SPIDER VENOM PEPTIDES FOR GENE THERAPY OF Chlamydia INFECTION 1

Vassili N. Lazareva,c*

, Nadezhda F. Polinaa, Marina M. Shkarupeta

a, Elena S. Kostrjukova

a, 2

Alexander A. Vassilevskib, Sergey A. Kozlov

b, Eugene V. Grishin

b, Vadim M. Govorun

a,b,c 3

4

aResearch Institute for Physico-Chemical Medicine of the Federal Medical-Biological Agency of 5

Russian Federation, 1a, Malaya Pirogovskaya st., 119435, Moscow, Russia 6

bShemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 7

Ul. Miklukho-Maklaya, 16/10, 117997, Moscow, Russia 8

cNational Research Centre "Kurchatov Institute", 1, Akademika Kurchatova pl., 123182, 9

Moscow, Russia 10

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*Corresponding author: Vassili N. Lazarev 21

1a, Malaya Pirogovskaya st., 119435, Moscow, Russia 22

Tel/Fax: +7 499 255 28 46 23

E-mail: lazar0@mail.ru 24

Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Antimicrob. Agents Chemother. doi:10.1128/AAC.00449-11 AAC Accepts, published online ahead of print on 29 August 2011

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ABSTRACT 25

Spider venoms are vast natural pharmacopoeias selected by evolution. Venom of the ant 26

spider Lachesana tarabaevi contains a wide variety of antimicrobial peptides. We tested six of 27

them (latarcins 1, 2a, 3a, 4b, 5, and cyto-insectotoxin 1a) for ability to suppress Chlamydia 28

trachomatis infection. HEK293 cells were transfected with plasmid vectors harboring genes of 29

the selected peptides. Controlled expression of the transgenes led to a significant decrease of 30

C. trachomatis viability inside the infected cells. 31

KEYWORDS 32

Chlamydia trachomatis, antichlamydial therapy, spider venom, Lachesana tarabaevi, 33

latarcin, cyto-insectotoxin 34

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Intensive usage of antibiotics in medical practice leads to inevitable emergence of 35

resistant bacterial strains (13). To overcome this problem, regular development and adoption of 36

new antibiotics are required. Among other candidates for novel antimicrobial agents are small 37

polypeptide molecules, the so-called antimicrobial peptides (AMPs) (14). Natural AMPs have 38

been isolated from different organisms, raging from bacteria to higher eukaryotes. In most cases, 39

AMPs are believed to directly bind to target cell membranes at micromolar concentrations and 40

lead to their functional and/or structural disturbance; this mechanism underlies low probability of 41

bacteria acquiring resistance to AMPs (14). Interestingly, AMPs are found in natural venoms. In 42

particular, spider venoms may simultaneously contain several dozen AMPs with different 43

structure and consequently variable spectrum of activity (4, 11, 12). 44

Intracellular parasitic bacteria of the genera Mycoplasma and Chlamydia cause infectious 45

diseases that are susceptible to treatment by antibiotics with varying degrees of success and gain 46

increased medical importance (1, 9). AMPs are able to repress Mycoplasma development in vitro 47

(2). However, active peptide concentrations are usually 0.1–10 µM, corresponding to rather high 48

therapeutic doses. Among major obstacles to successful commercialization of AMPs is the high 49

production cost, although there are initiatives to create competitive technologies (10). 50

Development of efficient methods for AMPs application is highly anticipated. One possibility is 51

AMP gene therapy that may substitute direct application of the peptides (3, 5). Our present work 52

proposes a novel method of AMPs application as gene therapy agents. The method is oriented on 53

tackling intracellular infection caused by Chlamydia. To avoid bio-ethic issues, however, we 54

should indicate that gene therapy of humans is not considered at this point. 55

Antichlamydial and antimycoplasmic effects have been previously demonstrated for the 56

AMP melittin in cell cultures and laboratory animals (mice and chicken) (5-7). Melittin, isolated 57

from the venom of the honey bee Apis mellifera, is one of the most famous and probably the best 58

studied AMP. However, strong hemolytic activity and allergic reactions in humans (8) make it of 59

little use in drug development. As mentioned above, spider venoms serve an attractive source of 60

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peptides with different type of activity. From the venom of the ant spider Lachesana tarabaevi 61

we purified a wide variety of AMPs (4, 12). Amino acid sequences of some of the most active 62

peptides and their minimal inhibitory concentrations (MICs) towards Escherichia coli are shown 63

in Table 1. Several peptides (latarcins Ltc 1, Ltc 3a, Ltc 4b) feature restricted activity profiles 64

being highly effective against bacteria, whereas others (latarcins Ltc 2a, Ltc 5, and cyto-65

insectotoxin CIT 1a) show broad spectrum cytolytic activity, reminiscent of melittin. We decided 66

to check the potencies of L. tarabaevi AMPs against Chlamydia and select for peptides that 67

would be both highly effective against intracellular parasites and not toxic to the host cells. 68

