Pradier

MD Brigitte Dunais
email: dunais@chu-nice.fr

MD Pascale Bruno

PharmD Hélène Carsenti

MD Pia Touboul

PhD. Christian Pradier

A decade-long surveillance of nasopharyngeal colonisation with Streptococcus pneumoniae among children attending day-care centres in South-Eastern France: 1999 -2008

    A decade-long surveillance of nasopharyngeal colonisation with among children attending day-care centres in south-eastern France: 1999-2008

Introduction

Since the seven valent pneumococcal conjugate vaccine became available, several studies in France and abroad have reported shifts in pneumococcal distribution as observed in blood or cerebrospinal fluid samples in the context of invasive disease, or in nasopharyngeal samples in that of acute otitis media [START_REF] Doit | Epidemiology of pediatric community-acquired bloodstream infections in a children hospital in Paris, France, 2001 to 2008[END_REF][START_REF] Cohen | Dynamic of pneumococcal nasopharyngeal carriage in children with acute otitis media following PCV7 introduction in France[END_REF]. Healthy day-care centre (DCC) attendees are known to be the major reservoirs and disseminators of pneumococci so that carriage surveillance in this particular setting provides a reasonably accurate estimate of the distribution of pneumococcal strains circulating within the community [START_REF] Sá-Leão | High rates of transmission of and colonization by Streptococcus pneumoniae and Haemophilus influenzae within a day care center revealed in a longitudinal study[END_REF][START_REF] Huang | Modeling community-and individual-level effects of child-care center attendance on pneumococcal carriage[END_REF]. Frequent nasopharyngeal carriage of penicillin-nonsusceptible Streptococcus pneumoniae (PDSP) and exposure to numerous antibiotic treatment courses were observed in the late 1990s among children attending DCCs in the Alpes Maritimes (AM), in South-eastern France [START_REF] Pradier | Nasopharyngeal carriage of Streptococcus pneumoniae in children's day-care centers: 10-month follow-up study in Nice, France[END_REF]. In an effort to curb one of the highest rates of pneumococcal resistance and antibiotic use in Europe [START_REF] Cars | Variation in antibiotic use in the European Union[END_REF], an intervention promoting prudent antibiotic use in the context of paediatric respiratory tract infections was implemented in AM in 2000, based on academic detailing visits to all local general practitioners and paediatricians [START_REF] Bruno-Bazureault | pour le Groupe d'étude et de prévention des infections de l'enfant (GEPIE). [Peer visits to community-based general practitioners and pediatricians as part of the "Antibiotics only when necessary" campaign in the Alpes-Maritimes district. Methodology, feasibility, and interest[END_REF]. It was followed by a nationwide media campaign initiated in 2002 [START_REF] Huttner | Antibiotics are not automatic anymore"--the French national campaign to cut antibiotic overuse[END_REF], while the 7-valent pneumococcal conjugate vaccine became available and its cost endorsed by the national health insurance early 2003. Pneumococcal carriage and antibiotic-resistance trends in DCCs were monitored in AM on an ongoing basis, from 1999 to 2008, allowing assessment of the combined impact of both these events on carriage in this particular setting [START_REF] Dunais | Trends in nasopharyngeal carriage of Streptococcus pneumoniae among children attending daycare centers in southeastern France from 1999 to 2006[END_REF]. We report trends in carriage, serotype distribution and antibiotic susceptibility of SP over the ten-year period from 1999 to 2008.

Methods

Cross-sectional surveys were conducted on a random sample of children attending group day-care from January to March 1999March , 2002March , 2004March , 2006March and 2008. Updated lists of all the DCCs were obtained from the local public health authorities for each survey. Care-center managers and attending physicians were informed of the survey and their eventual participation was requested in the event that they were randomly selected. For each survey, a two-stage cluster sample was obtained via a random sample of DCCs among which a random sample of 22 children was drawn, allowing for potential refusal by parents or absentees on the day of the survey. Only those children spending at least three days a week in the centre were included in the sampling frame. Refusal of a DCC centre to participate led to an offer of participation to the next centre on the list, in order to include 25 centres for each survey. In case parents agreed, they were asked to sign an informed consent document and to provide the child's health booklet, which is usually completed by the child's attending physician at each medical visit, and to complete a questionnaire concerning the child's recent medical history and antibiotic treatments over the previous three months. Only those treatments where parents could provide the name of the antibiotic were recorded. On the day of the survey the investigating physician recorded the immunization status for PCV7 from the children's health booklets and in some instances, when the booklet was not available, immunization status was provided by the DCC records. Since very few children received the vaccine before 2004, analysis of pneumococcal carriage and serotype distribution according to immunization status was conducted only for the 2004, 2006 and 2008 surveys.

Nasopharyngeal aspirates were obtained using a Vygon® 522.06 canula adapted on a 1 mL syringe. The canula was then transferred in a transport medium (Portagerm®, Biomérieux, Lyon) to the Laboratoire d'Antibiologie of Nice University Hospital.

The study protocol was approved by the Nice University Hospital Ethics committee.

Microbiology

Samples were submitted to direct microscopic examination and cultured on blood agar. Pneumococcal isolates were identified by Gram stain and susceptibility to optochin. Susceptibility of pneumococcal isolates to antibiotics was determined by disc diffusion to oxacillin (1µg and 5µg), erythromycin, clindamycine, chloramphenicol, and tetracycline. Susceptibility to trimethoprime/sulfamethoxazole combination was tested on separate lysed blood agar plates. Penicillin, amoxicillin and ceftriaxone minimal inhibitory concentrations (MICs) were determined for strains with an 1µg oxacillin diameter below 19mm and/or oxacillin 5µg below 26mm by E-test® (BMD, France) and strains were classified as penicillin-intermediate (MIC>0.064g/ml) or penicillin-resistant (MIC>1µg/ml) according to the recommendations put forward by the Antibiogram Committee of the French Microbiological Society (Comité de l'Antibiogramme de la Société Française de Microbiologie: http://www.sfm.asso.fr). Breakpoints for amoxicillin and ceftriaxone were >0.5µg/ml and >2µg/ml, respectively. Serotypes were analysed using the Quellung reaction with antisera provided by the Statens Serum Institute (Copenhagen, Denmark). These antisera concerned the following serotypes: 6A, 6B, 9V, 14, 19A, 19F, 23F, and 24. In 2004, strains not reacting with any of those antisera were sent to the National Pneumococcal Reference Center in Paris for identification. From 2006, all serotype determinations, including formerly unidentified serotypes of stored isolates from the previous surveys, were conducted in the Laboratoire d'Antibiologie of Nice University hospital with pneumococcal latex pool antisera and factor antisera from the Serum Statens Institute to identify specific serotypes included in the PCV7 formulation.

Regional epidemiology

Antibiotic prescription rates were confronted with regional sentinel surveillance data provided by the regional influenza surveillance sentinel physicians (Groupe Régionaux d'Observation de la Grippe). This surveillance is conducted every year from October 1 st (week 40) to April 15 th (Week 15), as participating physicians report every week the number of patients with influenza-like respiratory tract infection among children aged 0 to 4 years. The available aggregated regional data for the Provence Alpes Côte d'Azur region include the Alpes Maritimes area.

Sample size

In order to estimate the proportion of SP carriers within 5% and with 95% confidence, with a hypothesis 25% carriers of non-susceptible isolates, the required sample size for each survey was calculated to be 288.

Statistical analysis

Data were analyzed using Epi-Info 6c and SPSS © software. Univariate analysis was performed using the chi-square test with a 5% degree of significance. Means were compared using ANOVA or non-parametric Kruskall Wallis' test for unequal variances.

Results

Population characteristics (Table 1)

The AM in South-Eastern France has a population of approximately one million, and a birth rate of 11 per 1,000 inhabitants. In France, DCCs cater for children aged 3 months to 3 years of age. There were 58 DCCs in 1999 with a total capacity of 2,800 children. This increased gradually over the years to reach 97 DCCs with an overall capacity of approximately 5,000 attendees in 2008.

Over the five surveys, nasopharyngeal samples were obtained from a total of 1,604 children distributed among the 25 DCCs, aged 3 to 39 months. The number of participating children and their characteristics for each survey are described in Table 1. No major variation in distribution by age and gender was observed over the study period.

Antibiotic prescriptions

The proportion of children who received an antibiotic prescription within the three 2).

Antibiotic susceptibility

Overall, susceptibility to all tested antibiotics increased significantly over the study period, up to the 2006 survey (p<10 -3 for each tested antibiotic) and then stabilized (Figure 3). In particular, the proportion of isolates non-susceptible to erythromycin 4), and that year serotype 19A accounted for 57% of amoxicillin non-susceptible and 55% of ceftriaxone non-susceptible isolates (Figure 5).

dropped

Regional epidemiology

Data from the respiratory tract infection community-based sentinel surveillance group for the PACA region show a peak incidence rate in the 2001-2002 Winter season for the 0 to 4 year-old age group (Figure 5) although no increase in antibiotic prescriptions was observed among day care attendees that year. A higher mean rate of respiratory tract infection was observed in 2007-2008 compared to 2005-2006

(2.18±0.87 vs. 1.86±0.78, respectively). However, the difference was not significant.

Discussion

Our five consecutive surveys describe a ten-year trend in pneumococcal carriage among healthy children who attended a day-care centre at least 3 days a week in South-eastern France, covering a period which saw the introduction and wide use of pneumococcal conjugate vaccine, and the implementation of campaigns to reduce antibiotic prescriptions. Such surveys provide an insight into the epidemiology of Streptococcus pneumoniae within the community, the day-care setting being notorious for the high carriage rates among its young attendees [START_REF] Sá-Leão | High rates of transmission of and colonization by Streptococcus pneumoniae and Haemophilus influenzae within a day care center revealed in a longitudinal study[END_REF][START_REF] Huang | Modeling community-and individual-level effects of child-care center attendance on pneumococcal carriage[END_REF]. As major contributors to SP prevalence in the community, DCC attendees may be considered as sentinels providing an advanced warning system for trends in pneumococcal infection, through regular carriage monitoring in this setting. Huang et al. developed a model exploring both individual-and community-level effects of DCC attendance and suggested that the mere presence of DCCs within a community led to a 6 fold increase in the odds of carriage for non-attendees [START_REF] Huang | Modeling community-and individual-level effects of child-care center attendance on pneumococcal carriage[END_REF].

