TitleChlamydophila felis CF0218 is a novel TMH family protein withpotential as a diagnostic antigen for diagnosis of C. felisinfection( 本文(Fulltext) )

Author(s)

OHYA, Kenji; TAKAHARA, Yu; KURODA, Etsuko; KOYASU,Saori; HAGIWARA, Shigeyuki; SAKAMOTO, Maki; HISAKA,Mitsuaki; MORIZANE, Kazuko; ISHIGURO, Shinryou;YAMAGUCHI, Tsuyoshi; FUKUSHI, Hideto

Citation [Clinical and Vaccine Immunology] vol.[15] no.[10] p.[1606]-[1615]

Issue Date 2008-10

Rights American Society for Microbiology

Version 著者最終稿 (author final version) postprint

URL http://hdl.handle.net/20.500.12099/30645

※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。

1

Title: Chlamydophila felis CF0218 is a novel TMH-family protein with potential as a C. 1

felis infection-specific diagnostic antigen. 2

3

Runinng title: C. felis CF0218 as an infection-specific antigen. 4

5

Kenji OHYA,1 Yu TAKAHARA,1 Etsuko KURODA,1 Saori KOYASU,1 Shigeyuki 6

HAGIWARA,2 Maki SAKAMOTO,2 Mitsuaki HISAKA,2 Kazuko MORIZANE,2 Shinryou 7

ISHIGURO,2 Tsuyoshi YAMAGUCHI,1, 3 and Hideto FUKUSHI1, 3* 8

9

Laboratory of Veterinary Microbiology, Faculty of Applied Biological Sciences1 and 10

Department of Applied Veterinary Science, United Graduate School of Veterinary 11

Sciences, 3 Gifu University, Gifu 501-1193; and Advanced Technology Development 12

Center, Kyoritsu Seiyaku Corporation, Tsukuba 300-1252,2 Japan. 13

14

* Corresponding author 15

Mailing Address: Laboratory of Veterinary Microbiology, Faculty of Applied Biological 16

Sciences, Gifu University, Yanagido 1-1, Gifu, 501-1193, Japan. 17

Phone & Fax: +81-58-293-2946. 18

E-mail: hfukushi@gifu-u.ac.jp 19

20

2

Abstract 1

Chlamydophila felis is a causative agent of acute and chronic conjunctivitis and 2

pneumonia in cats (feline chlamydiosis). Also, C. felis is a suspected zoonotic agent 3

such as non-trachomatis conjunctivitis in humans although this is controversial. At 4

present, there is no serodiagnostic system that specifically detects C. felis infection 5

conveniently. Current systems use antigens such as lipopolysaccharide that 6

cross-react with all chlamydia species. In addition, it is difficult to distinguish cats that 7

are vaccinated with the commercial vaccine against C. felis and cats that are infected 8

with C. felis. Here, we describe a new candidate for diagnostic antigen of C. felis, 9

CF0218, that obtained by screening a genomic expression library of C. felis Fe/C-56 10

with C. felis-immunized serum. CF0218 was a putative transmembrane head 11

(TMH)-family protein with bi-lobed hydrophobic motifs at its N-terminus and 12

orthologues of CF0218 were not found in the C. pneumoniae or Chlamydia 13

trachomatis genomes. The recombinant CF0218 was not recognized by serum 14

against C. trachomatis, suggesting that CF0218 is C. felis specific. CF0218 15

transcription during the course of C. felis-infection was confirmed by RT-PCR. By 16

indirect immunofluorescence analysis, CF0218 was co-localized with the C. 17

felis-formed inclusion bodies in the infected cells. The antibody response against 18

CF0218 was elevated following C. felis infection but not by vaccination in 19

experimentally vaccinated and infected cats. These results suggest that CF0218, a 20

3

novel TMH-family protein of C. felis, possesses potential as a C. felis infection-specific 1

diagnostic antigen. 2

3

4

Introduction 1

The chlamydiae are obligate intracellular bacterial pathogens, possessing a 2

biphasic developmental cycle, consisting of a metabolically inactive infectious 3

elementary body (EB) and a metabolically active non-infectious reticulate body (RB). 4

The bacteria within host cells occupy vacuoles termed inclusions. Chlamydiae cause 5

a range of diseases in various animals, such as humans, birds and cats. The family 6

Chlamydiaceae is divided into two genera, Chlamydia and Chlamydophila (9). The 7

genus Chlamydia comprises Chlamydia trachomatis (a human conjunctivitis and 8

sexually transmitted disease agent), C. muridarum (a mouse pneumonia agent) and C. 9

suis (a pig conjunctivitis agent). The latter genus Chlamydophila includes 10

Chlamydophila pneumoniae (an agent for pneumonia and a suspected 11

atherosclerosis agent), C. psittaci (an agent for psittacosis), C. abortus (a ruminant 12

abortive agent), C. caviae (isolated from guinea pigs), C. pecorum (infects ruminants) 13

and C. felis (infects cats) (9). 14

C. felis is a causative agent of feline chlamydiosis, which is characterized by 15

acute and chronic conjunctivitis and pneumonia in cats (40). The prevalence of C. felis 16

in cats with ocular signs or upper respiratory tract diseases (URTD) has been 17

investigated by PCR or by detection of anti-chlamydial antibodies. The percentages of 18

cats positive for C. felis infection were 14.7% in Britain (29), 20.0% in Italy (32), 11.5% 19

in Switzerland (42),15.3% in Sweden (17), and 4.6% in the United States (26). In our 20

5

previous studies in Japan, the percentages were 26.3% in stray cats, 28.9% in 1

domestic cats, and 59.1% in cats with conjunctivitis and URTD (6, 31, 45). These 2

investigations indicate that C. felis is the common agent of feline conjunctivitis and 3

URTD in the world. 4

Since C. felis is susceptible to tetracylines, doxycycine is the first choice for 5

the treatment of feline chlamydiosis. Systemic administration of doxycycline for 3 6

weeks can effectively clear the pathogen (40). However, conjunctivitis and URTD in 7

cats are also caused by other pathogens such as feline calicivirus (FCV) and feline 8

herpesvirus-1 (FHV-1), and it is not possible to differentiate feline chlamydiosis from 9

viral conjunctivitis and URTD on the basis of clinical signs (40). Indeed, our previous 10

study showed that, in 66 domestic cats with conjunctivitis and URTD, 10.6% of cats 11

had C. felis and FHV-1, 15.2% of cats had C. felis and FCV and 1.5% of cats had C. 12

felis, FHV-1 and FCV (6). Therefore, to provide an adequate treatment and prevent 13

the spread of feline conjunctivitis and URTD, chlamydial infection in cats needs to be 14

differentiated from other viral conjunctivitis and URTD. 15

Current methods for diagnosing feline chlamydiosis are isolation of the 16

pathogen, immunofluorescence (IF) test using the infected cells or the purified EB as 17

antigens, or by the conventional PCR and the real-time PCR (15, 16, 40). However, 18

these methods require the use of the specialized culture techniques (isolation and the 19

IF test) and equipments (PCR and the real-time PCR). Therefore, microbiological 20

6

diagnosis of feline chlamydiosis can only be performed in well-equipped laboratories. 1

Another method to detect C. felis-infection is enzyme-linked immunosorbent assay 2

(ELISA). There is no ELISA system to specifically detect C. felis-infection since the 3

ELISA uses whole chlamydial EB and lipopolysaccharide (LPS) as antigens, which 4

are cross-reactive in all chlamydial species (EB and LPS) as well as other bacteria 5

