Inhibition of host cell cytokinesis by Chlamydiatrachomatis infection

Whitney Greene, Guangming Zhong*

Department of Microbiology, University of Texas Health Science Center at San Antonio,7703 Floyd Curl Drive, San Antonio, TX 78230, USA

KEYWORDSChlamydial inhibition of

cytokinesis

Summary Objectives. Chlamydia has to replicate in cytoplasmic vacuoles ofeukaryotic cells. To understand how chlamydia interacts with host cells during celldivision, we examined the effect of chlamydial infection on host cell cycle.

Methods. An immunofluorescence microscopic approach was used.Results. Although the number of cells was significantly reduced in chlamydia-

infected cultures, multiple nuclei were detected in the infected cells, indicating thatchlamydia inhibited host cell growth by selectively blocking cytokinesis while allowingmitosis to proceed. The chlamydial inhibition of host cell cytokinesis was confirmedwith various strains of Chlamydia trachomatis and in several types of host cells.Furthermore, the chlamydial inhibitory effect was found to depend on chlamydialprotein synthesis.

Discussions. These observations suggest that chlamydia may have evolved specificmechanisms for actively blocking host cell cytokinesis.Q 2003 The British Infection Society. Published by Elsevier Science Ltd. All rightsreserved.

Introduction

Chlamydia is an obligate intracellular bacterialpathogen that has to replicate inside a cytoplasmicvacuole of eukaryotic cells. The species of Chlamy-dia trachomatis, consisting of more than 15different serotypes, causes various human diseases,including trachoma (serovars A–C)1 and sexuallytransmitted diseases (serovars D–K). The severesequelae of urogenital tract infection withC. trachomatis include involuntary infertility andectopic pregnancy.2 Chlamydial persistence inhost cells is thought to be mainly responsible forthe chlamydia-induced diseases.3 –6 More recently,C. trachomatis alone or along with human

papillomavirus (HPV) has been associated with anincreased incidence of cervical cancer,7 – 10

suggesting that chlamydia may be able to alterthe normal process of the delicate host cell cycle.

The effects of chlamydial infection on host cellproliferation were noted several decades ago. Forexample, Horoschak and Moulder reported thatchlamydial infection suppressed host cell prolifer-ation based on the comparison of cell numbersbetween chlamydia-infected and uninfected cul-tures.11 However, Bose and Liebhaber found thatchlamydial infection did not significantly alter thegeneration time of host cells since both theinfected and uninfected cultures showed a similarrate of deoxyribonucleic acid synthesis.12 Toreconcile the apparently conflicting observations,we have carefully evaluated the interactionsbetween chlamydia and host cell cycle. We foundthat although the number of cells in the infectedculture was significantly lower than that in the

0163-4453/03/$ - see front matter Q 2003 The British Infection Society. Published by Elsevier Science Ltd. All rights reserved.doi:10.1016/S0163-4453(03)00039-2

Journal of Infection (2003) 47, 45–51

www.elsevierhealth.com/journals/jinf

*Corresponding author. Tel.: þ1-210-567-1169; Fax: þ1-210-567-0293.

E-mail address: zhongg@uthscsa.edu

Abbreviations: CPAF, chlamydial proteasome-like activityfactor; MOI, multiplicity of infection.

control culture, the total number of host nucleiremained to be similar between the infected anduninfected cultures. A close examination revealedthat most of the infected cells harbor two ormore nuclei per cell. These findings not onlyprovided an explanation for the previous conflictingobservations, but also demonstrated that chlamy-dial infection can selectively inhibit host cellcytokinesis without significantly affecting mitosis.

Materials and methods

Chlamydial organisms

The C. trachomatis strains used for the present studyinclude L2 (434/Bu), C (TW-3), D (UW3), E (UW-5) andK (UW-31), all of which were obtained from Dr C. Kuoat the University of Washington, Seattle. Thechlamydial organisms were grown, purified andtitrated as previously described.13 Aliquots ofchlamydial organisms were stored at 280 8C till use.

