbe a,*a Department de Genetica i Microbiologia, Universitat Autonoma de Barcelona (UAB), Bellaterra, 08193 Barcelona, Spainb Groupe de recherche sur les maladies infectieuses du porc and Centre de Recherche en Infectiologie Porcine,

Faculte de Medecine Veterinaire, Universite de Montreal, St-Hyacinthe, Quebec J2S 7C6, Canada

A R T I C L E I N F O

Article history:

Received 13 May 2009

Received in revised form 4 December 2009

Accepted 28 December 2009

Keywords:

Streptoccus suis

adcR

fur

Virulence

A B S T R A C T

In streptococci, the pleiotropic regulators AdcR and Fur control the transport of, zinc and

iron, respectively, which are essential components of many proteins. In this work, DadcR,

Dfur, and DadcR Dfur mutants of Streptococcus suis, a serious pathogen in pigs and humans,

were assayed in a mouse model to determine their involvement in the virulence of this

bacterium. The results showed, for the first time, that the virulence of S. suis mutants

carrying an inactivation of adcR, fur, or both genes is significantly attenuated compared to

the wild-type parent strain. Furthermore, all mutants were found to be more sensitive to

oxidative stress. Our data provide evidence that the adcR and fur genes play important

roles in the oxidative stress response of S. suis as well as in the full virulence of this

bacterium.

� 2010 Elsevier B.V. All rights reserved.

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Veterinary Microbiology

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1. Introduction

Streptococcus suis is a gram-positive bacterium consid-ered world-wide to be one of the most important pathogensin the swine industry (Gottschalk et al., 2007). S. suis

serotype 2, the most frequently isolated from infectionsproduced by this micro-organism (Staats et al., 1997), is alsoa zoonotic agent, affecting people in close contact with pigsor pork-derived products (Gottschalk et al., 2007). Althoughcurrent knowledge regarding the virulence of S. suis is stilllimited, several S. suis mutants for genes involved inregulation, metabolism, binding, and transport were foundto be poorly invasive for porcine brain microvascularendothelial cells (Vanier et al., 2009).

* Corresponding author at: Department de Genetica i Microbiologia,

Universitat Autonoma de Barcelona (UAB), Edifici C (Facultat de

Biosciencies), Bellaterra (Cerdanyola del Valles), 08193 Barcelona, Spain.

Tel.: +34 93 581 1837 fax: +34 93 581 2387.

E-mail address: jordi.barbe@uab.cat (J. Barbe).

0378-1135/$ – see front matter � 2010 Elsevier B.V. All rights reserved.

doi:10.1016/j.vetmic.2009.12.037

The streptococcal AdcR and Fur proteins have beenimplicated in the control of divalent-cation uptake,functioning as pleiotropic regulators of this process(Panina et al., 2003; Mahdi et al., 2008; Yuhara et al.,2008). AdcR is a streptococcal transcription factor analo-gous to the zinc-uptake regulator (Zur) (Panina et al.,2003). Very recent in vitro and in vivo experiments haveshown that S. suis AdcR acts by repressing the expression ofgenes encoding zinc/manganese transporters as well asputative virulence factors (Aranda et al., 2009). The ferricuptake regulator (Fur) is one of the most importanttranscription factors controlling iron metabolism and ispresent in almost all prokaryotes (Escolar et al., 1999).Interestingly, Fur has been shown to play a globalregulatory role in the detoxification of oxygen radicals,the acid shock response, the production of toxins andvirulence factors, and several other metabolic functions(Escolar et al., 1999). However, to our knowledge, the rolesof AdcR and Fur in the virulence of streptococcal pathogenshave not yet been investigated. Therefore, the aim of this

Fig. 1. Survival curve of mice inoculated with the WT (^), DadcR ( ), Dfur

(~), or DadcR Dfur (O) S. suis strains. Only groups of mice inoculated with

�107 cfu/mouse (n = 6 per group) are shown. Significant differences in

survival were noted in all mutants compared to the WT parent strain (x2

test, P< 0.05 in all cases).

J. Aranda et al. / Veterinary Microbiology 144 (2010) 246–249 247

work was to determine the importance of these twotranscriptional regulators in the virulence of S. suis.