(LPS) (5, 23, 44). Other serodiagnostic antigens include major outer membrane 6

protein (MOMP) and polymorphic membrane proteins (PMPs) which are highly 7

immunogenic and display intra-/ inter- species diversity (7, 24, 25, 39). For example, 8

Longbottom and colleagues developed the ELISA system by using POMP90 (one of 9

the PMPs in C. abortus) as an antigen. The ELISA can specifically detect anti C. 10

abortus antibodies in C. abortus infected-sheep (19, 21, 23). ELISA can be performed 11

conveniently in general laboratories, and can deal with many samples simultaneously. 12

Therefore, ELISA systems which can specifically detect C. felis (antibodies or 13

antigens) should be developed for diagnosing feline chlamydiosis (40). 14

Cases of C. felis infection in humans are rarely identified and whether such 15

cases exist is now under controversial (20). However, recently, C. felis was isolated 16

from a patient with non-trachomatis conjunctivitis and from one of the patient!s cats 17

(14). In addition, we also reported previously that 5.0% of small animal clinic 18

veterinarians were seropositive for C. felis (45). These results raise the possibility that 19

C. felis is a zoonotic agent, as is the case for C. psittaci and C. abortus (20). 20

7

Both live and inactivated vaccines for C. felis infection have been used in 1

Europe, the United States and Japan (only the inactivated form is approved in Japan). 2

While the vaccines for C. felis do not prevent infection completely, the vaccine can 3

enhance the humoral immune response and reduce severity in clinical signs in 4

vaccinated cats (22, 37, 40, 43). However, it is difficult to distinguish between the 5

vaccinated cats and the C. felis-infected cats by means of the current serological tests 6

(IF tests and the ELISA) because the antibody responses are observed in both cases. 7

In this study, to find out new diagnostic antigens of C. felis, the genomic 8

expression library of C. felis was screened by C. felis-immunized serum. One of the 9

positive clone was found to encode CF0218 (also named as mhcB2). Orthologues of 10

CF0218 were not present in other chlamydia species such as C. trachomatis and C. 11

pneumoniae genomes. The recombinant CF0218 was not recognized by C. 12

trachomatis-immunized serum. In addition, CF0218 was transcribed in C. 13

felis-infected cells and was co-localized with C. felis-formed inclusions. Finally, the 14

antibody response against CF0218 was elevated only following C. felis-infection but 15

not by the vaccination in experimentally vaccinated and infected cats. It is likely that 16

CF0218 possesses potential as diagnostic antigen of C. felis, which can specifically 17

detect C. felis-infection. 18

19

Materials and Methods 20

8

Chlamydial strains and infection of cultured cells. 1

C. felis Fe/C-56 isolated in Japan from a cat with conjunctivitis was used as a 2

standard strain of C. felis since this strain was already read full genomic DNA 3

sequencing (1, 6). For analyzing diversity of cf0218 obtained in this study, C. felis FP1 4

Baker (ATCC VR120; isolated in USA from a cat with pneumonia) (2, 12), Fe/B166 5

(isolated in UK from a cat with conjunctivitis) (30, 31) and Fe/C-38 (isolated in Japan 6

from a cat with conjunctivitis) (6) were used. C. felis were grown in HeLa cells. HeLa 7

cells were treated with minimal essential medium (MEM) ! (Wako Pure Chemical Ltd., 8

Osaka, Japan) containing 30 µg/ml DEAE-dextran at room temperature for 30 min 9

before inoculation. After inoculation of bacteria at a multiplicity of infection of up to 10, 10

flasks (or plates) were centrifuged at 700g for 60 min at room temperature, 11

subsequently incubated in the presence of 5% CO2 at 37°C for 60 min. Afterward, the 12

inoculums were exchanged to MEM! supplemented with 5% fetal bovine serum 13

(Invitrogen, Carlsbad, CA) and 1 µg/ml of cycloheximide in the presence of 5% CO2 at 14

37°C till the mature inclusion body formation or at the time indicated. C. felis EB was 15

purified from infected HeLa cells by sucrose-gradient centrifugation as described 16

previously (12). The purified EB was diluted at 2.0 mg/ml in 0.01 M Tris-HCl (pH 7.2) 17

and stored at -80°C until use. 18

19

Construction and immunoscreening of C. felis genomic DNA expression 20

9

library. 1

Genomic DNA of C. felis Fe/C-56, which was extracted from the purified EB by 2

sodium dodecyl sulfate (SDS), proteinase K and phenol/chloroform as previously 3

described (11), was partially digested with Eco RI in the presence of 2.5 mM Mn2+ and 4

ligated to Eco RI digested "-ZAPII phage arms (Stratagene, La Jolla, CA). The ligated 5

DNA was packaged in vitro with Gigapack extracts according to the manufacturer!s 6

instructions (Stratagene). Recombinant phage were plated on Escherichia coli XL-1 7

Blue MRF! cells (Stratagene) and incubated at 37°C for 6 hrs to allow development of 8

the plaques. The plates were sequentially overlaid with nitrocellulose membranes (GE 9

Healthcare, Buckinghamshire, United Kingdom), which were soaked in 20 mM 10

isopropyl-1-thio-#-D-galactopyranoside (IPTG), and incubated at 37°C for additional 4 11

hrs. The resulting membranes were reacted with C. felis hyper-immunized feline 12

serum (28) as the first antibody, subsequently reacted with anti-cat light 13

chain-horseradish peroxidase (HRP) (Bethyl, Montgomery, TX) as the secondary 14

antibody, and visualized by 3,3!-diaminobenzidine in the presence of 0.01% H2O2. 15

Several phage clones, which were confirmed as being positive by the secondary and 16

tertiary screening, were converted to the pBluescript SK(-) phagemid in E. coli SOLR 17

according to the manufacturer!s instructions (Strategene). The DNA from the insert in 18

each clone was sequenced in both directions by using M13 forward and reverse 19

primers, and analyzed by using the C. felis Fe/C-56 genomic DNA sequence data as 20

10

published previously (1). 1

2

Analysis of diversity and hydropathy profile of cf0218. 3

Diversity of cf0218 from different strains of C. felis was examined by sequencing of 4

the PCR products of cf0218 with primers 5

(5!-CGGGATCCATGACAACAAACTCATTG-3! and 5!- 6

GCGTCGACTTAATTAGCGTCATCATT-3!) from C. felis FP1 Baker, Fe/B166 and 7

Fe/C-38. The sequences of each cf0218 were deposited in the DDBJ/GenBank/EMBL 8

database under accession numbers AB444855 (FP1 Baker), AB444856 (Fe/B166) 9

and AB444857 (Fe/C-38). Hydropathy profiles were determined using the algorithm 10

by Kyte and Doolittle (18) with a window size of 11 on the Web source “ProtScale” 11

(available at http://www.expasy.ch/tools/protscale.html) and the DNA strider program 12

(27). 13

14

Recombinant CF0218 and antibody preparation. 15

The region encoding cf0218 was amplified by PCR with primers 16

(5!-CGGGATCCCGGACAACAAACTCATTGAAC-3! and 17

5!-GGAATTCCCATTAGTATGCTCTTCGCTGCCC-3!; the sites of restriction 18

endonucleases underlined), was cloned into the glutathione S-transferase (GST) 19

fusion protein expression vector pGEX-6P-1 (GE Healthcare). After checking the 20

11

correct nucleotide sequence and frame, the resulting plasmid was named as 1

pGST-CF0218. GST or GST-fused with CF0218 was expressed and purified 2

according to the manufacturer!s instructions. Briefly, logarithmic phase of E. coli BL21 3

