Tayeb Hamaizia

René Lozi
email: rlozi@unice.fr

Fast chaotic optimization algorithm based on locally averaged strategy and multifold chaotic attractor

Keywords: Chaos optimization algorithms, Nonlinear test functions, 2-D Discrete map, multifold chaotic attractor

Recently, chaos theory has been used in the development of novel techniques for global optimization , and particularly, in the specification of chaos optimization algorithms (COA) based on the use of numerical sequences generated by means of chaotic map.

In this paper, we present an improved chaotic optimization algorithm using a new two-dimensional discrete multifold mapping for optimizing nonlinear functions(ICOMM). The proposed method is a powerful optimization technique, which is demonstrated when three nonlinear functions of reference are minimized using the proposed technique.

Introduction

Application of chaos in industrial and applied problems is a very important and urgent research topic [START_REF] Li | Chaos optimization method and its application[END_REF][START_REF] Li | Optimizing complex function by chaos search[END_REF][START_REF] Choi | Chaotic local search algorithm[END_REF][START_REF] Zhang | Improved chaos optimization algorithm and its application in nonlinear constraint optimization problems[END_REF][START_REF] Yang | On the efficiency of chaos optimization algorithms for global optimization[END_REF] . In particulary in theory of control, cryptography and more recently in global optimization algorithms where introduction of chaotic numbers instead of random ones leads to better results [START_REF] Liu | Modified dynamic minimization algorithm for parameter estimation of chaotic system from a time series[END_REF]. In general, chaos has three main dynamics properties [START_REF] Tavazoei | An optimization algorithm based on chaotic behavior and fractal nature[END_REF]: sensitive dependence on initial conditions assessed by Lyapunov exponents [START_REF] Strogatz | Nonlinear Dynamics and Chaos. Massachussetts[END_REF][START_REF] Caponetto | Chaotic sequences to improve the performance of evolutionary algorithms[END_REF][START_REF] Wu | Introduction of Chaos Theory[END_REF], stochasticity and ergodicity. Taking advantage of properties as ergodicity and stochasticity of chaos, some new algorithms called chaos optimization algorithm (COA) and hybridization with other techniques are presented in the literature: gradient-based methods [START_REF] Ou-Yang | Combined BFGS-chaos method for solving geometric constraint[END_REF], genetic algorithms [START_REF] Fei | A novel chaotic optimization algorithm and its applications[END_REF][START_REF] Machado | Optimal tuning of fractional controllers using genetic algorithms[END_REF], particle swarm optimization [START_REF] Liu | Improved particle swarm optimization combined with chaos[END_REF][START_REF] Chen | Study and application of chaosparticle swarm optimization-based hybrid optimization algorithm[END_REF][START_REF] Meng | A hybrid particle swarm algorithm with embedded chaotic search[END_REF][START_REF] Alatas | Chaos embedded particle swarm optimization algorithms[END_REF][START_REF] Wei | Chaotic ant swarm for the traveling salesman problem[END_REF], differential evolution [START_REF] Guo | Chaos differential evolution algorithm with dynamically changing weighting factor and crossover factor[END_REF][START_REF] Ho | Parameter identification of chaotic systems using improved differential evolution algorithm[END_REF], clonal algorithms [START_REF] Du | Adaptive chaos clonal evolutionary programming algorithm[END_REF], artificial immune systems [START_REF] Zuo | Chaotic-search-based immune algorithm for function optimization[END_REF][START_REF] Zuo | The chaos artificial immune algorithm and its application to RBF neuro-fuzzy controller design[END_REF], bee colony algorithms [START_REF] Alatas | Chaotic bee colony algorithms for global numerical optimization[END_REF] and simulated annealing [START_REF] Mingjun | Application of chaos in simulated annealing[END_REF]. The aim of this paper is to present a new optimization algorithm chaotic based on new 2-D discrete chaotic system (map) with multifold attractor. The paper is organized as follows: in Sec.2 we present a new strategy based on locally averaged strategy of the global search and multifold chaotic attractor, in Sec. 3 we analyse the effectiveness of the proposed algorithm on a benchmark suite of 3 well-known nonlinear test functions which are optimized. Finally, we propose a conclusion.

Chaotic optimization method

Multifold chaotic attractor

Since the pioneer chaotic map introduced by Hénon [START_REF] Hénon | A two dimensional mapping with a strange attractor[END_REF] in 1976, many other chaotic maps have been studied [START_REF] Lozi | Un attracteur etrange? du type attracteur de Hénon[END_REF][START_REF] Sprott | chaos and Times-Series Analysis[END_REF][START_REF] Hunt | The Theory of Chaotic Attractorss[END_REF]. Among these maps , some display multifold patterns [START_REF] Zeraoulia | A two-dimensional discrete mapping with C ∞ -multifold chaotic attractors[END_REF][START_REF] Aziz-Alaoui | Dynamics of a Hénon-Lozi map[END_REF]. In this paper we have used the map recently introduced by Zeraoulia and Sprott, as a modification of Hénon map.

y 1 (k) = 1 -a(siny 1 (k -1)) + by(k -1) y(k) = y 1 (k -1) ( 1
)
where k is the iteration number.

The essential motivation to replace the quadratic term x 2 in the Hénon map by the nonlinear term in sinx. is to develop a C ∞ mapping that is capable of generating chaotic attractors with multifolds via a period-doubling bifurcation route to chaos which has not been studied before in the literature.

The fact that this map is C ∞ in some ways simplifies the study of the map and avoids some problems related to the lack of continuity or differentiability of the map. The choice of the term sinx has an important role in that it makes the solutions bounded for values of b such that |b| ≤ 1, and all values of a, while they are unbounded for |b| > 1. The chosen parameters values are a = 4 and b = 0.9 as suggested in [START_REF] Zeraoulia | A two-dimensional discrete mapping with C ∞ -multifold chaotic attractors[END_REF]. For this values the observed attractor (see Fig. 1) be- However, even if for all a in R and |b| < 1 and all initial conditions (y(0), y 1 (0)), the orbits of (1) are bounded (see cite26, theorem 5), this map exhibits very complicated dynamical behaviors with coexisting attractors. Hence in order to choose initial conditions for ICOMM for which the attractor is observed we choose it in a subset of D. The optimization algorithm needs to normalize the variable y(k) in the range [0, 1] using the transformation

z(k) = y(k) -α β -α . (2)
where Remark: Contrary to the theorical proof that Lozi map exhibits a strange chaotic attractor [START_REF] Misiurewicz | strange attractors for the lozi mappings[END_REF] there is only weak numerical evidence that (1) has chaotic attractors. It is possible that Fig. 1 displays only a transient regime wich leads eventually to a periodic orbit. Numerically, with any initial condition in the basin of attraction defined for a = 4 and b = 0.9 one finds a period 6 attractor when k ≥ 150, 000

y 1 (k + 6) = y 1 (k) = 10.9694028942956052. y 1 (k + 1) = 13.0613259136267086 y 1 (k + 2) = 8.97249334266406606 y 1 (k + 3) = 11.0071070225514713 y 1 (k + 4) = 13.0749780033934186 y 1 (k + 5) = 8
.95855079898761808 computation being done with double precision numbers. Again there is no proof that (1) possesses orbit shifted shadowing property as proved for generalized Lozi map [START_REF] Sakurai | orbit shifted shadowing property of generalized lozi maps[END_REF]. However, as for optimization algorithm only few iterates (i.e. k ≤ 10, 000) are needed, property of transient regime ( which is ergodic and stochastic within its range of value) worthes for ICOMM.

Locally averaged strategy

COMM is mainly COLM ( chaotic optimization method based on Lozi map) defined by Coelho [START_REF] Coelho | Tuning of PID Controller for an Automatic Regulator Voltage System using Chaotic Optimization Approach[END_REF] in which Lozi map is replaced by the map of Zeraoulia and Sprott. In order to improve COLM we have done [START_REF] Hamaizia | Improving Chaotic Optimization Algorithm using a new global locally averaged strategy[END_REF] some modification in the global step of reaserch. This new algorithm is called ICOLM ( Improved COLM). Now we introduce the modification to COMM in order the global search converges. The ICOMM algorithm is then defined as:

Find X to minimize f (X), X = [x 1 , x 2 , , x n ] subject to x i ∈ [L i ,U i ].
Where f is the objective function, and X is the decision solution vector consisting of n variables x i ∈ R n bounded by lower (L i ) and upper limits (U i ). The chaotic search procedure based on two-dimensional maps can be illustrated as follows [START_REF] Coelho | Tuning of PID Controller for an Automatic Regulator Voltage System using Chaotic Optimization Approach[END_REF][START_REF] Shayeghi | Robust PSS Design using Chaotic Optimization Algorithm for a Multimachine Power System[END_REF][START_REF] Shayeghi | A PSO based Unified Power Flow Controller for Damping of Power System Oscillations[END_REF] -Step 1 : Initialization of the numbers M g , M gl1 , M gl2 , M l of steps of chaotic search and initialization of parameters λ gl1 , λ gl2 , λ and initial conditions. Set k = 1, y(0), y 1 (0), a = 4 and b = 0.9 . Set the initial best objective function f = +∞ -Step 2: algorithm of chaotic global search:

while k ≤ M g do x i (k) = L i + z i (k).(U i -L i ) if f (X(k)) < f then X = X(k); f = f (x(k)) end if -Step 2-1: sub algorithm of first chaotic global-local search: while j ≤ M gl1 do for i = 0 to n do if r ≤ 0.5 then x i ( j) = xi + λ gl1 z i ( j).|(U i -L i )| else x i ( j) = xi -λ gl1 z i ( j).|(U i -L i )| end if end for if f (X( j)) < f then X = X( j); f = f (x( j)) end if j = j + 1 end while -Step 2-2: sub algorithm of second chaotic global- local search: while s ≤ M gl2 do for i = 0 to n do if r ≤ 0.5 then x i (s) = xi + λ gl2 z i (s).|(U i -L i )| else x i (s) = xi -λ gl2 z i (s).|(U i -L i )| end if end for if f (X(s)) < f then X = X(s); f = f (x(s)) end if s = s + 1 end while k = k + 1 end while -Step 3 : algorithm of chaotic local search: while k ≤ M g × (M gl1 + M gl2 ) + M l do for i = 0 to n do if r ≤ 0.5 then x i (k) = xi + λ z i (k).|(U i -L i )| else x i (k) = xi -λ z i (k).|(U i -L i )| end if end for if f (X(k)) < f then X = X(k); f = f (x(k)) end if k = k + 1 end while
Heuristics: the locally averaged strategy of ICOMM and ICOLM leads to better results than COMM or COLM as shown on Fig. 3. In this figure only three global search results are displayed x 1 , x 2 , x 3 with

f (x 2 ) < f (x 3 ) < f (x 1 ). (3)
The local search following global one starts from the best global result x 2 (from (3)) and gives x 2 . Instead the local-global search around x 1 , x 2 and x 3 , leads to x 1 , x 2 , x 3 which verify

f (x 1 ) < f (x 3 ) < f (x 2 ). (4)
The local search following the local-global one starts now from the best globally averaged result x 1 (from( 4)) and leads to x F f (x F ) < (x 1 ).

