research article small molecule inhibitor of type three ...research article small molecule inhibitor...

Research ArticleSmall Molecule Inhibitor of Type Three SecretionSuppresses Acute and Chronic Chlamydia trachomatisInfection in a Novel Urogenital Chlamydia Model

Ekaterina A Koroleva Natalia V Kobets Egor S ZayakinSergei I Luyksaar Ludmila A Shabalina and Naylia A Zigangirova

Gamaleya Institute of Epidemiology and Microbiology Ministry of Health Russian FederationGamaleya Street 18 Moscow 123098 Russia

Correspondence should be addressed to Natalia V Kobets nkobets2000yahoocom

Received 1 September 2014 Accepted 11 December 2014

Academic Editor Shigeru Kotake

Copyright copy 2015 Ekaterina A Koroleva et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Previously we reported that a compound from a group of thiohydrazides of oxamic acids CL-55 possessed antichlamydial activityin vitro that was accompanied by a decreased translocation of the type three secretion effector IncA into the host cell In thisstudy the antichlamydial activity of CL-55 was tested in vivo in DBA2 mice infected with C trachomatis serovar D We found thatintravaginal inoculation of DBA2 mice with the clinically relevant strain C trachomatis serovar D results in a course of infectionand pathology similar to that observed in humans The early stage of infection in this model was characterized by a shedding ofChlamydia in vaginal secretions followed by an ascending infection and inflammation in the upper genital tract We found thatCL-55 possessed antibacterial activity in vivo and was able to control C trachomatis vaginal shedding ascending infection andinflammation in the upper genital organs in DBA2 mice Our data provide a proof of concept for the protective effect of thethiadiazinon CL-55 against chlamydial infection in vivo and support the feasibility of further studies of its potential therapeuticapplications

1 Introduction

Chlamydia trachomatis is a leading cause of sexually transmit-ted diseases with an estimated 100million new cases reportedannually [1] This organism is responsible for a variety ofgenital syndromes in men and women with infertility as anoutcome in many cases [2]

Lack of an efficient antibacterial therapy for chronicchlamydia infection as well as the rise of antibiotic resistanceunderscores the importance of searching for new strategiesof antibacterial treatment [3 4] Among the new approachesis the development of small molecule virulence inhibitorsthat can disarm bacteria and allow the host to control theinfection

Recently we performed an identification of novel small-molecule type three secretion (T3S) inhibitors by chem-ical modification of thiohydrazides of oxamic acids and

experimental screening of their derivatives Selected com-pounds from a group of thiadiazinons were assessed forantichlamydial activity in vitro We demonstrated that thesecompounds inhibit chlamydial growth in vitro and that thiswas accompanied by the decreased translocation of the typethree secretion effector IncA [5] These results promptedus to proceed to evaluation of new inhibitors in an animalmodel

Human serovars ofC trachomatis have limited pathogen-icity in miceThus intravaginal infection in C3HHeJ mice isused in chemotherapy studies [6] and allows for the study ofbacterial shedding but not the upper genital tract pathologyConsistent establishment of infection in the upper genitaltract of mice with human serovars of C trachomatis requireshormonal manipulation and surgical intervention to placethe organisms directly into the upper genital tract via theuterus or ovarian bursa This procedure is time-consuming

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 484853 7 pageshttpdxdoiorg1011552015484853

2 BioMed Research International

but results in salpingitis altered tubal function and impairedfertility [7 8] and this model has been used successfullyto evaluate new antimicrobial agents [9] Infection can alsobe induced by intravaginal inoculation but development ofsalpingitis from this route has been reported to be serovardependent and irregular this model would therefore beunsuitable for studying the long-term effects of chemother-apy on upper genital tract inflammation [10]

Most studies tend to useC muridarum for chemotherapyand vaccine studies which is genetically and antigenicallydistinct from human biovars [11 12] In addition this strainwas reported to respond differently to some antibacterialagents [13]