Although the decline in pneumococcal carriage was moderate, our results show a significant overall drop in carriage rates of PDSP over the 10-year period concerned, along with fewer antibiotic prescriptions. Considering the high prevalence rates of PDSP initially recorded among this population in the late 1990s, these results are reassuring and confirm those observed both in France and abroad [START_REF] Lepoutre | Impact of infant pneumococcal vaccination on invasive pneumococcal diseases in France, 2001-2006[END_REF][START_REF] Grivea | Impact of heptavalent pneumococcal conjugate vaccine on nasopharyngeal carriage of penicillin-resistant Streptococcus pneumoniae among day-care center attendees in central Greece[END_REF]. In central Greece, wide coverage with PCV7 vaccination was followed by a significant reduction of carriage of highly-penicillin resistant pneumococci among DCC attendees [START_REF] Grivea | Impact of heptavalent pneumococcal conjugate vaccine on nasopharyngeal carriage of penicillin-resistant Streptococcus pneumoniae among day-care center attendees in central Greece[END_REF]. In France, widespread pneumococcal immunization combined with a local, followed by a nationwide campaign to curb antibiotic prescriptions, appears to have demonstrated its effects [START_REF] Sabuncu | Significant reduction of antibiotic use in the community after a nationwide campaign in France, 2002-2007[END_REF]. In a study focusing on children with acute otitis media in carriers in a study concerning children aged 3 to 84 months attending primary care centres in Massachusetts [START_REF] Huang | Continued impact of pneumococcal conjugate vaccine on carriage in young children[END_REF]. In our study, VT accounted for only 4% of carriage strains in 2008 with over 90% of children immunized with PCV7. However, although as elsewhere we found high prevalence rates of 19A strains, among which 96% were non-susceptible to penicillin in 2008, the predominant strain that year was serotype 15, of which 34% displayed non-susceptibility to penicillin. Due to the two-year delay between other carriage studies in France and Portugal and our own, it may be that serotype 15 is emerging as a major colonising serotype.

Our positive results should thus be considered with caution, as trends in both antibiotic prescription and carriage of non-susceptible pneumococci appear to have presently stabilized. Regarding antibiotic use, regional sentinel surveillance data do not suggest an increase in the incidence rate of respiratory tract infections in 2008 which would account for the lack of further progress in reducing prescriptions.

Whether the present prescription rate among children attending DCCs should be considered as the best we can achieve in France remains to be determined. It may be that sustained efforts are necessary to restrain antibiotic use, and the public health campaign that was implemented in France during 2003-2004 has not been sufficiently reinforced, nor specifically targeted towards DCC directors, whose exclusion policies have been shown, in a study conducted in Minnesota, to exert a major influence on antimicrobial prescription [START_REF] Nm | Child care center exclusion policies and directors' opinions on the use of antibiotics[END_REF].

Pneumococcal carriage rates remain high, and, as observed by others, the increasing prevalence of penicillin non-susceptible serotypes 19A and 6A (included in the newly available PCV13) and that of other non-vaccine serotypes exhibiting antibiotic-resistance, namely serotype 15 (not included in PCV13), are a cause for concern [START_REF] Dortet | Emergence of Streptococcus pneumoniae of serotype 19A in France: molecular capsular serotyping, antimicrobial susceptibilities, and epidemiology[END_REF]. Vaccine escape by genetic recombination at the capsular locus has been documented, with concomitant change of serotype and penicillin susceptibility. [START_REF] Brueggemann | Vaccine escape recombinants emerge after pneumococcal vaccination in the United States[END_REF] Surveillance of invasive pneumococcal disease reflects similar changes. In France, coverage by PCV7 of invasive pneumococcal isolates in children below 2 years of age dropped below 10% in 2008, while the newly licensed 13-valent conjugate vaccine (PCV-13) covered 68% of those strains [START_REF] Varon | Centre national de référence du pneumocoque[END_REF]. In a multicentre retrospective cohort study conducted in Northern France, the incidence rate of invasive pneumococcal disease observed among children below 2 years of age between 2005 and 2008 increased 6.5-fold, from 1.8/100,000 to 11.9/100,000, and was caused by non-vaccine serotypes [START_REF] Alexandre | Rebound in the incidence of pneumococcal meningitis in northern France: effect of serotype replacement[END_REF].

Regarding antimicrobial treatment, although fewer antimicrobials have been prescribed, those most frequently used may yet interfere with attempts to curb resistance. Indeed, French guidelines recommend treating acute otitis media in children below 2 years of age with an amoxicillin-clavulanate combination or 3 rd generation cephalosporins; declining prescriptions for other respiratory tract infections (i.e. common colds) may influence distribution of prescribed antibiotics in favour of these compounds. Ease of administration (twice daily) makes 3 rd generation cephalosporins more attractive to parents and day-care personnel and have thus been increasingly favoured; however, they are known to select for resistant strains [START_REF] Dabernat | Effects of cefixime or co-amoxiclav treatment on nasopharyngeal carriage of Streptococcus pneumoniae and Haemophilus influenzae in children with acute otitis media[END_REF][START_REF] Dagan | Will reduction of antibiotic use reduce antibiotic resistance? The pneumococcus paradigm[END_REF].

Conclusion

We have attempted to counter antibiotic-driven selective pressure with vaccine-driven selection, which has proved highly effective in reducing the incidence of invasive disease but also in modifying the nasopharyngeal pneumococcal niche by introducing new colonizing strains. At the same time, fewer antibiotic prescriptions have eased the burden of resistant organisms. However, previously infrequent replacement strains are now in turn developing antimicrobial resistance, demonstrating how easy it is to revert to the initial situation. A new, wider spectrum (13-valent) pneumococcal conjugate vaccine is now available in France, exerting yet more selective pressure. It is to be hoped that the benefits reaped in terms of reducing the burden of pneumococcal disease will not be outweighed by the potential virulence of replacement strains or the persistence of inappropriate antibiotic use.

Figure 2 :Figure 4 :Figure 5 :

 245 Figure 2: Distribution and penicillin-susceptibility of non vaccine types in 2008

  from 70.2% in 1999 to 42.9% in 2006 and 47.2% in 2008. But in 2008, fewer strains were susceptible to chloramphenicol and co-trimoxazole compared with 2006.

	The proportion of isolates non-susceptible to 3 or more antibiotics, which had
	dropped from 55.9% in 1999 to 29.2% in 2006, rose to 47.4% in 2008, a significant
	increase compared to the previous survey (p<10 -3 ), and these MDR strains were
	mainly serotypes 19A, 15 and 6A, representing 32.9%, 16.4% and 13.7% of MDR
	isolates, respectively, in 2008. In particular, 15 out of 32 serotype 15 isolates were
	non-susceptible to erythromycin, and 11, 3, and 4 isolates of this serotype were
	intermediately susceptible to penicillin, amoxicillin, and ceftriaxone, respectively.
	Among vaccine-types, penicillin susceptibility increased from 38.7% to 74.3% of
	strains from 1999 to 2006, while in 2008, among the 7 remaining vaccine-type
	isolates, 3 were not fully susceptible. In 2008 the majority of PSDP isolates were 19A
	(35.9%), 6A (12.5%), 23A/B (6.3%), and 15 (15.6%).
	Penicillin MICs of most PDSP isolates remained at the intermediate level and no
	significant change was observed regarding MIC distribution. None were resistant to

either amoxicillin or ceftriaxone over the study period, although 41% and 50% were not fully susceptible to these respective compounds in 2008 (Figure

Table 1 :

 1 Characteristics of the study population over previous 3 months as reported by parents; ** at least one dose; *** not applicable

			1999		2002	2004	2006		2008		p
	# children		298		294	334	335		343
	# DCC		25		25	25		25		25
	Girls		52,0%		41,2%	53,0%	49,3%		51,6%		0,056
	Age group:
	<12 months		15,1%		19,7%	14,7%	14,0%		10,5%
	12-24 months		40,6%		38,4%	40,4%	35,5%		39,9%
	>24 months		44,3%		41,8%	44,9%	50,4%		49,6%		ns
	SP carriers		161		172	182	168		155
	% SP carriers		54,0%		58,5%	54,5%	49,9%		45,2%		0,01
	PDSP carriers		101		111	86		57		64
	%PDSP carriers		33,9%		37,8%	25,7%	17,0%		18,7%		<0.001
	Antibiotic treatment*	62,7%		52,8%	48,0%	30,9%		37,9%		<0.001
	Mean number of treated episodes/child	0.84±0.87	0.78±0.91	0.65±0.82 0.38±0.62 0.46±0.67	<10 -3
	PCV7 Immunization
	immunized**		N/A***		N/A	37,1%	68,4%		90,1%		<0.001
	non-immunized		N/A		N/A	44,6%	17,9%		6,4%
	status unknown					18,3%	13,7%		3,5%
	Figure 1: Serotype distribution over time
								1999	2002	2004	2006	2008
	45%	Vaccine serotypes					Non-vaccine serotypes
	40%
	35%
	30%
	25%
	20%
	15%
	10%
	5%
	0%
	23F	6B	19F	14	9V	18C	6A	19A	15	23A/B	Other

*

Acknowledgements

The authors are grateful to the directors and staff of the day-care centres and to the parents and children who collaborated in the study. These surveys were supported by Produits Roche, Wyeth Pharmaceuticals and the Nice University Hospital Program for Clinical Research, Nice, France These results were presented at the 46 th InterScience Conference on Anti-microbial Agents and Chemotherapy in Washington, DC, 2008.