(5)

Experiments and analysis

In this section, the benchmark suite consists in three nonlinear multimodal functions that differ in terms of various characteristics. They are used to evaluate application performance of ICOMM. To examine the effectiveness of this method involving the multifold map, we apply ICOMM for each function. We use different values of steps size λ , λ gl1 and λ gl2 . For each trial we use 48 random initial points (48 runs); on a 3.2 GHz Pentium IV processor with 2 GB of RAM. For all the studied cases, the four configurations, numbered from C1 to C4, that are used are presented in Tab. 1 Table 1 The set of parameters values for every run on the benchmark suite defined in Sec. 2.2

Multimodal test functions

We test ICOMM in 2D optimization problem using :

3.1.1 Function f 1 (See Fig. 4)

The function f 1 is the Easom function [START_REF] Li | Optimizing complex function by chaos search[END_REF][START_REF] Yang | On the efficiency of chaos optimization algorithms for global optimization[END_REF]

f 1 = -cos(x 1 )cos(x 2 )e (-(x 1 -π) 2 -(x 2 -π) 2 ))
its characteristics are:

search domain:-10

≤ x i ≤ 10, i = 1, 2.
number of local minima: several local minima.

one global minimum: x = (π, π), f ( x) = -1.

3.1.2 Function f 2 (See Fig. 5)

The function f 2 is the Rosenbrock's function [START_REF] Li | Optimizing complex function by chaos search[END_REF][START_REF] Yang | On the efficiency of chaos optimization algorithms for global optimization[END_REF]

f 2 = 100(x 2 1 + x 2 ) 2 + (1 -x 1 )
2 its characteristics are defined as follows:

search domain: -2.048 ≤ x i ≤ 2.048, i = 1, 2.

number of local minima: several local minima.

-The global minimum: x = (0, 0), f ( x) = 0. The function f 3 is more complex [START_REF] Hamaizia | Improving Chaotic Optimization Algorithm using a new global locally averaged strategy[END_REF][START_REF] Cong | An Improved Algorithm of Chaos Optimization[END_REF] than f 1 and f 2

f 3 = x 4 1 -7x 2 1 -3x 1 + x 4 2 -9x 2 2 -5x 2 +11x 2 1 x 2 2 + 99sin(71x 1 ) + 137sin(97x 1 x 2 ) + 131sin(51x 2 )
.

search domain: -10 ≤ x i ≤ 10, i = 1, 2.

number of local minima: several local minima.

The essential feature of this benchmark function is that location of minima is not symmetric. In a forthcoming paper we will extend our numerical analysis in higher dimension with an extended benchmark suite [START_REF] Cong | An Improved Algorithm of Chaos Optimization[END_REF].

Numerical results

The numerical results are displayed in Tab.2. For both functions f 1 and f 2 the global minimum is easily reached in few steps. Configurations C1 and C2 are fast and efficient. Concerning f 3 which possesses hundreds of local minima, the best results are obtained using configurations C3 and C4. The global minima is not yet theoretically known, however extended numerical computations give some clues that the values of f 3 found using both C3 and C4 are not far from the value of f 3 on the global minimum. The locally averaged strategy of ICOMM is illustrated on Fig. 7 on which the result of every step 2-2 is plotted. method is fast and converges to a good optimum. because we used a sampling mechanism to coordinate the research methods based on chaos theory, and we refined the final solution using a second method of local search. Further research is needed to gain more confidence and better understanding of the proposed methodology. The proposed algorithm has to be evaluated for a large number of test functions in higher dimension.

1

  Chaotic multifold attractor of the map (1) obtained for a = 4 and b = 0.9. longs to the square (y 1 , y) ∈ [-8.588, 27.645] 2 = D ∈ R 2 .

1 0

 1 [α, β ] = [-8.588, 27.645] Numerical computation leads to the density d(s) of iterated values of y(k) displayed on Fig. 2. In this figure, the density is normalized to 1 over the whole interval [0, 1] i.e. d(s)ds = 1.

Fig. 2

 2 Fig. 2 density of iterated values of y(k) of equation (1) over the interval [0, 1] splitted in 100 boxes for 100,000 iterated values.

Fig. 3

 3 Fig. 3 Heuristics of locally-averaged strategy.

3. 1 . 3

 13 Function f 3 (See Fig.6)

1 - 2 Fig. 4

 124 Fig. 4 graph of test function f 1 in the search domain

Fig. 5 Fig. 6

 56 Fig. 5 graph of test function f 2 in the search domain

Fig. 7

 7 Fig. 7 Locally-averaged strategy of chaotic search. Results of every Step 2-2 for f 3

  : Inputs: M g : max number of iterations of chaotic Global search. Mgl 1 : max number of iterations of first chaotic Local search in Global search. Mgl 2 : max number of iterations of second chaotic Local search in Global search. M l : max number of iterations of chaotic Local search. Mt = Mg×(Mgl 1 +Mgl 2 )+Mg: stopping criterion of chaotic optimization method in iterations. λ gl1 : step size in first global-local search. λ gl2 : step size in second global-local search. λ : step size in chaotic local search.

	Outputs:
	X: best solution from current run of chaotic search.
	f : best objective function (minimization problem).

  Table 2 optimization results over 48 runs for 4 parameter configurations

	Best value	Mean	Std.Dev ( x,y)	T/s
		value
	f 1 -1.0000	-0.9999	0.0001 (3.1443, 3.1443)	1.9490
	-1.0000	-0.9998	0.0002 (3.1458, 3.1446)	1.9499
	-1.0000	-0.9999	0.0001 (3.1453, 3.1445)	27.8084
	-1.0000	-1.0000	0.0000 (3.1420, 3.1420)	55.5564
	f 2 0.0000	0.0000	0.0000 (0.9996, 0.9978)	1.8380
	0.0000	0.0000	0.0000 (0.9988,0.9977)	1.8386
	0.0000	0.0000	0.0000 (0.9999, 0.9998)	25.9905
	0.0000	0.0000	0.0000 (1.0001, 1.0002)	52.1532
	f 3 -373.2600	-362.8730	5.8505 (-0.2926,-2.6142)	2.1350
	-391.1240	-362.9798	10.7292 (-2.0556, -2.4995) 2.1736
	-395.5435	-390.5618	5.1234 (-0.2897, -0.2786) 31.3117
	-395.5870	-391.3068	4.7932 (-0.2034, 0.0920)	62.2426


Matthew Glover

Raynald Cossard

Damien Pichard

Kwasi Obiri-Danso

Agnès Rötig

Agnès Delahodde
email: agnes.delahodde@igmors.u-psud.fr

Caenorhabditis elegans, a pluricellular model organism to screen new genes involved in mitochondrial genome maintenance

Keywords: mitochondria, mtDNA maintenance, nematode / Caenorhabditis elegans

The inheritance of functional mitochondria depends on faithful replication and transmission of mitochondrial DNA (mtDNA). A large and heterogeneous group of human disorders is associated with mitochondrial genome quantitative and qualitative anomalies.

Several nuclear genes have been shown to account for these severe OXPHOS disorders.

However, in several cases the disease-causing mutations still remain unknown.

C. elegans has been largely used for studying various biological functions as this multicellular organism has short life cycle and is easy to grow in the laboratory. Mitochondrial functions are relatively well conserved between human and C. elegans and heteroplasmy exists in this organism as in human. C. elegans therefore represent a useful tool for studying mtDNA maintenance. Suppression by RNA interference of genes involved in mtDNA replication such as polg-1, encoding the mitochondrial DNA polymerase, results in reduced mtDNA copy number but in a normal phenotype of the F1 worms. By combining RNAi of genes involved in mtDNA maintenance and EtBr exposure we were able to reveal a strong and specific phenotype (developmental larval arrest) associated to a severe decrease of mtDNA copy number. Moreover, we tested and validated the screen efficiency for human orthologous genes encoding mitochondrial nucleoid proteins. This allowed us to identify several genes that seem to be closely related to mtDNA maintenance in C. elegans. This work reports a first step in the further development of a large-scale screening in C. elegans that should allow to identify new genes of mtDNA maintenance whose human orthologs will obviously constitute new candidate genes for patients with quantitative or qualitative mtDNA anomalies.

A large and heterogeneous group of disorders is associated with mitochondrial genome anomalies in human. Mutations in nuclear genes encoding proteins involved in mtDNA maintenance can result in large-scale mtDNA rearrangements (mtDNA deletion) and abnormal copy number (mtDNA depletion) of the mitochondrial genome. Autosomal dominant progressive external ophthalmoplegia (adPEO) is an adult-onset mitochondrial disorder characterized by ophthalmoparesis with exercise intolerance, ataxia, peripheral neuropathy, and multiple mtDNA deletions [START_REF] Spelbrink | Human mitochondrial DNA deletions associated with mutations in the gene encoding Twinkle, a phage T7 gene 4-like protein localized in mitochondria[END_REF][START_REF] Suomalainen | Autosomal dominant progressive external ophthalmoplegia with multiple deletions of mtDNA: clinical, biochemical, and molecular genetic features of the 10q-linked disease[END_REF]. Familial ad-PEO is genetically heterogeneous, and at least six nuclear genes account for this disease, namely, ANT1, encoding the adenosine diphosphate-triphosphate translocator [START_REF] Kaukonen | Role of adenine nucleotide translocator 1 in mtDNA maintenance[END_REF][START_REF] Agostino | Mutations of ANT1, Twinkle, and POLG1 in sporadic progressive external ophthalmoplegia (PEO)[END_REF], POLG1 [START_REF] Van Goethem | Mutation of POLG is associated with progressive external ophthalmoplegia characterized by mtDNA deletions[END_REF] and POLG2 [START_REF] Longley | Mutant POLG2 disrupts DNA polymerase gamma subunits and causes progressive external ophthalmoplegia[END_REF] encoding the catalytic and accessory subunits of the mtDNA polymerase, PEO1

encoding the Twinkle DNA helicase [START_REF] Spelbrink | Human mitochondrial DNA deletions associated with mutations in the gene encoding Twinkle, a phage T7 gene 4-like protein localized in mitochondria[END_REF], OPA1 encoding a mitochondrial dynamin-related GTPase [START_REF] Amati-Bonneau | OPA1 mutations induce mitochondrial DNA instability and optic atrophy 'plus' phenotypes[END_REF] and RRM2B encoding the p53-inducible small subunit of ribonucleotide reductase [START_REF] Tyynismaa | A heterozygous truncating mutation in RRM2B causes autosomal-dominant progressive external ophthalmoplegia with multiple mtDNA deletions[END_REF].

mtDNA depletion syndrome (MDS) is a clinically and genetically heterogeneous group of autosomal recessive diseases characterized by a reduction in mtDNA copy number [START_REF] Rotig | Genetic causes of mitochondrial DNA depletion in humans[END_REF].