We report here for the first time the developmentof an upper genital tract infection in progesterone-treatedsusceptible DBA2 mice caused by intravaginal inoculationwith clinically significant C trachomatis serovar D in whichsalpingitis and hydrosalpinx formation were observed Wealso have expanded our work on the antibacterial activityof T3S inhibitors on chlamydial shedding ascending uppergenital tract chlamydial infection and inflammatory sequelaein susceptible DBA2 mice and found that one T3S inhibitorefficiently suppressed both acute and chronic infection andameliorated the upper genital pathology in an animal model

2 Materials and Methods

21 Organisms C trachomatis serovar D DUW-3CX(ATCCVR 885) was grown in cycloheximide-treatedMcCoycells using conventional methods The growth medium wasminimal essential medium supplemented with 10 fetal calfserum 1 nonessential amino acids 2mM L-glutamine 1vitamins and 05 glucose Cell lysates containing approxi-mately 35 times 104 inclusion-forming units (IFU) per mL wereprepared and stored at minus70∘C

22 Animals Female DBA2 mice obtained from AnimalResource Center (Andreevka Moscow region) weighing 14to 16 g were used throughout and were given access to foodand water ad libitum

23 Inoculation The animals subcutaneously received 33mgper kg of the progesterone analog proligestone (CovinanIntervet Schering-Plough Animal Health Netherlands) Fivedays later the mice were infected with 106 CFUmouse ofChlamydia trachomatis serovarD in 40 120583L of PBSThe controlanimals received sterile PBS

24 Drug Treatment Azithromycin was given as a dailysingle oral dose of 10mgkg Treatment was administeredorally for 5 days to mimic the regimen often recommendedfor treatment in humans

4-(3-etoxy-4-hydrobenzil)-5-oxo-56-dehydro-4H-[1 34]-thiadiazol-2-(24-biftorphenyl)-carboxamid designatedas CL-55 was given as an intraperitoneal injection of 50 or100mgkg Inhibitor solutions were prepared by mixing theactive substance with Tween 80 and dissolving it in 1 starchin PBS The control infected mice received a solution of 1

starch in PBS with Tween 80 The early effects of therapywere assessed by measuring vaginal shedding of chlamydiaestarting at day three postinfection The long-term therapyoutcome was assessed using Real Time-PCR to measurechlamydial DNA in the ovaries and uteri 4 weeks afterinfection Genital tissues from the infected mice bothtreated and untreated were processed for histopathologicalexamination at days 28 to 30 after infection

25 Isolation of Chlamydia The canal and exocervix werevigorously scraped and swabbed material was transferredinto 2-sucrose phosphate transport medium and frozenimmediately at minus70∘C All specimens were processed on thesame day Thawed undiluted samples were plated in 05mLvolumes onto McCoy cells on coverslips (12mm diameter) in24-well plates and the infection was enhanced by centrifuga-tion Coverslips were incubated with FITC-conjugated mon-oclonal antibodies against chlamydial lipopolysaccharide(Nearmedic Plus Russia) for 48 h Inclusion-containing cellswere examined and photographed with a Nikon Eclipse 50ifluorescent microscope at 1350x magnification The resultswere expressed as lg IFU per milliliter of sample

26 Real Time PCR Samples for chlamydial DNA detec-tion were collected in phosphate-buffered saline (PBS) andfrozen at minus20∘C DNA was extracted from excised genitaltissues with an automated nucleic acid extractor NucliSENSeasyMAG (bioMerieux Netherland) Chlamydial DNA wasamplified with a C trachomatis PB screen kit (SyntolMoscowRussia) andReal TimePCRcycler CFX96 (Bio-RadUSA)

27 Histopathology Uteri and ovaries were frozen in theregimen of minus60∘C to minus20∘C temperature gradient in theelectronic Cryotome Shandon (USA) and serial 8120583m-thicksections weremade Sections were stained with haematoxylinand eosin (HampE) and examined without knowledge of theexperimental group to which they belonged

28 Statistical Analysis All datawerepresented as themean plusmnstandard deviation (SD) All statistics were performed usingGraphPad Prism version 600 (GraphPad Software Inc Cal-ifornia USA) Significance was set at 119875 lt 005 for alltests