Synthetic genes coding for the six AMPs were produced from oligonucleotides (Table 2) 69

by annealing and ligation and cloned into the pBI-EGFP vector (Clontech) at the PvuII site as 70

described earlier (7). The reverse tetracycline-controlled transactivator protein (rtTA) gene was 71

amplified using the vector pTet-On (Clontech) and cloned at the BglII site into the same pBI-72

EGFP. The resulting pBI/rtTA/EGFP/AMP vectors were used to transfect HEK 293 cells 73

vulnerable to Chlamydia infection. 74

Visualization of AMPs gene expression was possible due to concomitant expression of 75

the enhanced green fluorescent protein (EGFP) reporter gene (egfp). All the therapeutic vectors 76

displayed high gene expression levels; even at concentrations of the inducing agent doxycycline 77

as low as 0.002 µg/ml indicative fluorescence was observed. To further confirm AMP gene 78

expression total RNA was extracted from the induced cells and analyzed by RT-PCR (Fig. 1 79

presents data for CIT 1a). 80

Toxicity of recombinant EGFP and AMPs for the cells was minimal (cytotoxic effect on 81

host cells was evaluated by the LIVE/DEAD Kit (Invitrogen)); even if doxycycline 82

concentration was increased to 2 µg/ml, the number of living cells did not get lower than 95% 83

and was not significantly different from control non-transfected HEK 293 cells. 84

Recombinant constructs containing AMP genes were then tested for ability to suppress 85

Chlamydia infection in the HEK 293 cell line model. In those experiments we used plasmids 86

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with deletion of the egfp gene in order to detect Chlamydia inclusions by direct 87

immunofluorescence with fluorescein isothiocyanate (FITC)-labeled antibodies to the major 88

outer membrane protein (MOMP) of C. trachomatis (ChlaMonoScreen-2, Nearmedic Plus, 89

Russia) as described (5). 5 h after transfection of HEK 293 cells using the PolyFect Transfection 90

Reagent (Qiagen, Germany), doxycycline was added (0.02 µg/ml), and in 20 h cells were 91

infected by C. trachomatis. Control cells were transfected by the same plasmid vectors, but gene 92

expression was not induced. Calculation of Chlamydia inclusions was performed 24 h after 93

infection using a confocal microscope (C1; Nikon), at least 50 microscopic fields were analyzed 94

in each experiment. Inhibition of Chlamydia inclusion formation was defined as the ratio 95

between the number of inclusions in cells induced by doxycycline and control cells. Results are 96

shown in Fig. 2. Effects were considered significant at p<0.05. The most effective gene 97

(encoding CIT 1a) demonstrated a pronounced inhibitory effect (~85%) larger than that of 98

melittin (~60%) (5). Other peptide genes, except for ltc 5, also demonstrated high efficiency 99

(~60–70% inhibition). 100

To conclude, we have shown that controlled AMP gene expression can be exploited to 101

tackle Chlamydia infection. Moreover, a new possibility of exploiting the treasure trove of spider 102

venoms was presented. Therapeutic vectors containing AMP genes may be used for prophylaxis 103

of diseases caused by Chlamydia. One may also think that the same system would be effective 104

against diverse epithelial infections inflicted by different types of bacteria and fungi. We believe 105

that the vectors developed in this study may be used for control of intracellular parasites in 106

livestock production, and for protection or rescue of infected cell lines in laboratory practice. 107

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REFERENCES 108

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Microbiol Rev. 32: 956-73. 111

2. Fassi Fehri, L., H. Wroblewski, and A. Blanchard. 2007. Activities of antimicrobial 112

peptides and synergy with enrofloxacin against Mycoplasma pulmonis. Antimicrob 113

Agents Chemother 51:468-474. 114

3. Huang, G. T., H. B. Zhang, D. Kim, L. Liu, and T. Ganz. 2002. A model for 115

antimicrobial gene therapy: demonstration of human beta-defensin 2 antimicrobial 116

activities in vivo. Hum Gene Ther 13:2017-2025. 117

4. Kozlov, S. A., A. A. Vassilevski, A. V. Feofanov, A. Y. Surovoy, D. V. Karpunin, and E. 118

V. Grishin. 2006. Latarcins, antimicrobial and cytolytic peptides from the venom of the 119

spider Lachesana tarabaevi (Zodariidae) that exemplify biomolecular diversity. J Biol 120

