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Aygul Ishemgulova, Natalya Kraeva, Jana Hlaváčová, Sara L. Zimmer, Anzhelika Butenko, Lucie Podešvová, Tereza Leštinová, Julius Lukeš, Alexei Kostygov, Jan Votýpka, Petr Volf, Vyacheslav Yurchenko
A putative ATP/GTP binding protein affects Leishmania mexicana growth in insect vectors and vertebrate hosts
www.plosntds.org
www.plosntds.org
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(]:)Tj
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(i\))Tj
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(up-regulated)Tj
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(at)Tj
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(elevated)Tj
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(temperatures)Tj
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(in)Tj
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1.0318 0 Td
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-33.8227 -1.2982 Td
[()6()]TJ
1.5647 0 Td
[()-3()-5()-13()6()11()12()-9()]TJ
/F11 1 Tf
3.77 0 Td
(as)Tj
1.0148 0 Td
(a)Tj
0.6576 0 Td
(pre-adaptation)Tj
6.1455 0 Td
(to)Tj
1.0262 0 Td
(dixeny;)Tj
3.1351 0 Td
(ii\))Tj
1.0941 0 Td
(genes)Tj
2.4378 0 Td
(up-regulated)Tj
5.3348 0 Td
(in)Tj
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(a)Tj
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(isolate)Tj
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(of)Tj
/F16 1 Tf
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[()-3()2()-4()12()18()6()16()18()4()]TJ
4.7848 0 Td
[()6()16()3()11()]TJ
/F11 1 Tf
2.5682 0 Td
(LV561)Tj
2.88 0 Td
(compared)Tj
4.2462 0 Td
(to)Tj
1.0262 0 Td
(an)Tj
1.2018 0 Td
(attenuated)Tj
4.4334 0 Td
(isolate;)Tj
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(and)Tj
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(iii\))Tj
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(overexpressed)Tj
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(in)Tj
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(metacyclic)Tj
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(promastigotes)Tj
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(and)Tj
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[()6()1(\n)4()7()16()6()]TJ
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(shared)Tj
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(by)Tj
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(amastigote-gene)Tj
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(30)Tj
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[()]TJ
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[()6()1(\n)4()7()16()6()]TJ
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/F16 1 Tf
[()22(\n)]TJ
/F11 1 Tf
(.)Tj
/F16 1 Tf
[(\r)]TJ
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(.)Tj
/F16 1 Tf
[()]TJ
/F11 1 Tf
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[()-3()2()12()11()11()6()11()6()]TJ
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[()-3()-5()-13()6()11()12()-9()]TJ
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(35C)Tj
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[()]TJ
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[()6()1(\n)4()7()16()6()]TJ
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(18)Tj
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1.4683 0 Td
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2.1486 0 Td
(and)Tj
/F16 1 Tf
1.7235 0 Td
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1.0091 0 Td
[()-13()14()7()20()]TJ
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(and)Tj
1.7234 0 Td
(analyzed)Tj
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(its)Tj
1.1395 0 Td
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1.0318 0 Td
[()-3()2()-4()12()18()6()16()18()4()]TJ
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4.7792 0 Td
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5.4085 0 Td
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1.0261 0 Td
(insect)Tj