Several nuclear genes have been shown to account for these severe OXPHOS disorders.

Indeed, mutations in the deoxyguanosine kinase (DGUOK) and the thymidine kinase genes (TK2) have been reported in the hepatocerebral and myopathic forms of MDS [START_REF] Saada | Mutant mitochondrial thymidine kinase in mitochondrial DNA depletion myopathy[END_REF][START_REF] Mandel | The deoxyguanosine kinase gene is mutated in individuals with depleted hepatocerebral mitochondrial DNA[END_REF] respectively. Also, POLG1 mutations associated with Alpers' syndrome [START_REF] Ferrari | Infantile hepatocerebral syndromes associated with mutations in the mitochondrial DNA polymerase-gammaA[END_REF][START_REF] Naviaux | POLG mutations associated with Alpers' syndrome and mitochondrial DNA depletion[END_REF] as well as the hepatocerebral form lead to mtDNA depletion. Mutations in TP gene encoding the cytosolic thymidine phosphorylase result in mitochondrial neurogastrointestinal encephalopathy (MNGIE) syndrome with mtDNA deletion and depletion [START_REF] Nishino | Thymidine phosphorylase gene mutations in MNGIE, a human mitochondrial disorder[END_REF]. The succinyl coenzyme A-ligase subunits (SUCLA2, [START_REF] Elpeleg | Deficiency of the ADP-forming succinyl-CoA synthase activity is associated with encephalomyopathy and mitochondrial DNA depletion[END_REF] and SUCLG1 [START_REF] Ostergaard | Deficiency of the alpha subunit of succinate-coenzyme A ligase causes fatal infantile lactic acidosis with mitochondrial DNA depletion[END_REF]) and MPV17 [START_REF] Spinazzola | MPV17 encodes an inner mitochondrial membrane protein and is mutated in infantile hepatic mitochondrial DNA depletion[END_REF] genes have

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ACCEPTED MANUSCRIPT also been shown to account for mtDNA depletion in a few pedigrees. Finally, mutations of RRM2B encoding a small subunit of the cytosolic ribonucleotide reductase have been reported to cause severe muscle mtDNA depletion [START_REF] Bourdon | Mutation of RRM2B, encoding p53-controlled ribonucleotide reductase (p53R2), causes severe mitochondrial DNA depletion[END_REF]. All these genes are involved in mtDNA maintenance or dNTP metabolism. However, in several cases of ad-PEO and MDS the disease-causing mutations remain still unknown.

The suitability of the yeast Saccharomyces cerevisiae as a model for human mitochondrial studies has been well demonstrated. However notable differences in mtDNA structure and dynamics between yeast and human did not make it a perfect tool to study the mtDNA maintenance. Indeed, human cells contain10 2 -10 4 mtDNA copies whereas yeast cells contain only 20-100 copies. The mitochondrial genome, 16.6 kb in human and 85.8 kb in yeast, is predominantly linear in yeast but is circular in human [START_REF] Burger | Unique mitochondrial genome architecture in unicellular relatives of animals[END_REF][START_REF] Legros | Organization and dynamics of human mitochondrial DNA[END_REF][START_REF] Williamson | The curious history of yeast mitochondrial DNA[END_REF]. Finally heteroplasmy is very frequently observed for mtDNA mutations in human whereas yeast cannot normally maintain stably heteroplasmy [START_REF] Shoubridge | Mitochondrial encephalomyopathies[END_REF][START_REF] Zeng | The Saccharomyces cerevisiae ATP22 gene codes for the mitochondrial ATPase subunit 6-specific translation factor[END_REF]. Furthermore, because this yeast can grow robustly by fermentation in the absence of mtDNA, it loses its mitochondrial genome very rapidly upon inactivation of a large class of genes encoding mitochondrial proteins involved in almost all the mitochondrial biogenesis pathways (mitochondrial translation, ATP synthesis, iron homeostasis, mitochondrial import and morphology). As such it cannot be used easily to address the question of mtDNA transmission control [START_REF] Contamine | Maintenance and integrity of the mitochondrial genome: a plethora of nuclear genes in the budding yeast[END_REF].

The characteristics of Caenorhabditis elegans make it a perfect complement to the yeast system. While C. elegans has been largely used for studying various biological functions such as neuron development or apoptosis, few studies have focused on mitochondria. Its mitochondrial genome (13.7 kb) has been fully sequenced, it encodes 12 mitochondrial RC subunits and is similar in size and gene content to its human counterpart [START_REF] Okimoto | The mitochondrial genomes of two nematodes, Caenorhabditis elegans and Ascaris suum[END_REF]. The group of B. Lemire has brought a large contribution by showing that heteroplasmy exists in this organism as in human as far as mtDNA deletions are considered [START_REF] Tsang | Stable heteroplasmy but differential inheritance of a large mitochondrial DNA deletion in nematodes[END_REF]. This team also showed that mtDNA copy number is coordinated with the maturation steps of C. elegans life cycle. Indeed, mtDNA amplification is necessary for normal development as blocking mtDNA replication results in development arrest [START_REF] Tsang | Mitochondrial genome content is regulated during nematode development[END_REF]. The mtDNA copy number is strictly regulated during C. elegans development. Indeed, mtDNA content that is maternally derived remains essentially unchanged from embryo up to the L3 larval stage. The copy number then increases at least 3-fold in L4 larvae and is associated with somatic cells development and gonad formation. A further huge increase in mtDNA content takes place in adult hermaphrodites during oogenesis and high production of embryo [START_REF] Tsang | Mitochondrial genome content is regulated during nematode development[END_REF][START_REF] Bratic | Mitochondrial DNA level, but not active replicase, is essential for Caenorhabditis elegans development[END_REF]. Finally, blockage of mtDNA replication by ethidium bromide (EtBr), a well-known molecule acting as a DNA-intercaling dye and potent inhibitor of mtDNA transcription and replication [START_REF] Hayakawa | Ethidium bromide-induced inhibition of mitochondrial gene transcription suppresses glucose-stimulated insulin release in the mouse pancreatic beta-cell line betaHC9[END_REF][START_REF] Richardson | Mechanism of ethidium bromide inhibition of RNA polymerase[END_REF][START_REF] Richardson | Letter: Effect of ethidium bromide on transcription of linear and circular DNA templates[END_REF] results in developmental arrest [START_REF] Tsang | Mitochondrial genome content is regulated during nematode development[END_REF]. Surprisingly, inactivation by RNA interference (RNAi)

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ACCEPTED MANUSCRIPT

over one generation of genes encoding proteins essential for mtDNA replication such as polg-1 (DNA polymerase γ) or mtss-1 (mitochondrial single-stranded DNA-binding protein)

does not give rise to a strong phenotype [START_REF] Fernandez | New genes with roles in the C. elegans embryo revealed using RNAi of ovary-enriched ORFeome clones[END_REF][START_REF] Gonczy | Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III[END_REF][START_REF] Kamath | Systematic functional analysis of the Caenorhabditis elegans genome using RNAi[END_REF][START_REF] Rual | Toward improving Caenorhabditis elegans phenome mapping with an ORFeome-based RNAi library[END_REF][START_REF] Sonnichsen | Full-genome RNAi profiling of early embryogenesis in Caenorhabditis elegans[END_REF]. Only continuous exposure to RNAi over several generations revealed an abnormal phenotype (gonad protrusion and sterility, [START_REF] Sugimoto | Caenorhabditis elegans par2.1/mtssb-1 is essential for mitochondrial DNA replication and its defect causes comprehensive transcriptional alterations including a hypoxia response[END_REF]).

Furthermore, homozygous polg-1 knockout mutants had normal development rates indicating a high maternal contribution of polg-1. However, loss of polg-1 leads to complete sterility and shortened lifespan. Moreover during adult life, worms failed to maintain normal mtDNA levels [START_REF] Bratic | Mitochondrial DNA level, but not active replicase, is essential for Caenorhabditis elegans development[END_REF].

Several genes are known to be involved in mtDNA maintenance in various species.

Not only genes encoding mitochondrial proteins but also cytosolic proteins are involved in this mechanism such as RRM2B and TP in human. This suggests that unexpected genes could be required for normal mtDNA metabolism that have to be identified. In the present study, we designed an efficient and rapid screening for the identification of genes required for mtDNA maintenance in the worm C. elegans by combining RNAi and EtBr exposure.

Considering that mitochondrial functions are relatively well conserved during evolution, the systematic use of this screen for all C. elegans genes will certainly allow to identify new genes of mtDNA maintenance whose human orthologs could obviously constitute new candidate genes for patients.

Results

polg-1 and mtss-1 knock down results in mtDNA depletion

We first silenced two genes, polg-1 and mtss-1 in wild-type animals. These two genes are orthologs to the human POLG1 and SSBP1 genes encoding the mitochondrial DNA polymerase γ and the single strand DNA binding protein respectively, both genes being involved in mtDNA replication. Synchronized N2 embryos were subjected to RNAi feeding until the adult stage and during the F1 progeny. As expected, the F1 progeny developed into adulthood with no strong phenotype (Fig. 1A). We next estimated by quantitative PCR the mtDNA copy number in F1 adults having laid all their eggs to avoid any differences due to oocyte production. The mtDNA copy number in the hermaphrodites fed by bacteria expressing polg-1 or mtss-1 dsRNA was approximately 80% and 70% respectively lower than the control adult hermaphrodites (Fig. 1B) but similar to that of L4 worms treated with very high concentration of EtBr (125 µg/ml), concentration already known to lead to mtDNA depletion [START_REF] Tsang | Mitochondrial genome content is regulated during nematode development[END_REF].

