FEMS Immunology and Medical Microbiology 42 (2004) 291–297

www.fems-microbiology.org

A growth study of Coxiella burnetii Nine Mile Phase Iand Phase II in fibroblasts

J.D. Miller a,b, A.T. Curns c, H.A. Thompson a,b,*

a Q Fever Unit, Viral and Rickettsial Zoonoses Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases,

Centers for Disease Control and Prevention, 1600 Clifton Road, NE, Mailstop G-13, Atlanta, GA 30333, USAb Department of Microbiology, Immunology, and Cell Biology, West Virginia University, Morgantown, WV 26506, USA

c Office for the Director, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases,

Centers for Disease Control and Prevention, Atlanta, GA 30033, USA

Received 17 February 2004; received in revised form 20 May 2004; accepted 2 June 2004

First published online 17 June 2004

Abstract

Coxiella burnetii, a slow-growing, gram-negative, obligate intracellular bacterium, is the causative agent of Q fever in humans.

The avirulent Phase II C. burnetii Nine Mile strain can invade and establish persistent infections in a wide variety of laboratory cell

lines, and is generally considered to be easier to grow in culture than the wild-type Phase I organism. Efforts to improve Phase I

organism yield in the BHK-21 cell line demonstrated that high CO2 conditions and the use of Dulbecco’s modified Eagle’s medium

(DMEM) with 4.5 g/l glucose supplementation resulted in higher organism yields. Phase II organisms grown in the same cell line and

conditions showed lower growth rates. Analysis revealed that increased average numbers of C. burnetii Phase I organisms within

fibroblasts was due to higher growth rates within the hosts rather than to increased uptake or to increased cell-to-cell spreading.

Addition of the nucleoside cytidine to the growth medium stimulated growth of Phase II but not Phase I organisms.

� 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Coxiella burnetii; Q fever; BHK-21 fibroblasts; Cytidine

1. Introduction

Coxiella burnetii is an obligate intracellular parasite,

that grows slowly within acidified phagosomes, with a

generation time of 12–20 h [1]. The organism can infect a

broad range of animals, including humans, and is the

causative agent of Q fever. Q fever usually is an acute

disease, but in rare cases, persistent infection can result

in more significant illnesses, such as chronic Q fever

endocarditis [2]. Post-Q fever fatigue syndrome has alsobeen reported [2,3]. The C. burnetii Nine Mile wild-type

(Phase I clone 7) strain can undergo phase transition [4],

which results in a severe truncation of the lipopolysac-

charide (LPS) molecules and a subsequent loss of viru-

* Corresponding author. Tel.: +1-404-639-1083; fax: +1-404-639-

1056.

E-mail address: hct2@cdc.gov (H.A. Thompson).

0928-8244/$22.00 � 2004 Federation of European Microbiological Societies

doi:10.1016/j.femsim.2004.06.003

lence [5]. The resulting avirulent Phase II (clone 4)

organism has a 26-kb pair DNA deletion that spans oneLPS biosynthetic region of the chromosome [6,7]. It is

not known whether this deletion explains all of the

phenotypic differences between Phase I and II organisms

[7,8].

C. burnetii organisms infect a wide variety of host

cells in vitro, including human monocytes (THP-1 cells),

rodent fibroblasts (L-929 and BHK-21 cells), Vero cells,

chick endodermal (primary culture) cells, Chinesehamster ovary cells, and several mouse macrophage cell

lines [2,9–11]. In most cell lines that have been used for

study, Phase II organisms invade host cells faster than

Phase I organisms and are more successful at estab-

lishing persistent infections [11]. In earlier work, C3 was

found to bind to Phase II but not Phase I organisms [5].

In more recent work, Phase II uptake was found to be

mediated by leukocyte response integrin, avb3 (LRI),

. Published by Elsevier B.V. All rights reserved.

292 J.D. Miller et al. / FEMS Immunology and Medical Microbiology 42 (2004) 291–297

and CR3 [5,12]. Phase I organisms bind to and invade

host cells by using the avb3 (LRI) and integrin-associ-

ated protein complex, while not engaging CR3 [5,12,13].