(GE Healthcare) harboring pGST or pGST-CF0218 in Luria-Bertani broth 4

supplemented with 40 µg/ml ampicillin were further incubated at 30°C for 5 hrs in the 5

presence of 1.0 mM IPTG. The bacteria were suspended and sonicated in 6

phosphate-buffered saline (PBS) containing 1% (vol/vol) Triton X-100. After 7

centrifugation at 4°C, soluble form of GST or GST-CF0218 in the supernatant was 8

purified by Glutathione Sepharose 4B (GE Healthcare). Rabbit (Japanese white, 9

female, 12-wk-old) polyclonal antibody was raised against recombinant CF0218 10

removed of GST by PreScission protease (GE Healthcare). The antiserum obtained 11

was purified using the CF0218 blotted on nitrocellulose membrane (GE Healthcare). 12

Briefly, the blotted-CF0218 on nitrocellulose membrane stained by 1% Ponso S, was 13

excised and blocked in 1% polyvinylpyrrolidone for 1 hr at 37°C. After washing, the 14

membrane was incubated with the antiserum for 2 hrs at room temperature. The 15

binding antibody specific for CF0218 was eluted from the membrane with 0.1 M 16

Gly-HCl (pH 2.5) and neutralized with 2 M Tris. All animal experiments described in 17

this study were approved by the Commission for Animal Experiments in Gifu 18

University according to current guidelines. 19

20

12

Immunoblotting. 1

C. felis EB (1mg/ml) inactivated in PBS supplemented with 0.5% (vol/vol) Triton X-100 2

and 5% (vol/vol) 2-mercaptoehtanol, or each recombinant CF0218 product were 3

separated by SDS – polyacrylamide gel electrophoresis (PAGE) and transferred onto 4

polyvinylidene difluoride (PVDF) membrane (Immobilon-P; Millipore, Billerica, MA). 5

Following antiserum were used as the first antibodies diluted at 1:2,000 in PBS; rabbit 6

anti CF0218, rabbit C. psittaci Prk/daruma-hyperimmunized serum (Fukushi et al., 7

unpublished material), rabbit C. trachomatis L2/434/Bu-hyperimmunized serum (12), 8

and feline C. felis-hyperimmunized serum. The anti cat light chain-HRP and the anti 9

rabbit IgG-HRP (ICN Pharmaceuticals, Aurora, OH) were used as the secondary 10

antibodies diluted at 1:2,000 in PBS. The membranes were incubated in ECL western 11

blotting detection reagents (GE Healthcare) and exposed to X-ray film (Fujifilm, Tokyo, 12

Japan). 13

14

RT-PCR analysis 15

Total RNA was extracted from C. felis Fe/C-56 infected HeLa cells at each time after 16

infection by Trizol reagent (Invitrogen), and residual DNA contamination was removed 17

by treatment with amplification grade DNase I (Invitrogen), according to the 18

manufacturer!s instructions. For reverse transcription (RT)-PCR, cDNA was 19

synthesized from 1.0 µg total RNA by using random primer and Molony murine 20

13

leukemia virus reverse transcriptase for 60 min at 42°C according to the 1

manufacturer!s instructions (ReverTra Ace kit; Toyobo, Osaka, Japan). The cDNA 2

was amplified by PCR using each primer. Custom primer sets specific for C. felis 3

cf0218 (5!- CGGGATCCATGACAACAAACTCATTG-3! and 5!- 4

GCGTCGACTTAATTAGCGTCATCATT-3!) were used to detect cf0218-specific 5

message whereas previously described primers were used to examine levels of 6

chlamydophila ompA (CMGP-2F; 5!-GCCTTAAACATCTGGGATCG-3! and 7

CMGP-2R; 5!-GCACAACCACATTCCCATAAAG-3!) (8). C. felis genomic DNA was 8

used as positive controls, and total RNA samples without reverse transcription were 9

used as controls for DNA contamination. 10

11

Indirect immunofluolescence microscopy. 12

HeLa cells grown on coverslips were used for immunofluorescence study. 13

Seventy-two hrs after infection of C. felis Fe/C-56, cells were fixed with 4% 14

paraformaldehyde in PBS for 20 min at room temperature, subsequently, fixed again 15

with cold-methanol for 1 min on ice. The coverslips were blocked in 2% (wt/vol) bovine 16

serum albumin in PBS, and stained with rabbit anti CF0218 diluted at 1:50 in PBS, 17

anti rabbit IgG-fluorescein isothiocyanate (FITC) (ICN Pharmaceuticals) diluted at 18

1:200 in PBS, anti chlamydial LPS monoclonal antibody (13), anti mouse IgG-Alexa 19

594 (Invitrogen) diluted at 1:200 in PBS and 4!-6-diamidine-2-phenylindole 20

14

dihydrochloride (DAPI) (Dojindo Laboratory, Kumamoto, Japan) diluted at 1:5,000 in 1

PBS, respectively. The coverslips were mounted in SlowFade antifade reagent 2

(Invitrogen), and examined with an immunoflueorescence microscope (BZ-8000; 3

Keyence, Osaka, Japan) for deconvoluted fluorescence imaging. 4

5

Experimental vaccination and infection of cats 6

Six specific-pathogen-free female cats were used. They were 5 months old and 7

weighed 1.6 kg to 1.8 kg at the start of the experiments. They were divided into two 8

groups. On day 0 (0 wk) and 3 wks, cats in one group were vaccinated twice with the 5 9

antigens contained vaccine for cats, Fel-O-Vax 5 (Kyoritsu Seiyaku Corp., Tokyo, 10

Japan), which contains inactivated C. felis Cello strain EB, via the intramuscular route 11

according to the manufacturer!s instructions. Cats in another group were inoculated 12

with saline via the intramuscular route, on 0 wk and 3 wks. On 5 wks (2 wks after the 13

2nd vaccination), cats in both groups were inoculated with 104 50% embryo infectious 14

dose (EID50) of live C. felis Cello strain via the mucosal (conjunctival, oral and nasal) 15

route. Sera of all cats were collected from 0 to 8 wks. 16

17

ELISA 18

Antibody response against C. felis EB and CF0218 of infected cats was measured by 19

ELISA. For ELISA, C. felis EB (1 mg/ml) was inactivated in 1% Triton X-100 and 5% 20

15

2-mercaptoehtanol at 37°C for 30 min as described previously (28). To assess 1

antibody against CF0218 in feline sera, purified GST and GST-CF0218 were used as 2

antigens. Each antigen (EB, GST and GST-CF0218) was diluted at 1 µg/ml with 0.05 3

M carbonate-bicarbonate buffer (pH 9.6, 15 mM Na2CO3, 35 mM NaHCO3), 4

subsequently applied to the 96-well plates (100 ng/well) (F96 Maxisorp; Nunc, 5

Roskilde, Denmark) at 4°C overnight to coat wells. After blocking each well with 5% 6

(wt/vol) skim milk in PBS containing 0.05% Tween 20 (PBST), the sera diluted at 7

1:100 with PBS were added and incubated for 1 hr at room temperature. After 8

washing by PBST, the anti cat light chain-HRP diluted at 1:2,000 with PBS were 9

added as the secondary antibody. The plates were incubated for 1 hr at room 10

temperature and washed twice with PBST, and then twice with distilled water. Tablets 11

of 2,2!-azino-bis(3-ethlbenzothiazoline-6-sulfonic acid) (Sigma-Aldrich, St. Louis, MO) 12

dissolved in 0.05 M citrate buffer (pH 4.0) containing 0.0075% H2O2, was added to 13

each well according to the manufacturer!s instructions. The plates were incubated at 14