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(vectors)Tj
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Q
q
1 j
1 J
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BT
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1 TL
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(Materials)Tj
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(and)Tj
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Q
q
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(.)Tj
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[()]TJ
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6.1058 0 Td
(and)Tj
1.7291 0 Td
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(v.9.0)Tj
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(19)Tj
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(and)Tj
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(v.3.8.31)Tj
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(default)Tj
2.965 0 Td
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([)Tj
0.83 0.64 0.02 0 k
(20)Tj
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(].)Tj
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(visualized)Tj
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(using)Tj
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(Jalview)Tj
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(v.2.9)Tj
2.0807 0 Td
([)Tj
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(21)Tj
0 g
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Q
q
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1 J
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BT
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(axenic)Tj
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(and)Tj
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Q
q
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[()-3()2()-4()12()18()6()16()18()4()]TJ
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[()6()1(\n)4()7()16()6()]TJ
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(Louis,)Tj
2.6475 0 Td
(USA\))Tj
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[()]TJ
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4.0025 0 Td
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[()]TJ
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3.0444 0 Td
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2.296 0 Td
(Bioscience)Tj
4.4447 0 Td
(GmbH,)Tj
3.2542 0 Td
(Jena,)Tj
2.1826 0 Td
(Germany\),)Tj
4.5524 0 Td
(25mM)Tj
2.88 0 Td
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(50)Tj
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(units/ml)Tj
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(of)Tj
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(streptomycin)Tj
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(and)Tj
1.7291 0 Td
(10%)Tj
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(Bovine)Tj
3.0047 0 Td
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3.0614 0 Td
(FBS)Tj
1.8028 0 Td
(\(all)Tj
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(USA\))Tj
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(23C.)Tj
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(ALD1)Tj
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(knock-out)Tj
4.371 0 Td
(\(KO\))Tj
2.3244 0 Td
(and)Tj
1.7291 0 Td
(WT)Tj
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[()6()1(\n)4()7()16()6()]TJ
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(mice)Tj
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[()12()]TJ
1.0148 0 Td
[()-13()14()7()20()]TJ
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3.7077 0 Td
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3.3278 0 Td
(kinetics)Tj
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(comparison)Tj
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(6)Tj
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(the)Tj
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(starting)Tj
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(density)Tj