Cell culture and chlamydial infection

Various host cell lines were used for the presentstudy, including HeLa (ATCC, Manassas, VA 20108),Hep-2 (ATCC), Huve (kindly provided by Dr DeneeThomas at the University of Texas Health ScienceCenter at San Antonio) and CRL2481 (a primary cellline derived from human aorta wall; ATCC) andRHFL02 (a rat fibroblast cell line, kindly provided byDr Charles Gaunt at the University of Texas HeathScience Center at San Antonio). All cells weremaintained in DMEM with 10% FCS at 37 8C in a CO2

incubator supplied with 5% CO2. For infectionexperiments, cells were grown on glass coverslipsin 24 well plates overnight prior to chlamydialinoculation. Serovar L2 organisms diluted in DMEMwith 10% FCS were directly inoculated onto the cellmonolayers. However, for infection with otherserovars, the cell monolayers were pretreatedwith DEAE and chlamydial stocks were diluted inserum-free DMEM for inoculation. The chlamydialorganisms were allowed to attach to the mono-layers for 2 h at 37 8C. One ml of DMEM with 10% FCSwas then added to each well after the removal ofthe initial inocula. The uninfected cells weresimilarly treated. A multiplicity of infection (MOI)of 5 or as indicated in individual experiments wasused. For antibiotic treatment experiments, anti-biotics were added to the culture at the beginningof the infection and maintained during the infec-tion. The cell samples were cultured at 37 8C in aCO2 incubator and processed at various time points

after infection as indicated in individual experimentsfor microscopic observations as described below.

Immunofluorescence staining

For fluorescence staining, cells grown on coverslipswere fixed with 2% paraformaldehyde dissolved inPBS for 30 min at room temperature, followed bypermeabilization with 0.5% saponin for anadditional 30 min. After washing and blocking, arabbit antiserum recognizing either a chlamydialinclusion membrane protein IncG (kindly providedby Dr Ted Hackstadt at the National Institutes ofHealth)14 or chlamydial organisms (made in our ownlab; unpublished data) were used for stainingchlamydial inclusions. After washing away theunbound first antibodies, a cocktail consisting of agoat anti-rabbit IgG-conjugated with Cy2 (Caltag,Burlingame, CA), Bodipy 558/568 phalloidin (forvisualizing F-actin; Molecular Probes, Eugene,Oregon) and Hoechst dye (blue for DNA, Sigma,St Luis, MO) was applied to the cell samples. As aresult, the cell samples were triply stained forchlamydial inclusions (green), host cell cytoskele-ton F-actin (red) and DNA (blue). The F-actinstaining can clearly distinguish cell borders andthe Hoechst DNA dye staining can allow simult-aneous visualization of both host cell nucleus DNAand chlamydial inclusions. In some experiments,cell samples were stained with the DNA dye only ordually stained with the DNA dye plus an antibodyrecognizing a chlamydia-secreted protease-likeactivity factor (CPAF).15 The anti-CPAF antibodystaining can facilitate the identification of the cellborders of the infected cells.

Fluorescence microscopy

The appropriately stained cell samples were usedfor both image acquisitions and cell/nucleus count-ing with an Olympus AX-70 fluorescence micro-scope. For image acquisition and analysis, theimages of multi-color-stained samples wereacquired single color at a time using a Hamamatsudigital camera and superimposed with the softwareSimplePCI. For cell counting, five random views percoverslip were counted under the appropriateobjective lenses. In each experiment, the numberof cells/nuclei per view was calculated from 10random views of duplicate coverslips.

Statistical analysis

A two-tailed Student’s t-test was used for statisticanalysis of data (http://faculty.vassar.edu/lowry/tu.html).

W. Greene, G. Zhong46

Results

Chlamydial infection inhibits cellproliferation

To evaluate the effect of chlamydial infection onhost cell proliferation, we first compared thenumber of cells in cultures with or withoutchlamydial infection at various time points afterinfection (Fig. 1). Although the number of cells wassimilar between samples with or without chlamydialinfection at the initial and 10 h time point postinfection, the number of uninfected cells increasedas the culture continued while the number ofinfected cells remained at a similar level through-out the culture. By 20 h after infection, theuninfected cells showed obvious growth and therewas a significant difference in cell numbersbetween the uninfected (141 ^ 76) and infected(108 ^ 62) cultures ðp ¼ 0:016Þ: A similar differencebetween the uninfected (150 ^ 91) and infected(103 ^ 59) cultures ðp ¼ 0:013Þ was also noted at30 h after infection. The steady state level of theuninfected HeLa cells at 20 and 30 h may reflect thenormal replication cycle of HeLa cells with aduplication time of 18–24 h. It is worth nothingthat only about 50% HeLa cells in the uninfectedculture completed duplication (from the initial 92cells/field to 150 at 30 h post infection) under thepresent culture conditions. By 40 h after infection,a greater difference in cell numbers was foundbetween samples with (77 ^ 46) or without(210 ^ 114) chlamydial infection ðp ¼ 0:0012Þ: Asexpected, another 50% of the uninfected HeLa cellscompleted their duplication by this time (from 140

at 20 h to 210 at 40 h after infection) while thenumber of infected cells remained unchanged orslightly declined. The above observations togetherhave demonstrated that chlamydial infection cansuppress host cells from proliferation during boththe first and second replication cycles of HeLa cells.

