Polymorphic repetitive sequences in chlamydiae and uses thereof

BACKGROUND OF THE INVENTION

[0002] The invention relates to the field of diagnosis and treatment of infectious diseases.

[0003] The chlamydiae are obligate intracellular pathogens that cause a variety of diseases in animal species at virtually every phylogenetic level. Of these, Chlamydia (C.) trachomatis and C. pneumoniae are considered the most significant human pathogens. C. trachomatis is the leading cause of preventable blindness worldwide and the most common sexually transmitted bacterial species. C. pneumoniae causes 10% to 20% of community-acquired pneumonia worldwide and has recently been associated with coronary arteriosclerosis and multiple sclerosis. The chlamydiae undergo a developmental cycle unique among prokaryotes. The elementary body is infectious, but is metabolically inactive and cannot replicate. This form differentiates upon infection into the non-infectious reticulate body, a larger pleomorphic bacterium that is metabolically active and multiplies. Following uptake, chlamydiae develop and grow within an intracellular vacuole, called an inclusion, where they will differentiate from the elementary body to the reticulate body then to the elementary body.

[0004] Chlamydiae encode an abundant protein termed the major outer membrane protein (MOMP, or OmpA) that is surface exposed in C. psittaci and C. trachomatis and is the major determinant for serologic classification of chlamydial isolates. This protein is highly variable within its exposed domains except in ruminant invasive C. psittaci, feline strains of C. psittaci and C. pneumoniae, where they are extremely conserved.

[0005] Completion of the sequences of five chlamydial genomes (one C. trachomatis, three C. pneumoniae and one C. muridarum) has revealed the importance of a group of proteins unique to the chlamydiae, the polymorphic membrane proteins (Pmps). These proteins had been shown previously to be antigenic in C. psittaci. The genes encoding for these proteins belong to a complex family and span 13.6 and 17.5% of the C. trachomatis and C. pneumoniae genomes, respectively. There is a considerable expansion of these genes in C. pneumoniae; the C. trachomatis genome possesses 9 pmp genes (A to I) whereas the C. pneumoniae genome possesses 21 pmp genes. Pmps are characterized by two repeated tetrapeptidic motifs, almost never found outside chlamydiae: GGA(L/V/I) (SEQ ID NO: 1) and FXXN (SEQ ID NO: 2). The non-chlamydial proteins exhibiting these motifs have been implicated in the adherence to mammalian tissues. As the Pmps have been localized at the chlamydial cell surface, their role in adhesion, molecular transport, signaling, or some other cell wall associated function is likely.

[0006] Prokaryotic genomes are compact, with sizes ranging from less than 600 kb in Mycoplasma to more than 10 Mb in several cyanobacterial and myxobacterial species. Chlamydial genomes range from 1 to 1.2 Mb. These compact genomes have likely been maintained through selective pressure for rapid DNA replication and cell reproduction. Furthermore, the obligate intracellular way of life of the chlamydiae tends to minimize the length of the genome. It was therefore expected that repetitive sequences would be kept to a minimum under natural selection for rapid growth. Various classes of repetitive DNA elements have been recently discovered in many prokaryotes (Rocha et al., Mol. Biol. Evol. 16:1219-1230, 1999). Such repetitive sequences can be the cause or the hallmark of the plasticity of the genome. Thus, bacteria could have evolved mechanisms based on the presence of repeated sequences for increasing the frequency of random variations in a specific subset of genes. Molecular mechanisms at the basis of this variation are essentially based on slipped-mispair of replicating strands for close repeats and homologous recombination between long intra-chromosomal repeats. These highly mutable loci, sometimes called ‘contingency’ loci, would be involved in critical interactions with the environment, allowing certain phenotypic traits to respond rapidly, by natural selection, to unpredictable changes.

SUMMARY OF THE INVENTION

[0007] Using an in silico approach, we have examined repeats within the complete genomes of chlamydiae. This analysis focused on the search for repeats of statistically significant length, taking into account the genome size and composition. We then determined whether those repeats were sites for sequence variation in vivo.

[0008] We discovered that the repeated sequences in different strains of chlamydiae were polymorphic. The presence of a particular polymorphism can thus be used to detect the presence of a particular strain by detecting the presence of a polymorphic repeated sequence associated with that strain and not associated with other strains of chlamydiae.

[0009] Accordingly, in a first aspect, the invention features a method for determining the presence of a strain of chlamydia in a biological sample. The method includes the steps of (a) providing a biological sample; and (b) determining the presence of a polynucleotide containing a polymorphic repetitive sequence in a polynucleotide in the sample, wherein the polymorphic repetitive sequence is associated with one strain of chlamydia and not associated with other strains of chlamydiae. In this method, the presence of the polynucleotide containing the polymorphic repetitive sequence indicates the presence of that strain of chlamydia.

[0010] In a second, related aspect, the invention features a method for determining the presence of a plurality of strains of chlamydiae in a biological sample. This method includes the steps of: (a) providing a biological sample; and (b) determining the presence in the biological sample of a plurality of polynucleotides, each containing a polymorphic repetitive sequence, wherein each polymorphic repetitive sequence is associated with one strain of chlamydia and not associated with other strains of chlamydiae. In this method, presence of a polymorphic repetitive sequence indicates the presence of the strain of chlamydia associated with that polymorphic repetitive sequence, and the absence of that polymorphic repetitive sequence indicates absence of the associated strain of chlamydia.

[0011] In another aspect, the invention features a method for treating a chlamydial infection in a patient. This method includes the steps of (a) providing a biological sample from the patient; (b) determining the presence in the biological sample of a plurality of polynucleotides, each containing a polymorphic repetitive sequence, wherein each polymorphic repetitive sequence is associated with one strain of chlamydia and not associated with other strains of chlamydiae; and (c) administering to the patient anti-chlamydial agents that are effective against the strains of chlamydiae that are present in the biological sample.