3 Results

31 DBA2 Mice Intravaginally Infected with C trachomatisSerovar D Have Both Acute Chlamydia Shedding and UpperTract Infection and Pathology As existing C trachomatisinfection models in mice do not correspond to a humancourse of pathological sequelae we developed a new murineC trachomatis infection model For this we utilized theDBA2 strain of mice that have a range of genetic defects thatmake themmore susceptible to some bacterial infections andreproductive pathology [14 15]

In these studies progesterone-treated DBA2 micereceived 106 IFU of C trachomatis serovar D intravaginally

BioMed Research International 3

Days postinfection

IFU

per

swab

30000

20000

10000

0

3 6 9 13 17 22 25

(a)

Day 3 Day 6 Day 17 Day 25

(b)

Figure 1 (a) Chlamydia inclusion-forming units (IFUs) obtained from vaginal swabs of DBA2 mice infected with C trachomatis serovarD Shown are the IFU counts for vaginal swabs obtained at days 3 to 25 The median number of IFUs is indicated by the horizontal line (b)Microphotographs of McCoy cell cultures infected with C trachomatis from vaginal swabs of infected mice obtained at days 3 to 25

4

1

23

(a) (b) (c)

Figure 2 Features of pathology of the upper genital organs after intravaginal infection of DBA2 mice with C trachomatis serovar D (a)Uterine horns of infected mice at day 28 postinfection (1 hydrosalpinx 2 endometritis 3 endocervicitis 4 oophoritis) (b) Uterine tissue ofinfected mice (c) C trachomatis inclusions in uterine tissue of infected mice

Three days later a mean count of sim104 IFUmLwas recoveredfrom vaginal swabs from DBA2 mice (Figure 1) We regis-tered shedding until day 25 when it was 102 IFUmL thoughinfection partly ascended at that time and could also beregistered in the upper genital organs

Examination of the upper genital organs at day 28postinfection revealed inflammation of uterine horns mild

hydrosalpinx some features of endometritis endocervicitisand oophoritis (Figure 2(a)) Development of hydrosalpinxoccurred in more than 80 of the mice after 28 dayspostinfection

Histological examination of oviducts 28 days after infec-tion revealed swelling of the subepithelial connective tissueoviduct stenosis and vascular impairments


1

10

100

1000

10000

100000

Ovaries Uteri

Chla

myd

ia D

NA

copi

es

Figure 3 Chlamydial DNA detection in ovaries and uteri ofchlamydia-infected mice at day 28 postinfection Individual valuesfor each mouse per group are shown

Numerous polymorphonuclear leukocytes were also evi-dent (Figure 2(b)) Additionally Chlamydia inclusions werefound in the uterine tissue of the infected mice (Figure 2(c))

The presence of bacteria in the upper genital tract wasassessed using PCR Chlamydial DNA was detected in theovaries and uteri at day 28 postinfection and thus the PCRresults confirmed the development of chronic urogenitalinfection in DBA2 mice infected with clinically importantC trachomatis serovar D strain (Figure 3)

32 T3S Inhibitor Affects the Course of Infection In Vivo AsCL-55 showed promising results in the suppression of Ctrachomatis infection in vitro [5] in this study we analyzed itsantibacterial activity in vivo in DBA2 mice To this end weinfected progesterone-treated DBA2 mice with 106 IFU of Ctrachomatis serovar D To assess the inhibitor activity againstacute infection we treated mice with 50mgkg of CL-55 for 5days starting from day 1 of infection (early therapy) To assessthe inhibitor activity against chronic infection we treated themice with 50 or 100mgkg of CL-55 during 5 days startingfrom day 15 postinfection (late therapy) The efficiency ofour inhibitor was compared to the efficiency of azithromycinused either as an early (day 1 postinfection) or late (day 15postinfection) treatment