As POLG1 mutations in human can result in either quantitative or qualitative mtDNA anomalies, we looked for mtDNA deletions in worms subjected to polg-1 knock-down. Longrange PCR amplification of the nearly two-thirds of the genome in these worms revealed the

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same mtDNA length product (9 kb) in polg-1 treated animals and controls ruling out that polg-1 inactivation results in mtDNA deletion (Fig. 1C).

polg-1 silencing by RNAi was highly efficient and specific as polg-1 transcript was severely impaired in F1 progeny when subjected to RNAi feeding (Fig. 1D). We then analyzed the efficiency of mtss-1 RNAi in vivo. For this purpose, we constructed transgenic animals expressing a mtss-1::GFP fusion construct under the control of either the body-wall muscle specific myo-3 promoter (myo3-MTSS::GFP) or mtss-1 promoter and 3'-UTR regulatory sequences (par-MTSS::GFP). As shown in Figure 1E, the GFP signal appeared cytoplasmic and as punctuate foci. This signal is aligned and regularly spaced along myofibrilla within the cytoplasm of body-wall muscle cells in agreement with a mitochondrial localization. Feeding these transgenic worms with bacteria expressing mtss-1 dsRNA led to the loss of most of the MTSS::GFP punctuated signals (Fig. 1E), showing the efficiency of RNAi. Altogether, these results show that RNAi of polg-1 and mtss-1 over one generation lead to a specific knock-down of polg-1 and mtss-1, to a severe mtDNA depletion in somatic cells but does not result in major anomalies in worms development.

Ethidium bromide exposure combined with polg-1 and mtss-1 knock-down results in an obvious developmental phenotype

To design a large scale screening of genes involved in mitochondrial genome maintenance using RNAi feeding, we need a clear and obvious phenotype, appearing rapidly during the worms life.

Ethidium bromide (EtBr) is a well-known nucleic acid intercalating compound that preferentially inhibits mtDNA replication and transcription. Exposure to EtBr results in a total loss of mtDNA in mammalian cells (ρ° cells ) [START_REF] Hayashi | Effects of ethidium bromide treatment of mouse cells on expression and assembly of nuclear-coded subunits of complexes involved in the oxidative phosphorylation[END_REF][START_REF] King | Isolation of human cell lines lacking mitochondrial DNA[END_REF][START_REF] Park | Resistance of mitochondrial DNA-depleted cells against cell death: role of mitochondrial superoxide dismutase[END_REF] or diminished number of mtDNA copies in C. elegans [START_REF] Tsang | Mitochondrial genome content is regulated during nematode development[END_REF] and Fig. 1B). Concentration and timing of the EtBr treatment during C.

elegans development affects terminal developmental stages. Indeed, submitting L1 animals to high EtBr concentration (125 µg/ml) leads to a L3 stage arrest. Prolonged exposure to EtBr results in mtDNA depletion and L3 arrested larvae presented a 25-fold decrease of mtDNA content. Interestingly, the L3 arrest phenotype was reversible upon drug removal indicating that EtBr is not acting by causing mutations in either the mitochondrial or the nuclear genomes since these would be expected to be heritable and irreversible [START_REF] Tsang | Mitochondrial genome content is regulated during nematode development[END_REF].

We reasoned that animals with low mtDNA content should be more sensitive to EtBr than animals with normal mtDNA content. Therefore silencing of genes controlling mtDNA copy number combined with EtBr exposure should rapidly reveal a defective phenotype. We therefore performed polg-1 and mtss-1 RNAi experiments on wild-type animals from the L1 stage to the adult stage (3 days). The adults (F0) were then allowed to hatch 100 eggs (F1

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progeny) on NGM plates seeded with bacteria expressing polg-1 or mtss-1 dsRNA, with and without increasing concentrations of EtBr (10 to 80 µg/ml). We then analyzed the development of the F1 larvae and evaluated the concentration at which 100 % of the larvae were arrested at the L3 stage. We observed that 100% of control larvae were arrested at the L3 stage after treatment with 80 µg/ml EtBr as previously reported [START_REF] Tsang | Mitochondrial genome content is regulated during nematode development[END_REF] whereas as few as 20 and 60 µg/ml EtBr were sufficient to stop development of worms fed with dsRNA for polg-1

and mtss-1 respectively (Fig. 2A and2B). Concentration of 50 µg/ml of EtBr appeared to clearly discriminate genes important for mtDNA stability (90 to 100% L3 arrest) compared to the control (50% L3 arrest). To confirm that this L3 arrest phenotype is indeed related to mtDNA maintenance, we further examined silencing of rrt-2 gene encoding the arginyl-tRNA synthetase predicted to be involved in mitochondrial translation and of pmp-4 gene encoding a peroxisomal protein homologous to human ABCD1, which when mutated leads to X-linked adrenoleukodystrophy [START_REF] Mosser | Putative X-linked adrenoleukodystrophy gene shares unexpected homology with ABC transporters[END_REF]. The F1 progeny of worms submitted to RNAi of those genes shows 50-55% L3 arrest as control worms (Fig 2C). This clearly shows that the increased sensitivity towards EtBr exposure is specific to an effect on mtDNA replication. Altogether, these results show that combining RNAi of genes involved in mtDNA maintenance and EtBr exposure reveals a strong and specific phenotype that can be further used for large-scale screening in C. elegans.

Identification of new C. elegans genes involved in mtDNA stability

In human, the nucleoid consists of 5-7 mtDNA copies associated with several proteins. The precise protein composition of nucleoid is still under debate and depends of the procedure used to isolate these proteins [START_REF] Spelbrink | Functional organization of mammalian mitochondrial DNA in nucleoids: history, recent developments, and future challenges[END_REF]. Recent studies identify a set of proteins associated with human mtDNA nucleoids [START_REF] Bogenhagen | The layered structure of human mitochondrial DNA nucleoids[END_REF][START_REF] Wang | Human mitochondrial DNA nucleoids are linked to protein folding machinery and metabolic enzymes at the mitochondrial inner membrane[END_REF]. Among them, a set of 31 mtDNA crosslinked proteins constitutes the core nucleoid proteins (class I) that are supposed to be closely related to mtDNA replication and transcription. A second class of proteins (class II) is part of nucleoids but not crosslinked to mtDNA. Finally, class III proteins are not found in native nucleoids [START_REF] Bogenhagen | The layered structure of human mitochondrial DNA nucleoids[END_REF]. Considering the efficiency of our screen on C. elegans, we decided to successively inactivate the genes coding proteins of the mitochondrial nucleoid with the aim to reveal genes involved in mtDNA maintenance. The human protein sequences were used to explore the C. elegans genome database (C. elegans Sequencing Consortium 1998) by BLAST searching. Several open reading frames were identified, some of them being already known as true orthologous genes. Among those genes, we could study those that did not lead to embryonic lethality in large-scale RNAi screens and were present in the Ahringer RNAi feeding library. As a first screen, we retained 19 C. elegans genes homologous to 16 human mitochondrial nucleoid genes (Table 1). ANT2 and ANT3 were reported respectively

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as class I and III nucleoid proteins [START_REF] Bogenhagen | The layered structure of human mitochondrial DNA nucleoids[END_REF]. As there is no direct orthology between any C.

elegans ant gene and a particular human ANT gene, we decided to screen the five C. elegans mitochondrial ADP/ATP carrier proteins (ant-1.1, ant-1.2, ant-1.3, ant-1.4 and C47E12.2) as previously reported [START_REF] Farina | Differential expression pattern of the four mitochondrial adenine nucleotide transporter ant genes and their roles during the development of Caenorhabditis elegans[END_REF]. One of them ant-1.1 could not be included in our screen as its inactivation from L1 larval stage leads to sterility [START_REF] Farina | Differential expression pattern of the four mitochondrial adenine nucleotide transporter ant genes and their roles during the development of Caenorhabditis elegans[END_REF]. Moreover, due to the high sequence identities (84%) of ant-1.3 and ant-1.4 mRNA, RNAi of any of these two genes results in silencing of both ([45] and our results). Therefore results are presented for ant-1.4

only. On the same way, IMMT human gene (Mitofilin) presents two orthologs in C. elegans, immt-1 and immt-2 that were both tested.

Using the Ahringer library feeding RNAi clones, we inactivated expression of those genes beginning from synchronized L1 larvae. After 3 days, adults were then allowed to lay around 100 eggs onto new RNAi plates with 50 µg/ml EtBr. At day 4 the percentages of L3 arrested larvae versus gravid adults were counted (Fig. 3). This revealed a first group of genes for which inactivation resulted in 85 to 100% of L3 arrest. These genes are homologs of human genes encoding proteins of class I, mtss-1, polg-1, hmg-5, polrmt (Y105E8A.23), dnj-10, ANT (C47E12.2) and ant-1.4 but also of class II, atad-3, immt-1 and of class III, phi-37. To confirm that inactivation of these genes led to mtDNA depletion, we quantified the relative mtDNA copy number of worms submitted to the same RNAi without any EtBr exposure. As shown on Fig. 3, all the animals treated by RNAi, which led to more than 85%

EtBr sensitivity, presented obvious mtDNA depletion (10-40% of normal mtDNA content) except immt-1. None of these gene silencing resulted in a specific abnormal developmental phenotype (data not shown). To ascertain that the L3 arrest phenotype was due to specific inactivation of the targeted genes, we quantified by reverse transcriptase-PCR (RT-PCR) the expression of most of these genes during the RNAi experiments without EtBr exposure and consistently observed silencing of all genes (Fig. 4). These results therefore suggest that the proteins encoded by these genes are closely related to mtDNA replication.

A second group of genes was found for which inactivation did not result in any EtBr sensitivity (44 to 74% L3 arrest). Among them, mel-32, clpx (D2030.2), pyr-1, hsp-6 and ant1-2 encode proteins homologous of human proteins of the core nucleoid whereas dao-3, kat-1 and immt-2 are orthologs of the class II or III. Moreover, silencing of these genes does not result in major modification of the mtDNA copy number (Fig. 3). The same results were obtained with the inactivation of irk-1 and C34B7.

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at least in human, they seem not to be involved in mtDNA copy number control in C. elegans.