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(x)Tj
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(10)Tj
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Q
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1 J
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BT
/F11 1 Tf
1 TL
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(5)Tj
ET
Q
q
1 j
1 J
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BT
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1 TL
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(parasites)Tj
3.685 0 Td
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(100)Tj
/F17 1 Tf
1.6271 0 Td
[()]TJ
/F11 1 Tf
(g/ml)Tj
2.5625 0 Td
(of)Tj
1.0091 0 Td
(Sat)Tj
1.4117 0 Td
(\(Jena)Tj
2.2677 0 Td
(Bioscience)Tj
4.3993 0 Td
(GmbH\).)Tj
3.5602 0 Td
(The)Tj
/F16 1 Tf
1.7688 0 Td
[()]TJ
/F11 1 Tf
(.)Tj
/F16 1 Tf
0.9638 0 Td
[()6()1(\n)4()7()16()6()]TJ
3.9514 0 Td
[()22(\n)]TJ
/F11 1 Tf
(.)Tj
/F16 1 Tf
[(\r)]TJ
/F11 1 Tf
(.)Tj
/F16 1 Tf
[()]TJ
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 538.2992 484.1574 cm
BT
/F11 1 Tf
1 TL
-0.012 Tc
0 0 Td
(-/-)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 466.696 cm
BT
/F11 1 Tf
1 TL
-0.009 Tc
0 0 Td
(\(knock-out,)Tj
4.8868 0 Td
(KO\))Tj
1.9616 0 Td
(clones)Tj
2.6986 0 Td
(were)Tj
2.1033 0 Td
(selected)Tj
3.3108 0 Td
(on)Tj
1.2643 0 Td
(solid)Tj
2.1033 0 Td
(M199)Tj
2.5171 0 Td
(medium)Tj
3.5773 0 Td
(supplemented)Tj
5.794 0 Td
(as)Tj
1.0034 0 Td
(above)Tj
2.5229 0 Td
(with)Tj
-33.7434 -1.2982 Td
(additional)Tj
4.2235 0 Td
(100)Tj
/F17 1 Tf
1.6328 0 Td
[()]TJ
/F11 1 Tf
(g/ml)Tj
2.5625 0 Td
(of)Tj
1.0091 0 Td
(Hyg.)Tj
2.109 0 Td
(Correct)Tj
3.2428 0 Td
(integration)Tj
4.5978 0 Td
(was)Tj
1.6837 0 Td
(confirmed)Tj
4.3484 0 Td
(by)Tj
1.1735 0 Td
(PCR)Tj
2.0466 0 Td
(on)Tj
1.2642 0 Td
(genomic)Tj
3.6397 0 Td
(DNA)Tj
-33.5336 -1.3039 Td
(with)Tj
1.9785 0 Td
(specific)Tj
3.1465 0 Td
(primers)Tj
3.3165 0 Td
(and)Tj
1.7178 0 Td
(by)Tj
1.1678 0 Td
(Southern)Tj
3.8608 0 Td
(blot)Tj
1.7575 0 Td
([)Tj
0.83 0.64 0.02 0 k
(29)Tj
0 g
(].)Tj
2.0522 0 Td
(In)Tj
1.0999 0 Td
(brief,)Tj
2.2733 0 Td
(total)Tj
1.9786 0 Td
(genomic)Tj
3.6397 0 Td
(DNA)Tj
2.3584 0 Td
(was)Tj
1.6837 0 Td
(isolated)Tj
3.2485 0 Td
(using)Tj
-35.2797 -1.2983 Td
(DNeasy)Tj
3.3335 0 Td
(Blood)Tj
2.5625 0 Td
(&)Tj
0.9184 0 Td
(Tissue)Tj
2.7439 0 Td
(Kit)Tj
1.4457 0 Td
(\(Qiagen,)Tj
3.6283 0 Td
(Hilden,)Tj
3.1918 0 Td
(Germany\),)Tj
4.507 0 Td
(digested)Tj
3.4753 0 Td
(with)Tj
/F16 1 Tf
1.9786 0 Td
[(!)28()7()]TJ
/F11 1 Tf
1.7348 0 Td
(I)Tj
0.5556 0 Td
(overnight,)Tj
4.2576 0 Td
(sepa-)Tj
-34.333 -1.2983 Td
(rated)Tj
2.2506 0 Td
(on)Tj
1.2643 0 Td
(0.75%)Tj
2.6022 0 Td
(agarose)Tj
3.1804 0 Td
(gel,)Tj
1.5591 0 Td
(and)Tj
1.7178 0 Td
(transferred)Tj
4.5807 0 Td
(to)Tj
1.0205 0 Td
(a)Tj
0.6463 0 Td
(Zeta-Probe)Tj
4.6431 0 Td
(blotting)Tj
3.3165 0 Td
(membrane)Tj
4.5297 0 Td
(\(Bio-Rad,)Tj
4.0252 0 Td
(Her-)Tj
-35.3364 -1.2982 Td
(cules,)Tj
2.398 0 Td
(USA\).)Tj
2.6533 0 Td
(Blots)Tj
2.1883 0 Td
(were)Tj
2.1033 0 Td
(blocked)Tj
3.3165 0 Td
(and)Tj
1.7121 0 Td
(hybridized)Tj
4.456 0 Td
(with)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 408.0755 406.148 cm
BT
/F11 1 Tf
1 TL
-0.012 Tc
0 0 Td
(32)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 415.1055 401.7259 cm
BT
/F11 1 Tf
1 TL
-0.009 Tc
0 0 Td
(P-labeled)Tj
3.8947 0 Td
(PCR)Tj
2.0466 0 Td
(probes)Tj
2.8743 0 Td
(for)Tj
1.372 0 Td
(Sat,)Tj
1.6327 0 Td
(Hyg,)Tj
2.1147 0 Td
(5')Tj
-35.4441 -1.3039 Td
(UTR-,)Tj
2.7382 0 Td
(3')Tj
0.9015 0 Td
(UTR-,)Tj
2.7382 0 Td
(and)Tj
1.7178 0 Td
(ORF)Tj
2.0976 0 Td
(of)Tj
/F16 1 Tf
1.0091 0 Td
[()22(\n)]TJ
/F11 1 Tf
(.)Tj
/F16 1 Tf
[(\r)]TJ
/F11 1 Tf
(.)Tj
/F16 1 Tf
[()]TJ
/F11 1 Tf
6.0435 0 Td
(gene.)Tj
2.279 0 Td
(The)Tj
1.7688 0 Td
(following)Tj
3.9344 0 Td
(primers)Tj
3.3165 0 Td
(\()Tj
0.83 0.64 0.02 0 k
(S1)Tj
1.4967 0 Td
(Table)Tj
0 g
(\))Tj
2.7496 0 Td
(were)Tj
2.1033 0 Td
(used)Tj
-34.8942 -1.2982 Td
(to)Tj
1.0148 0 Td
(amplify)Tj
3.2088 0 Td
(probes:)Tj
3.1011 0 Td
(SBp_SAT_f)Tj
4.7962 0 Td
(and)Tj
1.7177 0 Td
(SBp_SAT_r)Tj