During attachment and invasion, Phase I organisms

induce activation of protein tyrosine kinases and tyro-sine phosphorylation of several substrates thought to

interfere with cytoskeleton organization, causing the

host cell to change morphology [14,15]. Differences in

the invasion process may explain why, under conven-

tional culture conditions, Phase II organisms establish

infection in host cells faster than do Phase I organisms.

However, in embryonated eggs, the growth yield of Nine

Mile Phase I organisms is consistently better than thatfor Phase II organisms [16,17].

Once C. burnetii organisms have entered the host cell

and are established in an acidified phagosome, they are

presumably positioned to take advantage of the concen-

trations of nucleosides, amino acids, and other metabolic

precursor molecules available within that subcellular

niche [18]. Past researchers in our laboratory have used

amino acid and nucleoside supplementation to improvemetabolic activity in vitro [10,19]. They also used higher

concentrations of CO2 and glucose to increase yields ofC.

burnetii Phase I organisms grown in Baby hamster kidney

(BHK-21) cells [10]. We have previously determined that

nucleosides but not nucleotides are transported by C.

burnetii organisms and incorporated into macromole-

cules [16]. In the present work, we sought to improve the

growth of Phase II organisms in BHK-21 cells by usingnucleoside supplements. We used cell staining and direct

enumeration techniques to determine the number of

bacteria within individual BHK-21 cells. For the first

time, we demonstrate the distribution ofC. burnetii Phase

I and Phase II organisms early in the infection process.

Inclusion of cytidine in the host cell growth medium im-

proved the net number of Phase II organisms 3-fold over

untreated or cytosine supplemented cultures. Cytidine didnot affect Phase I growth.

Concomitant with the transition from Nine Mile

Phase I to Phase II, several genes encoding nucleotide

sugar synthesis within LPS pathways are deleted from

the chromosome [7]. In the present study, we likewise

suspected that previously undetected deleted or point-

mutated gene(s) might explain the cytidine deficiency in

Phase II. In bacteria, cytidine auxotrophy is often ex-plained by dysfunction in CTP synthase. Thus, we also

examined the CTP synthase gene (pyrG) and enzyme

function in Phase II organisms.

2. Materials and methods

2.1. C. burnetii growth in BHK-21 cell culture

All procedures that involved use of viable C. burnetii

were performed under biosafety level 3 conditions as

recommended in Biosafety in Microbiological and Bio-

medical Laboratories [20]. BHK-21 cells (ATCC No.

CCL-10, American Type Culture Collection, Manassas,

VA) were cultured in complete growth medium (Dul-

becco’s modified Eagle’s medium (DMEM; withoutsodium pyruvate), containing 4.5 g/l glucose (Life

Technologies, Rockville, MD) and 5% fetal bovine se-

rum (FBS) (Life Technologies)) in 25-cm2 tissue culture

flasks (Corning, Corning, NY). During all cell culturing

procedures, the flasks were incubated at 37 �C in the

presence of 12% CO2 [10,19] until the cells reached 100%

confluence within the flask (approximately 5 days). On

the day of infection, 1.1 · 106 BHK-21 cells were placedinto each of two 25-cm2 flasks (paired experiments A

and B) and allowed to attach to their surfaces for 12

hours. The monolayers were infected for 12 h in in-

complete growth medium (i.e., without 5% FBS) con-

taining either 1.2� 106 C. burnetii Nine Mile Phase I

(clone 7) or Phase II (clone 4) organisms. Particle counts

were established by the use of a variation of the Gime-

nez staining technique described previously [16]. Theinocula were organisms from 50% (w/v) yolk sac seed

stock [16] diluted in phosphate-buffered saline (pH 7.2)

to achieve a multiplicity of infection (MOI) of one or-

ganism per host cell. After inoculation, the incomplete

medium was replaced with complete growth medium.

Three days after infection, the cells were treated with 1

ml of trypsin (1 mg/ml), and diluted 1/20 with complete

growth medium and then uniform samples of infectedcells were placed into six 25-cm2 tissue culture flasks

(Corning). For each infection, duplicate tissue culture

flasks were either left untreated, supplemented with 1

mM cytosine, or supplemented with 1 mM cytidine.