Discussion

We have designed an efficient screening method for the identification of genes involved in the mitochondrial genome stability in C. elegans. Indeed, by combining RNAi of polg-1 and mtss-1 genes already known to be involved in mtDNA maintenance and EtBr exposure of worms, we were able to rapidly reveal a L3 developmental arrest associated with a severe decrease in mtDNA copy number. It has been shown that there is no mtDNA replication during the early larval stages and that transition from L3 to adulthood is associated with an important increase of mtDNA replication [START_REF] Tsang | Mitochondrial genome content is regulated during nematode development[END_REF][START_REF] Bratic | Mitochondrial DNA level, but not active replicase, is essential for Caenorhabditis elegans development[END_REF]. Therefore we can hypothesize that EtBr exposure drastically inhibited replication of the low mtDNA content resulting from polg-1 or mtss-1 silencing. This developmental phenotype seems to be specific as silencing of other genes encoding either mitochondrial proteins not involved in mtDNA maintenance (C29H12.1, lrk-1) or non-mitochondrial proteins (pmp-4, C34B7.2) did not result in a L3 arrest and/or mtDNA depletion. Interestingly, with a notable exception (immt-1), only mtDNA quantitative anomalies were observed. This abnormal and specific phenotype is obvious after three days and therefore allows a very rapid screen that can be further used for large-scale screening in C. elegans.

We extended this screen to C. elegans genes encoding proteins homologous to human nucleoid proteins. The function of some of these proteins is clearly related to mtDNA stability. Indeed, the mitochondrial RNA polymerase, DNA polymerase, single strand DNA binding protein, Twinkle helicase or the transcription factor TFAM have been shown to directly interact with mtDNA in several species. We applied our screen to the C. elegans genes orthologous to human nucleoid encoding protein genes. This clearly showed us that inactivation of genes involved in mtDNA replication or transcription rapidly result in L3 developmental arrest and mtDNA depletion confirming the efficiency of our screen. However, other proteins identified as true components of the human nucleoid (core nucleoid) do not display obvious function related to mtDNA maintenance. This is the case for SHMT2, CPS1

or CLPX for example. SHMT2 (serine hydroxymethyltransferase) is primarily responsible for glycine synthesis, CPS1 (carbamoyl-phosphate synthetase 1) catalyzes the first committed step of the hepatic urea cycle and CLPX gene encodes a ClpX caseinolytic protease X homolog with no well-defined function. Whether these proteins are bifunctional proteins with a second function directly related to mtDNA maintenance or are structural components of the nucleoid with no direct function in mtDNA replication, transcription or translation is still under debate. Silencing of these genes by RNAi combined with EtBr exposure resulted in a normal mtDNA content, or an increased mtDNA content which can be the result of a general mitochondrial upregulation, and a L3 arrest level similar to that observed in control animals

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suggesting no functional interaction with the mitochondrial genome.

Furthermore, our screen distinguishes five new genes involved in mtDNA maintenance in a pluricellular organism, atad-3, phi-37, dnj-10, immt-1 and Y105E8A.23.

ATAD3 is an AAA-domain protein, a class of proteins that is known to have important roles in DNA and RNA transactions. In human, ATAD3 was reported to be an intrinsic mitochondrial membrane protein embedded in the inner membrane leaving its AAA-domain directed towards the matrix [START_REF] Bogenhagen | The layered structure of human mitochondrial DNA nucleoids[END_REF][START_REF] Cruz | Proteomic analysis of the mouse liver mitochondrial inner membrane[END_REF]. In C. elegans, atad-3, the homolog of vertebrate ATAD3, has been shown to be important for larval development and is required for proper organ function suggesting a crucial role of this protein for the up regulation of mitochondrial activity during the progression through larval stages [START_REF] Hoffmann | elegans ATAD-3 is essential for mitochondrial activity and development[END_REF]. Moreover, atad-3 (RNAi) animals showed drastic reduction of complex I and citrate synthase (CS) activities. The severe mtDNA depletion we observed after atad-3 silencing could explain this respiratory chain deficiency. Whether CS decrease is primary related to atad-3 suppression or secondary to mtDNA depletion has still to be demonstrated. It should be noted that CS is a class III protein of the nucleoid and that its decreased activity in atad-3 (RNAi) animals could therefore result from defective nucleoid assembly. Interestingly, in human and in flies it was shown that the ATAD3A protein does not bind mtDNA but rather participates in the connection between the inner and outer mitochondrial membranes regulating the mitochondrial dynamics [START_REF] Gilquin | The AAA+ ATPase ATAD3A controls mitochondrial dynamics at the interface of the inner and outer membrane[END_REF].

Interactions between the mitochondrial inner and outer membranes control a number of central mitochondrial functions such as channeling of metabolites, protein transport, coordinated fusion and fission, and mitochondrial DNA inheritance. The relationship between mitochondrial network and mtDNA maintenance has been already demonstrated as mutations in OPA1, encoding a dynamin-related GTPase and being mainly involved in the mitochondrial network organization [START_REF] Olichon | Mitochondrial dynamics and disease, OPA1[END_REF] results in multiple mtDNA deletions [START_REF] Amati-Bonneau | OPA1 mutations induce mitochondrial DNA instability and optic atrophy 'plus' phenotypes[END_REF]. Our results strengthen the idea that ATAD3A, through interactions with proteins of the inner membrane, directly contributes to mtDNA maintenance within nucleoids.

phi-37 encodes the alpha subunit of mitochondrial ATP synthase and the human ATP5A protein is a class III nucleoid protein. It is hypothesized that class III proteins were adventitiously crosslinked to other nucleoid proteins [START_REF] Bogenhagen | The layered structure of human mitochondrial DNA nucleoids[END_REF]. Nevertheless, suppression of phi-37 leads to highly sensitive worms to EtBr and mtDNA depletion. Whether this protein plays a direct role on mtDNA stability in addition to its role in the mitochondrial ATP synthase is very interesting and deserves more experiments. It can be also hypothesized that ATP synthase deficiency due to phi-37 silencing could induce cristae modifications and consequently avoid mtDNA attachment to the inner mitochondrial membrane resulting in a secondary mtDNA depletion [START_REF] Allen | Membrane tubulation and proton pumps[END_REF][START_REF] Lefebvre-Legendre | Failure to assemble the alpha 3 beta 3 subcomplex of the ATP synthase leads to accumulation of the alpha and beta subunits within inclusion bodies and the loss of mitochondrial cristae in Saccharomyces cerevisiae[END_REF][START_REF] Paumard | The ATP synthase is involved in generating mitochondrial cristae morphology[END_REF].

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DNAJA3 for mitochondrial biogenesis has been demonstrated in mice where a progressive RC deficiency and decreased copy number of mtDNA were reported in cardiomyocytes lacking DNAJA3 [START_REF] Hayashi | A crucial role of mitochondrial Hsp40 in preventing dilated cardiomyopathy[END_REF]. Hsp70 and PolG have been identified as interactors of DnaJ3 suggesting that DnaJ3, through its chaperone activity on PolG folding, thereby controls mtDNA replication. DNAJA3 function seems very well conserved through evolution since the invalidation of its homolog in yeast (Mdj1) that also interacts with the yeast mitochondrial DNA polymerase, causes mtDNA loss. Our results show that dnj-10 in C. elegans is also involved in mtDNA copy number control and could thus be a functional homolog of human

DNAJA3.

Mitofilin (IMMT) is a protein anchored to the mitochondrial inner membrane, with a small N-terminal domain protruding in the mitochondrial matrix [START_REF] Gieffers | Mitofilin is a transmembrane protein of the inner mitochondrial membrane expressed as two isoforms[END_REF]. A role in protein import related to maintenance of mitochondrial structure was suggested as mitofilin helps to regulate mitochondrial morphology [START_REF] Xie | The mitochondrial inner membrane protein mitofilin exists as a complex with SAM50, metaxins 1 and 2, coiled-coil-helix coiled-coil-helix domain-containing protein 3 and 6 and DnaJC11[END_REF]. Recently, mitofilin has been implicated in the maintenance of the mtDNA integrity as depletion of mitofilin in human cultured cells causes accumulation of mtDNA damage [START_REF] Rossi | Mitochondrial localization of PARP-1 requires interaction with mitofilin and is involved in the maintenance of mitochondrial DNA integrity[END_REF]. In C. elegans two genes encode the IMMT homolog, immt-1 and immt-2. Inactivation of immt-1 only, the closest homolog of IMMT, leads to EtBr sensitivity but not to mtDNA depletion (Fig. 3).

Y105E8A.23 encodes a protein highly homologous to the mitochondrial RNA polymerase (POLRMT). A direct relationship between RNA polymerase and mtDNA copy number has never been observed in animals. Only, the yeast mitochondrial RNA polymerase (RPO41) has been involved in the mitochondrial genome stability. The amino-terminal extension of the yeast mtRNA polymerase is required for a mtDNA maintenance function that is separable from the known RNA polymerization activity of the enzyme [START_REF] Wang | Stability of the mitochondrial genome requires an aminoterminal domain of yeast mitochondrial RNA polymerase[END_REF]. Our study adds new evidence of a role of the mitochondrial RNA polymerase on mtDNA stability in a pluricellular organism.

Finally, C. elegans genome contains five genes encoding ADP/ATP translocators. We found that only ant-1.2 (RNAi) worms resulted in a phenotype comparable to control animals.

Interestingly, the ant-1.2 gene is the most divergent C. elegans ant gene. On the contrary, down expression of the C47E12.2 gene, more distantly related to ant genes, leads to EtBr sensitivity and mtDNA depletion. The function of ANT proteins is not confined to exchanging ADP/ATP between mitochondria and cytosol. Human ANT1 also contributes to programmed cell death [START_REF] Belzacq | Adenine nucleotide translocator mediates the mitochondrial membrane permeabilization induced by lonidamine, arsenite and CD437[END_REF] and mtDNA stability as a constituent of the nucleoid [START_REF] Kaukonen | Role of adenine nucleotide translocator 1 in mtDNA maintenance[END_REF][START_REF] Agostino | Mutations of ANT1, Twinkle, and POLG1 in sporadic progressive external ophthalmoplegia (PEO)[END_REF]. Human ANT2 has been shown to also maintain ATP entry into mitochondria even when oxidative phosphorylation is impaired [START_REF] Giraud | Expression of human ANT2 gene in highly proliferative cells: GRBOX, a new transcriptional element, is involved in the regulation of glycolytic ATP import into mitochondria[END_REF]. These results highlight the differential contribution of these C. elegans ANT proteins on mtDNA maintenance.

In conclusion, our work allowed to develop a rapid and efficient screening in C.