2. Materials and methods

2.1. Bacterial strains and growth conditions

The wild-type (WT) S. suis strain used in this work was avirulent serotype 2 strain (P1/7) originally isolated in theUnited Kingdom from an ante-mortem blood cultureobtained from a pig dying of meningitis (Clifton-Hadley,1981). The three mutant strains used in this work, UA5000,UA5001, and UA5002, were derived from strain of S. suis

P1/7 carrying a deletion mutation in adcR, fur, or bothgenes, respectively (Aranda et al., 2009). All bacterialstrains were grown in THY [Todd–Hewitt (Difco) mediumwith 2% yeast extract (Difco)] at 37 8C without shaking.Growth was monitored by measuring the absorbance at600 nm (OD600). The growth rate (m) was calculated basedon the exponential segment of the growth curve anddefined as ln 2g�1, where g is the doubling time of anexponentially growing culture (Paredes-Lopez et al., 1976).

2.2. Virulence assays

Female BALB/cAnNHsd mice (8-weeks-old) obtainedfrom Harlan Iberica (Barcelona, Spain) were used forvirulence studies (Aranda et al., 2008). All animal experi-ments were approved by the UAB Animal Ethics Committee.Female 8-week-old BALB/cAnNHsd mice in four groups ofthree were used for each bacterial strain. Briefly, the micewere injected intraperitoneally with 0.1 ml of serial 10-folddilutions of WT, DadcR, Dfur, or DadcR Dfur mutant strains(�108 to 105 cfu/animal) prepared in THY broth supple-mented with 10% inactivated bovine serum (Invitrogen).The concentration of the original bacterial suspensions wasdetermined by enumeration after plating. The number ofanimals that survived 3 weeks post-inoculation wasrecorded, and the lethal dose 50 (LD50) was calculated aspreviously described (Reed and Muench, 1938).

2.3. Oxidative damage assays

Hydrogen peroxide sensitivity assays were performedas described elsewhere (Stohl et al., 2005), with somemodifications. Briefly, all strains were grown in THY brothuntil OD600 = 0.2 was reached. Aliquots of 5 ml were thenplaced in tubes, and hydrogen peroxide added to a finalconcentration of 30 mM (Stohl et al., 2005). The tubes werethen incubated for 30 min at 37 8C with shaking. Thesecultures were immediately serially diluted with THY brothcontaining 10 mg catalase/ml and plated onto THY agar.The survival percentage was calculated as the viable cfudivided by the total cfu (bacteria that did not undergo H2O2

treatment).

2.4. Statistical analysis

Unless otherwise specified, data were analyzed byKruskal–Wallis nonparametric test followed by Conovermultiple comparison procedure (Conover, 1980). For all

tests, a value of P< 0.05 was considered as the thresholdfor significance.

3. Results and discussion

Mutations in the fur gene are known to cause theattenuation of several pathogens, such as Salmonella

enterica serovar Typhimurium (Campoy et al., 2002a),Vibrio cholerae (Mey et al., 2005), Actinobacillus pleropneu-

moniae (Jacobsen et al., 2005), Bacillus cereus (Harvie et al.,2005), Agrobacterium tumefaciens (Kitphati et al., 2007),and Pseudomonas syringae (Cha et al., 2008). Similarly, zur

mutants of S. enterica serovar Typhimurium and ofXanthomonas oryzae are less virulent than their respectiveparental strains (Campoy et al., 2002b; Yang et al., 2007).By contrast, the virulence of fur-defective strains ofPasteurella multocida was the same as that of the parentalstrain (Bosch et al., 2001). In the present study, weexamined the roles of the AdcR and Fur proteins in S. suis

virulence, specifically, in the wild-type vs. the mutantstrains UA5000 (DadcR), UA5001 (Dfur), and UA5002(DadcR Dfur). The results showed that the inactivation ofadcR, fur, or both genes in S. suis WT strain P1/7 increasedthe LD50 from 4.4� 106 cfu/mouse to>2� 108,>2.5� 108,and >3� 108 cfu/animal, respectively (Fig. 1).