[0012] In any of the foregoing methods, the strain of chlamydia can be a strain of any chlamydial species (e.g., C. psittaci, C. trachomatis, C. pecorum, C. abortus, C. caviae, C. felis, C. suis, C. muridarum, Neochlamydia (N.) hartmannellae, Parachlamydia (P.) acanthamoebae, Simkania (S.) negevensis, and Waddlia (W.) chondrophila). In particular embodiments, the strain of chlamydia is C. pneumoniae strain CWL-029, C. pneumoniae strain AR 39, C. pneumoniae strain J138, or C. trachomatis strain D/UW-3/Cx.

[0013] The polymorphic repetitive sequence can be a simple sequence repeat (SSR); a small close or tandem repeat (TR); or a large repeat (LR). Exemplary SSRs and their locations are listed in Tables 1, 5, 9, 13, and 16, below. The locations of exemplary TRs and LRs are listed in Tables 2-4, 6-8, 10-12, 14, 15, and 17-19.

[0014] The biological sample can be a biopsy sample, blood, serum, peripheral blood mononuclear cells, cerebrospinal fluid, urine, nasal secretion, saliva, or any other biological sample that may contain chlamydiae. The method of detecting the presence of a polymorphic repetitive sequence can include any suitable polynucleotide detection step, e.g., by amplification of polynucleotide molecules that contain a polymorphic repetitive sequence.

[0015] By “chlamydia” or “chlamydiae” is meant organisms of the order Chlamydiales. Examples include, but are not limited to, C. psittaci, C. trachomatis, C. pecorum, C. abortus, C. caviae, C. felis, C. suis, C. muridarum, N. hartmannellae, P. acanthamoebae, S. negevensis, and W. chondrophila. By “chlamydial infection” is meant an infection of a cell or organism by an organism of the order Chlamydiales.

[0016] By “polypeptide” is meant any chain of more than two amino acids, regardless of post-translational modification such as glycosylation or phosphorylation.

[0017] In another aspect, the invention features a purified polypeptide that is substantially identical to a POMP2 polypeptide of SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, or a POMP4 polypeptide of SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.

[0018] In a related aspect, the invention features a purified polynucleotide encoding a polypeptide that is substantially identical to a POMP2 polypeptide of SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, or a POMP4 polypeptide of SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.

[0019] In still another aspect, the invention features a method of identifying a compound useful for treating or preventing an infection of C. pneumoniae. This method includes the steps of: (a) contacting a candidate compound and a POMP polypeptide; and (b) determining the specific binding of the candidate compound to the POMP polypeptide. A candidate compound that specifically binds to the POMP polypeptide is identified as a compound useful for treating or preventing an infection of C. pneumoniae.

[0020] The invention features another method of identifying a compound useful for treating or preventing an infection of C. pneumoniae. This method includes the steps of: (a) contacting a candidate compound and a POMP polynucleotide; and (b) determining the specific binding of the candidate compound to the polynucleotide, wherein a candidate compound that specifically binds to the polynucleotide is identified as a compound useful for treating or preventing an infection of C. pneumoniae.

[0021] The invention also features a method of immunizing a subject against an infection of C. pneumoniae by administering to the subject a purified POMP polypeptide or an immunogenic fragment thereof in an amount sufficient to induce an immune response to the POMP polypeptide or fragment thereof.

[0022] In still other aspects, the invention features a peptide fragment of a POMP2 or POMP4polypeptide, an isolated antibody that specifically binds a POMP2 or POMP4 polypeptide, an antigenic composition that includes a POMP2 or POMP4 polypeptide (or a fragment thereof) and a pharmaceutically acceptable carrier or diluent, and a pharmaceutical composition that includes an antibody that specifically binds a POMP2 or POMP4 polypeptide and a pharmaceutically acceptable carrier or diluent.

[0023] The invention also features a method of producing an immune response in an animal by immunizing the animal with an effective amount of a POMP polypeptide (e.g., a POMP2 or POMP4 polypeptide) or a peptide fragment of a POMP polypeptide.

[0024] POMP polypeptides that are a part of the invention include those that are substantially identical to C. pneumoniae POMP2 or POMP4 (FIGS. 2A-2C and 3A-3C, respectively). POMP polynucleotides that are a part of the invention include those encoding POMP polypeptides as defined above, as well as polynucleotides substantially identical to POMP1, POMP2, POMP3, POMP4, POMP5, POMP6, or POMP7 (FIG. 1).

[0025] By “substantially identical” is meant a polypeptide or polynucleotide exhibiting at least 95%, 99%, 99.5%, or 99.9%, identity to a reference amino acid or polynucleotide sequence. For polypeptides, the length of comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids. For polynucleotides, the length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides.

[0026] Sequence identity is typically measured using sequence analysis software with the default parameters specified therein (e.g., BLAST 2 (Tatusova et al., FEMS Microbiol Lett. 174:247-250, 1999); Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). These programs match similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

[0027] By “high stringency conditions” is meant hybridization in 2+ SSC at 40° C. with a DNA probe length of at least 40 nucleotides. For other definitions of high stringency conditions, see F. Ausubel et al., Current Protocols in Molecular Biology, pp. 6.3.1-6.3.6, John Wiley & Sons, New York, N.Y., 1994, hereby incorporated by reference.

DESCRIPTION OF THE DRAWINGS

[0028]
FIG. 1 is a schematic illustration showing a family of POMP elements, their positions in three strains of C. pneumoniae, the number of cytidines in the SSR, and the genes annotated for their region.

[0029]
FIG. 2A is a schematic illustration showing the amino acid sequence of POMP2 from C. pneumoniae strain CWL-029.

[0030]
FIG. 2B is a schematic illustration showing the amino acid sequence of POMP2 from C. pneumoniae strain J138.

[0031]
FIG. 2C is a schematic illustration showing the amino acid sequence of POMP2 from C. pneumoniae strain AR 39 .

[0032]
FIG. 3A is a schematic illustration showing the amino acid sequence of POMP4 from C. pneumoniae strain CWL-029 .

[0033]
FIG. 3B is a schematic illustration showing the amino acid sequence of POMP4 from C. pneumoniae strain J138.