To determine the short-term efficacy of each therapyvaginal swabs were taken from the DBA2 mice infectedwith 106 IFU of C trachomatis serovar D and treated with50mkgkg of CL-55 starting fromday 1 postinfection Vaginalswabs were taken from days 3 to 14 following infectionThe mice infected with Chlamydia without treatment orthe mice treated with azithromycin were used as controlsAll animals treated with azithromycin were culture negative(Figure 4)We also found a significant reduction of chlamydiainclusions at day 7 after infection in the mice treated withCL-55 starting from day 1 The level of inclusions droppedto zero in this group at day 11 postinfection (Figure 4) Thusthe effectiveness of our inhibitor in the treatment of acuteinfection was confirmed in these experiments

To assess the long-term effects of the therapy we analyzedthe ability of our inhibitor to suppress infection in theupper genital organs To this end we analyzed the levels

of chlamydial DNA in uteri of the infected mice at day30 postinfection In this set of experiments the mice wereinfected with 106 IFU of C trachomatis serovar D and treatedwith either 50 or 100mgkg of CL-55 intraperitoneally for5 days starting from day 15 postinfection Treatment of theinfected mice with T3S inhibitor CL-55 resulted in a decreaseofC trachomatisDNA in the upper genital tract of thesemiceand this effect was more pronounced in the group treatedwith 100mgkg of inhibitor (Figure 5) Thus we confirmedthat the T3S inhibitor CL-55 successfully inhibited chronicCtrachomatis infection in vivo

As we have previously reported that CL-55 has minimaltoxicity when tested in vitro using McCoy cells [5] in thisstudy we assessed the effects of this compound on the uppergenital tract pathology For this the progesterone-treatedmice were infected with 106 IFU of C trachomatis serovar Dand were inoculated ip with 100mkgkg of CL-55 or diluenteither at day 1 or at day 15 following infection The mice weregiven a daily treatment of T3S inhibitor for 5 days and 2 pairsof infected control and CL-55 treated mice were sacrificed atday 30 postinfection

We did not observe signs of pathology in the mice treatedwith the T3S inhibitor starting from day 1 (Figure 6(a))Mild salpingitis and inflammation of endocervix were stillobserved in the group treated with CL-55 starting fromday 15 however we did not observe hydrosalpinx in thisgroup (Figure 6(b)) Thus both early and late treatment withCL-55 decreased the features of pathology in upper genitalorgans of the DBA2 mice infected intravaginally with Ctrachomatis serovarDMild upper genital tract pathologywasalso observed in groups treated with azithromycin (data notshown)

4 Discussion

Although C muridarum reproduces human infection and itsinflammatory sequelae in the upper genital tract of mice itdoes not always share the C trachomatis pattern of responseto some antibacterial agents [13]

In this study we show that intravaginal inoculationof DBA2 mice with the clinically relevant C trachomatisserovar D gives a course of infection and pathology similarto that observed in humans The early stage of infection inthis model was characterized by the shedding of Chlamydiain vaginal secretions with numbers gradually declining overa 4-week period After this time no organisms could bedetected The later stage began from 4 to 5 weeks afterinfection when chlamydial DNA was detected in uteri andovariesThiswas accompanied by uterine horn inflammationendometritis endocervicitis hydrosalpinx and oophoritisdetected in a majority of the mice The course of events inthis model was similar to that observed after intravaginalinoculation with C muridarum or inoculation of humanserovars by the technically more demanding intrauterineor intrabursal routes [7 8] Thus many of the sequelae ofchlamydial infection in women have been reproduced rela-tively simply in our mouse model Moreover these findingsrepresent a significant contrast to other reports on the effect

BioMed Research International 5IF

Uv

agin

al sw

ab

Day 3

10000

8000

6000

4000

2000

0

(a)

IFU

vag

inal

swab

Day 7

40000

30000

20000

10000

0

(b)

IFU

vag

inal

swab

Day 11

8000

6000

4000

2000

0

(c)

IFU

vag

inal

swab

Day 14

10000

8000

6000

4000

2000

0

(d)

Figure 4 Chlamydia inclusion-forming units (IFUs) obtained from vaginal cultures for control (e or ⃝ 119899 = 8) CL-55-treated (◼ or ◻119899 = 8) and azithromycin-treated (998771 or 119899 = 8) mice Shown are the IFU counts for vaginal cultures obtained at the 3rd 7th 11th and 14thdays The median number of IFUs is indicated by the horizontal line Culture-negative ( ⃝ ◻ and ) mice and culture-positive (e ◼ and998771) mice are shown in each group