37°C for 30 min, optical density at 405 nm (OD405) was measured using a Microplate 15

Reader Model 550 (Bio-Rad, Hercules, CA). Net OD values for CF0218 were 16

calculated by subtracting the OD values of the GST wells from the GST-CF0218 wells 17

as described elsewhere (19). Each sample was measured in duplicate. 18

19

Statistical analysis 20

16

Overall antibody responses in cats were analyzed by using repeated measures 1

analysis of variance (ANOVA), and subsequently differences between control serum 2

(at 0 wk) and serum at each time point were analyzed by using a Bonferroni correction. 3

For all statistical analyses, P values of < 0.01 were considered statistically significant. 4

5

Results 6

Identification of cf0218 gene. 7

Seven positive clones were obtained by immunoscreening of the C. felis 8

genomic expression library (1.5 x 105 plaque forming unit) with the cat serum raised 9

against C. felis. Phagemids were excised from these clones and partially sequenced 10

with M13 primers (Supplementary Table). Among them, one clone (clone E in 11

Supplementary Table) was chosen for further analysis since this clone (insert size: 12

2,241 bp) contained one open reading frame [ORF, designated as cf0218 (also 13

named as mhcB2)], for which orthologous genes were not found in the C. pneumoniae 14

and C. trachomatis genomes (1). As shown in Fig. 1, cf0218 in the C. felis genomes is 15

placed at the syntenic region with the transmembrane head (TMH) locus in the C. 16

abortus (41) and the C. caviae (33) genomes, whereas this locus is not present in the 17

C. pneumoniae J138 genome (38) or the C. trachomatis D/UW-3/Cx genome (39). 18

cf0218 (1,146 bp) encodes 381 amino acid residues with a calculated molecular mass 19

of 42.0 kDa. CF0218 from different isolates of C. felis were highly conserved. For 20

17

example, CF0218 from C. felis FP1 Baker (isolated in USA; accession number 1

AB444855) and Fe/C-38 (isolated in Japan; accession number AB444857) exhibited 2

100% amino acids identity with that from Fe/C-56. Only CF0128 from Fe/B166 3

(isolated in UK; accession number AB444856) showed one amino acid difference at 4

position 110 (G) compared with that of other isolates (E). The TMH locus in the C. 5

abortus and the C. caviae genomes encodes several TMH-family proteins with paired 6

N-terminal transmembrane motifs. For example, C. abortus CAB764, CAB766 and C. 7

caviae CCA797 at the TMH locus, which are TMH-family proteins, had 25.0%, 27.4% 8

and 34.9% amino acid identity with CF0218, respectively (Fig. 2A). Furthermore, 9

CAB764, CAB766, CCA797 and CF0218 possess similar bi-lobed hydrophobic motifs 10

at their N-terminus (Fig. 2B), which implies that CF0218 is a TMH-family protein of C. 11

felis. 12

Expression of recombinant CF0218 and its immunogenicity. 13

Recombinant CF0218 was successfully expressed in E. coli as a soluble 14

fusion protein with GST (GST-CF0218) at the expected size (Fig. 3A). To generate 15

polyclonal antibodies against CF0218, recombinant CF0218 was cleaved from 16

GST-CF0218, and then injected into a rabbit. To evaluate the antigenicity of CF0218, 17

recombinant CF0218 and purified C. felis EB were subjected to western blot analysis. 18

The generated antiserum specifically reacted with GST-CF0218, purified recombinant 19

CF0218 and purified C. felis EB at the expected size but not GST alone (Fig. 3B). The 20

18

C. felis-hyperimmunized cat serum, which was used in the immunoscreening of the C. 1

felis library, reacted with GST-CF0218, purified CF0218 at the expected size, but it did 2

not react with GST alone (Fig. 3C). On the other hand, recombinant CF0218 was not 3

recognized by the C. trachomatis-hyperimmunized rabbit serum, which showed 4

cross-reactivity with C. felis EB(Fig. 3D). This result agreed with the fact that the 5

orthologues of CF0218 do not exist in the C. trachomatis genome (Fig. 1). In addition, 6

we also examined cross reactivity of CF0218 with non-C. felis chlamydiae. Since we 7

do not have available antisera against C. abortus and C. caviae, we used antiserum 8

against C. psittaci Prk/daruma which is genetically closely related to C. abortus than 9

other C. psittaci strains by southern hybridization analysis (11). This serum reacted 10

with C. felis EB but not with recombinant CF0218 (Fig. 3E). 11

Expression of CF0218 in C. felis-infected cells. 12

To determine whether CF0218 is expressed during C. felis infection in host 13

cells, RT-PCR analysis was performed. Total RNA from C. felis-infected or 14

mock-infected HeLa cells was isolated at 24, 48 and 72 hrs after inoculation of C. felis, 15

and subjected to RT-PCR using the primers specific for cf0218 and the gene 16

corresponding to MOMP (ompA) as a control for chlamydia-infection. As shown in Fig. 17

4, specific signals for cf0218 and ompA were detected at 24, 48 and 72 hrs after 18

inoculation of host cells with C. felis, revealed that cf0218 was transcribed in host cells 19

during C. felis infection. By western blot analysis, lysates of C. felis-infected HeLa 20

19

cells reacted weakly with anti chlamydial LPS, but no specific signal with anti CF0218 1

serum was detectable (data not shown). It may be due to low infection efficiency of C. 2

felis and/or the low expression level of CF0218 although we have no quantitative data 3

to describe the infection efficiency of C. felis compared with other chlamydia species. 4

We next performed indirect-immunofluorescent assay to visualize CF0218 in C. 5

felis-infected cells. C. felis-infected (72 hrs post inoculation) or mock-infected HeLa 6

cells were fixed and probed with anti CF0218 , anti chlamydial LPS and DAPI for host 7

nuclei and apparent chlamydial-inclusions (Fig. 5). The 72 hrs was chosen because 8

the inclusions are fully developed (white arrowheads in Fig. 5). Since the inclusions of 9

C. felis are relatively smaller than those of C. psittaci (data not shown), it is hard to 10

detect C. felis by DAPI staining except for the apparent inclusions. However, C. felis in 11

host cells can be visible by staining with anti chlamydial LPS (red in Fig. 5). Specific 12

signal for CF0218 (green in Fig.5) was co-localized with the chlamydial LPS (merged 13

images are yellow in Fig.5) as well as the apparent chlamydial inclusions (merged 14

images are white in Fig. 5). The immunofluorescence microscopic analysis clearly 15

showed that the subcellular localization of CF0218 is within the C. felis-formed 16

inclusions. 17

Antibody response against CF0218 in experimentally vaccinated and C. 18

felis-infected cats. 19

As mentioned in the Introduction section, the modified live and inactivated 20

20

vaccines against C. felis have been used in several countries including Japan (40). It 1

is difficult to differentiate vaccinated or infected cats since both vaccinated cats and 2

infected cats are assessed as positive by means of current serological tests. 3

Therefore, to assess potential of the CF0218 for diagnostic use, antibody response 4

against CF0218 in vaccinated and C. felis-infected cats were examined. 5

Specific-pathogen-free of cats were inoculated with the C. felis-inactivated vaccine 6

twice (at 0 and 3 wks), subsequently, challenged by C. felis at 5 wks (2 wks after the 7

2nd vaccination; for detail experimental design, see the Materials and Methods 8

section). Sera at each time point were collected and measured antibody response 9

against C. felis EB and the recombinant CF0218 by means of the ELISA, respectively 10