[0034]
FIG. 3C is a schematic illustration showing the amino acid sequence of POMP4 from C. pneumoniae strain AR 39.

[0035] Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof.

DETAILED DESCRIPTION OF THE INVENTION

[0036] Using algorithms designed to search for different types of repeats, we identified three classes of statistically significant repeats in the complete genomes of sequenced chlamydiae species (C. pneumoniae CWL-029, AR 39, J138, C. trachomatis D/UW-3/Cx, C. muridarum). These include (1) simple sequence repeats (SSRs); (2) small close or tandem repeats (TRs); and (3) large repeats (LRs). TRs and SSRs are thought to change by slipped-mispair at the time of replication or by single-strand annealing when the sequence faces double-strand breaks. Both mechanisms can result in conversion or deletion, but slipped mispair may also result in multiplication. LRs are thought to vary by homologous recombination, and this can lead to conversion or deletion. Additionally, recombination between direct LRs can result in multiplication, whereas recombination between inverted LRs may result in inversion. Hence, different repeats represent different recombination potentials that may result in substantially different outputs.

[0037] We have found when looking at a large collection of strains that these repeated sequences are polymorphic. Thus, the pmp—10.2 cytosine stretch has been shown to be variable within C. pneumoniae strains, resulting in a shift out of frame in CWL-029 but not in AR 39 or TW- 183 (Grimwood et al., Infect. Immun. 69:2383-2389, 2001). Another difference between C. pneumoniae strains is a 393 nucleotide sequence (coding for 131 amino acids) in the 5′ part of pmp—6, which is present three times in CWL-029 and J138 but only two times in AR 39.

[0038] These polymorphisms can be used as molecular markers that might differentiate strains bearing a conserved MOMP. The identification of subgroups within these groups (ruminant invasive C. psittaci, feline C. psittaci, and C. pneumoniae) should allow the search for correlations with the virulence and the different observed clinical syndromes.

[0039] Polymorphisms within simple and tandem repeats, according to their position in a coding or non-coding region, will generate a stop codon (if the modification in length is not a multiple of three) or a modification of the length of the promoter. Both mechanisms will lead to a modulation of the functional protein. The presence of polymorphisms allows for the identification of particular strains based on the presence of a particular polymorphism or pattern of polymorphisms.

[0040] Diagnostic Assays

[0041] As the presence of a particular polymorphic repetitive sequence is likely to correlate with the presence of a particular strain of chlamydiae, the invention features a method for determining the presence of one or more strains of chlamydiae in a patient. In the methods of the invention, a sample from an individual, such as an individual who is suspected of having a chlamydial infection or a disease associated with a chlamydial infection, is used. The test sample can include blood, serum, cerebrospinal fluid, urine, nasal secretion, saliva, or any other bodily fluid or tissue, or polynucleotides isolated from one of the foregoing samples.

[0042] The sample can be assayed for the presence or absence of the polymorphic repetitive sequence by Southern hybridization using a detectable probe for the appropriate polymorphic repetitive sequence. Alternatively, the test sample can be assayed using quantitative PCR or RT-PCR (e.g., by using a LightCycler™ (Idaho Technology Inc., Idaho Falls, Id.) and fluorescent LightCycler™ probes). The presence of the polymorphic repetitive sequence in the test sample is indicative of the presence of chlamydiae in the test sample. To facilitate assaying a test sample for the presence or absence of chlamydiae by detecting the presence or absence of a polymorphic repetitive sequence, the test sample can be subjected to methods to enhance isolation of chlamydia elementary bodies from the test sample and to release DNA from the elementary bodies. For example, elementary bodies have a tendency to adhere to the walls of a receptacle containing them; the elementary bodies can be removed from the receptacle by treating the receptacle containing the elementary bodies with trypsin/EDTA, thereby releasing elementary bodies that adhered to the receptacle; and then concentrating the released elementary bodies, such as by centrifugation or filtration. To release DNA from elementary bodies, the elementary bodies are incubated under disulfide reducing conditions, such as incubating the elementary bodies with a disulfide reducing agent such as dithiothreitol (DTT) or 2-mercaptoethanol; and digesting the elementary bodies with a protease (see, e.g., U.S. Pat. No. 6,258,532, hereby incorporated by reference).

[0043] The test sample can also be assayed for the presence of chlamydiae by detecting the presence of a polymorphic repetitive sequence in a protein from chlamydia. For example, the presence of a PMP protein having a particular polymorphic repetitive sequence in the test sample can be detected through the use of ELISA methodologies with an antibody that specifically recognizes the polymorphic repetitive sequence. Alternatively, the test sample may be assayed for the presence of chlamydiae by detecting the presence of human antibodies to polymorphic repetitive sequences in the test sample. The presence of a polymorphic repetitive sequence or antibodies to a polymorphic repetitive sequence in the test sample is indicative of the presence of chlamydiae in the test sample. The presence of proteins or antibodies may be detected by appropriate methods such as by ELISA, western blot, or isoelectric focusing.

[0044] The diagnostic methods described herein are useful for detecting or confirming the disease in a patient, as well as for monitoring the progress of the disease. Disease monitoring is useful, for example, for determining the efficacy of a particular therapy.

[0045] The invention will be further illustrated by the following non-limiting examples.

[0046] Identification of Polymorphic Repetitive Sequences

[0047] The tables containing the elements found in the five chlamydia genomes follow below ordered by genome and by repeat type. The GenBank accession identification numbers are as follows: AE001363 (C. pneumoniae CWL-029; Kalman et al., Nature Genet. 21:385-389, 1999); AE002161 (C. pneumoniae AR 39; Read et al., Nucleic Acids Res. 28:1397-1406, 2000); BA000008 (C. pneumoniae J138; Shirai et al., Nucleic Acids Res. 28:2311-2314, 2000); AE001273 (C. trachomatis; Stephens et al., Science 282:754-759, 1998); AE002160 (C. muridarum; Read et al., Nucleic Acids Res. 28:1397-1406, 2000). “ID” indicates an identification tag, “position” indicates the position of the start of the repeat in the respective genomes, “length” indicates the repeat length, “gene” indicates the gene where the repeat was found (if applicable), “sense” indicates if the repeat in the direct (d) or inverse (i) strand, “equivalent to” indicates the equivalent elements of the other genomes, “note” includes either the strand of the gene where the repeat stands or the flanking genes, in which case “D/C” stands for the position of the genes (direct or complement strands), and UFO indicates an unknown function ORF. For large repeats, “first” refers to the first occurrence of the repeat and “second” to the second occurrence. “Period” is in the form A×B, wherein “A” indicates the number of times the motif is repeated and “B” indicates the length of repeat. By “-” is meant that a consensus cannot be established to determine A and B with precision.