Multiple nuclei are detected inchlamydia-infected cells

To determine which step of the cell cycle wasblocked by chlamydial infection, we carefullycompared the cellular morphology of both normaland infected cell samples (Fig. 2). HeLa cells with orwithout chlamydial infection were triply stained asdescribed in Section 2. Both the host nuclei andchlamydial inclusions were simultaneously visual-ized with the Hoechst DNA dye staining (Fig. 2a, topleft panel). Most chlamydial inclusions appeared tobe surrounded with two or more host nuclei. Anadditional dual staining of F-actin and chlamydialinclusions showed that the adjacent host nucleisurrounding a given chlamydial inclusion belongedto a single cell. It is clear that most of chlamydia-uninfected cells contained two or more nuclei(Fig. 2a, top panels). However, most of uninfectedcells maintained a single nucleus per cell (Fig. 2a,lower panels). All nuclei in either normal orinfected cells appeared to be healthy and therewas no sign of apoptosis, which is consistent withmany previous observations.16 – 19 Formation ofmultiple nuclei in infected cells was also analyzedquantitatively (Fig. 2b). We found that about 17% ofinfected cells become multinucleated at 20 h afterinfection and the number of multinucleated cellscontinued to increase as the culture continued (37%at 30 and 81% at 40 h after infection). However, thecell samples without infection maintained a verylow level of multinucleated cells throughout theexperiment (,2%). To further determine whetherthe chlamydial induction of multiple nuclei is onlyrestricted within the infected cells, we examinedthe HeLa cell culture infected with chlamydia L2 ata lower infection rate in which only a portion ofcells were infected (data not shown; Fig. 2c, leftpanels). It clear that only the cells harboringchlamydial inclusions were multinucleated whilethe adjacent uninfected cells maintained singlenucleus per cell. The above observations togetherhave clearly demonstrated that chlamydia canprevent the infected cells from proliferation byselectively blocking cytokinesis. As a result of thecytokinesis blockade, the infected cells becamemultinucleated.

To test whether inhibition of host cell cytokinesisis a shared property of all C. trachomatis strains, we

Figure 1 Chlamydial inhibition of host cell prolifer-ation. HeLa cells with (solid bar) or without (open bar)C. trachomatis L2 infection at an MOI of 5 were processedand counted as described in Section 2 at various timepoints after injection (as shown along X-axis). The resultswere expressed as number of cells per field under a 40Xobjective lens (X ^ SD; Y-axis). The results shown in thefigure come from 6 to 8 experiments each with duplicatecoverslip samples. A two-tailed Student’s t-test was usedto analyze the statistical difference between experimen-tal groups (p . 0:05 between infected and uninfectedgroups at 0 and 10 h; p , 0:05 at 20 and 30 h and p , 0:01for 40 h).

Chlamydial inhibition of cytokinesis 47

Figure 2 Detection of multiple nuclei in chlamydia-infected cells. (a) HeLa cells with (top row) or without (bottom)C. trachomatis L2 infection at an MOI of 5 for 40 h were stained with Hoechst dye for DNA (blue), a rabbit antiserum forchlamydial IncG protein (green), phalloidin for F-actin (red) as described in Section 2. Note that most cells harboringchlamydial inclusions contain more than one nucleus. (b) At various time points after L2 infection, both infected

W. Greene, G. Zhong48

evaluated the ability of other strains of C. tracho-matis to induce multinucleated cells. HeLa cellsinfected with each of the tested serovarsincluding C, D, E and K uniformally became multi-nucleated (Fig. 2c), which suggests that inhibitionof host cell cytokinesis is a common property ofC. trachomatis species.

To exclude the possibility that C. trachomatisinhibition of host cytokinesis is unique to HeLacells, we evaluated several other cell lines for thechlamydial effect (data not shown). C. trachomatisinfection inhibited cytokinesis of all tested hostcells, including Hep2, Huve and CRL-2481.