4.8813 0 Td
(\(for)Tj
1.7064 0 Td
(Sat\),)Tj
1.9672 0 Td
(SBp_Hyg_f)Tj
4.7168 0 Td
(and)Tj
1.7178 0 Td
(SBp_Hyg_r)Tj
4.7961 0 Td
(\(for)Tj
1.7065 0 Td
(Hyg\),)Tj
-35.3307 -1.2983 Td
(SBp_)Tj
/F16 1 Tf
[()22(\n)]TJ
/F11 1 Tf
(.)Tj
/F16 1 Tf
[(\r)]TJ
/F11 1 Tf
(.)Tj
/F16 1 Tf
[()]TJ
/F11 1 Tf
(_5'f)Tj
9.547 0 Td
(and)Tj
1.7178 0 Td
(SBp_)Tj
/F16 1 Tf
[()22(\n)]TJ
/F11 1 Tf
(.)Tj
/F16 1 Tf
[(\r)]TJ
/F11 1 Tf
(.)Tj
/F16 1 Tf
[()]TJ
/F11 1 Tf
(_5'r)Tj
9.6321 0 Td
(\(for)Tj
1.7063 0 Td
(5')Tj
0.8958 0 Td
(UTR\),)Tj
2.7325 0 Td
(SBp_)Tj
/F16 1 Tf
[()22(\n)]TJ
/F11 1 Tf
(.)Tj
/F16 1 Tf
[(\r)]TJ
/F11 1 Tf
(.)Tj
/F16 1 Tf
[()]TJ
/F11 1 Tf
(_3'f)Tj
9.5471 0 Td
(and)Tj
-35.7786 -1.3039 Td
(SBp_)Tj
/F16 1 Tf
[()22(\n)]TJ
/F11 1 Tf
(.)Tj
/F16 1 Tf
[(\r)]TJ
/F11 1 Tf
(.)Tj
/F16 1 Tf
[()]TJ
/F11 1 Tf
(_3'r)Tj
9.6321 0 Td
(\(for)Tj
1.7064 0 Td
(3')Tj
0.9014 0 Td
(UTR\),)Tj
2.7269 0 Td
(SBp_)Tj
/F16 1 Tf
[()22(\n)]TJ
/F11 1 Tf
(.)Tj
/F16 1 Tf
[(\r)]TJ
/F11 1 Tf
(.)Tj
/F16 1 Tf
[()]TJ
/F11 1 Tf
(_f)Tj
8.8666 0 Td
(and)Tj
1.7178 0 Td
(SBp_)Tj
/F16 1 Tf
[()22(\n)]TJ
/F11 1 Tf
(.)Tj
/F16 1 Tf
[(\r)]TJ
/F11 1 Tf
(.)Tj
/F16 1 Tf
[()]TJ
/F11 1 Tf
(_r)Tj
8.9518 0 Td
(\(for)Tj
/F16 1 Tf
-34.503 -1.2983 Td
[()22(\n)]TJ
/F11 1 Tf
(.)Tj
/F16 1 Tf
[(\r)]TJ
/F11 1 Tf
(.)Tj
/F16 1 Tf
[()]TJ
/F11 1 Tf
6.0434 0 Td
(ORF\).)Tj
2.6589 0 Td
(Probes)Tj
2.914 0 Td
(were)Tj
2.1033 0 Td
(labeled)Tj
2.9934 0 Td
(with)Tj
1.9785 0 Td
(radioactive)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 432.6236 341.1212 cm
BT
/F11 1 Tf
1 TL
-0.012 Tc
0 0 Td
(32)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 439.6535 336.6992 cm
BT
/F11 1 Tf
1 TL
-0.009 Tc
0 0 Td
(P)Tj
0.7766 0 Td
(using)Tj
2.3641 0 Td
(the)Tj
1.4513 0 Td
(DecaLabel)Tj
4.3427 0 Td
(DNA)Tj
-32.8986 -1.2982 Td
(Labeling)Tj
3.6057 0 Td
(kit)Tj
1.2699 0 Td
(\(ThermoFisher)Tj
6.2305 0 Td
(Scientific,)Tj
4.0762 0 Td
(Waltham,)Tj
4.1272 0 Td
(USA\).)Tj
2.6589 0 Td
(PCR)Tj
2.0465 0 Td
(confirmation)Tj
5.4425 0 Td
(of)Tj
1.0034 0 Td
(the)Tj
1.457 0 Td
(correct)Tj
2.9991 0 Td
(inte-)Tj
-34.9169 -1.3039 Td
(gration)Tj
3.0727 0 Td
(was)Tj
1.6838 0 Td
(performed)Tj
4.4277 0 Td
(using)Tj
2.3641 0 Td
(primers)Tj
3.3165 0 Td
(pairs)Tj
2.143 0 Td
(SBp_Hyg_fSBp_Hy)Tj
8.6795 0 Td
(g_r)Tj
1.5364 0 Td
(\(expected)Tj
4.0195 0 Td
(size)Tj
1.6611 0 Td
(KO)Tj
1.6327 0 Td
(0.3)Tj
1.3833 0 Td
(kb\),)Tj
-35.9203 -1.2983 Td
(Hyg190_fHyg_3'r)Tj
8.2091 0 Td
(\(expected)Tj
4.0138 0 Td
(size)Tj
1.6668 0 Td
(KO)Tj
1.6271 0 Td
(0.8)Tj
1.3833 0 Td
(kb\),)Tj
1.7688 0 Td
(ASBp_Hyg_r)Tj
6.4005 0 Td
(\(expected)Tj
4.0138 0 Td
(size)Tj
1.6667 0 Td
(KO)Tj
1.6271 0 Td
(1.7)Tj
1.3833 0 Td
(kb\),)Tj
-33.7603 -1.2983 Td
(SBp_SAT_fSBp_SAT_r)Tj
10.3804 0 Td
(\(expected)Tj
4.0195 0 Td
(size)Tj
1.6611 0 Td
(KO)Tj
1.6328 0 Td
(0.3)Tj
1.3776 0 Td
(kb\).)Tj
1.7688 0 Td
(The)Tj
1.7688 0 Td
(complete)Tj
3.838 0 Td
(ablation)Tj
3.4242 0 Td
(of)Tj
1.0035 0 Td
(the)Tj
/F16 1 Tf
-30.8747 -1.3039 Td
[()22(\n)]TJ
/F11 1 Tf
(.)Tj
/F16 1 Tf
[(\r)]TJ
/F11 1 Tf
(.)Tj
/F16 1 Tf
[()]TJ
/F11 1 Tf
6.0434 0 Td
(gene)Tj
2.058 0 Td
(was)Tj
1.6837 0 Td
(confirmed)Tj
4.354 0 Td
(with)Tj
1.9786 0 Td
(primers)Tj
3.3165 0 Td
(pairs)Tj
2.143 0 Td
(ASBp_)Tj
/F16 1 Tf
[()22(\n)]TJ
/F11 1 Tf
(.)Tj
/F16 1 Tf
[(\r)]TJ
/F11 1 Tf
(.)Tj
/F16 1 Tf
[()]TJ
8.5548 0 Td
[()]TJ
/F11 1 Tf
(_r)Tj
1.9956 0 Td
(\(expected)Tj
-32.1276 -1.2982 Td
(size)Tj
1.6611 0 Td
(wild)Tj
1.9275 0 Td
(type,)Tj
2.1147 0 Td
(WT)Tj
1.7858 0 Td
(2.0)Tj
1.3833 0 Td
(kb\))Tj
1.5477 0 Td
(and)Tj
/F16 1 Tf
1.7121 0 Td
[()22(\n)]TJ
/F11 1 Tf
(.)Tj
/F16 1 Tf
[(\r)]TJ
/F11 1 Tf
(.)Tj
/F16 1 Tf
[()]TJ
/F11 1 Tf
(_f)Tj
/F16 1 Tf
[()22(\n)]TJ
/F11 1 Tf
(.)Tj
/F16 1 Tf
[(\r)]TJ
/F11 1 Tf
10.8793 0 Td
(.)Tj
/F16 1 Tf
[()]TJ
/F11 1 Tf
(_r)Tj
3.135 0 Td
(\(expected)Tj
4.0195 0 Td
(size)Tj
1.6611 0 Td
(WT)Tj
1.7915 0 Td
(0.25)Tj
1.8482 0 Td
(kb\).)Tj
-35.4668 -1.2983 Td
(See)Tj
0.83 0.64 0.02 0 k
1.525 0 Td
(S1)Tj
1.1622 0 Td
(Table)Tj
0 g
2.4151 0 Td
(for)Tj
1.372 0 Td
(primer)Tj
2.9593 0 Td
(sequences.)Tj
-0.004 Tc
-8.2374 -1.2983 Td
(For)Tj