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elegans for genes possibly involved in mtDNA maintenance. By applying this screen to genes homologous to human genes encoding proteins of the nucleoid, we identified new genes (Y105E8A.23, dnj-10, atad-3 and phi-37) that clearly show a direct control on mtDNA copy number. The human orthologs of these four genes (POLRMT, DNAJA3, ATAD3 and ATP5A1) should therefore be considered as candidate genes for patients with quantitative mtDNA anomalies. These results will now allow us to systematically invalidate all genes of the C. elegans genome with the aim to identify new components of the mitochondrial genome copy number control. It should be hypothesize that false positive will be obtained as it is the case for immt-1, the counterpart of fast high throughput screening being of course the risk of false positive or negative results. Obviously several of these genes would have been already identified as such but we hope to find new genes involved in mtDNA maintenance, to identify a yet unknown function of already described genes with no relation to any mitochondrial function or to identify transient nucleoid components in a temporal and/or spatial manner that could play important function on the stability and maintenance of mtDNA. This reservoir of new genes will represent a helpful tool and knowledge for mitochondrial diseases as they would be considered as candidate genes for patients.

Materials and Methods

Strains and growth conditions

The C. elegans wild type strain N2 Bristol and unc-119 (ed3)III were used in this work. The strains were maintained at 20°C on NGM plates seede d with OP50 E. coli strain.

Generation of transgenic worms

The entire mtss-1 gene with the promoter region or the mtss-1 coding sequence without stop codon were PCR amplified using mtss-1UP or mtss-1ATG as forward primers and mtss-1DW

as reverse primer (Table 2). The amplification product of the mtss-1 coding sequence was cloned into the pPD136.61 vector in fusion with the GFP sequence. The resulting plasmid encoded MTSS1-1::GFP under the control of the myo-3 promoter. The entire mtss-1 gene containing the promoter sequence was fused in frame with the GFP coding sequence by using the PCR-fusion method [START_REF] Hobert | PCR fusion-based approach to create reporter gene constructs for expression analysis in transgenic C. elegans[END_REF] and cloned into the pGEMT-easy plasmid. Both plasmids and the plasmid pDPMM016b carrying an unc119(+) gene, were linearized and used to perform microparticle bombardments of the unc-119 (ed3) strain using the Bio-Rad Biolistic PDS-1000/HE (Bio-Rad laboratories) as described [START_REF] Praitis | Creation of low-copy integrated transgenic lines in Caenorhabditis elegans[END_REF]. Following bombardment, worms were allowed to recover at 20°C and after 14 days, plates were examined for animals with wild type motility. Confirmation of the presence of the mtss-1::gfp transgenes was made by PCR. For each construct two independent transgenic worm lines were generated.

RNA interference and EtBr assay

The RNAi experiments were performed using the feeding procedure described by Kamath

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compared to the number of adults on the same plate. The phenotype was scored as sensitive to RNAi and EtBr if more than 80% of worms were arrested at the L3-stage on plates containing 50 µg/ml of EtBr. A gene was considered as positive for a given phenotype if the same result was observed in at least two independent feeding experiments.

The specificity and efficiency of RNAi inactivation of each gene were checked by semiquantitative RT-PCR analyses. Total RNA was isolated from N2 animals fed on RNAi bacteria using Trizol reagent (Invitrogen). Total RNA were treated with DNaseI, then subjected to cDNA synthesis using the reverse transcriptase SuperScript VILO TM (Invitrogen)

and PCR amplified. The primers used for each gene are listed in Table 2. The amplification of a 426 bp fragment of the ama-1 cDNA was used as internal control.

Quantification of mtDNA and investigation of mtDNA integrity

Extraction of total genomic and mitochondrial DNA was performed using the NucleoSpin Tissue extraction kit (Macherey-Nagel) from 10 F1-adult worms having laid all their eggs (day 9) after RNAi treatment or from control adults at the same stage. The mitochondrial cytb and the nuclear K01H12.2 genes were individually amplified by real-time PCR using primers cytbf/cytb-r and K01H12.2-f and K01H12.2-r (Table 2) as previously reported [START_REF] Sugimoto | Caenorhabditis elegans par2.1/mtssb-1 is essential for mitochondrial DNA replication and its defect causes comprehensive transcriptional alterations including a hypoxia response[END_REF]. The ratio of mtDNA copy number to nuclear DNA was used as a measure of mtDNA content in each specimen. Each Q-PCR measurement has been made in triplicate in at least two independent RNAi experiment. The integrity of mtDNA was checked by long-range PCR (Expand Long Template, Roche) using a pair of primers distant of 8561 bp (CE3 and CE4, Table 2).

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Legends to Figures

A

  C C E P T E D M A N U S C R I P T Introduction Mitochondria play an essential role in cellular energy production provided by the mitochondrial respiratory chain. The inheritance of functional mitochondria depends on faithful replication and transmission of mitochondrial DNA (mtDNA). The mitochondrial genome encodes only a few proteins of the respiratory chain (RC) complexes whereas all other mitochondrial proteins are encoded in the nucleus, synthesized on cytosolic ribosomes and imported into mitochondria. The mitochondrially encoded proteins are essential for the proper function of the respiratory chain and expression of those subunits requires that the mitochondrial genome be replicated, transcribed and translated. The newly synthesized subunits are subsequently assembled with nuclear encoded subunits. Over the past years, while key details of the content and expression of the mitochondrial genome have been elucidated, important questions remain regarding the maintenance and the transmission of mtDNA during cell proliferation and development.

2 ,

 2 two genes encoding proteins of unrelated mitochondrial function. irk-1 encodes a protein associated with the outer mitochondrial membrane homologous to human LRRK2 protein whom mutations result in Parkinson disease-8 and C34B7.2 encodes a putative cytosolic phosphoinositide phosphatase. Thus, whereas the human counterparts of the second group of genes are closely related to mtDNA, A C C E P T E D M A N U S C R I P T

dnj- 10

 10 is homologous to human DNAJA3, also known as Tid1. A crucial role of A C C E P T E D M A N U S C R I P T

  and Ahringer(2003) with slight modifications. Feeding RNAi clones were purchased from the Ahringer RNAi library (Geneservice Limited) and sequenced. A single colony of HT115 (DE3) bacteria of interest (RNase III-deficient E. coli strain, carrying IPTG-inducible T7-RNA polymerase) was first grown overnight at 37 o C in LB-ampicillin. The bacteria were then seeded onto NGM plates with 1 mM IPTG, 25 µg/ml carbenicillin and 50 µg/ml ampicillin and incubated at room temperature in a dark container at room temperature for 48 h to allow the expression of the double-stranded RNA (dsRNA). Worms feeding on HT115 bacteria carrying the empty vector (L4440) were used as controls in all the experiments. Synchronized L1-stage N2 worms were placed onto NGM plates seeded with bacteria expressing the dsRNA of interest and were incubated for 72 h at 20 o C. Four adult worms were independently picked up and transferred to fresh RNAi plates containing or not different concentrations of ethidium bromide (EtBr). Worms were allowed to lay 80 to 100 eggs before being removed. Eggs were immediately counted and the F1 progeny produced was analyzed after 3 and 4 days. At day 4, evaluation of the F1 progeny arrested at the L3 stage was A C C E P T E D M A N U S C R I P T

Figure 1 . 1 ( 1 (

 111 Figure 1. polg-1 (RNAi) and mtss-1 (RNAi) lead to mtDNA depletion. A, phenotype of polg-1 (RNAi) and mtss-1 (RNAi) F1 adult worms. B, mtDNA copy number of control (L4440), polg-1 (RNAi), mtss-1 (RNAi) and control adults treated from the L4 stage with 125 µg/ml EtBr (EtBr). The relative ratio of mtDNA copy number per adult is represented. C, Long-range PCR of the mtDNA extracted from polg-1 (RNAi) and L4440 control animals (C). M: molecular weight marker. D, polg-1 and ama-1 mRNA levels in N2 animals grown on bacteria expressing either polg-1 dsRNA or containing the empty L4440 RNAi vector (C) during larval development and adulthood. M: molecular weight. E, Expression of MTSS-1::GFP fusion protein in adult worms. MTSS-1::GFP was expressed either under its own promoter (par-MTSS-1::GFP, (1) Normaski and (2) fluorescence) or under the body-wall muscle specific myo-3 promoter (myo-3-MTSS-1::GFP, (3). RNAi of mtss-1 gene in myo3-MTSS-1::GFP transgenic animals (5) compared to control RNAi (4).

Figure 2 . 1 (

 21 Figure 2. polg-1 (RNAi) and mtss-1 (RNAi) mutants are hypersensitive to the EtBr. A, L3 larval development arrest after polg-1 (RNAi) and mtss-1 (RNAi) in the presence of 40 µg/ml of EtBr compared to control animals (L4440). B, Percentage of L3 larval arrest of polg-1 (RNAi) (square), mtss-1 (RNAi) (triangle) and control animals (L4440) (circle) grown with increasing concentration of EtBr. Each point corresponds to the analysis of n> 80 animals in two independent experiments. C, Percentage of L3 arrest of worms submitted to RNAi of polg-1, mtss-1, rrt-2 and pmp-4 genes and 50 µg/ml EtBr exposure. L4440: control RNAi.

Figure 3 .

 3 Figure 3. Correlation between L3 arrest and mtDNA copy number. mtDNA content of adult worms after 9 days of RNAi of various genes (black bars). Values are presented as ratio of mtDNA versus nuclear DNA relative to the control (L4440 RNAi). The error bars represent the SD. Percentage of L3 arrested animals after RNAi and 50 µg/ml EtBr exposure (gray bars). Experiments were performed in at least two independent RNAi for each gene.

Figure 4 .

 4 Figure 4. Efficiency and specificity of RNAi. The expression level of various genes was estimated by semiquantitative RT-PCR after RNAi of the corresponding genes and in control (L4440). ama-1 transcript level was used as internal control. M: molecular weight

  Figure 1

Figure 2 AFigure 4

 24 Figure 2

Figure 4 A

 4 Figure 4

Table 1 .

 1 List in crosslinked nucleoids but not found in native nucleoids. The two last columns show the Blast E-values between C. elegans and human genes.