1

10

100

1000

10000

Diluent

Chla

myd

ia D

NA

copi

es p

er u

teru

s

Day 30 postinfection50mgmL 100mgmL

Figure 5 CL-55 treatment decreases the levels of C trachomatisDNA in upper genital organs of DBA2 mice infected with Ctrachomatis serovar D Black bar infected DBA2 mice treated witha1cgk]aKgeSw light bar mice treated with 50mgmL of CL-55 greybar mice treated with 100mgmL of CL-55 lowast119875 le 005

of intravaginal inoculation with C trachomatis in which theinfection was described as mild and restricted only to thelower genital tract [6 16]

As we have previously shown C trachomatis was sus-ceptibly to our novel T3S inhibitor in vitro [5] and in thisreport we have further extended our study to evaluate thesusceptibility ofC trachomatis to our T3S inhibitor in vivo Toassess the efficacy of treatment with the T3S inhibitor in boththe early and later stages of infection we evaluatedChlamydiavaginal shedding Chlamydia DNA in ovaries and uteri andupper genital tract pathology after early (from day 1) or late(from day 15) treatment with our inhibitor Chlamydial shed-ding was abrogated in the group receiving early treatment byday 14 (Figure 4)This was also accompanied by abrogation ofthe upper genital tract pathology (Figure 6(a)) in this groupofmice A decrease in the number of Chlamydial DNA copieswas observed in the late treated group but though the uppergenital tract pathology was reduced in this group it was stillapparent which could be either due to the persistence of lownumbers of organisms or because the onset of inflammationwas triggered before the treatment Previously Slepenkinand colleagues reported on the antibacterial activity ofanother T3S inhibitor salicylidene acylhydrazide compoundin intravaginal chlamydial infection In this study the T3Sinhibitor was tested as a microbicide and decreased sheddingof Chlamydia in C3HHeJ mice intravaginally infected with


(a)

12

(b)

Figure 6 Treatment with CL-55 reduces uterine pathology in DBA2 mice infected with C trachomatis serovar D Uterine horns of infectedmice at day 30 postinfection are shown (a) Mice treated with CL-55 from day 1 postinfection (b) Mice treated with CL-55 from day 14postinfection (1 endometritis 2 endocervicitis)

C trachomatis serovar D [6] Later the same group assessedthe antibacterial activity of two salicylidene acylhydrazidecompounds administered systemically and again confirmedthe efficacy of the chosen T3S inhibitors in the control ofChlamydia vaginal shedding [17] however the impact of thecompound on the infection of the upper genital tract was notstudied In our study we confirmed for the first time thatthe antibacterial activity of a T3S inhibitor can be extendedto the control of ascending C trachomatis infection andaccompanying inflammatory sequelae in the upper genitalorgans

5 Conclusions

Our data provide a proof of concept for the protective effect ofone of the thiadiazinons CL-55 against chlamydial infectionin vivo and support the feasibility of further studies to test itspotential in therapeutic applications

Conflict of Interests

The authors confirm that they have no conflict of interestsregarding this paper

References

[1] WHO Initiative for Vaccine Research (IVR) Sexually Transmit-ted Diseases World Health Organization Geneva Switzerland2012

[2] WHO Global Strategy for the Prevention and Control of Sex-ually Transmitted Infections 2006mdash2015 Breaking the Chainof Transmission World Health Organization Geneva Switzer-land 2007

[3] F Blasi S Damato R Cosentini et al ldquoChlamydia pneumoniaeand chronic bronchitis association with severity and bacterialclearance following treatmentrdquo Thorax vol 57 no 8 pp 672ndash676 2002

[4] J D Carter L R Espinoza R D Inman et al ldquoCombinationantibiotics as a treatment for chronic Chlamydia-induced reac-tive arthritis a double-blind placebo-controlled prospectivetrialrdquo Arthritis and Rheumatism vol 62 no 5 pp 1298ndash13072010