1.6271 0 Td
(localization)Tj
4.8132 0 Td
(studies,)Tj
3.2145 0 Td
(the)Tj
/F16 1 Tf
1.4683 0 Td
[()22(\n)]TJ
/F11 1 Tf
(.)Tj
/F16 1 Tf
[(\r)]TJ
/F11 1 Tf
(.)Tj
/F16 1 Tf
[()]TJ
/F11 1 Tf
6.1058 0 Td
(gene)Tj
2.0806 0 Td
(was)Tj
1.6951 0 Td
(tagged)Tj
2.8345 0 Td
(with)Tj
1.9956 0 Td
(HA)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 484.8944 231.0236 cm
BT
/F11 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(3)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 490.6204 232.7244 cm
BT
/F11 1 Tf
1 TL
-0.004 Tc
0 0 Td
(and)Tj
1.7291 0 Td
(expressed)Tj
4.0932 0 Td
(from)Tj
-34.8829 -1.3039 Td
(the)Tj
1.4684 0 Td
(18S)Tj
1.6441 0 Td
(rRNA)Tj
2.6362 0 Td
(locus)Tj
2.2847 0 Td
(of)Tj
/F16 1 Tf
1.0148 0 Td
[()]TJ
/F11 1 Tf
(.)Tj
/F16 1 Tf
0.9751 0 Td
[()6()1(\n)4()7()16()6()]TJ
/F11 1 Tf
(.)Tj
4.2179 0 Td
(The)Tj
1.7802 0 Td
(open)Tj
2.2166 0 Td
(reading)Tj
3.2599 0 Td
(frame)Tj
2.5568 0 Td
(was)Tj
1.6951 0 Td
(amplified)Tj
4.0137 0 Td
(from)Tj
2.2054 0 Td
(the)Tj
1.4683 0 Td
(genomic)Tj
-33.4372 -1.2982 Td
(DNA)Tj
2.3755 0 Td
(using)Tj
2.3867 0 Td
(primers)Tj
3.3562 0 Td
(30.2090_)Tj
/F16 1 Tf
[(!)28()7()]TJ
/F11 1 Tf
(I_f30.2090_3xHA_)Tj
/F16 1 Tf
[(!)]TJ
14.3149 0 Td
[()12()]TJ
/F11 1 Tf
(I_r)Tj
2.1883 0 Td
(\()Tj
0.83 0.64 0.02 0 k
(S1)Tj
1.5137 0 Td
(Table)Tj
0 g
(\).)Tj
3.0046 0 Td
(The)Tj
1.7802 0 Td
(HA)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 523.7858 205.0582 cm
BT
/F11 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(3)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 529.5118 206.7023 cm
BT
/F11 1 Tf
1 TL
-0.004 Tc
0 0 Td
(tag)Tj
1.4173 0 Td
(was)Tj
-34.367 -1.2982 Td
(included)Tj
3.7134 0 Td
(in)Tj
1.0318 0 Td
(the)Tj
1.4683 0 Td
(reverse)Tj
3.0501 0 Td
(primer.)Tj
3.2145 0 Td
(The)Tj
1.7858 0 Td
(amplified)Tj
4.0081 0 Td
(fragment)Tj
3.8721 0 Td
(was)Tj
1.6952 0 Td
(cloned)Tj
2.8969 0 Td
(into)Tj
1.8426 0 Td
(pLEXSY-sat2)Tj
5.5274 0 Td
(\(Jena)Tj
-34.1062 -1.3039 Td
(Bioscience)Tj
4.4447 0 Td
(GmbH\).)Tj
3.5943 0 Td
(Five)Tj
/F17 1 Tf
1.8936 0 Td
[()]TJ
/F11 1 Tf
(g)Tj
1.1905 0 Td
(of)Tj
1.0148 0 Td
(the)Tj
1.4683 0 Td
(resulting)Tj
3.7304 0 Td
(plasmid)Tj
3.3959 0 Td
(were)Tj
2.1203 0 Td
(linearized)Tj
4.1442 0 Td
(with)Tj
/F16 1 Tf
1.9956 0 Td
[(")26(#)3()]TJ
/F11 1 Tf
(I)Tj
2.1657 0 Td
(and)Tj
1.7291 0 Td
(transfected)Tj
-32.8874 -1.2983 Td
(as)Tj
1.0148 0 Td
(above.)Tj
/F16 1 Tf
2.7723 0 Td
[()22(\n)]TJ
/F11 1 Tf
(.)Tj
/F16 1 Tf
[(\r)]TJ
/F11 1 Tf
(.)Tj
/F16 1 Tf
[()]TJ
/F11 1 Tf
(-HA)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 314.759 166.0535 cm
BT
/F11 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(3)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 320.485 167.6976 cm
BT
/F11 1 Tf
1 TL
-0.004 Tc
0 0 Td
(cell)Tj
1.559 0 Td
(line)Tj
1.7064 0 Td
(was)Tj
1.7008 0 Td
(isolated)Tj
3.2825 0 Td
(in)Tj
1.0375 0 Td
(complete)Tj
3.8778 0 Td
(M199)Tj
2.5398 0 Td
(medium)Tj
3.6056 0 Td
(containing)Tj
-31.3565 -1.2982 Td
(100)Tj
/F17 1 Tf
1.6441 0 Td
[()]TJ
/F11 1 Tf
(g/ml)Tj
2.5908 0 Td
(of)Tj
1.0148 0 Td
(Sat.)Tj
1.6498 0 Td
(PCR)Tj
2.0636 0 Td
(confirmation)Tj
5.5047 0 Td
(of)Tj
1.0148 0 Td
(the)Tj
1.4684 0 Td
(correct)Tj
3.0387 0 Td
(integration)Tj
4.6488 0 Td
(was)Tj
1.6951 0 Td
(performed)Tj
4.473 0 Td
(using)Tj
2.3868 0 Td
(primers)Tj
-33.1934 -1.3039 Td
(SSU_dir)Tj
3.5716 0 Td
(and)Tj
1.7291 0 Td
(SBp_)Tj
/F16 1 Tf
[()22(\n)]TJ
/F11 1 Tf
(.)Tj
/F16 1 Tf
[(\r)]TJ
/F11 1 Tf
(.)Tj
/F16 1 Tf
[()]TJ
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1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 613.2472 cm
BT
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1 TL
-0.0033 Tc
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5.4992 0 Td
(PCR)Tj
2.3103 0 Td
(analysis)Tj
3.7842 0 Td
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(parasites)Tj
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(insects)Tj
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Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 596.2393 cm
BT
/F11 1 Tf
1 TL
-0.004 Tc