Steve Peigneur

Bert Billen

Rita Derua

Etienne Waelkens

Sarah Debaveye

László Béress

Jan Tytgat
email: jan.tytgat@pharm.kuleuven.be

A BIFUNCTIONAL SEA ANEMONE PEPTIDE WITH KUNITZ TYPE PROTEASE AND POTASSIUM CHANNEL INHIBITING PROPERTIES

Keywords: Anthopleura elegantissima, K V channel inhibitor, sea anemone toxin, protease inhibitor

Sea anemone venom is a known source of interesting bioactive compounds, including peptide toxins which are invaluable tools for studying structure and function of voltage-gated potassium channels. APEKTx1 is a novel peptide isolated from the sea anemone Anthopleura elegantissima, containing 63 amino acids cross-linked by 3 disulfide bridges. Sequence alignment reveals that APEKTx1 is a new member of the type 2 sea anemone peptides targeting voltage-gated potassium channels (K V 's), which also include the kalicludines from Anemonia sulcata. Similar to the kalicludines, APEKTx1 shares structural homology with both the basic pancreatic trypsin inhibitor (BPTI), a very potent Kunitz-type protease inhibitor, and dendrotoxins which are powerful blockers of voltage-gated potassium channels. In this study, APEKTx1 has been subjected to a screening on a wide range of 23 ion channels expressed in Xenopus leavis oocytes: 13 cloned voltage-gated potassium channels (K

hERG, the insect channel Shaker IR), 2 cloned hyperpolarization-activated cyclic nucleotidesensitive cation non-selective channels (HCN1 and HCN2) and 8 cloned voltage-gated sodium channels (Na V 1.2-Na V 1.8 and the insect channel DmNa V 1). Our data shows that APEKTx1 selectively blocks K V 1.1 channels in a very potent manner with an IC 50 value of 0.9 nM. Furthermore, we compared the trypsin inhibitory activity of this toxin with BPTI. APEKTx1 inhibits trypsin with a dissociation constant of 124 nM. In conclusion, this study demonstrates that APEKTx1 has the unique feature to combine the dual functionality of a potent and selective blocker of K V 1.1 channels with that of a competitive inhibitor of trypsin.

Introduction

Among ion channels, potassium channels are the most diverse class of ion channels and they are key determinants of neuronal excitability. They regulate a variety of cellular processes and functions such as heart rate, neurotransmitter release and nerve conduction, insulin secretion, blood pressure and muscle contraction [START_REF] Gutman | International Union of Pharmacology. LIII. Nomenclature and molecular relationships of voltage-gated potassium channels[END_REF][START_REF] Lujan | Organisation of potassium channels on the neuronal surface[END_REF] . In the mammalian genome more than 100 genes are encoding the pore forming α subunits and auxiliary β subunits of K + channels. Together with these numerous genes, the presence of spliced variants, association with chaperone and scaffolding molecules and the formation of heteromultimeric channels contribute greatly to the diversity of the K + channel family [START_REF] Lujan | Organisation of potassium channels on the neuronal surface[END_REF]. This family has been divided into 15 subfamilies of which the voltage gated potassium channels (K V ) represent one of these subfamilies [START_REF] Yu | Overview of molecular relationships in the voltage-gated ion channel superfamily[END_REF]. They determine neuronal intrinsic electrical excitability by repolarization of the membrane after initiation of the action potential [START_REF] Pongs | Voltage-gated potassium channels: from hyperexcitability to excitement[END_REF]. Upon depolarization of the membrane, K V channels will open within 1 ms, allowing the flux of K + ions driven by their electrochemical gradient [START_REF] Gutman | International Union of Pharmacology. LIII. Nomenclature and molecular relationships of voltage-gated potassium channels[END_REF][START_REF] Lujan | Organisation of potassium channels on the neuronal surface[END_REF]. Functional K V channels are formed by a complex of four pore forming α subunits associated with one or A c c e p t e d M a n u s c r i p t 2 more auxiliary β subunits. Each α subunit consists of six transmembrane spanning segments (S1-S6). The first four transmembrane segments form the voltage sensing domain with S4 serving as the voltage sensor. This S4 segment has been modeled using the crystal structure of the bacterial K V channel K V AP and contains 4 conserved Arg residues. These positively charged basic residues display a net movement outwards upon depolarization allowing conformational changes resulting in the opening of the pore [START_REF] Bezanilla | The voltage sensor in voltage-dependent ion channels[END_REF][START_REF] Swartz | Towards a structural view of gating in potassium channels[END_REF]. The pore region of the channels is formed by the S5 and S6 segments which are connected by a re-entrant P-loop. The P-loop contains an extremely well conserved domain among the K V channels, the so-called signature sequence (-TXGYGD-) and comprises the selectivity filter, the pore helix and the turret region [START_REF] Jiang | Endogenous Kv channels in human embryonic kidney (HEK-293) cells[END_REF]. The auxiliary β subunits of K V channels (K V β) are cytoplasmic proteins, capable of modifying K V channel biophysical properties. Since the beginning of last century sea anemones have been studied with an increasing interest. Although a number of sea anemone toxins have been isolated and characterized, these animals remain poorly studied in comparison with other venomous animals such as scorpions, spiders, cone snails or snakes. Sea anemones are a known pharmacological treasure of biological active compounds acting upon a diverse panel of ion channels such as TRPV1, voltage-gated sodium and potassium channels [START_REF] Ouadid-Ahidouch | KV1.1 K(+) channels identification in human breast carcinoma cells: involvement in cell proliferation[END_REF][9][START_REF] Honma | Peptide toxins in sea anemones: structural and functional aspects[END_REF][START_REF] Castaneda | Discovery and characterization of cnidarian peptide toxins that affect neuronal potassium ion channels[END_REF]. Of these different toxins those that target sodium channels are the best studied group with more then 100 known toxins [START_REF] Bosmans | Sea anemone venom as a source of insecticidal peptides acting on voltage-gated Na+ channels[END_REF]. In contrast, no more then 12 potassium channel toxins have been characterized to date. Based on structural differences and activity profile, these potassium channel toxins can be divided into 4 structural classes [START_REF] Castaneda | Discovery and characterization of cnidarian peptide toxins that affect neuronal potassium ion channels[END_REF][START_REF] Oliveira | BcIV, a new paralyzing peptide obtained from the venom of the sea anemone Bunodosoma caissarum. A comparison with the Na+ channel toxin BcIII[END_REF][START_REF] Diochot | Sea anemone toxins affecting potassium channels[END_REF][START_REF] Diochot | A new sea anemone peptide, APETx2, inhibits ASIC3, a major acid-sensitive channel in sensory neurons[END_REF]. Up to date 6 toxins from A. elegantissima have been isolated and characterized: APE1-1, APE1-2, APE2-2 and ApC which are type 1 sodium channel toxins; APETx1 a selective modifier of the human ether a go-go related gene K + channel (hERG) and APETx2 which specifically inhibits the Acid Sensing Ion Channel (ASIC3) [START_REF] Diochot | A new sea anemone peptide, APETx2, inhibits ASIC3, a major acid-sensitive channel in sensory neurons[END_REF][START_REF] Bruhn | Isolation and characterisation of five neurotoxic and cardiotoxic polypeptides from the sea anemone Anthopleura elegantissima[END_REF][START_REF] Diochot | APETx1, a new toxin from the sea anemone Anthopleura elegantissima, blocks voltage-gated human ether-a-go-go-related gene potassium channels[END_REF][START_REF] Salceda | Effects of ApC, a sea anemone toxin, on sodium currents of mammalian neurons[END_REF]. In this work we present the purification, biochemical analysis and electrophysiological characterization of a very potent and selective K V 1.1 blocker which represents the newest member of the sea anemone type 2 potassium channel toxins.

Material and methods

Toxin purification.

The toxin APEKTx1 was purified as described previously [START_REF] Bruhn | Isolation and characterisation of five neurotoxic and cardiotoxic polypeptides from the sea anemone Anthopleura elegantissima[END_REF][START_REF] Diochot | APETx1, a new toxin from the sea anemone Anthopleura elegantissima, blocks voltage-gated human ether-a-go-go-related gene potassium channels[END_REF][START_REF] Schaller | Isolation and amino acid sequence of a morphogenetic peptide from hydra[END_REF]. The fraction containing APEKTx1 was further purificated by RP-HPLC with a semi preparative Vydac C18 column (4.6X250 mm). Solvent A was 0.1% TFA in water, solvent B was 0.085% TFA in acetonitrile. A linear gradient from 0 to 80% solution B was developed for 80 min at a flow rate of 1ml/min. A second purification was performed in the same conditions as described above. All final fractions were dried by speed-vac evaporation and stored at -20°C.

Biochemical characterization of toxins.

The mass of the intact toxin was measured by MALDI-TOF (4800 Analyzer, Applied Biosystems, USA) as well as by ESI-QUAD analysis (4000QTRAP, Applied Biosystems, USA). The toxin peptide was subjected to Edman degradation on an Applied Biosystems Procise instrument. The toxin was digested with legumain (R&D Systems, USA). A MALDI TOF/TOF instrument (4800 Analyzer, Applied Biosystems, USA) was used for de novo sequencing analysis of the obtained fragments. These fragments were subjected to MS/MS analysis and the spectra were manually interpreted.

Expression of voltage-gated ion channels in Xenopus laevis oocytes.