1TABLE 1Chlamydia pneumoniae strain CWL-029 SSRsIDPositionlengthgenesensenoteequivalent toC(G)N(N>11) C11080614INTdD/UFO/CPn0007 D/UFO/CPn0008J1, A4 C21335014INTdD/UFO/CPn0009 D/UFO/CPn0010J2 C32058814pmp_2dDA3, J3 C45847414CPn0043dDA2, J4 C58533614CPn0069dD— C650720013pmp_10.2dCJ5, A1 C7120706113CPn1054dDJ6 C8120960912INTdD/UFO/CPn1054 D/UFO/CPn1055A5, J7ACCN /CACN(N>3) C962840014CPn0542dD/ABC transporterJ8, A9TCCN (N>4)C10115053015ftsHdD/proteaseJ10, A8TTCN (N>4)C1195621215yphCdC/GTPaseJ11, A7CGTN/GTCN (N>3)C1260726013CPn0525D/UFOJ9, A6, TR4, M7ATGCTN(N>2)C1325815815ypdPdD/UFOJ12, A11ATTAAN (N>2)C1440792915INTdC/sigma/rpsD C/flagelar secretion/flhAJ13TTTCTN (N>2)C1539638715CPn0352dD/UFOJ14, A10

[0048]

2

TABLE 2

Chlamydia pneumoniae
strain CWL-029 TRs

id
Position
length
period
genes
note
equivalent to

C1
7547
937
3 × 330
INT
C/UFO/CPn0006 D/UFO/CPn0007
A12

C2
10807
178
2 × 89
INT
D/UFO/CPn0007 D/UFO/CPn0008
- (A12?)

C3
240764
45
2 × 13
INT
D/oppA_4 D/oppB_1
J2, A11

C4
255447
35
2 × 14
INT
C/UFO/CPn0214
A9, J4

C5
278045
26
⅔ × 8
INT
C/CPn0240 C/CPn0241
A8, J5

C6
341108
32
2 × 15
lpxD
D/UDP-acyltransferase
J6, A7

C7
379100
40
—
INT
many erased, C/CPn0333 D/CPn0334
A6, J7

C8
432020
330
—
hctB
many erased, C/histone like
J8, A5, M1, TR1

C9
451458
30
2 × 15
CPn0405
C/UFO
J9, A4

C10
492298
20
4 × 6
pmp_6
D
J10, A3

C11
568858
55
2 × 18
CPn0487
C/UFO
A2, J11

C12
662224
80
—
INT
many erased, C/murA D/UFOCPn0572
J12 A1 M9 TR4

C13
916873
70
—
CPn0809
many erased C/UFO
J13, A16, M11,

TR7

C14
984289
30
2 × 13
rodA
D/rod shape protein
J14, A15

C15
1028449
26
2 × 13
CPn0897
C/phosphohydrolase
J15, A14

C16
1085124
18
3 × 6
glgA
C/glycogen synthase
J16, A13

[0049]

3

TABLE 3

Chlamydia pneumoniae
strain CWL-029 LRs (inverse)

id
First
second
length
first
Second
equivalent to

C1
207095
208884
35
D/CPnO165/UFO
C/CPnO169IUFO
J1 A1

C2
493543
506266
23
D/pmp_6
INT
J3 A3

C3
954974
955029
32
C/CPn0843
C/CPn0843
J2 A2

[0050]

4

TABLE 4

Chlamydia pneumoniae strain CWL-029 LRs (direct)

id
First
second
length
first
Second
equivalent to

C1
26238
29415
23
D/pmp_4.2
D/pmp_5.2
—

C2
234959
236693
27
D/oppA_1
D/oppA_2
J1 A10

C3
259232
259385
26
INT
D/tgt/tRNA transferase
J2 A9

C4
290023
292838
40
C/CPn0255/UFO
INT
J3 A8

C5
415142
416513
31
D/CPn0369/UFO
D/CPn0370/UFO
J4 A6

C6
495909
498766
23
D/pmp_7
D/pmp_8
J6 A4

C7
501979
514804
24
D/pmp_9
D/pmp_13
J7 A3

C8
522778
525176
28
C/CPn0457/UFO
C/CPn0458/UFO
J8 A2

C9
528528
530945
29
C/CPn0461/UFO
C/CPn0462/UFO
J9 A1

C10
1111630
1113279
1650
D/glmS/amynotransferase
D/yccA_transport trunc
A13

D/tyrP_1/transport
D/tyrP_2/transport

[0051]

5

TABLE 5

Chlamydia pneumoniae
strain AR 39 SSRs

id
Position
length
gene
sense
Note
equivalent to

G(C)N(N>11)

A1
334377
13
CP0303
d
D/PmpG
C6

A2
782709
14
CP0730
d
C/UFO
C4 J4

A3
820588
14
CP0761
d
C/PmpG
C3 J3

A4
830377
14
INT
d
C/CP0766/UFO C/CP0767/UFO
J1 C1

A5
861807
15
INT

C/CP0795 C/CP0796
C8 J7

ACGN/GACN (N>3)

A6
234314
13
CP0228
d
C/UFO
J9 C12 TR4 M7

GAAN (N>4)

A7
1115211
15
CP1025
d
D/GTP_binding
J11 C11

GGAN (N>4)

A8
920865
15
CP0857
d
C/FtsH
C10 J10

GGTN/GTGN (N>3)