Previous studies have shown that S. suis and othermicro-organisms deficient in cation-uptake regulatorsoverexpress certain virulence factors (Harvie et al.,2005; Aranda et al., 2009). However, in our mouse modelof infection, the LD50 of S. suis mutants deficient in genesencoding either AdcR or Fur proteins increased by aminimum of 45-fold compared to the WT parental strain.The differences in these results can be attributed, in part, todifferences in the in vitro growth rates of the mutants. Infact, the growth rates of the S. suis mutants DadcR

(m = 0.259 h�1), Dfur (m = 0.294 h�1) and DadcR Dfur

(m = 0.222 h�1) were dramatically and significantly lower(P< 0.01) in all strains than that of the WT P1/7 strain(m = 0.338 h�1). In addition, the duration of the lag phase

Fig. 2. Growth curves of WT ( ), DadcR (^), Dfur (&), and DadcR Dfur ( )

S. suis strains in THY medium. The means from three independent

experiments (for each growth curve) are shown. To simplify, error bars

have been omitted; however, in all cases the standard deviation was

<10%.

J. Aranda et al. / Veterinary Microbiology 144 (2010) 246–249248

increased in all mutant strains, especially in the double-mutant UA5002 (Fig. 2).

Among the many host defense mechanisms encoun-tered by bacteria is superoxide, which is produced by host-cell oxidases during aerobic metabolism (Janssen et al.,2003). Reactive oxygen species such as superoxide areknown to cause oxidative damage in living cells (Dubracand Touati, 2000). Although the pathogenesis of S. suis

infection has not been completely defined, it has beenreported that this bacterial species is able to survive withinnon-phagocytic host cells (Vanier et al., 2004) due to thefact that, like other streptococci, it lacks cytochromes andcatalase, is aerotolerant, and is able to resist oxidativestress (Niven et al., 1999). Zinc acts as an antioxidant thatconfers protection from oxidative stress and serves as acofactor for many enzymes, including superoxide dismu-tase (Gaballa and Helmann, 2002). Here, the ability of the S.suis DadcR-mutant strain to survive H2O2 challenge wasexamined using oxidative damage assays. As seen in Fig. 3,significant differences (P = 0.001) in survival wereobserved between the WT (80%) and the DadcR-mutant(36.5%) after H2O2 treatment. As previously reported in

Fig. 3. Hydrogen peroxide resistance of WT, DadcR, Dfur, and DadcR Dfur

strains of S. suis. Bacterial cells were treated with 30 mM H2O2 for 30 min

and serially diluted into THY media containing catalase. Error bars

represent the standard error of the mean of three independent

experiments. *P< 0.05 relative to the parent strain P1/7.

studies of a Corynebacterium diphtheriae zur mutant (Smithet al., 2009), the S. suis UA5000 DadcR strain demonstratedan increased susceptibility to peroxide stress, suggestingthat AdcR is required in oxidative stress defenses.

As noted above, most bacteria employ several metal-dependent transcriptional regulators, each controllingdifferent regulons. By responding to diverse inducers,these regulatory proteins maintain metal homeostasis inaddition to controlling other metabolic and cellularfunctions, including those related to virulence. Inactivationof the fur gene results in the deregulation of ironmetabolism and thus increases bacterial sensitivity toredox stress (Andrews et al., 2003). Therefore, we testedthe ability of S. suis Dfur- and DadcR Dfur-mutant strains tosurvive H2O2 challenge (Fig. 3). The results showedsignificant survival differences (P = 0.049) between theDfur mutant and the WT strain, although the mutant wasless sensitive (56%) than the DadcR mutant to H2O2

challenge. Moreover, even greater differences were foundbetween the double-mutant strain (DadcR Dfur) and theWT parent strain, with 71% of DadcR Dfur-mutant cellskilled by H2O2 treatment (P< 0.001). Although further, in

vivo experiments are needed to fully validate thesefindings, our data support the hypothesis that AdcR andFur proteins are involved in the resistance to oxidativestress in S. suis and thus contribute to the in vivo protectionof this pathogen against peroxide-induced oxidativedamage. Accordingly, both proteins are necessary for thefull virulence of this bacterial species.

Acknowledgements

This work was funded by grants AGL2005-03574 fromthe Ministerio de Educacion (MEC) de Espana and2009SGR1106 from the Departament d’Universitats,Recerca i Societat de la Informacio (DURSI) de la General-itat de Catalunya. We acknowledge Joan Ruiz and AnitaFernandez for their excellent assistance.

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