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1.3379 0 Td
(quantify)Tj
3.5433 0 Td
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(of)Tj
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1.0148 0 Td
[()-3()2()-4()12()18()6()16()18()4()]TJ
/F11 1 Tf
4.7905 0 Td
(parasites)Tj
3.685 0 Td
(in)Tj
1.0375 0 Td
(the)Tj
1.4684 0 Td
(guts)Tj
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(of)Tj
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(female)Tj
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(sand)Tj
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(days)Tj
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(1)Tj
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(and)Tj
-35.7333 -1.2982 Td
(7)Tj
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(p.i.,)Tj
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(RT-qPCR)Tj
4.1555 0 Td
(with)Tj
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1.9956 0 Td
[()-3()2()-4()12()18()6()16()18()4()]TJ
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4.7905 0 Td
(kinetoplast)Tj
4.6148 0 Td
(DNA-specific)Tj
5.6976 0 Td
(primers)Tj
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([)Tj
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(33)Tj
0 g
(].)Tj
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(DNA)Tj
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(females)Tj
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(extracted)Tj
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(a)Tj
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(\(Roche)Tj
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1.4683 0 Td
(manufacturer's)Tj
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(evaluated)Tj
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3.7758 0 Td
(v.)Tj
0.9014 0 Td
(6.1.)Tj
ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 515.1118 cm
BT
/F0 1 Tf
1 TL
-0.0033 Tc
0 0 Td
(Mice)Tj
2.3102 0 Td
(infection)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 498.1606 cm
BT
/F11 1 Tf
1 TL
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1.627 0 Td
(mice)Tj
2.1487 0 Td
(infections,)Tj
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(sand)Tj
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(fly)Tj
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(females)Tj
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(infected)Tj
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(with)Tj
/F16 1 Tf
1.9956 0 Td
[()]TJ
/F11 1 Tf
(.)Tj
/F16 1 Tf
0.9751 0 Td
[()6()1(\n)4()7()16()6()]TJ
/F11 1 Tf
3.9912 0 Td
(WT)Tj
1.7971 0 Td
(or)Tj
1.0999 0 Td
(KO)Tj
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(strains)Tj
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(described)Tj
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(above)Tj
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(and)Tj
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(checked)Tj
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(presence)Tj
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(localization)Tj
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(89)Tj
1.6894 0 Td
(d.p.i.)Tj
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(freshly)Tj
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3.8324 0 Td
(thoracic)Tj
3.4752 0 Td
(midguts)Tj
3.4866 0 Td
(\(TM\),)Tj
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(with)Tj
2.0012 0 Td
(colonized)Tj
4.0819 0 Td
(stomodeal)Tj
4.3427 0 Td
(valve)Tj
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(and)Tj
1.7292 0 Td
(high)Tj
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(homogenized)Tj
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(sterile)Tj
2.6135 0 Td
(saline)Tj
2.5001 0 Td
(solution.)Tj
3.7077 0 Td
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5.4992 0 Td
(5)Tj
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0.6973 0 Td
[()]TJ
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(l)Tj
0.9695 0 Td
(of)Tj
1.0148 0 Td
(the)Tj
1.474 0 Td
(suspension)Tj
