From the Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio 43210-1093
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ABSTRACT |
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Human granulocytic ehrlichiosis (HGE) is caused
by infection with an obligatory intracellular bacterium, the HGE agent.
We previously cloned a gene encoding HGE agent 44-kDa major outer membrane protein and designated it p44. In this study, we
(i) identified five different mRNAs that are transcribed from
p44-homologous genes in the HGE agent cultivated in HL-60
cells; (ii) cloned genes corresponding to the mRNAs from the
genomic DNA of the HGE agent; (iii) showed that the genes being
expressed were not clustered in the HGE agent genome; (iv) estimated
that a minimum copy number of the p44-homologous genes in
the genome is 18; (v) detected two different P44-homologous proteins
expressed by the HGE agent; and (vi) demonstrated existence of
antibodies specific to the two proteins in sera from patients with HGE.
These findings showed that p44 multigenes have several
active expression sites and the expression is regulated at
transcriptional level, suggesting a potentially unique mechanism for
generating the diversity in major antigenic outer membrane proteins of
the HGE agent. Characterization of p44-homologous genes
expressed by the HGE agent in a tissue culture would assist in
understanding a role of the p44 multigene family in
pathogenesis and immune response in HGE.
Human granulocytic ehrlichiosis
(HGE),1 a tick-borne
zoonosis, was first reported in 1994 and increasingly recognized in the United States (1-3). Serologic and PCR analyses suggest that HGE also
exists in Europe (4-6). HGE is characterized by chills, headache,
myalgia, and hematological abnormalities, including leukopenia and
thrombocytopenia. It frequently requires prolonged hospitalization, and
when the treatment is delayed due to misdiagnosis or in
immunocompromized patients, HGE can be fatal (7). HGE is caused by
infection with an obligatory intracellular bacterium, HGE agent.
Comparison of 16 S rRNA gene sequences (2) and ultrastructure (8)
indicates that the HGE agent is closely related to Ehrlichia phagocytophila, the agent of tick-borne fever, and Ehrlichia
equi, the agent of equine ehrlichiosis. The HGE agent is
transmitted by the Ixodes sp. tick (9), and the white-footed
mouse is considered to be the major reservoir of the HGE agent in the
United States (9).
Although the successful culture isolation of the organism was
accomplished in 1995, little is known about the pathogenesis and
intracellular parasitism of the HGE agent at the molecular level.
Recent studies of the HGE agent showed that 38-49-kDa proteins of this
organism are dominant antigens recognized by the sera from patients
with HGE (10-13). We demonstrated that these proteins are present in
the outer membrane fraction of our five human patient isolates of the
HGE agent and of a tick isolate (USG3) of the granulocytic
Ehrlichia (GE) sp. (12). More recently, we cloned, sequenced, and expressed in Escherichia coli a gene encoding
a 44-kDa protein of the HGE agent (HZ isolate (8), also called isolate
13 (12-14)), and designated it p44 (13). Another ORF homologous to the p44 gene was found in the area downstream
from the p44. This ORF lacked the universal start codon
(AUG) and had two conserved sequences consisting of 59 and 65 amino
acid residues identical to those of the p44 gene, which
flanked a central hypervariable region. Genomic Southern blot analysis
revealed more than 10 bands bound to the p44 gene probe,
suggesting the existence of additional p44-homologous genes
in the HGE agent genome. By Western blot analysis, a mouse antiserum
against the recombinant P44 (rP44) protein recognized two to six
P44-homologous proteins in each of the five isolates of the HGE agent
and in USG3 isolate. Three monoclonal antibodies that react to the rP44
recognized one to four 44-kDa-range proteins in the whole organism as
well as in the outer membrane fraction from these six isolates (14).
Passive immunization with these antibodies induced a partial protection against infection with the live HGE agent in mice. These studies suggest that multiple antigenically cross-reactive proteins of 38-49
kDa are expressed in each isolate of the HGE and USG3 isolate in HL-60
cell culture and are potential protective antigens in HGE infection.
Murphy et al. (15) cloned three p44-homologous
genes (msp-2a, msp-2b, and msp-2c) of
USG3 isolate. The chemically determined amino acid sequences at N
termini or internal segments of native 43- and 45-kDa proteins of the
isolate approximately match with the segments of amino acid sequences
predicted from one to two each of the three cloned genes. However, due
to existence of highly conserved amino acid sequences among these
P44-homologous proteins, whether these genes are actually expressed by
the isolate was not conclusive in that study. Ijdo et al.
(16) cloned a p44-homologous gene (hge-44) from
the NCH-1 isolate of HGE agent and showed by RT-PCR and sequencing the
product that this gene is expressed by the HGE agent in HL-60 cells.