For the expression of the VGPCs (rK

V 1.1 (Accesion number: NM173095), rK V 1.2 (NM012970), hK V 1.3 (L23499), rK V 1.4 (NM012971), rK V 1.5 (NM012972), rK V 1.6 (NM575671), Shaker IR (CG12348), rK V 2.1 (NM013186), hK V 3.1 (NM004976), rK V 4.2 (NM031730), rK V 4.3 (NM031739), hERG (NM000238), mHCN 1 (NM010408), mHCN 2 (NM008226) and the VGSCs (rNa V 1.2 (NM012647), A c c e p t e d M a n u s c r i p t 3 rNa V 1.3 (NM012647), rNa V 1.4 (M26643), hNa V 1.5 (NM198056), mNa V 1.6 (L39018), rNa V 1.7 (AF000368), rNa V 1.8 (NM017247), rβ1 (
NM017288), hβ1 (NM001037) and the insect channel DmNa V 1 (NC004354)) in Xenopus oocytes, the linearized plasmids were transcribed using the T7 or SP6 mMESSAGE-mMACHINE transcription kit (Ambion, USA). The harvesting of stage V-VI oocytes from anaesthetized female Xenopus laevis frog was previously described [START_REF] Liman | Subunit stoichiometry of a mammalian K+ channel determined by construction of multimeric cDNAs[END_REF]. Oocytes were injected with 50 nl of cRNA at a concentration of 1 ng/nl using a micro-injector (Drummond Scientific, USA). The oocytes were incubated in a solution containing (in mM): NaCl, 96; KCl, 2; CaCl 2 , 1.8; MgCl 2 , 2 and HEPES, 5 (pH 7.4), supplemented with 50 mg/l gentamycin sulfate.

Electrophysiological recordings.

Two-electrode voltage-clamp recordings were performed at room temperature (18-22°C) using a Geneclamp 500 amplifier (Molecular Devices, USA) controlled by a pClamp data acquisition system (Axon Instruments, USA). Whole cell currents from oocytes were recorded 1-4 days after injection. Bath solution composition was ND96 (in mM): NaCl, 96; KCl, 2; CaCl 2 , 1.8; MgCl 2 , 2 and HEPES, 5 (pH 7.4) or HK (in mM): NaCl, 2; KCl, 96; CaCl 2 , 1.8; MgCl 2 , 2 and HEPES, 5 (pH 7.4). Voltage and current electrodes were filled with 3M KCl. Resistances of both electrodes were kept between 0.5-1.5 MΩ. The elicited currents were filtered at 1 kHz and sampled at 0.5 kHz (for potassium currents) or at 20 kHz (for sodium currents) using a four-pole low-pass Bessel filter. Leak subtraction was performed using a -P/4 protocol. K V 1.1-K V 1.6 and Shaker currents were evoked by 500 ms depolarizations to 0 mV followed by a 500 ms pulse to -50 mV, from a holding potential of -90 mV. Current traces of hERG channels were elicited by applying a +40 mV prepulse for 2 s followed by a step to -120 mV for 2 s. K V 2.1, K V 3.1 and K V 4.2, K V 4.3 currents were elicited by 500 ms pulses to +20mV from a holding potential of -90 mV. Sodium current traces were, from a holding potential of -90 mV, evoked by 100 ms depolariarions to V max (the voltage corresponding to maximal sodium current in control conditions). In order to investigate the current-voltage relationship, current traces were evoked by 10 mV depolarization steps from a holding potential of -90 mV. gV curves were calculated from IV relationships as follows:

g K =I K /(E m -E K ) with E k = (RT/zF)ln[K #0] o /[K #0] i .
In these equations g K represents the conductance, I K the potassium current, E m the membrane potential, E K the reversal potential, R the gas constant (8.31 J/K.mol), T the temperature, z the charge of the ion (for K + ions: z = 1), F the Faraday's constant (96.500 C/mol), [K #0] o and [K #0] I respectively are the extracellular and intracellular K + ion concentrations. The values of I K or g K were plotted as function of voltage and fitted using the Boltzmann equation: g K /g max = [1 + (exp(Vg -V)/k] -1 , were gmax represents maximal gK, Vg is the voltage corresponding to half-maximal conductance, and k is the slope factor. To assess the concentration dependency of the APEKTx1 induced inhibitory effects, a concentration-response curve was constructed, in which the percentage of current inhibition was plotted as a function of toxin concentration. Data were fitted with the Hill equation: y = 100/[1 +(IC 50 /[toxin]) h ], were y is the amplitude of the toxin-induced effect, IC 50 is the toxin concentration at half-maximal efficacy, [toxin] is the toxin concentration, and h is the Hill coefficient. To investigate the bimolecular kinetics of toxin inhibition, oocytes expressing wild type K V 1.1 channels were depolarized to o mV for 0.5 s from a holding potential of -90 mV every 5 s, both in the absence and presence of different concentrations of toxin. The obtained current values were plotted as a function of time. Data were fitted with the following exponential equation: y = Ae -t/τ , where t represents the time, A is the amplitude of the current and τ is the time constant for toxin binding (τ on ) or toxin unbinding (τ off ). The first-order association rate constant (k on ) was calculated using following equation: τ on = (k on + k off ) -1 . The first-order dissociation constant (k off ) was calculated using the equation:

τ off = k off -1
. Comparison of two sample means was made using a paired Student's t test (p < 0.05). All data represent at least 3 independent experiments (n ≥ 3) and are presented as mean ± standard error.

Kunitz-type inhibition activity.

A c c e p t e d M a n u s c r i p t 4

The possible trypsin inhibition activity of APEKTx1 was measured spectrophotometrically as described previosly [START_REF] Schweitz | Two different classes of sea anemone toxins for voltage sensitive K+ channels[END_REF]. Briefly, aliquots containing a final concentration of 3 µM trypsin (from bovine pancreas, TPCK treated, Sigma-Aldrich, USA) were incubated with various concentrations of peptides in 5 mM CaCl 2 and 50 nM Tris-HCl, pH 7.8. After incubation for 3 h at 25°C the remaining trypsin activity was determined by addition of 5 µl of a 5 mM Nα-benzoyl-DL-arginine p-nitroanilide (BAPNA, Sigma-Aldrich,USA). Paranitroanaline release was measured at 405 nm.

2.6. Molecular modeling.

APEKTx1

was modeled using the publicly available program MODELLER9v8 (http://www.salilab.org/modeller) using textilinin-1, a Kunitz-type serine protease inhibitor from the venom of the Australian common brown snake Pseudonaja textilis (PDB code 3BYB; http://www.pdb.org/pdb) as template.

Results

Purification of APEKTx1.

The screening of fractions obtained from the sea anemone A. elegantissima, after anion and cation exchange and gel filtration, yielded one fraction which was able to fully block K V 1.1 channels. This fraction was further purified in 2 steps using reversed-phase HPLC. A linear gradient from 0 to 80% solution B was developed for 80 min at a flow rate of 1ml/min. A second purification was performed in the same conditions as described above (fig. 1).

Biochemical properties.

With MALDI analysis in the linear mode, a broad mass peak with an apex mass of 7468.8 Da was measured. This experimental mass of 7475 Da corresponds well with the theoretical mass of 7468.5 Da. During ESI analysis, a mass of 7484.8 Da was observed, suggesting an oxidation of Met32. The intact toxin was subjected to Edman degradation. Since only the 20 first N-terminal amino acids could be reliably sequenced, we subjected the toxin to a proteolytic treatment in order to obtain fragments amenable to de novo sequencing analysis by MALDI TOF/TOF instrument. Digestion of the toxin by trypsin, gluC, thermolysin turned out to be unsuccessful. Finally, an asparaginylendopeptidase was found to cut the toxin peptide into smaller fragments. The digestion was executed according to the protocol of the manufacturer. After digestion, the resulting peptides were separated and desalted by HPLC. The HPLC fractions were dried by speed-vac evaporation and resolubilized in alfa-cyano 4-hydrocinnamic acid matrix solution before analysis by MALDI TOF/TOF. The protein sequence data reported in this paper will appear in the UniProt Knowledgebase under the accession number P86862.

Electrophysiological experiments.

Sequence alignment (fig. 2A) indicated that APEKTx1 is a new member of the type 2 sea anemone peptides directing against voltage-gated potassium channels, which also include the kalicludines from A. sulcata and SHTX II from Stichodactyla haddoni [START_REF] Schweitz | Two different classes of sea anemone toxins for voltage sensitive K+ channels[END_REF][START_REF] Honma | Novel peptide toxins from the sea anemone Stichodactyla haddoni[END_REF]. From figure 2B it can be seen that APEKTx1 also shares homology with protease inhibitors from other sea anemones and with the potent Kunitz-type protease inhibitor Bovine Pancreatic Trypsin Inhibitor (BPTI). Previous electrophysiological experiments in Xenopus leavis oocytes have shown that the kalicludines block K V 1.2 channels with IC 50 values around 1 µM. SHTX II was reported to inhibit the binding of 125 I-α-DTX to synaptosomal membranes with an Kd value of 650 nM [START_REF] Schweitz | Two different classes of sea anemone toxins for voltage sensitive K+ channels[END_REF][START_REF] Honma | Novel peptide toxins from the sea anemone Stichodactyla haddoni[END_REF]. As shown in figure 3A, the pure peptide APEKTx1 was subject of a wide screening on 23 ion channels: 12 cloned voltage-gated potassium channels (K