A9
213173
13
INT
d
C/CP0209 C/CP0211 ABC transporters
C9 J8

AGAAAN(N>2)

A10
444793
15
INT
d
C/CP0406/UFO C/CP0408/ATP carrier
J14 C15

AGCATN (N>2)

A11
583107
15
CP0548
d
C/UFO
J12 C13

TTAATN (N>2)

A12
433252
15
CP0415
d
D/reductoisomerase
—

[0052]

6

TABLE 6

Chlamydia pneumoniae
strain AR 39 TRs

id
Position
length
period
genes
Note
equivalent to

A1
179248
135
—
INT
C/CP0177/UFO D/CP0178/transferase
J12 C12 M9 TR4

A2
272675
43
⅔ × 13
CP0267
D/UFO
C11 J11

A3
349273
18
3 × 6
CP0309
C/PmpG
J10 C10

A4
389712
30
2 × 15
CP0350
D/UFO
C9 J9

A5
408951
235
—
CP0371
D/Nucleoprotein
J8 C8 M1 TR1

A6
462041
145
—
INT
C/UFO/CP0424 D/UFO/CP0425
C7 J7

A7
500140
33
2 × 15
CP0456
C/UDP-transferase
C6 J6

A8
563145
200
—
INT
D/UFO/CP0521 D/UFO/CP0522
C5 J5

A9
585797
35
2 × 14
CP0551
D/UFO
C4 J4

A10
587034
24
¾ × 7
INT
D/UFO/CP0551 D/UFO/CP0553
(J3?)

A11
600477
46
2 × 13
INT
C/UFO/CP0568 C/UFO/CP0569
C3 J2

A12
832710
990
3 × 330
CP0769
C/UFO
C1

A13
986268
18
3 × 6
CP0911
D/glycogen synthase
J16 C16

A14
1042952
37
2 × 13
CP0969
D/UFO
J15 C15

A15
1087106
43
2 × 13
CP1002
C/MrdB
J14 C14

A16
1154483
40
—
CP1062
D/UFO
C13 J13 M11 TR7

[0053]

7

TABLE 7

Chlamydia pneumoniae
strain AR 39 LRs (inverse)

id
First
second
length
First
second
equivalent to

A1
632368
634157
35
D/CP0602/UFO
C/CP0606/UFO
J1 C1

A2
1116377
1116432
32
D/CP1026
D/CP1026 frameshifted
C3 J2

A3
335302
348025
23
D/CP0303/pmpG
C/CP0309/pmpG
C2 J3

[0054]

8

TABLE 8

Chlamydia pneumoniae
strain AR 39 LRs (direct)

id
first
second
length
First
second
equivalent to

A1
310615
313032
29
D/CP0290/UFO
D/CP0291/UFO
C9 J9

A2
316385
318783
28
D/CP0294/UFO
D/CP0295/UFO
C8 J8

A3
326762
339588
24
C/CP0299/pmpG
C/CP0306/pmpG
J7 C7

A4
342802
345659
23
C/CP0307/pmpG
C/CP0308/pmpG
J6 C6

A5
349362
349755
47
C/CP0309/pmpG
C/CP0309/pmpG
J5

A6
424652
426023
31
C/CP0387/UFO
C/CP0388/UFO
J4 C5

A7
540623
541657
449/365
C/CP0493/UFO
C/CP0495/UFO
—

A8
548403
551218
40
INT
D/CP0506/UFO
J3 C4

A9
581869
582022
26
C/CP0546/tRNA transferase
INT
J2 C3

A10
604567
606301
27
C/CP0571/ABC tr
C/CP0572/ABC tr
J1 C2

A11
811759
814936
24
INT
INT
—

A12
947563
948860
1144
C/CP0878/UFO
C/CP0879/UFO
—

A13
956482
958131
1650
C/CP0888/UFO
C/CP0891/permease
C10

C/CP0889/permease

[0055]

9

TABLE 9

Chlamydia pneumoniae
strain J138 SSRs

id
position
length
gene
sense
note
equivalent to

C(G)N(N>11)

J1
10806
14
INT
d
D/UFO/CPj0007 D/UFO/CPj0008
C1 A4

J2
13350
13
INT
d
D/UFO/CPj0009 D/UFO/CPj0010
C2

J3
20597
13
pmp_2_1
d
D
C3 A3

J4
58475
14
CPj0043
d
D/UFO
C4 A2

J5
506847
14
pmp_10
d
C
C6 A1

J6
1205090
12
CPj1054
d
D/UFO
C7

J7
1207641
16
INT
d
D/UFO/CPj1054 D/UFO/CPj1055 grey hol
C8 A5

ACCN/CACN (N>3)

J8
628050
14
CPj0542
d
D/ABC transp
C9 A9

CGTN/GTCN (N>3)

J9
606910
14
CPj0525
d
D/UFO
C12 A6, TR4, M7

TCCN (N>4)

J10
1148562
15
ftsH
d
D
C10 A8

TTCN/TTCN (N>4)

J11
955863
15
yphC
d
C/GTPase
C11 A7

ATGCTN(N>2)

J12
258110
15
ypdP
d
D/UFO
C13 A11

ATTAAN (N>2)

J13
407968
15
flhA
d
C
C14

TTTCTN (N>2)

J14
396425
15
CPj0352
d
D
C15 A10

[0056]

10

TABLE 10

Chlamydia pneumoniae
strain J138 TRs

id
position
length
period
genes
note
equivalent to

J1
127027
80
2 × 40
htrB_1
C/acyltransferase
—

J2
240709
38
2 × 13
INT
D/oppA_4 D/oppB_1
C3 A11

J3
254172
28
4 × 7
CPj0213
C/UFO
- (A10?)