(Fig. 6). As shown in Fig. 6A, antibody response against C. felis EB was elevated 11

following the C. felis vaccination from 4 wks after the 1st vaccination in the vaccinated 12

group (closed squares) but not in the non-vaccinated group (closed circles). 13

Thereafter, C. felis was challenged to both groups (at 5 wks), antibody against EB 14

was elevated from 7wks (2wks after the challenge) in both groups. These results 15

confirmed that antibody response due to the vaccination and the infection correctly 16

occurred in the experiments. Antibody response against CF0218 in the same samples 17

was measured by means of the ELISA using the recombinant CF0218 as an antigen 18

(Fig. 6B). Antibody against CF0218 was not increased till 5 wks in the vaccinated 19

group (Fig. 6B, closed squares and solid line) although antibody against EB was 20

21

elevated at this time point in the vaccinated group (Fig. 6A). Whereas, antibody 1

against CF0218 was increased following C. felis infection from 7 wks (2 wks after the 2

challenge) in the vaccinated group and even in the non-vaccinated group (Fig. 6B, 3

closed circles and dotted line). These results suggest that antibody against CF0218 4

was not elevated by the vaccination but elevated only by live-C. felis infection. 5

6

Discussion 7

In this study, we describe the cloning and molecular characterization of C. 8

felis CF0218 as a new diagnostic antigen. Our results show as follows: (I) CF0218 9

was obtained by immunoscreening of C. felis genomic library with the C. 10

felis-immunized cat serum; (II) the location of cf0218 in the C. felis genome is on the 11

syntenic region of the TMH locus in the C. abortus genome, which was not present in 12

the genomes of C. trachomatis or C. pneumoniae; (III) recombinant CF0218 was 13

recognized by serum against C. felis but not by serum against C. trachomatis and C. 14

psittaci; (IV) CF0218 was expressed in C. felis-infected HeLa cells and was 15

co-localized in C. felis-formed inclusion bodies; (V) antibody against CF0218 was 16

elevated by C. felis infection but not by the vaccination in experimentally vaccinated 17

and infected cats. 18

The TMH locus is a newly identified gene cluster showing limited distribution 19

among chlamydial species. Thomson et al. reported that this region is present in C. 20

22

abortus and C. caviae, but the syntenic regions in C. trachomatis, C. pneumonia and 1

C. muridarum revealed significant levels of variation in gene content (41). The cf0218 2

obtained in this study by immunoscreening is present in the putative TMH locus in the 3

C. felis genome (Fig. 1). The TMH locus is characterized by encoding several ORFs 4

with paired N-terminal TM motifs with varying length, which are termed TMH-family 5

proteins. CF0218 shows similarity with the C. abortus and C. caviae TMH-family 6

protein CAB764, CAB766 and CCA797 especially at the N-terminus, and possesses a 7

bi-lobed hydrophobic motif at the N-terminus as well as CAB764, CAB766 and 8

CCA797 (Fig. 2), suggesting that CF0218 is a TMH-family protein of C. felis. 9

At this time, there has been no report describing the role of the TMH-family 10

proteins during the course of chlamydia infection. However, the presence of paired 11

N-terminal TM helices suggests that TMH family proteins may belong to the Inc-family 12

proteins (41). Inc-family proteins play a major role in the formation of chlamydial 13

inclusion membranes, and may participate in the chlamydial developmental process 14

including growth and survival within the host cells (35). TMH-family proteins may play 15

a similar role to Inc-family proteins in the chlamydial developmental process. 16

In C. felis-infected HeLa cells, CF0218 is distributed throughout the 17

chlamydial inclusion bodies (Fig. 5). In contrast, other researchers showed that a 18

large number of chlamydial Inc proteins are localized to the inclusion membranes (3, 19

35). In addition, unexpectedly, CF0218 was also detected by immunoblotting in the 20

23

purified C. felis EB (Fig. 3B) although several Inc proteins of C. caviae (formerly C. 1

psittaci GPIC) and C. trachomatis were not reported to be detected in purified EBs (4, 2

36). On the other hand, C. trachomatis IncA was detected in purified EB even a lesser 3

extent (36) and CopN (a component of the type III secretion system) was also 4

detected in purified EB (10). Both IncA and CopN play pivotal roles in chlamydial 5

infection process. It remains to be elucidated whether CF0218 is a structural 6

component of EB or acts as an Inc-like protein in infected cells during the C. felis 7

infection process. 8

In general, serological diagnosis of chlamydia is exclusively performed by 9

using EB or chlamydial cell surface components (LPS, MOMP and PMPs) as antigens 10

(see the Introduction section), while other chlamydial products represent 11

immunogenicity in infected animals or human patients. For example, C. caviae IncA 12

and IncC were initially identified by sera from C. caviae-infected guinea pigs (4, 34). 13

Additionally, several recombinant putative Inc proteins are recognized by sera from C. 14

trachomatis and C. pneumoniae-infected patients (3). These reports indicate that 15

chlamydial products other than LPS, MOMP and PMPs are immunogenic and can be 16

used as diagnostic antigens. CF0218 is highly conserved at amino acids level among 17

different C. felis isolates. Antigenicity of CF0218 was examined by using the serum 18

against C. felis was confirmed (Fig. 3C). Furthermore, antibody against CF0218 was 19

increased in experimentally C. felis-infected cats (Fig. 6B). These results indicate that 20

24

CF0218 is immunogenic with potential as a diagnostic antigen of C. felis. 1

Interestingly, the TMH locus has been identified in the C. abortus, C. caviae 2

(41) and C. felis (this study) genomes but not in the C. trachomatis and C. 3

pneumoniae genomes, and orthologues of CF0218 have not been identified in the C. 4

trachomatis and C. pneumoniae genomes (1), suggesting that the TMH-family 5

proteins may be specific for non-human chlamydophila. There is possibility of 6

cross-reactivity of CF0218 with closely related non-C. felis chlamydiae possessing 7

TMH-family proteins since C. abortus CAB764 CAB766 and C. caviae CCA797 8

exhibited 25 to 35% identity with CF0218. Since we do not have antisera against C. 9

abortus and C. caviae, we examined cross-reactivity of CF0218 with antiserum 10

against C. psittaci Prk/daruma and this serum did not react with CF0218 (Fig. 3E). 11

Currently genome sequence of C. psittaci is unavailable, however, C. psittaci 12

Prk/daruma is genetically closely related to C. abortus rather than other C. psittaci 13

strains (11). This result may suggest that CF0218 with potential as a diagnostic 14

antigen specific for C. felis. As far as we know, there is no report that C. abortus and C. 15

caviae infect cats. However, von Bomhar et al. reported that Neochlamydia 16

hartmannellae can be a causative agent for feline chlamydiosis (42). Therefore, 17

further study is needed to examine cross-reactivity of CF0218 with N. hartmannellae. 18

As described in the Introduction section, C. felis is a suspected zoonotic agent (20). 19

The fact that the recombinant CF0218 was not recognized by the serum against C. 20

25

trachomatis (Fig. 3D) raise the possibility of using CF0218 to clarify whether C. felis 1

infection in humans is the cause of non-trachomatis conjunctivitis. 2

Finally, we examined antibody response against CF0218 in experimentally 3

vaccinated cats since the vaccine against C. felis leads to be difficult to distinguish 4

vaccinated and infected cats by means of the current serodiagnostic methods (see the 5

Introduction section). Our result (shown in Fig. 6B) suggests that it is possible to 6

differentiate the vaccinated and the infected cats by measuring antibody against 7