Throughout this report, the day that supplements were

added to the complete growth medium is referred to as

day 0. Two tissue culture flasks containing uninfected

BHK-21 cells were maintained as negative controls.

2.2. Cell counting

Samples were taken at 5-day intervals from each flask

throughout the 25-day experiment. Complete growth

medium was exchanged every third day after sampling.Infected cultures were split every 5 days, at a 1/20 di-

lution. To prepare samples, 50 ll of trypsin-treated cells

in medium from Phase I- and Phase II-infected cells was

placed onto duplicate microscope slides and uniformly

spread, by dragging a clean slide across the sample, to

obtain a thin film. The smears were air-dried, heat-fixed,

and immersed in acetone for 15 min at room tempera-

ture. The slides were stained with the Gimenez technique[21]. Prepared microscope slides were stored in the dark

at room temperature until examined. C. burnetii Phase I

and Phase II organism counts were determined by

scanning the slide for fields of diffuse, stained host cells

on a Nikon Eclipse E800 microscope (Nikon USA,

J.D. Miller et al. / FEMS Immunology and Medical Microbiology 42 (2004) 291–297 293

Melville, NY). Stained cells were centered in a field

under 1000� magnification, and the red-stained organ-

isms within the blue-green host cell cytoplasm were

enumerated with the aid of a digital counter. Cells were

rejected from the count only when the BHK-21 cellswere unstained or poorly stained. Dense concentrations

of organisms that could not be accurately counted were

recorded as ‘‘too numerous to count’’ (TNTC). Eighty

BHK-21 fibroblasts were counted per treatment condi-

tion (i.e., untreated, cytosine supplemented, or cytidine

supplemented) at each time point to obtain a represen-

tative sampling of host cells. Data from paired experi-

ments (A and B) of both Phase I and Phase II organismswere compiled and analyzed by the number of C. bur-

netii organisms (Phase I or Phase II) per host cell per

growth condition (untreated or cytosine or cytidine

supplement) (Fig. 1). Three-dimensional bar graphs

were used to organize and depict the number of host

cells infected, the total number of organisms, and the

number of Phase I and Phase II organisms in each cell

when cultured in the specified growth conditions overtime (days 0–25) (Figs. 2–4). Data were graphed using

Sigma Plot 2001 (SPSS, Inc., Chicago, IL).

2.3. Statistical analyses

Growth differences in C. burnetii by experiment (A or

B) at each time period (days 0, 5, 10, 15, 20, 25) were

assessed by analysis of variance [22]. Since multiplecomparisons by experiment and treatment type were

conducted for each time period, p-values were adjusted

using the Tukey–Kramer method to be more conserva-

tive in rejecting the null hypothesis of no significant

Time (Days)0 5 10 15 20 25

# C

oxie

lla/

Hos

t Cel

l

0

5

10

15

20

25

30

35Exp A NoneExp B NoneExp A CytosineExp B CytosineExp A CytidineExp B Cytidine

*

(a) (

Fig. 1. Growth curves of C. burnetii variants in BHK-21 host cells. C. burneti

in BHK-21 cells in tissue culture medium left untreated (circles), in the presenc

A data are represented by closed symbols, while Experiment B data are rep

signifies the number of C. burnetii organisms in the acidified phagosomes of B

per host cell in 80 randomly counted BHK-21 cells. Error bars represent stand

dense to count accurately.

differences in growth density (organisms per cell) of C.

burnetii by the presence or absence of supplement [23].

Differences in mean values by group that resulted in p-values <0.05 were considered statistically significant.

Multiple regression analysis was used to evaluate trendsin growth density over time by experiment and supple-

ment [22]. Time-squared terms were evaluated for im-

proved model fit as were interaction terms between

supplement type and time. All statistical analyses were

performed using SAS software (Version 8.2; SAS Insti-

tute, Cary, NC).