4.6374 0 Td
(\(from)Tj
-35.2457 -1.3039 Td
(~)Tj
0.7993 0 Td
(10)Tj
1.1679 0 Td
(sand)Tj
2.0919 0 Td
(fly)Tj
1.2133 0 Td
(TMs\))Tj
2.4207 0 Td
(was)Tj
1.7008 0 Td
(intra-dermally)Tj
6.0151 0 Td
(injected)Tj
3.3845 0 Td
(into)Tj
1.8425 0 Td
(the)Tj
1.4684 0 Td
(ear)Tj
1.4513 0 Td
(pinnae)Tj
2.9594 0 Td
(of)Tj
1.0148 0 Td
(a)Tj
0.6519 0 Td
(ketamin/xylazin)Tj
6.6671 0 Td
(anes-)Tj
-34.8489 -1.2983 Td
(thetized)Tj
3.3958 0 Td
(BALB/c)Tj
3.3562 0 Td
(mouse.)Tj
3.0841 0 Td
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3.2201 0 Td
(of)Tj
1.0148 0 Td
(four)Tj
1.9163 0 Td
(BALB/c)Tj
3.3562 0 Td
(mice)Tj
2.1486 0 Td
(of)Tj
1.0148 0 Td
(each)Tj
2.0296 0 Td
(studied)Tj
3.1521 0 Td
(group)Tj
2.6135 0 Td
(\(WT)Tj
2.1373 0 Td
(vs.)Tj
1.2586 0 Td
(KO\))Tj
1.9786 0 Td
(were)Tj
-35.6766 -1.2982 Td
(used)Tj
2.0579 0 Td
(in)Tj
1.0375 0 Td
(two)Tj
1.7064 0 Td
(independent)Tj
5.2781 0 Td
(experiments.)Tj
5.3631 0 Td
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3.2258 0 Td
(development)Tj
5.4085 0 Td
(was)Tj
1.7008 0 Td
(monitored)Tj
4.49 0 Td
(weekly.)Tj
3.1748 0 Td
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2.228 0 Td
(were)Tj
-35.6709 -1.304 Td
(sacrificed)Tj
4.0024 0 Td
(at)Tj
0.9525 0 Td
(the)Tj
1.474 0 Td
(15)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 273.77 411.5905 cm
BT
/F11 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(th)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 282.1606 407.1118 cm
BT
/F11 1 Tf
1 TL
-0.004 Tc
0 0 Td
(week)Tj
2.2506 0 Td
(\(experiment)Tj
5.1137 0 Td
(14)Tj
1.6895 0 Td
(mice)Tj
2.1543 0 Td
(per)Tj
1.5307 0 Td
(each)Tj
2.0296 0 Td
(group\))Tj
2.948 0 Td
(or)Tj
1.0998 0 Td
(13)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 479.8488 411.5905 cm
BT
/F11 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(th)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 488.2393 407.1118 cm
BT
/F11 1 Tf
1 TL
-0.004 Tc
0 0 Td
(week)Tj
2.245 0 Td
(\(experiment)Tj
-31.0674 -1.2982 Td
(24)Tj
1.6894 0 Td
(mice)Tj
2.1543 0 Td
(per)Tj
1.5307 0 Td
(each)Tj
2.0296 0 Td
(group\))Tj
2.948 0 Td
(p.i.)Tj
1.4513 0 Td
(and)Tj
1.7348 0 Td
(infected)Tj
3.4186 0 Td
(ears)Tj
1.8085 0 Td
(were)Tj
2.1203 0 Td
(used)Tj
2.0636 0 Td
(for)Tj
1.3833 0 Td
(both)Tj
/F16 1 Tf
2.0636 0 Td
[()-3()2()-4()12()18()6()16()18()4()]TJ
/F11 1 Tf
4.7848 0 Td
(re-isolation)Tj
4.8018 0 Td
(by)Tj
-35.9826 -1.2983 Td
(cultivation)Tj
4.49 0 Td
(and)Tj
1.7291 0 Td
(quantification)Tj
5.8394 0 Td
(of)Tj
1.0148 0 Td
(parasite)Tj
3.3278 0 Td
(infestation)Tj
4.4617 0 Td
(by)Tj
1.1792 0 Td
(RT-qPCR)Tj
4.1556 0 Td
(as)Tj
1.0148 0 Td
(described)Tj
4.0477 0 Td
(above.)Tj
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-30.0639 -1.3039 Td
(Ethics)Tj
2.7836 0 Td
(statement.)Tj
/F11 1 Tf
5.1817 0 Td
(Animals)Tj
3.5829 0 Td
(were)Tj
2.1203 0 Td
(maintained)Tj
4.7906 0 Td
(and)Tj
1.7291 0 Td
(handled)Tj
3.4526 0 Td
(in)Tj
1.0374 0 Td
(the)Tj
1.4684 0 Td
(animal)Tj
2.9706 0 Td
(facility)Tj
2.897 0 Td
(of)Tj
1.0148 0 Td
(Charles)Tj
-34.2252 -1.2983 Td
(University)Tj
4.3993 0 Td
(in)Tj
1.0375 0 Td
(Prague)Tj
2.999 0 Td
(in)Tj
1.0375 0 Td
(accordance)Tj
4.7395 0 Td
(with)Tj
1.9956 0 Td
(institutional)Tj
5.0909 0 Td
(guidelines)Tj
4.2747 0 Td
(and)Tj
1.7291 0 Td
(Czech)Tj
2.6701 0 Td
(legislation)Tj
4.286 0 Td
(\(Act)Tj
-34.2592 -1.2982 Td
(No.)Tj
1.6894 0 Td
(246/1992)Tj
3.8778 0 Td
(and)Tj
1.7291 0 Td
(359/2012)Tj
3.8778 0 Td
(coll.)Tj
1.8708 0 Td
(on)Tj
1.2756 0 Td
(protection)Tj
4.3823 0 Td
(of)Tj
1.0148 0 Td
(animals)Tj
3.3279 0 Td
(against)Tj
3.0274 0 Td
(cruelty)Tj
2.9649 0 Td
(in)Tj
1.0375 0 Td
(present)Tj
3.1634 0 Td
(statutes)Tj
3.2201 0 Td
(at)Tj
-36.4588 -1.2983 Td
(large\),)Tj
2.7269 0 Td
(which)Tj
2.6475 0 Td
(complies)Tj
3.7871 0 Td
(with)Tj
1.9955 0 Td
(all)Tj
1.1452 0 Td
(relevant)Tj
3.4243 0 Td
(European)Tj
4.1158 0 Td
(Union)Tj
2.829 0 Td
(and)Tj
1.7291 0 Td
(international)Tj
5.4141 0 Td
(guidelines)Tj
4.2746 0 Td
(for)Tj
-34.0891 -1.3039 Td
(experimental)Tj
5.4594 0 Td
(animals.)Tj
3.5547 0 Td
(All)Tj
1.3946 0 Td
(the)Tj
1.4683 0 Td