Chem 281:20983-20992. 121

5. Lazarev, V. N., T. M. Parfenova, S. K. Gularyan, O. Y. Misyurina, T. A. Akopian, and V. 122

M. Govorun. 2002. Induced expression of melittin, an antimicrobial peptide, inhibits 123

infection by Chlamydia trachomatis and Mycoplasma hominis in a HeLa cell line. Int J 124

Antimicrob Agents 19:133-137. 125

6. Lazarev, V. N., M. M. Shkarupeta, G. A. Titova, E. S. Kostrjukova, T. A. Akopian, and 126

V. M. Govorun. 2005. Effect of induced expression of an antimicrobial peptide melittin 127

on Chlamydia trachomatis and Mycoplasma hominis infections in vivo. Biochem Biophys 128

Res Commun 338:946-950. 129

7. Lazarev, V. N., L. Stipkovits, J. Biro, D. Miklodi, M. M. Shkarupeta, G. A. Titova, T. A. 130

Akopian, and V. M. Govorun. 2004. Induced expression of the antimicrobial peptide 131

melittin inhibits experimental infection by Mycoplasma gallisepticum in chickens. 132

Microbes Infect 6:536-541. 133

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8. Raghuraman, H., and A. Chattopadhyay. 2007. Melittin: a membrane-active peptide with 134

diverse functions. Biosci Rep 27:189-223. 135

9. Senn L, Hammerschlag MR, Greub G. 2005. Therapeutic approaches to Chlamydia 136

infections. Expert Opin Pharmacother. 6: 2281-90. 137

10. Vassilevski, A. A., S. A. Kozlov, and E. V. Grishin. 2008. Antimicrobial peptide 138

precursor structures suggest effective production strategies. Recent Pat Inflamm Allergy 139

Drug Discov 2:58-63. 140

11. Vassilevski, A. A., S. A. Kozlov, and E. V. Grishin. 2009. Molecular diversity of spider 141

venom. Biochemistry (Mosc) 74:1505-1534. 142

12. Vassilevski, A. A., S. A. Kozlov, O. V. Samsonova, N. S. Egorova, D. V. Karpunin, K. 143

A. Pluzhnikov, A. V. Feofanov, and E. V. Grishin. 2008. Cyto-insectotoxins, a novel 144

class of cytolytic and insecticidal peptides from spider venom. Biochem J 411:687-696. 145

13. Wright GD. 2007 Nat Rev Microbiol. 5: 175-86. The antibiotic resistome: the nexus of 146

chemical and genetic diversity. 147

14. Yeaman, M.R., Yount, N.Y., 2003. Mechanisms of antimicrobial peptide action and 148

resistance. Pharmacol. Rev. 55, 27-55. 149

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Figure 1. Cit 1a gene expression in HEK 293 cells. (A) Optical/fluorescent merged image of cells

induced with doxycycline (0.02 µg/ml) in 24 h after transfection by the plasmid vector

pBI/rtTA/EGFP/CIT 1a. Green fluorescence indicates egfp expression. (B) RT-PCR analysis of cit 1a gene

expression. Total RNA was extracted from the cells in 12 h after addition of doxycycline. 1 – RT-PCR

product for the non-transfected HEK 293 cells. 2 – negative control (reverse transcriptase was not added to

the PCR mixture). 3 – RT-PCR product for the transfected HEK 293 cells. 4 – positive control (the vector

pBI/rtTA/GFP/CIT 1a was used as matrix for PCR). M – molecular markers (DNA Ladder Mix, Thermo

Scientific).

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Figure 2. Antichlamydial activity of L. tarabaevi AMPs. HEK 293 cells harboring AMP

genes were infected by C. trachomatis in 20 h after gene expression induction. (А) Direct

immunofluorescence staining of Chlamydia inclusions (green) in 24 h after transfection of cells

by the vector pBI/rtTA/CIT 1a. Cells were also stained by the Evans blue dye (red).

(B) Inhibition of Chlamydia inclusions formation in cells expressing various AMP genes. Data

are expressed as mean ± standard error of mean.

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Table 1. Amino acid sequences and activity of AMPs from L. tarabaevi spider venom,

used to create regulated therapeutic genes.