3. Results and discussion

C. burnetii Nine Mile strain Phase I and Phase II

organisms were introduced to BHK-21 fibroblasts at a

low MOI (from 0.9� 0.1 to 1.0� 0.1 organisms per cell)

to examine the initial stages of infection. The growth

increases measured are net increases in the population of

organisms within an expanding BHK-21 population.During the 25-day period examined (Fig. 1), Phase I

organisms with or without nucleoside supplement dis-

played increases in growth until the numbers of organ-

isms within the acidified phagosomes of the BHK-21

cells became too dense to count accurately (signified by

an � on day 25, Fig. 1(a)). Phase I organism counts were

statistically similar for both experiments throughout the

25-day experiment, with two exceptions (day 5 experi-ment A, cytidine¼ 4.3� 0.3 organisms per cell, p¼ 0.02;

day 15 experiment B, cytidine¼ 14.9� 1.2 organisms per

cell, p¼ 0.0026). For all other days, there were no sta-

tistically significant differences in Phase I growth density

Time (Days)0 5 10 15 20 25

# C

oxie

lla/

Hos

t Cel

l

0

5

10

15

20

25

30

35Exp A NoneExp B NoneExp A CytosineExp B CytosineExp A CytidineExp B Cytidine

b)

i Nine Mile strain Phase I (1a) and Phase II (1b) organisms were grown

e of 1 mM cytosine (triangles), or 1 mM cytidine (squares). Experiment

resented by open symbols. The x-axis represents days, and the y-axisHK-21 cells. Each point represents the mean number of bacteria cells

ard error. � signifies that the number of C. burnetii organisms were too

Fig. 2. C. burnetiiNine Mile strain untreated Phase I (a) and Phase II (b) distribution in BHK-21 cells. Representative samples of the data are shown.

The x-axis signifies the number of C. burnetii organisms counted in each randomly selected host cell. The y-axis represents the number of BHK-21

cells in each bar. The z-axis represents the days on which organisms were stained and enumerated in host cells (20 days for Phase I organisms and 25

days for Phase II organisms). Graphs are shown as an alternating black and white pattern to make the data bars easier to distinguish.

Fig. 3. C. burnetii Nine Mile strain Phase I (a) and Phase II (b) distribution in BHK-21 cells treated with 1 mM cytosine. Representative samples of

the data are shown. The x-axis signifies the number of C. burnetii organisms counted in each randomly selected host cell. The y-axis represents the

number of BHK-21 cells in each bar. The z-axis represents the days on which organisms were stained and enumerated in host cells (20 days for Phase

I organisms and 25 days for Phase II organisms). Graphs are shown as an alternating black and white pattern to make the data bars easier to

distinguish.

294 J.D. Miller et al. / FEMS Immunology and Medical Microbiology 42 (2004) 291–297

by experiment, indicating that cytidine had either no

effect or a minor effect on Phase I growth in BHK-21

cells.

When the net growth rate of Phase II organisms inuntreated and supplemented culture conditions was ex-

amined, it was found that Phase II cultures did not ex-

hibit significantly different growth until day 10 (Fig.

1(b)). Thus, under conditions used here, we obtained no

evidence for more rapid uptake of Phase II organisms.

From day 10 until day 25, the cytidine-supplemented

cultures displayed significantly higher growth densities

than the untreated and cytosine-supplemented cultures

at every time point (p6 0:001). Phase II organisms in-

fecting BHK-21 cultures supplemented with cytidinegrew to between 9 and 11 organisms per cell on day 20,

which was the peak of Phase II growth in the presence of

cytidine. This was a higher concentration of organisms

per cell than observed in untreated or cytosine-supple-

mented conditions (from 3.3� 0.2 to 4.4� 0.3 organisms

per BHK-21 cell). Phase II experiments A and B with

Fig. 4. C. burnetii Nine Mile strain Phase I (a) and Phase II (b) distribution in BHK-21 cells treated with 1 mM cytidine. Representative samples of

the data are shown. The x-axis signifies the number of C. burnetii organisms counted in each randomly-selected host cell. The y-axis represents thenumber of BHK-21 cells in each bar. The z-axis represents the days on which organisms were stained and enumerated in host cells (20 days for Phase

I organisms and 25 days for Phase II organisms). Graphs are shown as an alternating black and white pattern to make the data bars easier to

distinguish.