(experiments)Tj
5.1421 0 Td
(were)Tj
2.1203 0 Td
(approved)Tj
3.9741 0 Td
(by)Tj
1.1792 0 Td
(the)Tj
1.474 0 Td
(Committee)Tj
4.7394 0 Td
(on)Tj
1.2756 0 Td
(the)Tj
1.4683 0 Td
(Ethics)Tj
2.6646 0 Td
(of)Tj
-35.9146 -1.2983 Td
(Laboratory)Tj
4.6431 0 Td
(Experiments)Tj
5.2837 0 Td
(of)Tj
1.0148 0 Td
(the)Tj
1.4684 0 Td
(Charles)Tj
3.2484 0 Td
(University)Tj
4.3994 0 Td
(in)Tj
1.0375 0 Td
(Prague)Tj
2.999 0 Td
(and)Tj
1.7348 0 Td
(were)Tj
2.1203 0 Td
(performed)Tj
4.4673 0 Td
(under)Tj
2.6192 0 Td
(per-)Tj
-35.0359 -1.2982 Td
(mission)Tj
3.3391 0 Td
(No.)Tj
1.6838 0 Td
(MSMT-31114/201513)Tj
9.513 0 Td
(of)Tj
1.0035 0 Td
(the)Tj
1.4627 0 Td
(Ministry)Tj
3.668 0 Td
(of)Tj
1.0091 0 Td
(the)Tj
1.457 0 Td
(Environment)Tj
5.5729 0 Td
(of)Tj
1.0033 0 Td
(the)Tj
1.4627 0 Td
(Czech)Tj
2.6589 0 Td
(Republic.)Tj
-33.834 -1.304 Td
(Investigators)Tj
5.3461 0 Td
(are)Tj
1.4513 0 Td
(certificated)Tj
4.6658 0 Td
(for)Tj
1.389 0 Td
(experimentation)Tj
6.8314 0 Td
(with)Tj
1.9956 0 Td
(animals)Tj
3.3335 0 Td
(by)Tj
1.1792 0 Td
(the)Tj
1.4684 0 Td
(Ministry)Tj
3.6792 0 Td
(of)Tj
1.0148 0 Td
(Agriculture)Tj
-32.3543 -1.2982 Td
(of)Tj
1.0148 0 Td
(the)Tj
1.4683 0 Td
(Czech)Tj
2.6702 0 Td
(Republic.)Tj
3.9798 0 Td
(All)Tj
1.3947 0 Td
(efforts)Tj
2.7609 0 Td
(were)Tj
2.1203 0 Td
(made)Tj
2.4264 0 Td
(to)Tj
1.0262 0 Td
(minimize)Tj
4.0421 0 Td
(the)Tj
1.4741 0 Td
(number)Tj
3.4128 0 Td
(and)Tj
1.7347 0 Td
(the)Tj
1.4684 0 Td
(suffering)Tj
3.7587 0 Td
(of)Tj
-34.7524 -1.2983 Td
(experimental)Tj
5.4594 0 Td
(animals)Tj
3.3279 0 Td
(during)Tj
2.9253 0 Td
(the)Tj
1.474 0 Td
(study.)Tj
ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 222.0094 cm
BT
/F3 1 Tf
1 TL
-0.0033 Tc
0 0 Td
(Results)Tj
/F9 1 Tf
0 -1.5023 Td
(LmxM)Tj
/F0 1 Tf
(.)Tj
/F9 1 Tf
(30)Tj
/F0 1 Tf
(.)Tj
/F9 1 Tf
(2090)Tj
/F0 1 Tf
6.7654 0 Td
(encodes)Tj
3.9543 0 Td
(a)Tj
0.7701 0 Td
(cytosolic)Tj
4.0111 0 Td
(putative)Tj
3.6756 0 Td
(ATP/GTP-binding)Tj
7.989 0 Td
(protein)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 186.9732 cm
BT
/F11 1 Tf
1 TL
-0.004 Tc
0 0 Td
(TriTryp)Tj
3.4242 0 Td
(database)Tj
3.6396 0 Td
(annotation)Tj
4.6091 0 Td
(of)Tj
1.0148 0 Td
(the)Tj
/F16 1 Tf
1.4684 0 Td
[()22(\n)]TJ
/F11 1 Tf
(.)Tj
/F16 1 Tf
[(\r)]TJ
/F11 1 Tf
(.)Tj
/F16 1 Tf
[()]TJ
/F11 1 Tf
6.1058 0 Td
([)Tj
0.83 0.64 0.02 0 k
(19)Tj
0 g
(])Tj
1.8538 0 Td
(revealed)Tj
3.5206 0 Td
(no)Tj
1.2756 0 Td
(conserved)Tj
4.2633 0 Td
(motifs)Tj
2.7609 0 Td
(or)Tj
-33.9361 -1.2982 Td
(domains)Tj
3.685 0 Td
(for)Tj
1.3833 0 Td
(the)Tj
1.4683 0 Td
(predicted)Tj
4.0025 0 Td
(382)Tj
1.6497 0 Td
(amino)Tj
2.795 0 Td
(acid)Tj
1.8708 0 Td
(\(aa\))Tj
1.7689 0 Td
(long)Tj
1.9842 0 Td
(protein.)Tj
3.3789 0 Td
(However,)Tj
4.0762 0 Td
(we)Tj
1.3322 0 Td
(have)Tj
2.058 0 Td
(previously)Tj
-31.453 -1.2983 Td
(shown)Tj
2.8459 0 Td
(that)Tj
1.7802 0 Td
(affiliation)Tj
4.0648 0 Td
(to)Tj
1.0262 0 Td
(protein)Tj
3.1521 0 Td
(families)Tj
3.3278 0 Td
(can)Tj
1.6271 0 Td
(be)Tj
1.1452 0 Td
(identified)Tj
4.0592 0 Td
(by)Tj
1.1848 0 Td
(manual)Tj
3.2315 0 Td
(visual)Tj
2.5058 0 Td
(analysis)Tj
3.3279 0 Td
([)Tj
0.83 0.64 0.02 0 k
(35)Tj
0 g
(],)Tj
2.0749 0 Td
(so)Tj
1.0885 0 Td
(we)Tj
-36.4419 -1.2982 Td
(inspected)Tj
4.0081 0 Td
(the)Tj
1.4684 0 Td
(sequence)Tj
3.8607 0 Td
(further.)Tj
3.2542 0 Td
(DELTA-BLAST)Tj
6.599 0 Td
(search)Tj
2.7609 0 Td
(with)Tj
1.9956 0 Td
(default)Tj
2.965 0 Td
(parameters)Tj
4.6771 0 Td
([)Tj
0.83 0.64 0.02 0 k
(36)Tj
0 g
(])Tj
1.8482 0 Td
(produced)Tj
-33.4372 -1.304 Td
(no)Tj
1.2755 0 Td
(known)Tj
3.0047 0 Td
(protein)Tj
3.1521 0 Td
(hits,)Tj
1.8992 0 Td
(but)Tj
1.5534 0 Td
(gave)Tj
1.9956 0 Td
(high)Tj
2.0069 0 Td
(E-value)Tj
3.2258 0 Td
(\(range)Tj
2.8063 0 Td
(from)Tj
2.2053 0 Td
(10)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 440.7874 139.4645 cm
BT
/F17 1 Tf
1 TL
-0.0053 Tc
0 0 Td
[()]TJ
/F11 1 Tf
(7)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 450.7653 134.9858 cm
BT
/F11 1 Tf
1 TL
-0.004 Tc
0 0 Td
(to)Tj
1.0261 0 Td
(10)Tj
ET
Q
q
1 j
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