V 1.1-K V 1.6, K V 1.1 mutant, K V 2.1, K V 3.1, K V 4.2, K V 4.3,

hERG, the insect channel Shaker IR); 2 cloned hyperpolarization-activated

A c c e p t e d M a n u s c r i p t 5 cyclic nucleotide-sensitive cation non-selective channels (HCN1 and HCN2) and 8 cloned voltage-gated sodium channels (Na V 1.2-Na V 1.8 and the insect channel DmNa V 1). Surprisingly, at concentrations up to 1 µM, no significant effect could be observed on the other ion channel isoforms tested. Figure 3B shows original traces for wild type K V 1.1 channels with application of 0.3, 1 and 3 nM APEKTx1 and for mutant K V 1.1 channels with application of 100 µM APEKTx1. A concentration response curve was constructed in order to determine the concentration at which half of the channels were blocked by APEKTx1. The IC 50 value yielded 0.9 ± 0.1 nM (fig. 4A). Thus APEKTx1 inhibits K V 1.1 channels with the same potency as DTX I and α-DTX and is approximately 700 to 3000 times more potent than other known 2 sea anemone peptides directed against K V channels. Moreover, this also means that at concentrations which are a 1000-fold the IC 50 value of APETx1 on K V 1.1, this toxin still discriminates between subtype isoforms. The inhibition of K V 1.1 channels by APEKTx1 was not voltage-dependent as in a range of test potentials from -30 mV to +40 mV no difference in the degree of APEKTx1 induced block could be observed (n=8) (fig. 4B). In order to investigate if the observed current inhibition is due to pore blockage or rather to altered channel gating upon APEKTx1 binding, the IV and gV curves in ND96 and HK solution were constructed (fig. 4C andD). Application of 10 nM APEKTX1 caused 80.45 ± 2.23% and 84.21 ± 3.74% inhibition of the potassium current in ND96 and HK, respectively (n=10). In ND96, the g max curve in control and in the presence of 10 nM toxin was characterized by a V 1/2 value of -15.7 ± 1.23 mV and -12.71 ± 0.5 mV (n=8) respectively. In HK, the V 1/2 was respectively, -25.84 ± 2.53 mV and -19.89 ± 3.2 mV (n=8). It can be concluded that in both solutions no significant difference in V 1/2 values occurred (P>0.05). Furthermore, the IV relationship in HK solution shows that the reversal potential, E K is not significantly influenced by APEKTx1 (P>0.05; n=8). E K values yielded -7.56 +/-2.45 mV in control and -7.96 +/-1.92 mV after application of toxin. All together, these experiments imply that current inhibition upon APEKTx1 binding does not result from changes in the voltage-dependence of channel gating. Presumably, APEKTx1 acts by blocking the pore in the open state of the K V channel. Blockage of K V 1.1 channels occurred rapidly and its binding was reversible since the current recovered quickly and completely upon washout (n=10) (fig. 4E). Both characteristics suggest an extracellular site of action. The reversibility of current inhibition is in contrast with DTX K binding. Toxin K binding is reversible at low concentrations but becomes irreversibly at concentrations higher than its IC 50 value. However, alanine substitution of the Lys3 or Lys26 rendered DTX K binding reversible, possibly explaining the reversible binding of APEKTx1 since this toxin also possess neutral residues at these positions. Another observed difference between both toxins is that DTX K cannot fully inhibit the potassium current, even at very high concentrations. APEKTx1 however, is able to completely inhibit the current. Because APEKTx1 did not alter channel gating and binding was reversible we investigated whether APEKTx1 blockage followed the kinetic behavior of a bimolecular reaction [START_REF] Tytgat | The alpha-dendrotoxin footprint on a mammalian potassium channel[END_REF]. Figure 4F shows the effects of increasing concentrations APEKTx1 on a K V 1.1 channel. The apparent first order association rate constant k on (open circles) increased linearly with toxin concentration, while the second order dissociation constant k off (closed circles) remained constant. Both values are in concordance with the scheme of a bimolecular reaction. The influence on toxin-channel binding by through-space electrostatic forces between charged residues of the K V 1.1 channel and of APEKTx1 was also examined. This was done by measuring the effectiveness of APEKTx1 binding on K V 1.1 channels in solutions of different ionic strength. As described above, the IC 50 value of APEKTx1 for K V 1.1 homomeric channels was 0.9 ± 0.1 nM. However, when using an iso-osmotic solution with a 48 mM NaCl concentration (sucrose substitution) the obtained IC 50 value was 0.43 ± 0.07 nM. This 2 fold increase in APEKTx1 potency underlines that through-space electrostatic interaction is involved in APEKTx1 binding. These results are in corroboration with previously described similar observations for charybdotoxin binding to Shaker channels and for α-DTX binding to K V 1.1 channels [START_REF] Tytgat | The alpha-dendrotoxin footprint on a mammalian potassium channel[END_REF][START_REF] Hurst | Identification of amino acid residues involved in dendrotoxin block of rat voltage-dependent potassium channels[END_REF][START_REF] Mackinnon | Mutant potassium channels with altered binding of charybdotoxin, a pore-blocking peptide inhibitor[END_REF]. In order to define the α-dendrotoxin footprint on mammalian potassium channels, 3 mutations (A352P, E353S and Y379H) were introduced in the dendrotoxin binding site, located in the H5 loop between the A c c e p t e d M a n u s c r i p t 6 transmembrane domains S5 and S6 of K V 1.1 subunits (fig. 5). By mutating these 3 particular residues, the pore region of the K V 1.1 triple mutant closely resembles this one of K V 1.3 channels [START_REF] Tytgat | The alpha-dendrotoxin footprint on a mammalian potassium channel[END_REF][START_REF] Hurst | Identification of amino acid residues involved in dendrotoxin block of rat voltage-dependent potassium channels[END_REF]. We investigated whether the sensitivity of APEKTx1 for K V 1.1 could be affected by mutating these 3 critical residues. Interestingly, even at extreme high concentrations, APEKTx1 was unable to induce full blockage of current through these mutated K V 1.1 channels. Application of 100 µM APEKTx1 caused only 53 % inhibition (n=4) and the obtained IC 50 yielded 10.8 ± 0.6 µM (fig. 4 A and Fig. 3B). This dramatic loss of affinity underlines the crucial interaction of these mutated residues with the toxin. Since it was reported that DTX K only needs one K V 1.1 subunit to cause inhibition of the potassium current through wild type heteromultimeric channels [START_REF] Akhtar | Characteristics of brain Kv1 channels tailored to mimic native counterparts by tandem linkage of alpha subunits: implications for K+ channelopathies[END_REF], we investigated also if DTX K could block the triple mutant. Concentration response curves were constructed for both K V 1.1 wild type and triple mutant channels (fig. 4A). The observed IC 50 value for wild type channels was 0.51 ± 0.064 nM which is in good accordance with previous reported values [START_REF] Robertson | Novel effects of dendrotoxin homologues on subtypes of mammalian Kv1 potassium channels expressed in Xenopus oocytes[END_REF]. For mutant channels the IC 50 was 5.28 ± 0.23 nM. This means a 10-fold decrease in sensitivity compared to wild type channels.

Trypsin inhibition activity.

As all members of the type 2 sea anemone toxins APEKTx1 share sequence homology with the well characterized and potent Kunitz-type protease inhibitor BPTI, we also investigated if APEKTx1 could block trypsin activity. In order to do this, trypsin was incubated with different concentrations of APEKTx1 at room temperature for 3 hours to reach equilibrium. In the same conditions, trypsin was also incubated with DTX I, DTX K or BPTI. The protease inhibition activity was determined spectrophotomatrically after incubation by measuring the ability of the remaining free trypsin to release paranitroaniline from BAPNA. It has been shown before that the kalicludines have a very similar inhibition profile as BPTI [START_REF] Schweitz | Two different classes of sea anemone toxins for voltage sensitive K+ channels[END_REF]. Figure 6 shows that APEKTx1 is a trypsin inhibitor with a K d = 124 nM. Presumably, APEKTx1 is a competitive trypsin inhibitor. A 3-fold molecular excess of APEKTx1 over trypsin completely inhibited the paranitroanaline release. However, this is almost 3 times the amount of BPTI or the kalicludines necessary to reach total inhibition of trypsin activity. The same figure also indicates that DTX I and DTX K, could not inhibit the trypsin activity, even at a 6:1 ratio of toxin over trypsin.