Chlamydial inhibition of host cell cytokinesisis dependent on chlamydial protein synthesis

To test whether chlamydial protein synthesis isrequired for chlamydial inhibition of host cellcytokinesis, we first evaluated the impact ofinfection dose on the chlamydial inhibitory effectusing the formation of multinucleated cells as aread-out (Fig. 3A). We found that as chlamydialinfection dose increased, a higher proportion ofcells became multinucleated. When the MOI wasincreased to 5, the number of cells with multiplenuclei reached a plateau. This is because at an MOIof 5, .95% cells were infected and the number ofinfected cells already reached a plateau. Thecorrelation between infection doses and the num-ber of multinucleated cells demonstrated thatchlamydial inhibition of host cell cytokinesisrequired chlamydial growth.

We further used antibiotics that can selectivelyblock either prokaryotic or eukaryotic proteinsynthesis to determine whether newly synthesizedchlamydial proteins are required for the chlamydialinhibition of cytokinesis (Fig. 3B). Treatment withchloramphenicol at a concentration of 60 mg/mlcompletely prevented the chlamydia-inducedmultinucleated cells while such treatment did notalter the ability of normal HeLa cells to proliferate.Since chloramphenicol is able to inhibit chlamydialprotein synthesis without affecting host proteinbiosynthesis, we can conclude that chlamydialprotein synthesis is required for the inhibition ofhost cell cytokinesis. Furthermore, treatment ofthe cultures with cycloheximide, an antibiotic that

(solid bar) and normal HeLa cells (open bar) with single nucleus or multiple nuclei were counted separately under a 60Xobjective lens as described in Section 2. The results were expressed as percentage of cells with multiple nuclei (X ^ SD;Y-axis). The results shown in the figure come from 4 to 5 experiments each with duplicate samples. (c) Induction ofmultiple nuclei by various C. trachomatis serovars (L2, C, D, E and K) as indicated at the top of each panel. The cellsamples were infected with chlamydia and dually stained as described in Section 2. CPAF is a chlamydial protein that issecreted into host cell cytosol (red). Note that multiple nuclei were only found in cells harboring chlamydial inclusions.

Figure 3 Chlamydial protein synthesis is required forthe inhibition of host cell cytokinesis. (A) Correlationbetween chlamydial infection doses and host cell multi-nucleation. HeLa cells were infected with L2 at variousMOI as shown along the X-axis and, 40 h after infection,the cells were stained as described in Section 2. Theresult was expressed as percentage of cells carryingmultiple nuclei counted under a 60X lens (Y-axis). Thedata in the figure represents the mean values from twoindependent experiments each with duplicate coverslips.The variation between two experiments is about 20%.Note that the percentage of multinucleated cellsincreased in proportion to the infection doses. (B) Theeffect of antibiotics on chlamydia-induced multinuclea-tion. HeLa cells were infected with L2 at an MOI of 5.Either chloramphenicol (CLF; at a final concentration of60 mg/ml) or cycloheximide (CHX; 2 mg/ml) was added tothe culture at the beginning of and throughout theculture. Normal HeLa cells were similarly treated ascontrols. The percentage of multinucleated cells fromboth infected (solid bar) and normal (open bar) HeLa cellswas calculated from 3 to 5 independent experiments eachwith duplicate coverslips (counted under a 60 X lens) anddisplayed along the Y-axis (X ^ SD). Note that chloram-phenicol completely prevented while cycloheximidesignificantly reduced the chlamydia-induced multi-nucleation of host cells.

Chlamydial inhibition of cytokinesis 49

selectively inhibits host protein synthesis and isconventionally used in chlamydial cultures, signifi-cantly reduced the number of multinucleated cellsinduced by chlamydia. This observation not onlyprovides an explanation for the failure to detectthe chlamydia-induced host cell multinucleationin our conventional infection experiments, butalso more importantly demonstrates that thechlamydia-induced multinucleation is an activecellular process that requires host cell biosynthesis.