[5] N A Zigangirova E S Zayakin L N Kapotina et al ldquoDevel-opment of chlamydial type III secretion system inhibitors forsuppression of acute and chronic forms of chlamydial infectionrdquoActa Naturae vol 4 no 2 pp 87ndash97 2012

[6] A Slepenkin H Chu M Elofsson P Keyser and E MPeterson ldquoProtection of mice from a Chlamydia trachomatisvaginal infection using a salicylidene acylhydrazide a potentialmicrobiciderdquo The Journal of Infectious Diseases vol 204 no 9pp 1313ndash1320 2011

[7] M Tuffrey P Falder J Gale andD Taylor-Robinson ldquoSalpingi-tis inmice induced by human strains ofChlamydia trachomatisrdquoBritish Journal of Experimental Pathology vol 67 no 4 pp 605ndash616 1986

[8] M Tuffrey F Alexander C Inman and M E Ward ldquoCorrela-tion of infertility with altered tubalmorphology and function inmice with salpingitis induced by a human genital-tract isolate ofChlamydia trachomatisrdquo Journal of ReproductionampFertility vol88 no 1 pp 295ndash305 1990

[9] J Zana M Muffat-Joly D Thomas J Orfila J Salat-Barouxand J-J Pocidalo ldquoRoxithromycin treatment of mouse chlamy-dial salpingitis and protective effect on fertilityrdquo AntimicrobialAgents and Chemotherapy vol 35 no 3 pp 430ndash435 1991

[10] M Tuffrey and D Taylor-Robinson ldquoProgesterone as a keyfactor in the development of a mouse model for genital-tractinfection with Chlamydia trachomatisrdquo FEMS MicrobiologyLetters vol 12 no 2 pp 111ndash115 1981


[11] A S Beale and P A Upshon ldquoCharacteristics of murine modelof genital infection with Chlamydia trachomatis and effectsof therapy with tetracyclines amoxicillin-clavulanic acid orazithromycinrdquoAntimicrobial Agents and Chemotherapy vol 38no 9 pp 1937ndash1943 1994

[12] E Weiss S Schramek N N Wilson and L W NewmanldquoDeoxyribonucleic acid heterogeneity between human andmurine strains of Chlamydia trachomatisrdquo Infection and Immu-nity vol 2 no 1 pp 24ndash28 1970

[13] J Coers I Bernstein-Hanley D Grotsky et al ldquoChlamydiamuridarum evades growth restriction by the IFN-120574-induciblehost resistance factor Irgb10rdquo The Journal of Immunology vol180 no 9 pp 6237ndash6245 2008

[14] K RWilson J M Napper J Denvir V E Sollars and H D YuldquoDefect in early lung defence against Pseudomonas aeruginosain DBA2 mice is associated with acute inflammatory lunginjury and reduced bactericidal activity in naıve macrophagesrdquoMicrobiology vol 153 no 4 pp 968ndash979 2007

[15] M Zhou J Fu W Huang et al ldquoIncreased cystic fibrosis trans-membrane conductance regulators expression and decreasedepithelial sodium channel alpha subunits expression in earlyabortion findings from a mouse model and clinical cases ofabortionrdquo PLoS ONE vol 9 no 6 Article ID e99521 2014

[16] A W Olsen P Andersen and F Follmann ldquoCharacterizationof protective immune responses promoted by human antigentargets in a urogenital Chlamydia trachomatis mouse modelrdquoVaccine vol 32 no 6 pp 685ndash692 2014

[17] T Ur-Rehman A Slepenkin H Chu et al ldquoPre-clinicalpharmacokinetics and anti-chlamydial activity of salicylideneacylhydrazide inhibitors of bacterial type III secretionrdquo Journalof Antibiotics vol 65 no 8 pp 397ndash404 2012

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Stem CellsInternational



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Microbiology


but results in salpingitis altered tubal function and impairedfertility [7 8] and this model has been used successfullyto evaluate new antimicrobial agents [9] Infection can alsobe induced by intravaginal inoculation but development ofsalpingitis from this route has been reported to be serovardependent and irregular this model would therefore beunsuitable for studying the long-term effects of chemother-apy on upper genital tract inflammation [10]