Discussion

The unique feature of a dual functionality within one molecule is intriguing. However, since the production of toxins is an energy demanding task for an organism, the dual effects contained within one molecule can be seen as an efficient and economic manner to save energy with a maximal pharmacological output. Furthermore, the dual effect has clearly advantages for the sea anemone in both an offensive and defensive point of view: the presence of a trypsin inhibitor will prolong the half-life of the peptides in the venom and therefore optimize the strategy of the venom. APEKTx1 is the newest member of the type 2 sea anemone toxins directed towards voltage-gated potassium channels. To date, this family exists of only 4 other toxins, 3 kalicludines and SHTX II. Although it is known that these toxins are effective on K V channels, little is known about their subtype selectivity. Here we report for the first time a screening of a type 2 toxin on a wide range of 23 different ion channels. This broad screening not only reveals a potent activity against K V 1.1 (IC 50 = 0.9 ± 0.1 nM) but also points out an impressive selectivity for these channels over the other isoforms tested. Even at concentrations 1000-fold higher than the IC 50 value for K V 1.1 channels, APEKTx1 has the ability to selectively inhibit this isoform subtype over others within the K V 1 family. Moreover, A352P, E353S and Y379H substitutions in the K V 1.1 subunit made these channels insensitive for APEKTx1 indicating that these 3 residues are critical for the toxin-channel interaction. Further electrophysiological characterization showed that this toxin does not modulate the voltage dependence of gating of K V channels. The IV relationship in HK solution showed that ion selectivity was not changed after toxin binding. The observation that there is no difference in the percentage induced block either in ND96 or HK leads to the A c c e p t e d M a n u s c r i p t 7 conclusion that channel blockage is independent of the direction of the potassium current flux and is not influenced by the extracellular concentration of K + ions. Moreover, APEKTx1 did not show voltage dependence in its blockage of channels in a wide voltage range. The site of toxin binding is presumably located at the extracellular side since the inhibition of current through K V 1.1 channels occurred rapidly and binding was reversible upon washout. We have shown that APEKTx1 shares not only sequence and structure homology with dendrotoxins (fig. 7) but also a similar activity profile with a comparable potency as these snake toxins. The APEKTx1 blockage followed the kinetic behavior of a bimolecular reaction as previously described for α-DTX. The dendrotoxins have been extensively studied. The X-ray structure of α-DTX and the NMR structure of DTX K have been reported [START_REF] Skarzynski | Crystal structure of alpha-dendrotoxin from the green mamba venom and its comparison with the structure of bovine pancreatic trypsin inhibitor[END_REF][START_REF] Berndt | Nuclear magnetic resonance solution structure of dendrotoxin K from the venom of Dendroaspis polylepis polylepis[END_REF]. They both possess structures which resemble very well the Kunitz-type serine protease inhibitors such as BPTI. The dendrotoxins have an N-terminal 3 10 -helix, a β hairpin and a C-terminal α-helix. The residues important for their potent K V blocking activity have been characterized. For DTX K, site-directed mutagenesis studies have shown that Lys3, Tyr4, Lys6, Pro8, Arg10, Trp25, Lys26 and Lys28 are the main residues involved in the interaction with the K V 1.1 channels [START_REF] Harvey | Dendrotoxins: structure-activity relationships and effects on potassium ion channels[END_REF][START_REF] Gasparini | Delineation of the functional site of alphadendrotoxin. The functional topographies of dendrotoxins are different but share a conserved core with those of other Kv1 potassium channel-blocking toxins[END_REF][START_REF] Imredy | Energetic and structural interactions between delta-dendrotoxin and a voltage-gated potassium channel[END_REF]. Among these, the basic residues in the 3 10helix, Lys3 and Lys6, have been pointed out as the key residues for toxin binding since mutation to an alanine led to an dramatically decrease in the ability to inhibit the K + current [START_REF] Wang | Identification of residues in dendrotoxin K responsible for its discrimination between neuronal K+ channels containing Kv1.1 and 1.2 alpha subunits[END_REF]. Mutating the Pro8 and the Arg10 caused a 50-and 140-fold increase in IC 50 value, respectively. The dramatic loss of affinity observed for DTX K binding to synaptic membranes when Trp25 and Lys26 were mutated to a alanine underlines that these residues are critical in recognition of K V 1.1 channels [START_REF] Wang | Identification of residues in dendrotoxin K responsible for its discrimination between neuronal K+ channels containing Kv1.1 and 1.2 alpha subunits[END_REF][START_REF] Smith | Site-directed mutagenesis of dendrotoxin K reveals amino acids critical for its interaction with neuronal K+ channels[END_REF]. Among these key residues, Pro8, Arg10 and Arg28 are conserved in APEKTx1. For α-DTX a mutational approach based on site-directed mutagenesis and chemical synthesis in which 31 out of 59 amino acids were mutated, revealed that Arg1, Arg2, Lys3, Leu4, Ile6, Leu7 were crucial for channel blockage activity of this toxin. Substituting Arg1 by alanine resulted in a less than 10-fold decreased affinity, whereas Arg2, Leu4 and Ile6 substitution lowered the affinity 10 to 30 times. The Lys3 and Leu6 seem to be the most critical residues since mutating these amino acids resulted in a thousand times less potent toxins [START_REF] Harvey | Dendrotoxins: structure-activity relationships and effects on potassium ion channels[END_REF][START_REF] Gasparini | Delineation of the functional site of alphadendrotoxin. The functional topographies of dendrotoxins are different but share a conserved core with those of other Kv1 potassium channel-blocking toxins[END_REF][START_REF] Danse | On the site by which alpha-dendrotoxin binds to voltage-dependent potassium channels: site-directed mutagenesis reveals that the lysine triplet 28-30 is not essential for binding[END_REF][START_REF] Harvey | Recent studies on dendrotoxins and potassium ion channels[END_REF]. Of these residues critical for α-DTX binding, the leu4 and Ile6 are replaced by a homologous residue in APEKTx1. These Lys3 and Leu6 residues in α-DTX form the so-called dyad. It has been proposed that most toxins that block K V channels possess a conserved functional core composed of a key basic residue (Lys or Arg) associated with a 6.6 ± 1 Å distant key hydrophobic residue (Leu, Tyr or Phe). Such a functional dyad can be found in a broad range of structurally unrelated peptides from various animals such as scorpions, cone snails, snakes and sea anemones [START_REF] Dauplais | On the convergent evolution of animal toxins. Conservation of a diad of functional residues in potassium channel-blocking toxins with unrelated structures[END_REF][START_REF] Mouhat | The 'functional' dyad of scorpion toxin Pi1 is not itself a prerequisite for toxin binding to the voltage-gated Kv1.2 potassium channels[END_REF]. However, it has been reported that besides this dyad, other toxin determinants are required for a high affinity interaction between the toxin and his target [START_REF] Mackinnon | Structural conservation in prokaryotic and eukaryotic potassium channels[END_REF]. Known examples of toxins lacking a dyad but still capable of blocking the K V channel (or the other way around toxins with a dyad but incapable of blocking) strongly suggest that the functional dyad on its own cannot represent the minimal pharmacophore or prerequisite for Kv1 binding [START_REF] Shon | kappa-Conotoxin PVIIA is a peptide inhibiting the shaker K+ channel[END_REF][START_REF] Huys | BmTx3, a scorpion toxin with two putative functional faces separately active on A-type K+ and HERG currents[END_REF]. Docking experiments with the scorpion toxin Pi1 and its homologs have highlighted that a ring of basic residues interacts with acidic residues of the K + channel turret located in the most extracellular part of the channel outer vestibule. Pi1 analogs, in which the basic residues from the ring were replaced by alanines, show a 50-480-fold reduction in peptide affinity for their target but were still able to block the potassium current [START_REF] Mouhat | The 'functional' dyad of scorpion toxin Pi1 is not itself a prerequisite for toxin binding to the voltage-gated Kv1.2 potassium channels[END_REF]. In general, it is assumed that toxins recognize the Kv1 subtypes through the interaction of various toxin residues, among which the basic ring, with certain residues of the K V 1 channel turret. These interactions can be sufficient to inhibit the potassium current. Moreover, these specific molecular contacts determine toxin selectivity towards particular K V 1 channel isoforms. The functional dyad can then be viewed as a secondary anchoring point, providing a higher toxin affinity without altering toxin selectivity. The side chain of the basic key residue enters the ion channel pore and is surrounded by four Asp residues of the Ploop selectivity filter. This Asp residue is highly conserved among all the K V 1 channel isoforms. The carbonyl oxygen atoms of these Asp residues will form a stabilizing interaction with the side chain of the basic residue of the dyad. The key hydrophobic residue of the dyad will interact through both hydrophobic forces and hydrogen bonding with a cluster of aromatic residues in the P-loop. The specific positioning of the 2 residues from the dyad results in a physical barrier, especially because of the A c c e p t e d M a n u s c r i p t 8 positively charged side chain which is opposed to the K + flux [START_REF] Mouhat | Contribution of the functional dyad of animal toxins acting on voltage-gated Kv1-type channels[END_REF][START_REF] Jouirou | Toxin determinants required for interaction with voltage-gated K+ channels[END_REF]. Because of the low identity with other K V channel toxins and because none of the key residues critical for the K V blockage of DTX K or α-DTX are conserved in APEKTx1, we tried to hypothesize the key residues of APEKTx1 with the help of structural models. As it can be seen from figure 7, the role of a possible dyad in APEKTx1 can be fulfilled by Arg15 and Phe13, a basic and hydrophobic residues which are 6.2 Å separated. Assumably, the side chain of Arg15 enters the K V 1.1 channel pore and interacts with the carbonyl oxygen atoms of the Asp377 residues. The Phe13 will most likely interact through hydrophobic forces with the aromatic cluster formed by Phe356, Trp364, Trp365, and Tyr375. Previous work has highlighted Tyr375 in K V 1.1 as a crucial residue for the selectivity of scorpion and sea anemone toxins [START_REF] Gilquin | A variable residue in the pore of Kv1 channels is critical for the high affinity of blockers from sea anemones and scorpions[END_REF]. The amino acid at this position is highly variable between K V 1 isoforms and the nature of the side chain of this residue contributes significantly to the subtype selectivity of toxins. The observed 12000-fold decrease in affinity of APEKTx1 for mutated K V 1.1 channels can be explained partially by the Y379H mutation. The same mutation prevented the K V 1.1 binding of another sea anemone toxin BgK, which binds to wild type channels with an IC 50 value of 0.7 nM [START_REF] Jouirou | Toxin determinants required for interaction with voltage-gated K+ channels[END_REF][START_REF] Gilquin | A variable residue in the pore of Kv1 channels is critical for the high affinity of blockers from sea anemones and scorpions[END_REF][START_REF] Visan | Mapping of maurotoxin binding sites on hKv1.2, hKv1.3, and hIKCa1 channels[END_REF][START_REF] Alessandri-Haber | Mapping the functional anatomy of BgK on Kv1.1, Kv1.2, and Kv1.3. Clues to design analogs with enhanced selectivity[END_REF]. The E353 also seems to be critical for interaction with APEKTx1. Hurst et al have shown that each of the 3 mutations on its own decreased dendrotoxin potency and that the removal of a negative charge at position 353 caused the greatest decrease in affinity [START_REF] Hurst | Identification of amino acid residues involved in dendrotoxin block of rat voltage-dependent potassium channels[END_REF]. However, simultaneous substitution of all 3 residues caused a much stronger decrease in DTX sensitivity than the single mutations. Further site-directed mutagenesis studies or ideally co-crystallization studies are required to elucidate if such subtle changes in sequence of the pore region are responsible for the dramatically loss of sensitivity of K V 1.1 for this toxin. Also shown in figure 7 are the basic residues which are conserved in DTX K and α-DTX. These residues possibly contribute to the basic ring important for recognition, interaction and correct positioning of the toxin on the channel. As mentioned above, previous work has shown that mutating these conserved residues leads to affinity loss in DTX K or α-DTX [START_REF] Harvey | Dendrotoxins: structure-activity relationships and effects on potassium ion channels[END_REF]. However, this is only a hypothesis which requires further conformation from site-directed mutagenesis studies, docking experiments and structure analysis. The bovine pancreatic trypsin inhibitor belongs to the Kunitz-type inhibitors and is a potent inhibitor of serine proteases. The crystal structures of BPTI with bovine trypsin and with bovine chymotrypsin have been determined [START_REF] Helland | The crystal structures of the complexes between bovine beta-trypsin and ten P1 variants of BPTI[END_REF][START_REF] Capasso | Crystal structure of the bovine alpha-chymotrypsin:Kunitz inhibitor complex. An example of multiple protein:protein recognition sites[END_REF][START_REF] Scheidig | Crystal structures of bovine chymotrypsin and trypsin complexed to the inhibitor domain of Alzheimer's amyloid beta-protein precursor (APPI) and basic pancreatic trypsin inhibitor (BPTI): engineering of inhibitors with altered specificities[END_REF]. Analysis of these complexes have shown that there are 10-13 residues on the inhibitor forming less than 4 Å contacts with 20-25 residues of the enzyme. The 13 key residues of the inhibitor form a structural epitope seen as two extended and exposed loops. The first loop which is the primary contact site is composed of the residues Thr11 to Ile19. The second loop is more C-terminal situated and is formed by residues Gly36 to Arg39 [START_REF] Krowarsch | Interscaffolding additivity: binding of P1 variants of bovine pancreatic trypsin inhibitor to four serine proteases[END_REF][START_REF] Buczek | Analysis of serine proteinase-inhibitor interaction by alanine shaving[END_REF]. The Lys15 has been pinpointed as a single residue hot spot responsible for a major part of the enzyme-inhibitor contacts. The side chain of this basic amino acid penetrates deeply into the specificity binding pocket of the protease where it electrostaticly interacts with the Asp189 of the protease. This Lys15-Asp189 interaction delivers the major contribution to the association energy for the BPTI-trypsin complex. The role of Lys15 as a crucial player in the energetics of the recognition was demonstrated by substituting this residue with Gly. The point mutation reduced the total association energy with 70% leading to an association constant decreased by 9 orders of magnitude. Structure determination of α-DTX provided an explanation why this toxin and its homologue DTX I cannot inhibit proteases. The basic amino acid in BPTI has been replaced in α-DTX by a Tyr incapable of interacting effectively with binding pocket of trypsin [START_REF] Czapinska | Structural and energetic determinants of the S1-site specificity in serine proteases[END_REF][START_REF] Lancelin | Proteinase inhibitor homologues as potassium channel blockers[END_REF][START_REF] Krowarsch | Amino-acid substitutions at the fully exposed P1 site of bovine pancreatic trypsin inhibitor affect its stability[END_REF]. Further explanation for the lack of protease inhibition by the dendrotoxins can be found in the presence of Asp17 in α-DTX, Glu17 in DTX I and Arg16 in DTX K. The existence of these longer amino acid residues in dendrotoxins instead of the smaller alanine in BPTI causes a most unfavorable complex with the trypsin binding site. Moreover, the Pro19 in DTX K and Pro21 in DTX I and α-DTX instead of the Ile19 in BPTI greatly impedes a good interaction with the side chain of the Tyr39 of trypsin. The residues essential for kunitz-type inhibition in the primary contact site (Gly12, Cys14, Lys15 and Ala16) are all conserved in APEKTx1 except for the Arg15 (fig. 7). However, previous residue-specific modification studies have shown that mutating the Lys 15 to Arg did not alter the association constant. The lower protease inhibition activity of APEKTx1 compared to BPTI (fig. 5) can be explained by the presence of Phe13 and Pro19. A double BPTI mutant with a phenylalanine at position 13 and an arginine at position 15, exactly as in APEKTx1, resulted in a A c c e p t e d M a n u s c r i p t 9 100-fold decreased association constant compared to the wild type [START_REF] Krowarsch | Amino-acid substitutions at the fully exposed P1 site of bovine pancreatic trypsin inhibitor affect its stability[END_REF]. The unfavorable interaction of Pro19 with trypsin has been described above.