Peptide

name

Uniprot

accession

number

Amino acid sequence MIC*,

µM

CIT 1a P85253 GFFGNTWKKIKGKADKIMLKKAVKIMVKKEGISKEEAQAK

VDAMSKKQIRLYLLKYYGKKALQKASEKL-OH

1

Ltc 1 Q1ELT9 SMWSGMWRRKLKKLRNALKKKLKGE-OH 1

Ltc 2a Q1ELU1 GLFGKLIKKFGRKAISYAVKKARGKH-OH 0.5

Ltc 3a Q1ELU3 SWKSMAKKLKEYMEKLKQRA-NH2 2.5

Ltc 4b Q1ELU4 SLKDKVKSMGEKLKQYIQTWKAKF-NH2 4.5

Ltc 5 Q1ELU9 GFFGKMKEYFKKFGASFKRRFANLKKRL-NH2 0.5

*against E. coli DH5α. MICs were determined using the conventional microtitre broth

dilution assay.

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Table 2. Oligonucleotides used for AMPs gene synthesis.

AMP

name

GenBank Primer

name

Sequence (5’→3’)

CIT 1a FM165474

CIT1a1

CIT1a2

CIT1a3

CIT1a4

CIT1a1-rev

CIT1a2-rev

CIT1a3-rev

CIT1a4-rev

ATGGGCTTCTTCGGCAACACCTGGAAGAAGATCAAGGGCAAGGCCGACAAGATCATG

CTGAAGAAGGCCGTGAAGATCATGGTGAAGAAGGAGGGCATCAGCAAGGAGGAGGCCCAGG

CCAAGGTGGACGCCATGAGCAAGAAGCAGATCCGGCTGTACCTGCTGAAGT

ACTACGGCAAGAAGGCCCTGCAGAAGGCCAGCGAGAAGCTGTGA

GGCCTTCTTCAGCATGATCTTGTCGGCCTTGCCCTTGATCTTCTTCCAGGTGTTGCCGAAGAAGCCCAT

CATGGCGTCCACCTTGGCCTGGGCCTCCTCCTTGCTGATGCCCTCCTTCTTCACCATGATCTTCAC

GCCTTCTTGCCGTAGTACTTCAGCAGGTACAGCCGGATCTGCTTCTTGCT

TCACAGCTTCTCGCTGGCCTTCTGCAGG

Ltc 1 AM232700 Ltc1a

Ltc1b

Ltc1a-rev

Ltc1b-rev

ATGTCCATGTGGTCCGGCATGTGGAGAAGAAAGCTGAAGAAGCTGCGAAA

CGCTTTGAAGAAGAAGCTGAAGGGCGAGTGA

AGCTTTCTTCTCCACATGCCGGACCACATGGACAT

TCACTCGCCCTTCAGCTTCTTCTTCAAAGCGTTTCGCAGCTTCTTC

Ltc 2a AM232698 Ltc2a1

Ltc2a2

Ltc2a1-rev

Ltc2a2-rev

ATGGGCCTGTTCGGCAAGCTGATCAAGAAGTTTGGAAGAAAGGCTATCTCCTAC

GCCGTGAAGAAGGCTAGAGGAAAGCACTGA

CTTCCAAACTTCTTGATCAGCTTGCCGAACAGGCCCAT

TCAGTGCTTTCCTCTAGCCTTCTTCACGGCGTAGGAGATAGCCTTT

Ltc 3a AM232696 Ltc3a

Ltc3a-rev

ATGAGCTGGAAGAGCATGGCCAAGAAGCTGAAGGAGTACATGGAGAAGCTGAAGCAGCGGGCCTGA

TCAGGCCCGCTGCTTCAGCTTCTCCATGTACTCCTTCAGCTTCTTGGCCATGCTCTTCCAGCTCAT

Ltc 4b AM232695 Ltc4b1

Ltc4b2

Ltc4b1-rev

Ltc4b2-rev

ATGAGCCTGAAGGACAAGGTGAAGAGCATGGGCGAGAAGCTGAAG

CAGTACATCCAGACCTGGAAGGCCAAGTTCTGA

ATGCTCTTCACCTTGTCCTTCAGGCTCAT

TCAGAACTTGGCCTTCCAGGTCTGGATGTACTGCTTCAGCTTCTCGCCC

Ltc 5 AM232690 Ltc5a

Ltc5b

Ltc5a-rev

Ltc5b-rev

ATGGGCTTCTTCGGCAAGATGAAGGAGTACTTCAAGAAGTTCGGCGCCAGCTTC

AAGCGGCGGTTCGCCAACCTGAAGAAGCGGCTGTGA

TTCTTGAAGTACTCCTTCATCTTGCCGAAGAAGCCCAT

TCACAGCCGCTTCTTCAGGTTGGCGAACCGCCGCTTGAAGCTGGCGCCGAAC

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