J.D. Miller et al. / FEMS Immunology and Medical Microbiology 42 (2004) 291–297 295

cytidine supplement were not significantly different fromone another over time. We used multiple regression

analysis to compare the growth curves of Phase II or-

ganisms in the untreated or supplemented culture con-

ditions: over the 25-day time period, organisms grown

with cytidine supplementation replicated to a signifi-

cantly higher density than those grown with 1 mM cy-

tosine or without supplementation (p < 0:0001).To better understand the effect phase variation has on

the growth of C. burnetii Phase I and Phase II organ-

isms, we examined the net increase of Phase I or Phase II

organisms in each host cell and compared the numbers

of infected host cells by the bacterial load in each cell to

search for growth trends in the experiments. The growth

data of Phase I organisms were statistically similar when

results of the paired experiments were compared. The

Phase II growth data were also statistically similar in thepaired experiments. Data from a Phase I and Phase II

experiment were used as representative samples in Figs.

2–4. It was found that on day 0 both Phase I and Phase

II organisms had infected similar numbers of randomly

selected BHK-21 cells (50% of BHK-21 cells infected

with Phase I vs. 52.5% infected with Phase II). On day 5

of the Phase I infections, 60–70% of fibroblasts were

infected (all variables), and by day 10, all fibroblastscontained at least some coxiellae (data not shown). In

Phase II infections, the corresponding infection rates

were 65–78% (day 5) and 95% on day 10 (data not

shown). Phase I organisms, regardless of supplement

condition, increased in total bacterial numbers and

bacterial load within the phagosomes of the host cells

over time (Figs. 2(a), 3(a), and 4(a)). All Phase I con-

ditions (untreated or supplemented) in both experimentsresulted in high total bacterial numbers in observed

BHK-21 cells (n ¼ 80) by day 20 (Fig. 2(a): untreated¼2212� 72 bacteria; Fig. 3(a): 1 mM cytosine¼ 2077� 55

bacteria; Fig. 4(a): 1 mM cytidine¼ 2278� 36 bacteria).

Each Phase I growth condition (untreated or supple-

mented) on day 20 also resulted in >12% of the BHK-21

cell population displaying heavy infections (>40 bacte-

ria per cell, Figs. 2(a), 3(a), and 4(a)). Thus the shift inPhase I cultures from lower to higher numbers of in-

tracellular coxiellae between day 15 and 20 was partic-

ularly noticeable, as was the striking appearance of a

subpopulation of fibroblasts containing >40 organisms

(i.e., TNTC) (Figs. 2(a), 3(a), and 4(a) – notice popu-

lation bar patterns in 20-day columns). Untreated and

cytosine-supplemented Phase II cultures showed poor

growth and low total numbers of bacteria in BHK-21cells compared with those for Phase I cultures (Fig. 2(b):

untreated day 25¼ 227� 21 bacteria; Fig. 3(b): 1 mM

cytosine day 20¼ 192� 6 bacteria). Throughout the

experiment most untreated or cytosine-supplemented

BHK-21 cells were infected with 2 to 4 Phase II organ-

isms/cell, and no BHK-21 cell was observed to contain

>15 Phase II organisms (Figs. 2(b) and 3(b)). When 1

mM cytidine was added to growth medium, the Phase IIorganisms displayed a 3-fold increase in total numbers

of bacteria (Fig. 4(b): day 25¼ 687� 29 Phase II or-

ganisms). A comparison of data in Figs. 2(b) and 4(b)

clearly demonstrates the nature of the growth rate shift

caused by cytidine in Phase II cultures. The average

infection in each BHK-21 cell shifted from 2-4 Phase II

organisms per cell to 6–10 Phase II organisms per cell

296 J.D. Miller et al. / FEMS Immunology and Medical Microbiology 42 (2004) 291–297

(Fig. 4(b)). Furthermore, the cultures treated with 1 mM

cytidine had more BHK-21 cells infected with P 15

Phase II organisms (Fig. 4(b)). This effect was not due to

greater cell-to-cell spreading or to a higher rate of initial

infection: it was clearly a reflection of the intracellularreplication rate of Phase II organisms. Of interest, this

growth effect requires several days of culturing with

cytidine before becoming evident.