Conclusion

We have demonstrated the unique dual function of APEKTx1, which is a competitive Kunitz-type protease inhibitor and a very potent and selective K V 1.1 channel blocker. Moreover, several amino acid residues have been suggested to play a functionally critical role in the high potency and unique selectivity of this toxin. Further structure-function analysis involving site-directed mutagenesis studies, docking experiments and NMR are required in order to confirm their role. Because of its high affinity and selectivity, APEKTx1 might be a very interesting probe for further characterization of the binding sites of K V 1.1 channels. Finally, APEKTx1 represents a valuable tool to examine the exact role of K V 1.1 in various channelopathies, and might serve as a lead compound in the development of novel therapeutical agents for these diseases.

A c c e p t e d M a n u s c r i p t A, the crude fraction found to be active in the electrophysiological screening was loaded on a C18 column with a linear gradient of 0.085% TFA in acetonitrile. Only the fraction containing the largest peak indicated by the arrow was active on K V 1.1 channels. B, this active fraction was further purified using the same conditions as in A.

Fig. 1 :

 1 Fig. 1: Purification of APEKTx1.A, the crude fraction found to be active in the electrophysiological screening was loaded on a C18 column with a linear gradient of 0.085% TFA in acetonitrile. Only the fraction containing the largest peak indicated by the arrow was active on K V 1.1 channels. B, this active fraction was further purified using the same conditions as in A. Fig. 2: Alignment. Amino acid sequence of APEKTx1 and alignment with the other members of the type 2 sea anemone toxins, dendrotoxins, protease inhibitors from sea anemones and BPTI. Amino acid residues identical with APEKTx1 are shown on grey background, cysteine residues are printed in red. Disulfide bridge pattern based on AsKC3 are indicated. AsKC1-3 from Anemonia sulcata, AFAPI-I and AFAPI-III from Anthopleura fuscoviridis, AXPI-II from Anthopleura xanthogrammica, DTX E from Dendroaspis

Fig. 2 :Fig. 3 :Fig. 4 :Fig. 6 : 7 :

 23467 Fig. 1: Purification of APEKTx1.A, the crude fraction found to be active in the electrophysiological screening was loaded on a C18 column with a linear gradient of 0.085% TFA in acetonitrile. Only the fraction containing the largest peak indicated by the arrow was active on K V 1.1 channels. B, this active fraction was further purified using the same conditions as in A. Fig. 2: Alignment. Amino acid sequence of APEKTx1 and alignment with the other members of the type 2 sea anemone toxins, dendrotoxins, protease inhibitors from sea anemones and BPTI. Amino acid residues identical with APEKTx1 are shown on grey background, cysteine residues are printed in red. Disulfide bridge pattern based on AsKC3 are indicated. AsKC1-3 from Anemonia sulcata, AFAPI-I and AFAPI-III from Anthopleura fuscoviridis, AXPI-II from Anthopleura xanthogrammica, DTX E from Dendroaspis

Figure 2 Page 17 of 22 A c c e p t e d M a n u s c r i p

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Acknowledgments

We would like to thank O. Pongs for sharing the rK V 1.2, rK V 1.4, and rKV1.5 and rK V 1.6 cDNA. We are grateful to M.L. Garcia for sharing the hK V 1.3 clone and to DJ Snyders for sharing the rK V 2.1, hK V 3.1, rKV4.2 and rK V 4.3. The Shaker IR clone was kindly provided by G. Yellen. We thank M. Keating for sharing hERG, John N. Wood for sharing rNa V 1.8, A.L. Goldin for sharing rNa V 1.2, rNa V 1.3 and mNa V 1.6, G. Mandel for sharing rNa V 1.4, R.G. Kallen for sharing hNa V 1.5, S.H. Heinemann for sharing the ratβ1 subunit, S.C. Cannon for sharing the hβ1 subunit and Martin S. Williamson for providing the Para and tipE clone. The Na V 1.7 clone was kindly provided by Roche. The authors would like to thank Prof. Dr. Dr.h.c. Wolf-Georg Forssmann, Dr. Eva Cuypers for the useful discussions, Dr. Elia Diego-García for helpful suggestions and Annelies Van Der Haegen for help with the modeling. This work was supported by the following grants: G.0257.08 and G.0330.06 (F.W.O. Vlaanderen), OT-05-64 (K.U. Leuven), UA P6/31 (IAP), P6/28 (IAP), GOA 08/16 (K.U.Leuven).

Conflict of interest

The authors declare that they have no conflict of interest.


Wenjun Li

Didier Raoult

Jean-Marc Rolain

Bernard La Scola
email: bernard.lascola@medecine.univ-mrs.fr

Evidence of circulation of an epidemic strain of Francisella tularensis in France by Multi Spacer Typing

Keywords: Francisella tularensis, genotyping, epidemiology, France

Multispacer typing (MST) was used to type 10 Francisella tularensis strains detected in French patients. Incorporating 79 Swedish F. tularensis strains, phylogenetic analysis demonstrated that that in spite tularemia appears as a sporadic disease in France, it is caused by an epidemic cluster of strains.

facultative intracellular bacterial species. Four subspecies of F. tularensis, subsp. tularensis, subsp. holarctica, subsp.novicida, and subsp. mediasiatica, have been identified and each of them has distinct virulence and geographic distribution [START_REF] Svensson | Evolution of subspecies of Francisella tularensis[END_REF]. Human tularemia is endemic in certain areas of the northern hemisphere and may be present in various clinical forms depending on the route of inoculation and the virulence of the F. tularensis strains involved [START_REF] Oyston | Tularaemia: bioterrorism defence renews interest in Francisella tularensis[END_REF]. The less frequent but more severe clinical form, respiratory tularemia (type A tularemia), is caused by inhalation of highly virulent F. tularensis subsp. tularensis and has mainly been identified in Northern America [START_REF] Oyston | Tularaemia: bioterrorism defence renews interest in Francisella tularensis[END_REF]. The most common clinical manifestation of F. tularensis infection, type B tularemia, is caused by F. tularensis subsp. holarctica. It is usually contracted from the bite of an arthropod vector that previously fed on an infected animal or contact with infected animals. Type B tularemia is characterized by the development of a local painful lesion at the site of inoculation, tender regional lymphadenopathy, fever and other constitutional symptoms including malaise, headache, anorexia, and myalgia (3). Type B tularemia is endemic to countries of the Northern Hemisphere and is rarely life threatening but may lead to long-lasting complications (e.g., lymph node suppuration). In France, tularemia is a rare and mostly sporadic disease and only type B tularemia has been reported (4). Several animal outbreaks have been recorded [START_REF] Vaissaire | Tularemia. The disease and its epidemiology in France][END_REF]. In 2004, an outbreak of airborne tularemia caused by F. tularensis subsp. holarctica occured in western France, which suggested a transmission from dog to humans by inhalation of contaminated particles from the dog's fur [START_REF] Siret | An outbreak of airborne tularaemia in France, August 2004[END_REF].