J4
255396
38
2 × 17
CPj0214
C/UFO
A9 C4

J5
277997
60
—
INT
C/UFO/CPj0240 C/UFO/CPj0241
C5 A8

J6
341060
30
2 × 15
lpxD
D/UDP-acyltransferase
C6 A7

J7
379052
140
—
INT
C//ltuB/CPj0333 D/CPj0334
A6 C7

J8
432059
300
—
hctB
C/histone like
C8 A5 M1 TR1

J9
451497
30
2 × 15
CPj0405
C/UFO
C9 A4

J10
491945
25
¾ × 6
pmp_6
D
C10 A3

J11
568506
56
2 × 18
CPj0487
C/UFO
A2 C11

J12
661875
120
—
INT
C/murA D/CPj0572
C12 A1 M9 TR4

J13
916525
80
—
CPj0809
C/UFO
A16 C13 M11 TR9

J14
983940
43
2 × 13
rodA
D
C14 A15

J15
1028100
37
2 × 13
CPj0897
C/phosphoydrolase
C15 A14

J16
1084803
18
3 × 6
glgA
C/glycogen synthase
C16 A13

[0057]

11

TABLE 11

Chlamydia pneumoniae
strain J138 LRs (inverse)

id
First
second
length
first
second
equivalent to

J1
207048
208837
35
D/CPj0165/UFO
C/CPj0169/UFO
C1 A1

J2
954625
954680
32
INT-C/CPj0843/UFO
C/CPj0843/UFO
C3 A2

J3
493190
505913
23
D/pmp_6
C/pmp_10
C2 A3

[0058]

12

TABLE 12

Chlamydia pneumoniae
strain J138 LRs (direct)

id
first
second
length
first
second
equivalent to

J1
234904
236638
27
D/oppA_1
D/oppA_2
C2 A10

J2
259184
259337
26
INT
D/tgt
C3 A9

J3
289975
292790
40
C/CPj0255/UFO
C/CPj0259/UFO
C4 A8

J4
415181
416552
31
D/CPj0369/UFO
D/CPj0370/UFO
C5 A6

J5
491436
491829
47
D/pmp_6
D/pmp_6
A5

J6
495556
498413
23
D/pmp_7
D/pmp_8
C6 A4

J7
501626
514452
24
D/pmp_9
D/pmp_9 (2pmp9...)
C7 A3

J8
522427
524825
28
C/CPj0457/UFO
C/CPj0458/UFO
C8 A2

J9
528177
530594
29
C/CPj0461/UFO
C/CPj0462/UFO
C9 A1

[0059]

13

TABLE 13

Chlamydia trachomatis
strain D/UW-3/Cx SSRs

id
position
length
gene
sense
note
equivalent to

C(G)N(N>11)

TR1
291810
12
INT
d
C/CT259 D/CT260
—

GTN (N>5)

TR2
964233
12
ftsY
d
C/cell division
—

ATTN (N>4)

TR3
1008839
15
CT857
d
D/UFO
—

CGTN (N>3)

TR4
456967
15
CT398
d
D/UFO
M7, J9 A6, C12

GCAN (N>4)

TR5
531772
15
CT456

D/UFO
—

TGCAAN (N>2)

TR6
687502
15
uvrD
d
D
—

[0060]

14

TABLE 14

Chlamydia trachomatis
strain D/UW-3/Cx TRs

id
position
Length
period
Genes
note
equivalent to

TR1
51545
400
15 bp -
hctB
many erased, D/histone like
M1 J8 A5 C8

TR2
511072
53
2 × 15
tsp
D/protease
—

TR3
527891
53
2 × 17
argS
D/tRNA transferase
—

TR4
531363
450
3 × 150
CT456
D/UFO
J12 A1 C12 M9

TR5
532487
18
3 × 6
CT456
D/UFO
—

TR6
613891
18
3 × 6
dnaE
D/DNA pol III
—

TR7
650720
140
—
CT578
D/UFO
M11 J13 A16 C13

TR8
657611
58
2 × 13
gp6D
D/UFO/plasmid paralog
—

TR9
861061
40
2 × 16
CT741
C/UFO
—

TR10
984536
45
2 × 13
INT
D/tRNASer_4 D/CT837
—

[0061]

15

TABLE 15

Chlamydia trachomatis
strain D/UW-3/Cx LRs (direct)

id
first
second
Length
first
second

TR1
485249
574902
25
tRNASer_3
TRNASer_2

TR2
853782
875828
5474
rRNA + tRNA
rRNA + tRNA

[0062]

16

TABLE 16

Chlamydia muridarum
SSRs

id
position
length
gene
sense
Note
equivalent to

C(G)N(N>11)

M1
501838
12
TC0436
d
C/phospholipase
—

M2
496505
12
INT
i
C/TC0432 C/TC0433 phospholipases
—

M3
542159
13
TC0447
i
D/phospholipase
—

ACN (N>5)

M4
787670
12
TC0662
d
D/UFO
—

ACAN (N>4)

M5
1001176
15
TC0868
d
D/UFO
—

AGCN (N>4)

M6
212303
17
TC0181
d
C
—

CGTN (N>3)

M7
807276
15
TC0677
d
D/UFO
J9 C12 TR4 A6

CCTCCN (N>2)

M8
889812
15
TC0750
d
D/UFO
—

GAGAGN (N>2)

M9
272616
15
TC0235
d
C/UFO
—

[0063]

17

TABLE 17

Chlamydia muridarum
TRs

id
position
length
period
genes
note
similar to

M1
369850
450
—
TC0337
C/UFO
TR1 J8 A5 C8

M2
452977
50
4 × 14
TC0392
D/UFO
—

M3
602448
48
2 × 21
TC0500
C/UFO
—

M4
715922
975
5 × 201
TC0602
C/helicase
—

M5
738193
37
2 × 17
TC0618
C/dehydrogenase
—

M6
756226
55
2 × 13
TC0634
C/UFO
—

M7
758966
22
2 × 13
TC0635
C/UFO
—

M8
871834
44
2 × 13
TC0733
C/SecDF
—

M9
881447
930
3 × 330
TC0741
D/UFO
J12 A1 TR4 C12

M10
985017
23
2 × 13
INT
D/TC0850/type III secretion
—

D/TC0853/type III membrane

M11
1000266
80
—
TC0867
D/UFO
TR7 J13 C13 A16

M12
1036721
18
3 × 6
TC0898
D/helicase/uvrD
—

[0064]