CF0218. 8

It is noteworthy that CF0218 was detected by immunoblotting in the purified 9

C. felis EB of which the component of the vaccine is formalin-inactivated form (Fig. 10

3B). The reason why the antibody against CF0218 is not elevated by vaccination is 11

unclear. However, Shewen et al. reported that formalin-inactivated C. felis vaccines 12

did not induce the complement fixing (CF) antibodies in experimentally vaccinated 13

cats, but the vaccines reduced the clinical severities of subsequent C. felis infection 14

(37). Like CF antibodies, antibody against CF0218 might not be increased by the 15

vaccination. In addition, although the vaccine used in this study contains 16

formalin-inactivated C. felis Cello EB, antibody against CF0218 was elevated only 17

after challenging with live-C. felis Cello strain. In this case, it may be that the antigen 18

(CF0218) recognition by the host immune system requires live organisms infection. It 19

is noteworthy that modified-live vaccines for C. felis, although used in other countries 20

26

(22, 40), are not approved in Japan, so we did not test them in this study. According to 1

our marketing research (Ishiguro S, unpublished data), percentages of the inactivated 2

C. felis vaccine among a total sale of C. felis vaccine in the United States are around 3

50% (50% in 2005 and 47% in 2006, respectively). Further studies are needed to 4

determine whether modified-live vaccines induce an antibody response to CF0218. 5

In conclusions, we identified CF0218, a novel TMH-family protein of C. feis, 6

which can be used as a diagnostic antigen specific for C. felis infection. The precise 7

role of CF0218 during the course of C. felis infection should be explored. This is the 8

first report to describe the molecular characteristics of a C. felis TMH-family protein. 9

We are currently determining the seroprevalence of CF0218 in Japanese cats. 10

11

Acknowledgments 12

This work was supported in part by grants-in-aid for the Zoonosis Control Project from 13

the Ministry of Agriculture, Forestry and Fisheries of Japan (grant ZCP-40) and from 14

the Japan Society for the Promotion of Science (grant 18780229 and 20780218). 15

16

17

27

References 1

1. Azuma, Y., H. Hirakawa, A. Yamashita, Y. Cai, M. A. Rahman, H. Suzuki, S. 2

Mitaku, H. Toh, S. Goto, T. Murakami, K. Sugi, H. Hayashi, H. Fukushi, M. 3

Hattori, S. Kuhara, and M. Shirai. 2006. Genome sequence of the cat 4

pathogen, Chlamydophila felis. DNA Res. 13:15-23. 5

2. Baker, J. A. 1942. A Virus Obtained from a Pneumonia of Cats and Its 6

Possible Relation to the Cause of Atypical Pneumonia in Man. Science 7

96:475-476. 8

3. Bannantine, J. P., R. S. Griffiths, W. Viratyosin, W. J. Brown, and D. D. 9

Rockey. 2000. A secondary structure motif predictive of protein localization to 10

the chlamydial inclusion membrane. Cell. Microbiol. 2:35-47. 11

4. Bannantine, J. P., D. D. Rockey, and T. Hackstadt. 1998. Tandem genes of 12

Chlamydia psittaci that encode proteins localized to the inclusion membrane. 13

Mol. Microbiol. 28:1017-1026. 14

5. Brade, L., O. Holst, P. Kosma, Y. X. Zhang, H. Paulsen, R. Krausse, and H. 15

Brade. 1990. Characterization of murine monoclonal and murine, rabbit, and 16

human polyclonal antibodies against chlamydial lipopolysaccharide. Infect. 17

Immun. 58:205-213. 18

6. Cai, Y., H. Fukushi, S. Koyasu, E. Kuroda, T. Yamaguchi, and K. Hirai. 19

2002. An etiological investigation of domestic cats with conjunctivitis and upper 20

28

respiratory tract disease in Japan. J. Vet. Med. Sci. 64:215-219. 1

7. Caldwell, H. D., and J. Schachter. 1982. Antigenic analysis of the major outer 2

membrane protein of Chlamydia spp. Infect. Immun. 35:1024-1031. 3

8. Chahota, R., H. Ogawa, Y. Mitsuhashi, K. Ohya, T. Yamaguchi, and H. 4

Fukushi. 2006. Genetic diversity and epizootiology of Chlamydophila psittaci 5

prevalent among the captive and feral avian species based on VD2 region of 6

ompA gene. Microbiol. Immunol. 50:663-678. 7

9. Everett, K. D., R. M. Bush, and A. A. Andersen. 1999. Emended description 8

of the order Chlamydiales, proposal of Parachlamydiaceae fam. nov. and 9

Simkaniaceae fam. nov., each containing one monotypic genus, revised 10

taxonomy of the family Chlamydiaceae, including a new genus and five new 11

species, and standards for the identification of organisms. Int. J. Syst. Bacteriol. 12