While each subspecies of F. tularensis has distinct virulence and geographic distribution, the genetic difference among them is minor, making individual discrimination of F. tularensis strains and epidemiological surveillance of tularemia extremely difficult [START_REF] Broekhuijsen | Genome-wide DNA microarray analysis of Francisella tularensis strains demonstrates extensive genetic conservation within the species but identifies regions that are unique to the highly virulent F. tularensis subsp. tularensis[END_REF]. Multispacer typing (MST) incorporating four highly variable intergenic spacers which were selected by genome comparison exhibited more powerful capacity of individual discrimination of F. tularensis strains and more reasonable subspecies classification than multilocus variable number tandem repeat analysis (MLVA) [START_REF] Johansson | Worldwide genetic relationships among Francisella tularensis isolates determined by multiple-locus variable-number tandem repeat analysis[END_REF]. In this study, MST was tested to direct typing of F. tularensis strains detected in human specimens in France.

This study was performed after agreement of the ethical committee of IFR 48 under 08-014. Ten French patients with swollen lymph nodes and with diagnosis of type B tularemia on the basis of clinical manifestations and serological test in 2008 were included in this study.

Lymph node biopsies from these patients were frozen at -20°C then referred to our laboratory.

Total genomic DNA was extracted from the lymph nodes using QIAamp Tissue kit (QIAGEN, Hilden, Germany) according to the manufacturer's recommendations. Specific PCR detection of F. tularensis on the DNA extracts was performed using a real-time quantitative PCR assays targeting the 17kDa major membrane protein precursor of the bacterium. This PCR included the primers Tul4F (ATTACAATGGCAGGCTCCAGA) and Tul4R (TGCCCAAGTTTTATCGTTCTTCT) and a Taqman probe (Tul4, 6-FAM-TTCTAAGTGCCATGATACAAGCTTCCCAATTACTAA-TAMRA). PCR was done in a LightCycler instrument (Roche Biochemicals, Mannheim, Germany).The PCR mixture included a final volume of 20μl with 10μl of the Probe Master kit (Qiagen), 0.5μl (10pmol/μl) of each primer, 5μl (2μmol/μl) of probe, 3μl of distilled water, and 2μl of extracted DNA. The amplification conditions were as follows: an initial denaturation step at 95°C for 15 min, followed by 40 cycles of denaturation at 95°C, annealing and elongation at 60°C for 60s, with fluorescence acquisition in single mode. After every five patients samples, negative controls (sterile distilled water, PCR mix, and biopsy samples from healthy humans) were inserted. MST incorporating 4 highly variable intergenic spacers, S1, S2, S3, and S4, which were selected by genome comparison was performed as previously described [START_REF] Scola | Rapid comparative genomic analysis for clinical microbiology: the Francisella tularensis paradigm[END_REF]. Briefly, these 4 highly variable intergenic spacers were amplified and sequenced from the genomic DNA of each lymph node biopsy. The sequence variations of concatenation of these four intergenic spacers were then used for discrimination of F. tularensis strains detected in these lymph node biopsies. A MST genotype is defined as unique sequence variation of concatenation of these four intergenic spacers. Phylogenetic organization of the 10 French strains in this study and the 79 strains collected in Sweden and one strain collected in France in previous study was inferred on the basis of concatenation of these 4 intergenic spacer sequences using the UPGMA, Neighbor-Joining and Maximum Parsimony methods within the MEGA 4.1 software. We added to the present analysis the 4 intergenic spacer sequences of F. tularensis subsp. tularensis strain Schu 4, F. tularensis subsp.novicida strain GA99-3549, and F. tularensis subsp. mediasiatica strain FSC147 with GenBank accession numbers, NC_006570, NZ_AAYF00000000, and NC_010677, respectively. MST, a DNA sequence-based genotyping method using highly variable intergenic spacers as typing markers which are selected on the basis of genome comparison, is a byproduct of genome biology in the genomic era [START_REF] Li | Bacterial strain typing in the genomic era[END_REF]. Since 2004, MST has been applied to several important human pathogenic bacteria and exhibited great power for strain typing. In our previous study, MST represented by four highly variable spacers showed good discriminatory power and gave a consistent phylogenetic classification of the studied strains [START_REF] Scola | Rapid comparative genomic analysis for clinical microbiology: the Francisella tularensis paradigm[END_REF]. Using this technique, we identified 4 new genotypes of spacer S1, types 19-22, but no new genotype of spacers S2, S3, and S4 (Figure 1). Newly identified genotypes of each spacer identified in the present study were deposited in GenBank (accession number: EU877932-EU877935). In total, 4 new MST genotypes were identified among the 10 French strains (Figure 1). The sequence identity of concatenation of 4 intergenic spacers of 11 French strains is higher as compared to that of 79 Swedish strains with 97.39% versus 88.62% respectively (Table 1). Phylogenetic analysis based on different methods exhibits similar organization. The 31 MST genotypes identified in French and Sweden strains of subsp. holarctica were grouped into 3 clusters and F. tularensis subsp. tularensis, F. tularensis subsp. mediasiatica, and F. tularensis subsp. novicida formed outgroups, respectively (Figure 1). Interestingly, the eleven French strains grouped tightly into one cluster and all the 79 Sweden strains were grouped into 2 other Swedish clusters, respectively (Figure 1).

In our analysis of F. tularensis subsp. holarctica, we found that one cluster is unique to French strains which exhibit relatively lower genetic diversity than previously typed Swedish strains (Table 1 and Figure 1). This confirms that French cases of tularemia that appear as rare and sporadic, is in fact an epidemic due to closely related strains as previously observed in central and western Europe [START_REF] Pilo | Identification of Francisella tularensis cluster in central and western Europe[END_REF]. In France, tularemia is a notifiable disease. Every year, between 20 and 40 cases are reported (http://invs.sante.fr/surveillance/tularemie/default.htm).

Direct diagnosis is done in approximately 20% of cases, mostly by PCR but only 1-3 F. tularensis strains are isolated yearly due to the fastidious cultural properties of F. tularensis. The difficulty of isolation and culture of F. tularensis from human specimens further hinders epidemiological surveillance because many molecular typing approaches are based on cultivated strains [START_REF] Li | Bacterial strain typing in the genomic era[END_REF]. Dempsey et. al identified a 1.59kb genomic deletion specific to F. tularensis subsp. holarctica isolates from France and Spain and classified these isolates into an emerging subclone in France and Iberian Peninsula [START_REF] Dempsey | Genomic deletion marking an emerging subclone of Francisella tularensis subsp. holarctica in France and the Iberian Peninsula[END_REF]. The same picture was observed for strains from Switzerland and France, countries where the epidemic clone was present before the tularemia * also see Figure 1 ** The DNA sequence identity and difference of concatenation of 4 intergenic spacers were generated by sequence alignment using Clustal W program (http://npsa-pbil.ibcp.fr/cgibin/npsa_automat.pl?page=/NPSA/npsa_clustalwan.html). of F. tularensis subsp holarctica with 80 strains of F. tularensis subsp holarctica from previous study [START_REF] Scola | Rapid comparative genomic analysis for clinical microbiology: the Francisella tularensis paradigm[END_REF] and F. tularensis subsp. tularensis strain Schu 4, F. tularensis subsp. mediasiatica strain GA99-3549, and F. tularensis subsp.novicida strain FSC147 which have genome sequences in GenBank. Sequences from the four intergenic spacers were concatenated.

Figure 1 .

 1 Figure 1. Dendrogram showing the phylogenetic organization of the studied 10 French strains

Table 1 .

 1 Comparison of genetic diversity of French and Swedish strains of F. tularensis subsp. holarctica by DNA sequence variation, number of MST genotype, and number of cluster based on four highly variable intergenic spacers.

	F. tularensis subsp.	Strain number* Number of MST	Number of	Sequence	Sequence
	holarctica strains		genotype*	cluster*	identity**	difference**
	French strains	11	5	1	97.39%	2.61%
	Swedish strains	79	26	2	88.62%	11.38%

tularensis subsp. holarctica strains in France are an emerging subclone. Identification of several subclusters among F. tularensis subsp. holarctica also confirms that it is a dynamic subspecies rather than a homogeneous group as observed in F. tularensis subsp. tularensis in which two subpopulations were identified [START_REF] Kugeler | Molecular Epidemiology of Francisella tularensis in the United States[END_REF]. The non homogeneous structure of F. tularensis subsp. holarctica population was also recently observed in Japan by using MLVA and in a global study using microarray SNP genotyping (14;15). This study confirms the usefulness of MST in epidemiological study of F. tularensis infection in France and even in Europe where tularemia is mainly caused by subsp holarctica, a particularly conserved subspecies [START_REF] Broekhuijsen | Genome-wide DNA microarray analysis of Francisella tularensis strains demonstrates extensive genetic conservation within the species but identifies regions that are unique to the highly virulent F. tularensis subsp. tularensis[END_REF]. To obtain a clearer population structure of F. tularensis subsp. holarctica, MST, that requires performing four short sequences for powerful discrimination, may be useful in the future large scale epidemiologic studies incorporating a large number of strains.