Most studies tend to useC muridarum for chemotherapyand vaccine studies which is genetically and antigenicallydistinct from human biovars [11 12] In addition this strainwas reported to respond differently to some antibacterialagents [13]

We report here for the first time the developmentof an upper genital tract infection in progesterone-treatedsusceptible DBA2 mice caused by intravaginal inoculationwith clinically significant C trachomatis serovar D in whichsalpingitis and hydrosalpinx formation were observed Wealso have expanded our work on the antibacterial activityof T3S inhibitors on chlamydial shedding ascending uppergenital tract chlamydial infection and inflammatory sequelaein susceptible DBA2 mice and found that one T3S inhibitorefficiently suppressed both acute and chronic infection andameliorated the upper genital pathology in an animal model

2 Materials and Methods

21 Organisms C trachomatis serovar D DUW-3CX(ATCCVR 885) was grown in cycloheximide-treatedMcCoycells using conventional methods The growth medium wasminimal essential medium supplemented with 10 fetal calfserum 1 nonessential amino acids 2mM L-glutamine 1vitamins and 05 glucose Cell lysates containing approxi-mately 35 times 104 inclusion-forming units (IFU) per mL wereprepared and stored at minus70∘C

22 Animals Female DBA2 mice obtained from AnimalResource Center (Andreevka Moscow region) weighing 14to 16 g were used throughout and were given access to foodand water ad libitum

23 Inoculation The animals subcutaneously received 33mgper kg of the progesterone analog proligestone (CovinanIntervet Schering-Plough Animal Health Netherlands) Fivedays later the mice were infected with 106 CFUmouse ofChlamydia trachomatis serovarD in 40 120583L of PBSThe controlanimals received sterile PBS

24 Drug Treatment Azithromycin was given as a dailysingle oral dose of 10mgkg Treatment was administeredorally for 5 days to mimic the regimen often recommendedfor treatment in humans

4-(3-etoxy-4-hydrobenzil)-5-oxo-56-dehydro-4H-[1 34]-thiadiazol-2-(24-biftorphenyl)-carboxamid designatedas CL-55 was given as an intraperitoneal injection of 50 or100mgkg Inhibitor solutions were prepared by mixing theactive substance with Tween 80 and dissolving it in 1 starchin PBS The control infected mice received a solution of 1

starch in PBS with Tween 80 The early effects of therapywere assessed by measuring vaginal shedding of chlamydiaestarting at day three postinfection The long-term therapyoutcome was assessed using Real Time-PCR to measurechlamydial DNA in the ovaries and uteri 4 weeks afterinfection Genital tissues from the infected mice bothtreated and untreated were processed for histopathologicalexamination at days 28 to 30 after infection

25 Isolation of Chlamydia The canal and exocervix werevigorously scraped and swabbed material was transferredinto 2-sucrose phosphate transport medium and frozenimmediately at minus70∘C All specimens were processed on thesame day Thawed undiluted samples were plated in 05mLvolumes onto McCoy cells on coverslips (12mm diameter) in24-well plates and the infection was enhanced by centrifuga-tion Coverslips were incubated with FITC-conjugated mon-oclonal antibodies against chlamydial lipopolysaccharide(Nearmedic Plus Russia) for 48 h Inclusion-containing cellswere examined and photographed with a Nikon Eclipse 50ifluorescent microscope at 1350x magnification The resultswere expressed as lg IFU per milliliter of sample

26 Real Time PCR Samples for chlamydial DNA detec-tion were collected in phosphate-buffered saline (PBS) andfrozen at minus20∘C DNA was extracted from excised genitaltissues with an automated nucleic acid extractor NucliSENSeasyMAG (bioMerieux Netherland) Chlamydial DNA wasamplified with a C trachomatis PB screen kit (SyntolMoscowRussia) andReal TimePCRcycler CFX96 (Bio-RadUSA)