49:415-440. 13

10. Fields, K. A., and T. Hackstadt. 2000. Evidence for the secretion of 14

Chlamydia trachomatis CopN by a type III secretion mechanism. Mol. Microbiol. 15

38:1048-1060. 16

11. Fukushi, H., and K. Hirai. 1989. Genetic diversity of avian and mammalian 17

Chlamydia psittaci strains and relation to host origin. J. Bacteriol. 18

171:2850-2855. 19

12. Fukushi, H., and K. Hirai. 1988. Immunochemical diversity of the major outer 20

29

membrane protein of avian and mammalian Chlamydia psittaci. J. Clin. 1

Microbiol. 26:675-680. 2

13. Fukushi, H., K. Nojiri, and K. Hirai. 1987. Monoclonal antibody typing of 3

Chlamydia psittaci strains derived from avian and mammalian species. J. Clin. 4

Microbiol. 25:1978-1981. 5

14. Hartley, J. C., S. Stevenson, A. J. Robinson, J. D. Littlewood, C. Carder, J. 6

Cartledge, C. Clark, and G. L. Ridgway. 2001. Conjunctivitis due to 7

Chlamydophila felis (Chlamydia psittaci feline pneumonitis agent) acquired 8

from a cat: case report with molecular characterization of isolates from the 9

patient and cat. J. Infect. 43:7-11. 10

15. Helps, C., N. Reeves, K. Egan, P. Howard, and D. Harbour. 2003. Detection 11

of Chlamydophila felis and feline herpesvirus by multiplex real-time PCR 12

analysis. J. Clin. Microbiol. 41:2734-2736. 13

16. Helps, C., N. Reeves, S. Tasker, and D. Harbour. 2001. Use of real-time 14

quantitative PCR to detect Chlamydophila felis infection. J. Clin. Microbiol. 15

39:2675-2676. 16

17. Holst, B. S., L. Englund, S. Palacios, L. Renstrom, and L. T. Berndtsson. 17

2006. Prevalence of antibodies against feline coronavirus and Chlamydophila 18

felis in Swedish cats. J. Feline Med. Surg. 8:207-211. 19

18. Kyte, J., and R. F. Doolittle. 1982. A simple method for displaying the 20

30

hydropathic character of a protein. J. Mol. Biol. 157:105-132. 1

19. Livingstone, M., G. Entrican, S. Wattegedera, D. Buxton, I. J. McKendrick, 2

and D. Longbottom. 2005. Antibody responses to recombinant protein 3

fragments of the major outer membrane protein and polymorphic outer 4

membrane protein POMP90 in Chlamydophila abortus-infected pregnant 5

sheep. Clin. Diagn. Lab. Immunol. 12:770-777. 6

20. Longbottom, D., and L. J. Coulter. 2003. Animal chlamydioses and zoonotic 7

implications. J. Comp. Pathol. 128:217-244. 8

21. Longbottom, D., S. Fairley, S. Chapman, E. Psarrou, E. Vretou, and M. 9

Livingstone. 2002. Serological diagnosis of ovine enzootic abortion by 10

enzyme-linked immunosorbent assay with a recombinant protein fragment of 11

the polymorphic outer membrane protein POMP90 of Chlamydophila abortus. J. 12

Clin. Microbiol. 40:4235-4243. 13

22. Longbottom, D., and M. Livingstone. 2006. Vaccination against chlamydial 14

infections of man and animals. Vet. J. 171:263-275. 15

23. Longbottom, D., E. Psarrou, M. Livingstone, and E. Vretou. 2001. 16

Diagnosis of ovine enzootic abortion using an indirect ELISA (rOMP91B 17

iELISA) based on a recombinant protein fragment of the polymorphic outer 18

membrane protein POMP91B of Chlamydophila abortus. FEMS Microbiol. Lett. 19

195:157-161. 20

31

24. Longbottom, D., M. Russell, S. M. Dunbar, G. E. Jones, and A. J. Herring. 1

1998. Molecular cloning and characterization of the genes coding for the highly 2

immunogenic cluster of 90-kilodalton envelope proteins from the Chlamydia 3

psittaci subtype that causes abortion in sheep. Infect. Immun. 66:1317-1324. 4

25. Longbottom, D., M. Russell, G. E. Jones, F. A. Lainson, and A. J. Herring. 5

1996. Identification of a multigene family coding for the 90 kDa proteins of the 6

ovine abortion subtype of Chlamydia psittaci. FEMS Microbiol. Lett. 7

142:277-281. 8

26. Low, H. C., C. C. Powell, J. K. Veir, J. R. Hawley, and M. R. Lappin. 2007. 9

Prevalence of feline herpesvirus 1, Chlamydophila felis, and Mycoplasma spp 10

DNA in conjunctival cells collected from cats with and without conjunctivitis. Am. 11

J. Vet. Res. 68:643-648. 12

27. Marck, C. 1988. 'DNA Strider': a 'C' program for the fast analysis of DNA and 13

protein sequences on the Apple Macintosh family of computers. Nucleic Acids 14

Res. 16:1829-1836. 15

28. Matsuno, H., H. Fukushi, T. Yamaguchi, and K. Hirai. 1991. Antigenic 16

analysis of feline and bovine Chlamydia psittaci with monoclonal antibodies. J. 17

Vet. Med. Sci. 53:173-179. 18

29. McDonald, M., B. J. Willett, O. Jarrett, and D. D. Addie. 1998. A comparison 19

of DNA amplification, isolation and serology for the detection of Chlamydia 20

32

psittaci infection in cats. Vet. Rec. 143:97-101. 1

30. Pudjiatmoko, H. Fukushi, Y. Ochiai, T. Yamaguchi, and K. Hirai. 1997. 2

Diversity of feline Chlamydia psittaci revealed by random amplification of 3

polymorphic DNA. Vet. Microbiol. 54:73-83. 4

31. Pudjiatmoko, H. Fukushi, Y. Ochiai, T. Yamaguchi, and K. Hirai. 1996. 5

Seroepidemiology of feline chlamydiosis by microimmunofluorescence assay 6

with multiple strains as antigens. Microbiol. Immunol. 40:755-759. 7

32. Rampazzo, A., S. Appino, P. Pregel, A. Tarducci, E. Zini, and B. Biolatti. 8

2003. Prevalence of Chlamydophila felis and feline herpesvirus 1 in cats with 9

conjunctivitis in northern Italy. J. Vet. Intern. Med. 17:799-807. 10

33. Read, T. D., G. S. Myers, R. C. Brunham, W. C. Nelson, I. T. Paulsen, J. 11

Heidelberg, E. Holtzapple, H. Khouri, N. B. Federova, H. A. Carty, L. A. 12

Umayam, D. H. Haft, J. Peterson, M. J. Beanan, O. White, S. L. Salzberg, 13

R. C. Hsia, G. McClarty, R. G. Rank, P. M. Bavoil, and C. M. Fraser. 2003. 14

Genome sequence of Chlamydophila caviae (Chlamydia psittaci GPIC): 15

examining the role of niche-specific genes in the evolution of the 16

Chlamydiaceae. Nucleic Acids Res. 31:2134-2147. 17

34. Rockey, D. D., R. A. Heinzen, and T. Hackstadt. 1995. Cloning and 18

characterization of a Chlamydia psittaci gene coding for a protein localized in 19

the inclusion membrane of infected cells. Mol. Microbiol. 15:617-626. 20

33

35. Rockey, D. D., M. A. Scidmore, J. P. Bannantine, and W. J. Brown. 2002. 1

Proteins in the chlamydial inclusion membrane. Microbes Infect. 4:333-340. 2

36. Scidmore-Carlson, M. A., E. I. Shaw, C. A. Dooley, E. R. Fischer, and T. 3

Hackstadt. 1999. Identification and characterization of a Chlamydia 4

trachomatis early operon encoding four novel inclusion membrane proteins. 5

Mol. Microbiol. 33:753-765. 6

37. Shewen, P. E., R. C. Povey, and M. R. Wilson. 1980. A comparison of the 7

efficacy of a live and four inactivated vaccine preparations for the protection of 8

cats against experimental challenge with Chlamydia psittaci. Can. J. Comp. 9

Med. 44:244-251. 10

38. Shirai, M., H. Hirakawa, M. Kimoto, M. Tabuchi, F. Kishi, K. Ouchi, T. 11

Shiba, K. Ishii, M. Hattori, S. Kuhara, and T. Nakazawa. 2000. Comparison 12

of whole genome sequences of Chlamydia pneumoniae J138 from Japan and 13

CWL029 from USA. Nucleic Acids Res. 28:2311-2314. 14

39. Stephens, R. S., S. Kalman, C. Lammel, J. Fan, R. Marathe, L. Aravind, W. 15

Mitchell, L. Olinger, R. L. Tatusov, Q. Zhao, E. V. Koonin, and R. W. Davis. 16

1998. Genome sequence of an obligate intracellular pathogen of humans: 17

Chlamydia trachomatis. Science 282:754-759. 18

40. Sykes, J. E. 2005. Feline chlamydiosis. Clin. Tech. Small Anim. Pract. 19

20:129-134. 20

34

41. Thomson, N. R., C. Yeats, K. Bell, M. T. Holden, S. D. Bentley, M. 1

Livingstone, A. M. Cerdeno-Tarraga, B. Harris, J. Doggett, D. Ormond, K. 2

Mungall, K. Clarke, T. Feltwell, Z. Hance, M. Sanders, M. A. Quail, C. Price, 3

B. G. Barrell, J. Parkhill, and D. Longbottom. 2005. The Chlamydophila 4

abortus genome sequence reveals an array of variable proteins that contribute 5

to interspecies variation. Genome Res. 15:629-640. 6

42. von Bomhard, W., A. Polkinghorne, Z. H. Lu, L. Vaughan, A. Vogtlin, D. R. 7

Zimmermann, B. Spiess, and A. Pospischil. 2003. Detection of novel 8

chlamydiae in cats with ocular disease. Am. J. Vet. Res. 64:1421-1428. 9

43. Wills, J. M., T. J. Gruffydd-Jones, S. J. Richmond, R. M. Gaskell, and F. J. 10

Bourne. 1987. Effect of vaccination on feline Chlamydia psittaci infection. 11

Infect. Immun. 55:2653-2657. 12

44. Wills, J. M., W. G. Millard, and P. E. Howard. 1986. Evaluation of a 13

monoclonal antibody based ELISA for detection of feline Chlamydia psittaci. 14

Vet. Rec. 119:418-420. 15

45. Yan, C., H. Fukushi, H. Matsudate, K. Ishihara, K. Yasuda, H. Kitagawa, T. 16

Yamaguchi, and K. Hirai. 2000. Seroepidemiological investigation of feline 17

chlamydiosis in cats and humans in Japan. Microbiol. Immunol. 44:155-160. 18

19

20

21

35

Figure legends 1

FIG. 1. Gene arrangement of the TMH loci in the C. felis (Fe/C-56) C. abortus (S26/3) 2