18

TABLE 18

Chlamydia muridarum
LRs (inverse)

first
second
length
First
Second

M1
93051
985895
23
C/TC0080/trigger factor
D/TC0853/type III

M2
495386
533316
1150
C/TC0432/phospholipase
D/TC0440/phospholipase

M3
497071
533316
978
C/TC0433/phospholipase
D/TC0440/phospholipase

[0065]

19

TABLE 19

LRs (direct)

first
second
length
First
Second

M1
133478
151897
1050
D/TC0113/UFO-INT
INT-rRNA

M2
134545
156503
800
C/TC0114/UFO-INT
C/TC0130/UFO-INT

M3
236729
238122
25
D/TC0204/permease
D/TC0205/permease

M4
495294
496810
1244
C/TC0432/phospholipase
C/TC0433/phospholipase

M5
503667
513542
23
D/TC0437/adherence
D/TC0438/adherence

M6
539556
540091
119
D/TC0444/UFO
C/TC0445/UFO

M7
834344
923737
1050
C/tRNA-Ser-3
C/tRNA-Ser-4

D/TC0696/ABC transport
D/TC0784/helicase

[0066] Some interesting features can be observed from these data. First, repeated sequences are more frequent in C. pneumoniae strains than in non-C. pneumoniae strains. This is true for SSRs, TRs, and direct LRs (t-student test, P<0.05 for SSR and TR and P<0. 1 for LDR) (Table 20).

20TABLE 20SSRTRLDRLIRMultipletsCpn CW15161031Cpn A12161330Cpn J1416931Ctr610200Cmu91273

[0067] Second, several of these repeated sequences fall within the pmp locus. Indeed, even if larger numbers of LRs in C. pneumoniae can be attributed to the Pmp proteins, the larger numbers of SSRs and TRs are typically outside of these elements and possibly reflect other variation strategies. Third, this approach allowed us to discover a new family of seven genes encoding proteins that we call POMPs for polymorphic outer membrane proteins (FIG. 1).

[0068] Characterization of the POMPs

[0069] We performed a similarity search, motif analysis, and detection of transmembrane domains.

[0070] BLAST searches on the complete GenBank/EMBL/DDBJ database provided for no significant hits at E<10−10, except for C. pneumoniae sequences. The same result was observed when we performed a full search for orthologues in completely sequenced genomes (including C. trachomatis and C. muridarum). Finally, we carried out BLAST searches on the TIGR database of unfinished genomes, and against the fully sequenced, but still non-annotated genome of C. psitacci, also without positive results. Based on our searches, we concluded that POMP elements were specific to C. pneumoniae, perhaps having horizontally transferred after divergence with the other fully sequenced chlamydiae.

[0071] The analysis of the amino acid content revealed an excess of some residues, including cysteine, a residue that is characteristic of outer membrane proteins of C. pneumoniae (e.g. in Pmp; Melgosa et al, FEMS Lett., 112:199-204). We then determined whether the hydrophobicity profile, presence of putative transmembrane domains, and von Heijne's method for signal sequence recognition agreed in the prediction of a signal peptide. These methods indicated a signal peptide domain that would be cleaved at residue 51. We then used Klein's method for transmembrane region allocation, which predicted a transmembrane domain in residues 68-84. A similar result was obtained by using Top-pred. Using MTOP, we then predicted the membrane topology of the peptide. Results indicated that the N-terminal side should be inside, and the C-terminus outside. Thus, bioinformatic analyses consistently suggested that the POMP peptide was a membrane protein with one transmembrane segment, and a cytoplasmic N-terminus.

[0072] The putative amino acid sequences of POMP2 and POMP4 polypeptides are depicted in FIGS. 2A-2C and 3A-3C, respectively. The corresponding polynucleotide sequences are found at the region of the annotated sequence indicated in FIG. 1.

[0073] The identification of POMPs as a multigenic family restricted to C. pneumoniae strains implicates the POMP polynucleotides and polypeptides as being useful in the development of therapeutic and diagnostic agents, as described below.

[0074] Antibodies

[0075] The POMP polypeptides and polynucleotides of the invention (or variants thereof) or cells expressing the same can be used as immunogens to produce antibodies immunospecific for such polypeptides or polynucleotides respectively. Antibodies generated against POMP polypeptides or polynucleotides can be obtained by administering the polypeptides and/or polynucleotides, or epitope-bearing fragments of either or both, analogues of either or both, or cells expressing either or both, to an animal, preferably a nonhuman, using routine protocols. For preparation of monoclonal antibodies, any technique known in the art that provides antibodies produced by continuous cell line cultures can be used. Techniques for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce single chain antibodies to polypeptides or polynucleotides of this invention. Additionally, transgenic mice, or other organisms such as other mammals, may be used to express humanized antibodies immunospecific to the POMP polypeptides or polynucleotides of the invention. Phage display technology may be also utilized to select antibody genes with binding activities towards a POMP polypeptide of the invention, either from repertoires of PCR amplified v-genes of lymphocytes from humans screened for possessing anti-POMP, or from naive libraries. The affinity of these antibodies can also be improved by, for example, chain shuffling.

[0076] The above-described antibodies may be employed to isolate or to identify clones expressing a POMP polypeptide or polynucleotide of the invention to purify the polypeptide or polynucleotide by, for example, affinity chromatography. Antibodies against a POMP polypeptide or POMP polynucleotide may be employed to treat infections of C. pneumoniae.

[0077] In accordance with an aspect of the invention, there is provided the use of a POMP polynucleotide of the invention for therapeutic or prophylactic purposes, in particular genetic immunization. Among the particularly preferred embodiments of the invention are naturally occurring allelic variants of POMP polynucleotides and polypeptides encoded thereby. The use of a polynucleotide of the invention in genetic immunization will preferably employ a suitable delivery method such as direct injection of plasmid DNA into muscles, delivery of DNA complexed with specific protein carriers, coprecipitation of DNA with calcium phosphate, encapsulation of DNA in various forms of liposomes, particle bombardment, or in vivo infection using cloned retroviral vectors.