27 Histopathology Uteri and ovaries were frozen in theregimen of minus60∘C to minus20∘C temperature gradient in theelectronic Cryotome Shandon (USA) and serial 8120583m-thicksections weremade Sections were stained with haematoxylinand eosin (HampE) and examined without knowledge of theexperimental group to which they belonged

28 Statistical Analysis All datawerepresented as themean plusmnstandard deviation (SD) All statistics were performed usingGraphPad Prism version 600 (GraphPad Software Inc Cal-ifornia USA) Significance was set at 119875 lt 005 for alltests

3 Results

31 DBA2 Mice Intravaginally Infected with C trachomatisSerovar D Have Both Acute Chlamydia Shedding and UpperTract Infection and Pathology As existing C trachomatisinfection models in mice do not correspond to a humancourse of pathological sequelae we developed a new murineC trachomatis infection model For this we utilized theDBA2 strain of mice that have a range of genetic defects thatmake themmore susceptible to some bacterial infections andreproductive pathology [14 15]

In these studies progesterone-treated DBA2 micereceived 106 IFU of C trachomatis serovar D intravaginally


Days postinfection

IFU

per

swab

30000

20000

10000

0

3 6 9 13 17 22 25

(a)


(b)


4

1

23

(a) (b) (c)







1

10

100

1000

10000

100000

Ovaries Uteri

Chla

myd

ia D

NA

copi

es










4 Discussion




Uv

agin

al sw

ab

Day 3

10000

8000

6000

4000

2000

0

(a)

IFU

vag

inal

swab

Day 7

40000

30000

20000

10000

0

(b)

IFU

vag

inal

swab

Day 11

8000

6000

4000

2000

0

(c)

IFU

vag

inal

swab

Day 14

10000

8000

6000

4000

2000

0

(d)


1

10

100

1000

10000

Diluent

Chla

myd

ia D

NA

copi

es p

er u

teru

s






(a)

12

(b)



5 Conclusions




References


























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Days postinfection

IFU

per

swab

30000

20000

10000

0

3 6 9 13 17 22 25

(a)


(b)


4

1

23

(a) (b) (c)







1

10

100

1000

10000

100000

Ovaries Uteri

Chla

myd

ia D

NA

copi

es










4 Discussion




Uv

agin

al sw

ab

Day 3

10000

8000

6000

4000

2000

0

(a)

IFU

vag

inal

swab

Day 7

40000

30000

20000

10000

0

(b)

IFU

vag

inal

swab

Day 11

8000

6000

4000

2000

0

(c)

IFU

vag

inal

swab

Day 14

10000

8000

6000

4000

2000

0

(d)


1

10

100

1000

10000

Diluent

Chla

myd

ia D

NA

copi

es p

er u

teru

s






(a)

12

(b)



5 Conclusions




References


























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


1

10

100

1000

10000

100000

Ovaries Uteri

Chla

myd

ia D

NA

copi

es










4 Discussion




Uv

agin

al sw

ab

Day 3

10000

8000

6000

4000

2000

0

(a)

IFU

vag

inal

swab

Day 7

40000

30000

20000

10000

0

(b)

IFU

vag

inal

swab

Day 11

8000

6000

4000

2000

0

(c)

IFU

vag

inal

swab

Day 14

10000

8000

6000

4000

2000

0

(d)


1

10

100

1000

10000

Diluent

Chla

myd

ia D

NA

copi

es p

er u

teru

s






(a)

12

(b)



5 Conclusions




References


























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Uv

agin

al sw

ab

Day 3

10000

8000

6000

4000

2000

0

(a)

IFU

vag

inal

swab

Day 7

40000

30000

20000

10000

0

(b)

IFU

vag

inal

swab

Day 11

8000

6000

4000

2000

0

(c)

IFU

vag

inal

swab

Day 14

10000

8000

6000

4000

2000

0

(d)


1

10

100

1000

10000

Diluent

Chla

myd

ia D

NA

copi

es p

er u

teru

s






(a)

12

(b)



5 Conclusions




References


























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a)

12

(b)



5 Conclusions




References


























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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