and C. caviae (GPIC) genomes, and the analogous regions in the C. pneumoniae 3

(J138) and C. trachomatis (D/UW-3/Cx) genomes. 4

By screening of the C. felis genome library with anti C. felis serum, the obtained 5

positive clone was placed at the TMH locus (between lpxB and pmp genes; an arrow 6

at both ends) in the C. felis genome. The TMH loci are present in the C. felis, the C. 7

abortus and the C. caviae genomes, but not present in the C. pneumoniae and the C. 8

trachomatis genomes. Representative locus-tags (and gene names) are shown on 9

each ORF (arrows). Black arrows: putative TMH-family proteins. 10

11

FIG. 2. Comparative analysis of the predicted amino acid sequences of CF0218, 12

CAB764, CAB766 and CCA797. 13

(A) Multiple alignment of CF0218, CAB764, CAB766 and CCA797. Boxes indicate 14

identical residues. (B) Comparison of hydropathy profiles of CF0218, CAB764, 15

CAB766 and CCA797. Profiles were determined using the algorithm developed by 16

Kyte and Doolittle (18) with a window size of eleven amino acids. The relative 17

hydrophobicity of each protein is shown on the vertical axis. Negative numbers 18

indicate relative hydrophilicity. The bilobed hydrophobic region present in each protein 19

is boxed. 20

36

1

FIG. 3. Production of recombinant CF0218 and its Immunogenicity. 2

(A) Purified GST alone, GST-CF0218, and GST-cleaved CF0218 were separated by 3

SDS-PAGE and stained with Coomassie brilliant blue (CBB). (B) Equal amounts of 4

recombinant CF0218 shown in (A) and purified C. felis EB were separated by 5

SDS-PAGE and analyzed by immunoblotting using rabbit antiserum raised against 6

CF0218 (B), C. felis -hyperimmunized antiserum (C), C. trachomatis -hyperimmunized 7

antiserum (D) and C. psittaci-hyperimmunized antiserum (E). Molecular weight 8

standards are indicated in kilodaltons on the left side of panels. 9

10

FIG. 4. RT-PCR analysis of cf0218 expression in infected cells. 11

Specific messages for ompA and cf0218 were detected from total RNA of HeLa cells 12

infected with C. felis at the times indicated (including non-infected cells as negative 13

control). DNase I-treated total RNA was applied to reactions in the absence (-) or 14

presence (+) of reverse transcriptase (RT). Message for ompA was amplified as a 15

control for chlamydial infection. C. felis genomic DNA was amplified as a PCR control. 16

17

FIG. 5. Localization of CF0218 in C. felis-infected cells. 18

HeLa cells infected with C. felis for 72 hrs were fixed and stained with DAPI for host 19

cell nuclei and apparent chlamydial inclusion bodies (blue; white arrowheads for large 20

37

chlamydial inclusion bodies), anti chlamydial LPS (red) and anti-CF0218 (green). The 1

right columns indicate merged triple fluorescence images. Scale bars represent 10 2

µm. 3

4

FIG. 6. Antibody response against EB and CF0218 in experimentally vaccinated and 5

C. felis-infected cats. 6

Female-specific-pathogen-free of cats were inoculated with the vaccine containing 7

formalin-inactivated C. felis EB (closed squares, solid line) or with saline (closed 8

circles, dotted line) via the intramuscular route twice (at 0 and 3 wks; open triangles). 9

Subsequently, 5 wks after the first vaccination (black triangles), all cats were 10

inoculated with 104 EID50 of C. felis via the mucosal (conjunctival, oral and nasal) route. 11

To assess antibody response against C. felis EB (A) and CF0218 (B) from sera at 12

each time point, ELISAs were performed using detergent-inactivated C. felis EB and 13

the recombinant CF0218 as antigens. Each sample was measured in duplicate. Data 14

represent the OD value of the mean ± SD. Significant differences (P < 0.01) between 15

control serum (at 0 wk) and at each time point are indicated as asterisks (*: in the 16

vaccinated cats; **: in the non-vaccinated cats). 17

18

C. felis

C. abortus

C. pneumoniae J138

C. trachomatis D/UW-3/Cx

cf0218(mhcB2)

cf0221(lpxB)

cf0222(pcnB2)

cf0224(mrsA1)

cf0225(glmS)

cf0217(mhcB1)

cf0209(pmp21)

cf0208(plsX)

cf0220(mhcA3)

cab756(glmS)cab757cab758

cab759(lpxB)cab760

cab761cab764cab766cab771cab772

cab776(pmp18D)cab777

cpj968(glmS)

cpj967(pgm)

cpj966(pcnB2)

cpj965(lpxB)

cpj963(pmp21)

cpj962(plsX)

ct816(glmS)

ct815(mrsA2)

ct812(pmpD)

ct811(plsX)

TMH locus

The positive clone

Fig. 1

C. caviaecca788(glmS)

cca789(glmM)

cca792(lpxB)cca793

cca794cca795

cca797cca799cca801cca806

cca807(plsX) cca800

CF0218

CAB764

100

100

200

200

300

300

4 4

3 3

2 2

1 1

0 0

-1 -1

-2 -2

-3 -3

100

100

200

200

300

300

400

400

3 3

2 2

1 1

0 0

-1 -1

-2 -2

-3 -3

CAB766100

100

200

200

4 4

3 3

2 2

1 1

0 0

-1 -1

-2 -2

-3 -3

A

B

Hyd

rop

ho

bic

ity

Fig. 2

100

100

200

200

3 3

2 2

1 1

0 0

-1 -1

-2 -2

CCA797

GST

GST-

CF0

218

CF0

218

CBB stain

GST

GST-

CF0

218

CF0

218

IB: anti CF0218

C. felis EBA B

C D

GST

GST-

CF0

218

CF0

218C. felis EB

GST

GST-

CF0

218

CF0

218C. felis EB

IB: anti C. felis IB: anti C. trachomatis

25

37

50

75

25

37

50

75

75

50

37

25

75

50

37

25

75

50

37

25

75

50

37

25

75

50

37

25

Fig. 3

E

75

50

37

25

75

50

37

25

C. felis EB

GST

GST-

CF0

218

CF0

218

IB: anti C. psittaci

ompA

cf0218

RT - + - +- +- + -

non-infected 24 hr 48 hr 72 hr

Fig. 4

C. felis

DNA

DAPI Chlamydia CF0218 MergeC

. fe

lis-i

nfe

cte

dm

ock

Fig. 5

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

0 1 2 3 4 5 6 7 8

0

0.2

0.4

0.6

0.8

1

1.2

0 1 2 3 4 5 6 7 8

A

B

vaccination C. felis challenge

vaccination C. felis challenge

weeks post vaccination

weeks post vaccination

OD

at

405 n

mO

D a

t 405 n

m

Fig. 6

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