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	M A N U S C R I P T
	A C C E P T E D

of C. elegans genes homologous to human nucleoid genes. The three classes correspond to the nucleoid class of proteins described in Bogenhagen et al., 2008. Class I: proteins isolated in both native and crosslinked nucleoid preparations, class II: proteins observed in native nucleoids but not found crosslinked to mtDNA and class III: proteins identified

Table 2

 2 Oligonucleotides used in this study.

Table 1

 1

Table 1 A

 1

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	M A N U S C R I P T
	C C E P T E D

Table 2

 2

		target genes	primers	PCR length
	mtss1	mtss-1UP	GAAGTTTTCTTGTGAAGAAGC
		mtss-11ATG	GGAAGATCTTAGAAAATGCTTCGTTCACTT
		mtss-1DW	CGGGATATCGGAAACAGTTATGTTTGG
	RT-PCR	ama-1-f (95-112)	CAGTGGCTCATGTCGAGT
		ama-1-r (559-577)	CGACCTTCTTTCCATCAT	482 bp
		ant1-2-f (46-65)	CTCGCCTCCGGAGGCACTGC
		ant1-2-r (606-627)	GCCCGTCAGTTGAGTACAATG	581 bp
		atad-3-f (291-311)	GGCCAATATGAAATCAGAGCA
		atad-3-r (626-647)	CGGTTCTCTTCTTCGTGAAGC	356 bp
		dnj-10-f (692-711)	GTAATCGATGCAGAGGAAGT
		dnj-10-r (1142-1162)	CGCAGCCCAAGCCAACATAA	470 bp
		C47E12.2-f (251-271)	GGAGAGGAAACATGACAAATG
		C47E12.2-r (717-737)	GTGTCCCAAGGGTAGGTTAA	486 bp
		immt-1-f (385-404)	CCAAGAGAGCCCACACATGT
		immt-1-r (814-834)	GATCTCATCAAGCTGATGAG	449 bp
		D2030.2-f (663-682)	GCAGCAGCAGAGTAATAATC
		D2030.2-r (1164-1184)	CCAGAAGACGTACCAAATCC	521 bp
		hmg-5-f (52-71)	CGTGCTTCTGTCGCAGCTTC
		hmg-5-r (563-583)	CCCATTTCTGGAGGACGACA	531 bp
		polg-1-f (19-39)	ATGCAAATTTTCCACGTATCC
		polg-1-r (619-638)	CCCGACATCTGGCTCGATC	619 bp
		phi-37-f (53-73)	ATGCCGGAATCGCCACCACCG
		phi-37-r (507-528)	GATGGTGTCAATGGCAATGGC	475 bp
	q-PCR	cyb-f (158-177)	CGCCCGATAGGTTAATAGCA
		cyb-r (234-253)	TGGCCCCATTAAAATGAAAA	95 bp
		K01H12.2-f (502-522)	CGTGAATTCAAAGGTCTGGCT
		K01H12.2-r (602-622)	AGTAAGCGGCACGGTAGATGA	120 bp
	deletion	CE3 (1840-1860)	GAGCGTCATTTATTGGGAAG
		CE4 (10529-10550)	CACAAAGGTCGACATATCAAC	8710 bp

Table 2

 2

Acknowledgments

We would like to thank Drs Monique Bolotin-Fukuhara and Emmanuel Dassa for discussions and reading the manuscript. This work was supported by ANR GENOPAT (D.P) and French government fellowship for M.G.A.

A C C E P T E D

M A N U S C R I P T


E Margo Molhoek

Albert Van Dijk

Edwin J A Veldhuizen

Helma Dijk-Knijnenburg

Roos H Mars-Groenendijk

Linda C L Boele

Wendy E Kaman-Van

Henk P Zanten

Floris J Haagsman

Bikker

Wendy E Kaman-Van Zanten

Henk P Haagsman

Floris J Bikker

Helma Dijk- Knijnenburg

Chicken cathelicidin-2-derived peptides

Keywords:

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This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. E-mail address: h.p.haagsman@uu.nl (H.P. Haagsman).

A c c e p t e d M a n u s c r i p t 3 ABSTRACT Host defence peptides (HDPs) are considered to be excellent candidates for the development of novel therapeutic agents. Recently, it was demonstrated that the peptide C1-15, an N-terminal segment of chicken HDP cathelicidin-2, exhibits potent antibacterial activity while lacking cytotoxicity towards eukaryotic cells. In the present study, we report that C1-15 is active against bacteria such as Bacillus anthracis and Yersinia pestis that may potentially be used by bioterrorists. Substitution of single and multiple phenylalanine (Phe) residues to tryptophan (Trp) in C1-15 resulted in variants with improved antibacterial activity against B. anthracis and Y. pestis as well as decreased salt sensitivity. In addition, these peptides exhibited enhanced neutralisation of lipopolysaccharide (LPS)-induced release of pro-inflammatory cytokines in human peripheral blood mononuclear cells (PBMCs). The antibacterial and LPS-neutralising activities of these C1-15-derived peptides are exerted at concentrations far below the concentrations that are toxic to human PBMCs. Taken together, we show that PheTrp substitutions in C1-15 variants enhances the antibacterial and LPS-neutralising activities against pathogenic bacteria, including those that may potentially be used as biological warfare agents.

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

Introduction

Exposure to biological warfare agents may cause a highly progressive, acute infection that may be lethal in some forms. In these cases, use of broad-spectrum antibiotics might be life saving. In addition, the number of antibiotic-resistant microorganisms is increasing, underlining the growing need for novel antimicrobial compounds.

Host defence peptides (HDPs) are excellent candidates for the development of novel broad-spectrum antibiotics. In general, HDPs exhibit potent broad-spectrum antibacterial and/or immunomodulatory properties. Moreover, HDPs have a low tendency to select resistant strains [START_REF] Brown | Cationic host defense (antimicrobial) peptides[END_REF][START_REF] Zasloff | Antimicrobial peptides of multicellular organisms[END_REF].

We previously demonstrated that the N-terminal fragment of the HDP chicken cathelicidin-2 (CATH-2), namely the peptide C1-15, possesses broad-spectrum antibacterial activity while being non-haemolytic against erythrocytes and is therefore a promising lead for the development of novel antimicrobial drugs with broad-spectrum activity [START_REF] Van Dijk | Identification of chicken cathelicidin-2 core elements involved in antibacterial and immunomodulatory activities[END_REF].

In this study, we explored the use of C1-15 as a potential novel lead compound to combat biowarfare agents. In doing so, we aimed at broadening the antibacterial activity of C1-15 by substitution of phenylalanine (Phe) residues on the non-polar face by tryptophan (Trp) residues. The antibacterial activity and toxicity against human peripheral blood mononuclear cells (PBMCs) of C1-15 variants were evaluated. In addition, the anti-inflammatory activity of C1-15 variants was examined. (RWGRWLRKIRRWRPK) were synthesised by Fmoc chemistry using a SYRO peptide synthesiser (MultiSynTech, Bochum, Germany) as described by Bikker et al. [START_REF] Bikker | Evaluation of the antibacterial spectrum of drosocin analogues[END_REF].

Antibacterial activity

The antimicrobial activity of C1-15 variants was evaluated against Gram-positive The antimicrobial activity of C1-15 and C1-15 variants was determined using colony counting assays as described previously [START_REF] Van Dijk | Identification of chicken cathelicidin-2 core elements involved in antibacterial and immunomodulatory activities[END_REF].

Bacillus anthracis

A c c e p t e d M a n u s c r i p t 6

Isolation and culture of peripheral blood mononuclear cells

Human PBMCs were isolated from buffy coats (Sanquin, Rotterdam, The Netherlands) as reported previously [START_REF] Van Dijk | Identification of chicken cathelicidin-2 core elements involved in antibacterial and immunomodulatory activities[END_REF] and were seeded at 0.5  10 6 cells/well into a Corning Costar 96-well flat-bottom culture plate (Corning Inc., Corning, NY) in RPMI-1640 (Lonza BioWhittaker, Basel, Switzerland) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (FBS Gold; PAA, Pasching, Austria), 100 U/mL penicillin and 100 g/mL streptomycin (Lonza BioWhittaker).

Cytotoxicity assay

The toxic effect of C1-15 variants on PBMCs was evaluated using WST reagents (Roche Diagnostics, Mannheim, Germany). Cells were incubated for 24 h with culture medium and C1-15 variants at 37 C and 5% CO 2 . Following centrifugation, supernatants were removed and 100 L of fresh RPMI-1640 supplemented with 10% FBS and 10 L of WST-1 reagents was added and the cells were further incubated.

After 60 min incubation time, absorbance was determined at 450 nm, with a reference wavelength at 650 nm.

Neutralisation of the lipopolysaccharide (LPS)-induced cytokine response by C1-15 variants

PBMCs were used to study the modulation of LPS-induced cytokine secretion by C1-15 variants. PBMCs were stimulated with 1 ng/mL ultrapure LPS from Escherichia coli O111:B4 (InvivoGen, San Diego, CA) for 24 h in the absence or presence of various

Cytokine measurements

Levels of interleukin (IL)-6 were determined by enzyme-linked immunosorbent assay (ELISA) using the commercial PeliKine Compact™ Human ELISA Kit (Sanquin, Amsterdam, The Netherlands) following the manufacturer's recommendations.

LPS binding by C1-15 variants

C1-15 variants were examined for LPS-neutralising activity in a limulus amoebocyte lysate (LAL) assay. Briefly, 25 L of peptide at concentrations from 0 to 20 M diluted in RPMI-1640 (Lonza BioWhittaker) were incubated with 25 L of a 2 ng/mL ultrapure LPS from E. coli O111:B4 (InvivoGen) for 60 min at 37 C in a 96-well endotoxin-free plate (Hycult Biotechnology, Uden, The Netherlands). Residual LPS activity was detected using the LAL assay according to the manufacturer's recommendations (Hycult Biotechnology).

Statistical analysis

Results

Antibacterial activity of C1-15 and C1-15 variants

The N-terminal segment of CATH-2, peptide C1-15, affected the survival of all tested bacteria (Fig. 1)

To enhance the antimicrobial activity of C1-15, a panel of C1-15 variants with PheTrp substitutions was designed. In comparison with Phe, Trp contains a more bulky hydrophobic side chain consisting of an indole chain. Since there are three Phe residues on the non-polar face of C1-15, variants were synthesised with PheTrp substitutions at positions 2 (F2W), 5 (F5W) and 12 (F12W). In addition, a triple PheTrp variant was synthesised (F2,5,12W).

Compared with C1-15, a single PheTrp substitution (F2W, F5W and F12W) improved the antibacterial activity against Y. pestis and B. anthracis. Survival of Y. pestis was reduced with 50% by 5 M F2W, F5W or F12W compared with 20 M for C1-15 (Fig. 1B). Moreover, 2 M of F2W, F5W or F12W led to a 50% decline in B. anthracis survival compared with 4 M for C1-15 (Fig. 1AD. Antibacterial activity against Y. pestis and B. anthracis was further improved by multiple substitutions, with 2.5 M and 1 M, respectively, of F2,5,12W needed to reduce the survival of the bacteria by 50% (Fig.