Discussion

We have demonstrated that the C. trachomatis-induced inhibition of host cell proliferation is due tothe chlamydial blockade of cytokinesis. As a result,multiple nuclei are formed in the infected cells. It isunlikely that the inhibition of host cell proliferationis a result of either cell fusion or cell death in theinfected cultures. First, we did not note any cellfusion intermediates or cell apoptosis at any timepoints after infection that we have monitored(Fig. 2a and c). Second, active mitosis events canbe found in chlamydia-infected cells (Fig. 2a, topleft panel), suggesting that the multiple nuclei in aninfected cell are the result of nuclear division.Third, it has been previously demonstrated thatchlamydia-infected cells can undergo DNA synthesisat a similar rate as the uninfected cells do,12

suggesting that the infected cells still maintain theability to synthesize DNA for mitosis. Fourth,chlamydia-infected cells, even at a very late stageof infection, can synthesize and secrete variouscytokines,20 suggesting that chlamydial infectiondoes not affect host cell de novo biosynthesispathways that are required for mitosis. We haveindeed found that the chlamydia-induced multi-nucleation required host cell protein synthesissince cycloheximide treatment significantly inhib-ited the multinucleation (Fig. 3B). Finally, we havepreviously shown that cells infected withC. trachomatis not only do not undergo apoptosisbut also are resistant to apoptosis induction byexternal stimuli,16 which is consistent with thecurrent observation that no apoptosis was found inthe infected cells (Fig. 2a and c). One caution isthat cell lysis does occur at the late stage of L2infection. The slight decline in the number of cellsin the infected culture at 40 h post infection (Fig. 1)may be due to lysis of overly infected cells. SinceHeLa cells were infected at an MOI of 5 for mostexperiments (to ensure that .95% cells areinfected), a small portion of cells was inevitablyinfected with many organisms. Although most of

the infected cells can live up to 72–96 h under thecurrent infection conditions, the portion of cellsinfected with more organisms may be lysed as earlyas 40 h post infection. However, the slight declinein cell number at 40 h as a result of cell lysis did notcontribute to the statistical difference between theinfected and uninfected samples. This is becausethere is a significant statistical difference in cellnumbers between the uninfected sample at 40 hand the infected samples at either the initial or 10 htime points post infection when there is no celllysis. More importantly, there is no significantstatistical difference in cell numbers between anyof the infected samples, suggesting that theinfected cell samples maintained a relativelysteady level of cell numbers throughout the cultureperiod. Therefore, we can conclude that formationof multiple nuclei in the infected cells is mostlikely due to the selective inhibition of host cellcytokinesis by chlamydia.

It is known that blockade of mammalian cellcytokinesis can lead to either apoptosis or multi-nucleation.21 Since we and others have previouslydemonstrated that chlamydia can actively inhibithost cell apoptosis,16 –19 inhibition of cytokinesis ofthe chlamydia-infected cells can then only lead tothe formation of multiple nuclei without apoptosis.However, we do not yet know how chlamydiaselectively inhibits cytokinesis while allowing mito-sis to proceed. Although alteration of many cellularprocesses and components such as syntaxin 1, Ral,Rho, Rab and myosin II may lead to the disruption ofcytokinesis, we hypothesize that chlamydia mayhave evolved specific mechanisms for the selectiveinhibition of cytokinesis. It has been shown thatmicroinjection of Botulinum neurotoxin C1 can leadto proteolysis of syntaxin and cause blockage ofcytokinesis in sea urchin embryos,22 suggesting thatmicrobes can alter host cell cycle by secretingtoxins to selectively modify components involved inhost cell cytokinesis. Chlamydia-infected cells arecapable of performing many normal cellular pro-cesses, including cytokine secretion23,24 and DNAreplication.12 It appears that only the cellularfunctions that are critical for controlling chlamydialsurvival are selectively disrupted by chlamydia viaspecific mechanisms.15,16,25,26 Interestingly, boththe chlamydial antiapoptotic activity16 and inhi-bition of cytokinesis (Fig. 3 of current study) aredependent on chlamydial protein synthesis,suggesting that these two chlamydial propertiesmay be actively acquired by chlamydia throughevolution. It has been shown that Bartonella-associated endothelial proliferation depends oninhibition of apoptosis.27 However, it is not clearat this moment whether the chlamydial alterations

W. Greene, G. Zhong50

of host cell cycle and apoptosis are also directlyrelated. Efforts are under way to address therelationship between chlamydial inhibition ofcytokinesis and antiapoptotic activity and tofurther understand the mechanisms of chlamydialinhibition of host cell cytokinesis.

Acknowledgements

This work was supported in part by grants (toG. Zhong) from the National Institutes of Health(R01 HL64883 & R01 AI47997).

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