[0078] Drug Screening

[0079] POMP polypeptides and polynucleotides of the invention may also be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures. These substrates and ligands may be naturally occurring or may be structural or functional mimetics. In general, antagonists of POMP function may be employed for therapeutic and prophylactic purposes for treating infections of C. pneumoniae. The screening methods may simply measure the binding of a candidate compound to a POMP polypeptide or polynucleotide, or to cells or membranes bearing the polypeptide or polynucleotide, or a fusion protein of the polypeptide by means of a label directly or indirectly associated with the candidate compound. Alternatively, the screening method may involve competition with a labeled competitor. Further, these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the POMP polypeptide, using detection systems appropriate to the cells expressing the POMP polypeptide. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed.

[0080] POMP polypeptides may be used to identify membrane bound or soluble receptors, if any, for such polypeptide, through standard receptor binding techniques known in the art. These techniques include, but are not limited to, ligand binding and crosslinking assays in which the polypeptide is labeled with a radioactive isotope (for instance, 125I), chemically modified (for instance, biotinylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (e.g., cells, cell membranes, cell supernatants, tissue extracts, bodily materials). Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy. These screening methods may also be used to identify agonists and antagonists of the polypeptide that compete with the binding of the polypeptide to its receptor(s), if any. Standard methods for conducting such assays are well understood in the art.

[0081] Vaccines

[0082] The invention provides a method for inducing an immunological response in an individual, particularly a mammal, by inoculating the individual with a POMP polynucleotide and/or polypeptide, or a fragment or variant thereof, adequate to produce antibody and/or T cell immune response to protect that individual from an infection of C. pneumoniae.

[0083] A polypeptide of the invention may be used as an antigen for vaccination of a host to produce specific antibodies which protect against invasion of C. pneumoniae, for example by blocking adherence of bacteria to damaged tissue. Examples of tissue damage include wounds in skin or connective tissue caused, for example, by mechanical, chemical, thermal or radiation damage or by implantation of indwelling devices, or wounds in the mucous membranes, such as the mouth, throat, mammary glands, urethra, or vagina.

[0084] The invention also includes a vaccine formulation that includes an immunogenic recombinant polypeptide and/or polynucleotide of the invention together with a suitable carrier, such as a pharmaceutically acceptable carrier. Since the polypeptides and polynucleotides may be broken down in the stomach, each is preferably administered parenterally, including, for example, administration that is subcutaneous, intramuscular, intravenous, or intradermal. Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostatic compounds and solutes which render the formulation isotonic with the bodily fluid, preferably the blood, of the individual; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use. The vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.

[0085] Diagnostics

[0086] Antibodies that specifically bind a POMP polypeptide may be used for the diagnosis of an infection of C. pneumoniae, or in assays to monitor patients being treated for an infection of C. pneumoniae. The antibodies useful for diagnostic purposes may be prepared in the same manner as those described above for therapeutics. Diagnostic assays for POMP polypeptides include methods that utilize the antibody and a label to detect POMP polypeptides in human body fluids or extracts of cells or tissues. The antibodies may be used with or without modification, and may be labeled by joining them, either covalently or non-covalently, with a reporter molecule. A wide variety of reporter molecules known in the art may be used. A variety of detection protocols (e.g., ELISA, RIA, and FACS) are also known in the art and provide a basis for diagnosing an infection of C. pneumoniae on the basis of detection of a POMP polypeptide.

[0087] POMP polynucleotides may also be used for diagnostic purposes. POMP polynucleotide sequences that may be used include antisense RNA and DNA molecules, and oligonucleotide sequences. The POMP polynucleotides may be used to detect and quantitate POMP expression in biopsied tissues. The diagnostic assay may be used to monitor an infection of C. pneumoniae during therapeutic intervention.

[0088] POMP polynucleotides may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; or in dip stick, pIN, ELISA or chip assays utilizing fluids or tissues from patient biopsies to detect an infection of C. pneumoniae. Such qualitative or quantitative methods are well known in the art.

[0089] POMP polynucleotides may be labeled by standard methods, and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantitated and compared with a standard value. If the amount of signal in the biopsied or extracted sample is significantly altered from that of a comparable control sample, the labeled POMP polynucleotides have hybridized with polynucleotide sequences in the sample, indicating the presence of C. pneumoniae in the sample. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or in monitoring the treatment of an individual patient.

[0090] Once disease is established and a treatment protocol is initiated, hybridization assays may be repeated on a regular basis to evaluate whether the expression in the patient is eliminated. The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.

[0091] The foregoing results were obtained using the following methods.

[0092] Methods

[0093] An SSR is a strictly tandem repeat with n elements of a motif X (e.g., 3 CG in CGCGCG). Considering L, the length of the genome, the probability of not finding Xn anywhere is given by the formula:

P
=(1−fXn)L

[0094] where fX is the relative frequency of the motif X in the genome. We used a threshold p-value of 0.01 and searched for significant SSR elements with motifs ranging in length from 1 to 5 nucleotides, in all genomes of chlamydiae using standard pattern matching methods.

[0095] LR minimal length was defined through the use of a statistic of extremes that takes into account the composition in nucleotides and the length of the genome. For chlamydiae, this value is in the range of 25 nucleotides, which coincides with the minimal region of strict homology required for homologous recombination in E. coli and B. subtilis.

[0096] A small repeat was kept if it had at least the minimal significant length and if its two or more copies occur at short distances (<1 kbp). We searched for such repeats in sliding windows of 1000 bp, and for each window we computed the extreme statistics that allowed the definition of the length threshold in the window. These values varied slightly from window to window (in function of the window composition), but typically ranged from 12 to 14 bp. Then we inspected for more distinctive tandem repeats, by identifying repeats with occurrences at less than 50 bp apart, and for those with copies distant less than three times their length and by eye-checking all the others using dot-plots.

Polymorphic repetitive sequences in chlamydiae and uses thereof

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CPC

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