1A,D.

A c c e p t e d M a n u s c r i p t 9

Antibacterial activity in the presence of salts

The antibacterial activity of CATH-2 against MRSA was not affected by 100 mM NaCl (data not shown). However, C1-15 lost its antibacterial activity in the presence of 100 mM NaCl. Even the highest peptide concentration used (40 M) could only reduce the survival by 1 log unit. However, Trp substitutions resulting in F2,5,12W partly restored the antibacterial activity in the presence of NaCl (Fig. 2).

Cytotoxicity of C1-15 variants

C1-15 variants were evaluated for their toxicity to human PBMCs with and without the presence of 10% FBS. Cytotoxicity of C1-15 to PBMCs in the presence of FBS was negligible. Substitution of Phe by Trp residues increased the cytotoxicity to PBMCs up to 75% at 40 M after multiple substitutions (F2,5,12W) (Fig. 3). Similar experiments in the absence of FBS showed that FBS only had a marginal protective effect against peptide cytotoxicity (data not shown).

Neutralisation of the LPS-induced cytokine response

PBMCs were stimulated with LPS in the presence of various concentrations of C1-15 variants. The concentration of the pro-inflammatory mediator IL-6 was measured in culture supernatants (Fig. 4A).

Substitution of Phe residues in C1-15 by Trp residues improved the LPS-neutralising capacity by improving inhibition of the LPS-induced release of IL-6. Multiple PheTrp

A c c e p t e d M a n u s c r i p t 10 substitutions in C1-15 resulted in even higher LPS-neutralising activity; 10 M of F2,5,12W significantly inhibited 85% of the IL-6 LPS response (Fig. 4A).

LPS binding by C1-15 variants

To verify whether the improved LPS-neutralising activities of C1-15 variants could be contributed to improved LPS binding, C1-15 variants were evaluated for their ability to bind LPS in a LAL assay. PheTrp substitution in C1-15 enhanced the LPS-binding capacity of the peptides (Fig. 4B).

Discussion

During military operations or bioterrorist attacks, immediate diagnosis or identification of the bacterial agent is not always possible. In case of exposure from an unknown source, the availability and usability of broad-spectrum antimicrobials can be vital. Furthermore, owing to the use and misuse of current antimicrobials, growing numbers of multiresistant bacteria have arisen, including those that might potentially be used for offensive purposes, implying that there is a need for new broad-spectrum antimicrobials.

Recently, it was reported that the HDP C1-15, a truncated variant of CATH-2, possesses strong broad-spectrum antibacterial activity. In addition, this peptide is non-haemolytic against erythrocytes [START_REF] Van Dijk | Identification of chicken cathelicidin-2 core elements involved in antibacterial and immunomodulatory activities[END_REF]. In this study, we extended the earlier reported broad antibacterial activity with more bacteria, including bacteria often related to bioterrorist Substitutions of C1-15 Phe to Trp residues resulted in a peptide containing a large number of Arg and Trp residues. Previously it was reported by others that Arg and Trp complement each other well for the purpose of antimicrobial activity [START_REF] Chan | Tryptophan-and arginine-rich antimicrobial peptides: structures and mechanisms of action[END_REF]. The cationic charge of Arg provides an effective means of attraction of the peptides to the target membranes, and hydrogen bonding facilitates the interaction with the negatively charged surfaces of bacterial membranes. The hydrophobic bulk of Trp residues play a role in membrane association because it preferentially interacts with the interfacial region of membranes [START_REF] Yau | The preference of tryptophan for membrane interfaces[END_REF]. In combination, Arg and Trp residues are capable of participating in stronger cation- interactions compared with Arg and Phe residues [START_REF] Dougherty | Cation-pi interactions in chemistry and biology: a new view of benzene, Phe, Tyr, and Trp[END_REF].

Such interactions would facilitate deeper embedding of the peptides in the membrane and disrupt the membrane structure [START_REF] Chan | Tryptophan-and arginine-rich antimicrobial peptides: structures and mechanisms of action[END_REF].

In line with these data, we observed that single Phe substitutions (F2W, F5W and F12W) augmented the antibacterial activity by a factor of four against Y. pestis and by a factor of two against B. anthracis. Multiple substitutions (F2,5,12W) further enhanced antibacterial activity: the peptide was eight and four times more potent compared with C1-15 in reducing the survival of Y. pestis and B. anthracis, respectively.

The ability to resist salt is highly relevant for HDPs in order to function under physiological conditions. As reported earlier by Xiao et al. [START_REF] Xiao | The central kink region of fowlicidin-2, an -helical host defense peptide, is critically involved in bacterial killing and endotoxin neutralization[END_REF] and confirmed by us, Overall, these findings show that the insertion of Trp residues can make peptides less salt-sensitive and more potent in bacterial killing.

Bacterial killing by antimicrobial compounds may cause a release of endotoxin (LPS) from Gram-negative bacteria, which subsequently might lead to an exaggerated immune response. This can result in early sepsis, in which high levels of cytokines and inflammatory mediators become destructive, causing organ failure, cardiovascular shock and even resulting in death [START_REF] Cohen | The immunopathogenesis of sepsis[END_REF]. Some HDPs are found to counteract the development of septic shock by neutralising endotoxin [10]. Although CATH-2 is able to inhibit the LPSinduced pro-inflammatory cytokine responses, the N-terminal part, C1-15, showed only moderate LPS-neutralising activities [START_REF] Van Dijk | Identification of chicken cathelicidin-2 core elements involved in antibacterial and immunomodulatory activities[END_REF]. PheTrp substitutions in C1-15, resulting in peptide F2,5,12W, significantly increased the LPS-neutralising capacity. Recently, we [START_REF] Molhoek | Structure-function relationship of the human antimicrobial peptide LL-37 and LL-37 fragments in the modulation of TLR responses[END_REF] and others [START_REF] Nan | Investigating the effects of positive charge and hydrophobicity on the cell selectivity, mechanism of action and anti-inflammatory activity of a Trp-rich antimicrobial peptide indolicidin[END_REF] reported the importance of hydrophobicity in neutralising LPS cytokine responses. In line with our observations, Nan et al. [START_REF] Nan | Investigating the effects of positive charge and hydrophobicity on the cell selectivity, mechanism of action and anti-inflammatory activity of a Trp-rich antimicrobial peptide indolicidin[END_REF] showed that substitution of Trp by Lys residues in the Trp-rich peptide indolicidin resulted in decreased hydrophobicity and loss of LPS-neutralising activity. Previously it was shown that inhibition of the pro-inflammatory response is mainly established by direct binding of cationic HDPs to LPS [START_REF] Molhoek | Structure-function relationship of the human antimicrobial peptide LL-37 and LL-37 fragments in the modulation of TLR responses[END_REF][START_REF] Rosenfeld | Endotoxin (lipopolysaccharide) neutralization by innate immunity host-defense peptides. Peptide properties and plausible modes of action[END_REF]. In line with these observations, we found a correlation between LPS-neutralising activities and LPS binding.

*

  Corresponding author. Tel.: +31 30 253 53 54; fax: +31 30 253 23 33.

A c c e p t e d M a n u s c r i p t 5 2.

 5 Material and methods2.1. Peptide synthesisC1-15 (RFGRFLRKIRRFRPK) and the C1-15 variants F2W (RWGRFLRKIRRFRPK), F5W (RFGRWLRKIRRFRPK), F12W (RFGRFLRKIRRWRPK) and F2,5,12W

  Vollum strain ATCC 14578 (American Type Culture Collection, Manassas, VA) and meticillin-resistant Staphylococcus aureus (MRSA) ATCC 43300 as well as against the Gram-negative virulent Yersinia pestis NCTC 08775 (National Collection of Type Cultures, London, UK) and Vibrio cholerae (clinical isolate, Slotervaart Hospital, Amsterdam, The Netherlands). All bacteria were grown and maintained in tryptic soy broth (TSB) (bioTRADING, Mijdrecht, The Netherlands) under aerobic conditions at 35 C (B. anthracis, MRSA and V. cholera) or 26 C (Y. pestis).

  A c c e p t e d M a n u s c r i p t 7 concentrations of C1-15 variants. Following incubation, cell culture supernatants were collected and stored at -20 C until further analysis.

  Statistical significance was determined by two-way analysis of variance (ANOVA) followed by post hoc testing by the Bonferroni method. A P-value of <0.05 was considered statistically significant. Values shown are expressed as mean  standard error of the mean.

  . anthracis, V. cholera and Y. pestis). In addition, we aimed to enhance the antibacterial activity of C1-15 by substituting Phe residues by Trp residues.

A c c e p t e d M a n u s c r i p t 12 CATH- 2

 122 is resistant to salt, but C1-15 almost completely lost its antibacterial activity in the presence of 100 mM NaCl. However, we could restore the antibacterial activity in the presence of salt by Phe to Trp substitutions.

A c c e p t e d M a n u s c r i p t 13 It

 13 has been demonstrated that high concentrations of cationic HDPs containing a high amount of Trp residues may be toxic to the host[START_REF] Andrushchenko | Thermodynamics of the interactions of tryptophan-rich cathelicidin antimicrobial peptides with model and natural membranes[END_REF]. In the present study, we observed that an increased number of Trp residues results in increased cytotoxicity against mammalian cells such as PBMCs. However, the Trp substitutes exerted broad antibacterial and LPS-neutralising activities at much lower concentrations than their toxic effects on human PBMCs.In summary, we successfully increased the antibacterial activity of C1-15 and made it less salt sensitive by replacement of Phe by Trp residues. In addition, F2,5,12W showed good neutralising activities of LPS cytokine responses.

Fig. 1 .

 1 Fig. 1. Bactericidal activity of C1-15 (○), F2W (■), F5W (•), F12W (▼) and F2,5,12W ()

Fig. 2 .

 2 Fig. 2. Salt sensitivity of C1-15 and F2,5,12W: bactericidal activity against meticillin-

Fig. 3 .

 3 Fig. 3. Cytotoxicity of C1-15 (○), F2W (■), F5W (•), F12W (▼) and F2,5,12W () on

Fig. 4 .

 4 Fig. 4. (A) Comparison of the lipopolysaccharide (LPS)-neutralising activity of C1-15 (○),

Funding

This work was financially supported by the V502 programme of the Dutch Ministry of Defence and the Immuno Valley ALTANT programme of the Dutch Ministry of Agriculture, Nature and Food Quality.

Competing interests

None declared.

Ethical approval

Not required.