Itwas known frompreviouswork thatNineMile Phase

II is a deletionmutant [7,8].Assuming that cytidinewithin

the supplemented media would contribute to an increase

in host cell nucleoside pools, and thereby be available to

the intracellular coxiellae, we reasoned that the resultsmay indicate a genetic deficiency in the cytidine pathway

of the Phase II strain. A critical step in de novo synthesis

of cytidine is the conversion, in nucleotide form, from

UTP to CTP by CTP synthase. Cloning and character-

ization of theCTP synthase gene (pyrG) revealed however

that it is present without sequence defects in Phase II or-

ganisms [24]. Enzyme function experiments conducted

with dialyzed, crude extracts from freshly prepared,highly purified Phase I and II organisms grown in yolk

sacs revealed that both extracts possessed at least some

CTP synthase activity [24]. Since CTP synthase is a ho-

motetrameric enzyme in active form and is allosterically

regulated, a regulatory explanation also had to be con-

sidered. Thus we used a recombinant C. burnetii CTP

synthase expressed in and purified from Escherichia coli

[24] in attempts to rule out the presence of any small orlarge molecule inhibitors in Phase II cells. This was ac-

complished by the addition of Phase I and II extracts to

purified, hexahistidine-tagged enzyme; such experiments

showed no inhibitory effects (data not shown).

Thus, we have no concrete explanation for the stim-

ulatory effect of cytidine in these culture experiments.

Because the addition of cytidine to Phase I-infected cells

does not result in an effect similar to that seen in PhaseII cultures, it seems doubtful that this is solely a host

response that is resulting in a different signal to Phase II

compared with Phase I organisms. It may instead be a

difference in response by the different phase organisms

to a host signal or condition.

In conclusion, we have described here a method of

achieving differential growth that can distinguish between

C. burnetii Phase I and II organisms. This method is morespecific than established PCR methods [25] in that it can

differentiate between a large number of host cells infected

with a very few bacteria or a few host cells infected with a

large number of bacteria. As methods of genetic manip-

ulation of coxiellae develop, the application of this tool in

genetic complementation experiments may make it pos-

sible to predict the success of gene recombinants in re-

storing virulence to Phase II organisms. This system couldbe employed prior to testing such recombinants in more

expensive and difficult animal virulence models. At the

very least, this will prove to be an interesting way to

characterize new isolates as well as some poorly charac-

terized strains of lower virulence. Presently, we do not

completely understand why, under these conditions,

Phase I organisms grow preferentially well, to the exclu-

sion of Phase II. However, it is likely that the increasedCO2 levels (12%) and the very high glucose concentration

(4.5 g/l) used in the culture medium causes rapid acidifi-

cation of host cell cytoplasms, as has been described be-

fore in animal cell physiology studies [26,27]. This could

certainly affect the ease of parasitophorous vacuole

acidification, whichmay be beneficial to Phase I organism

growth. The organism is a moderate acidophile [9]. The

mechanism underlying the differential effect that thesegrowth conditions have upon Phase I and Phase II cul-

tures remains to be satisfactorily explained. In this regard,

it is pertinent to understand that the lysosomal pathway

taken by the Phase I organism likely differs from that of

Phase II. As discussed, this is believed to be a consequence

of their different cell entry mechanisms [12,13]. The sub-

sequent differences in intracellular trafficking may lead to

a vacuole of differing pH for Phase I or Phase II organ-isms, with the Phase II vesicle having a lower pH than the

Phase I vesicle. C. burnetii acidified phagosomes become

more alkaline by treating THP-1 monocytes with IFN-c[28]. It may be that Phase II acidified phagosomes are

affected by BHK-21 cells grown in 12% CO2, to the det-

riment of the organisms. Thus, in the present experiments,

Phase II growthmight be almost completely abrogated by

overacidification, whereas in Phase I culturing the effect isstimulatory or of no consequence.

Acknowledgements

We thank Dr. Edward Shaw and Kara Bennett for

their discussions and advice during this project. We

acknowledge Dr. Emilio Weiss for his suggestion in the1970s that increased levels of CO2 during culturing

might stimulate C. burnetii growth. This work was

partially supported by a National Institutes of Health

grant (AI-34984-03) to H.A. Thompson during his ten-

ure at West Virginia University.

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