So far, little is known about the relationship between the diversity of
antigenically cross-reactive proteins of 38-49 kDa and multiple
p44-homologous genes in the HGE agent. In this study, we
characterized the structure, distribution, and expression of the
p44-homologous genes of the HGE agent cultivated in HL-60 cells. Thereby, the study is expected to facilitate understanding of a
role of the p44 multigene family in pathogenesis and immune responses in HGE infection and also to be helpful in designing a
vaccine candidate by using these gene products.
Culture and Purification of the HGE Agent--
The HGE agent (HZ
isolate (8), also called isolate 13 (12-14)) was cultivated in HL-60
cells (human promyelocytic leukemia cell line) and purified by the
Sephacryl S-1000 chromatography method as described elsewhere (17).
RT-PCR and Cloning of the cDNA--
A pair of
oligonucleotides used for RT-PCR (p3708 and p4257 as shown in Fig.
1 and Table
I) was designed based on the conserved regions between DNA sequences of the p44 gene and a
truncated p44-homologous gene downstream from the
p44. Total RNA was extracted from HL-60 cells infected with
the HGE agent by using TRIzol reagent (Life Technologies, Inc.). The
isolated RNA (3 µg) was heated at 70 °C for 10 min and
reverse-transcribed in a 20-µl reaction mixture (0.5 mM
deoxynuleoside triphophate mixture (dNTP), 200 units of SuperScript II
reverse transcriptase (Life Technologies, Inc.), 2 pmol of p4257
primer, and 3 mM MgCl2) at 42 °C for 50 min.
PCR was performed in a 100-µl reaction mixture containing 2 µl of
the cDNA product, 10 pmol each of p3708 and p4257 primers, 0.2 mM dNTP mixture, 5 units of Taq DNA polymerase,
and 1.5 mM MgCl2, with 3 min of denaturation at
94 °C followed by 30 cycles consisting of 1 min of denaturation at
94 °C, 1 min of annealing at 52 °C, and 2 min of extension at
72 °C. To rule out contamination of DNA in the RNA preparation,
RT-PCR without reverse transcriptase was carried out (negative
control). The amplified RT-PCR products were cloned in a pCRII vector
by using the TA Cloning Kit (Invitrogen Co., San Diego, CA).
Twenty-five cDNA clones, which were randomly selected from the
transformants, were sequenced by dideoxy chain termination method with
an Applied Biosystems 373 DNA sequencer.
Genomic Southern Blot Analysis--
DNA probes specific to each
of the cDNAs for Southern blotting were designed based on a
comparison of deduced amino acid sequences among these cDNAs. The
central hypervariable regions (approximately 94 amino acid residues) in
each cDNA and in the p44 gene were amplified by PCR with
primer pairs as shown in Table I. The amplicons were cloned into a
pCRII vector. The DNA insert excised from each recombinant plasmid was
labeled with [ Genomic Cloning of p44-homologous Genes of the HGE
Agent--
The XbaI DNA fragments of the HGE agent, which
were detected by genomic Southern blot analysis, were inserted into a
pBluescript II KS (+) vector, and the recombinant plasmids were
introduced into E. coli DH5 Northern Blot Analysis--
Total RNA (15 µg) from the HGE
agent-infected cells was separated on 1.2% denaturing agarose gel
containing 0.22 M formaldehyde and transferred to Hybond-N+
nylon membrane (Amersham Pharmacia Biotech). The
[ Pulsed-field Gel Electrophoresis (PFGE)--
The purified HGE
agent (25 µg of protein) was suspended in 10 mM Tris-HCl
(pH 7.2), 20 mM NaCl, and 20 mM EDTA. The
suspension was mixed with an equal volume of 2% CleanCut agarose
(Bio-Rad) at 45 °C to make a sample plug for PFGE. The plug (5 × 5 mm) was incubated for 16 h at 50 °C in 100 mM
EDTA (pH 8.0), 1 mg/ml proteinase K, and 1% sodium lauroyl sarcosine.
After being washed twice with 20 mM Tris-HCl (pH 8.0), 50 mM EDTA containing 1 mM phenylmethylsulfonyl fluoride, the plug was washed twice in the same solution without phenylmethylsulfonyl fluoride. Then, the DNA in the plug was digested with 100 units of BamHI and EagI for 16 h at
37 °C and was electrophoresed in 1% agarose gel in 0.5× Tris
borate-EDTA buffer (45 mM Tris, 45 mM borate,
and 1 mM EDTA (pH 8.0)) by using CHEF DRIII (Bio-Rad) apparatus at 14 °C. Electrophoretic conditions were set at 6 V/cm with ramped pulse time from 1 to 5 s for 12 h, then from 1 to 10 s for 8 h, and finally from 10 to 15 s for 6 h.
The DNA bands in a gel were visualized by ethidium bromide staining and
transferred to nylon membrane. Southern blotting was performed with a
mixture of five 32P-labeled probes specific to each of the
cDNAs (p44-2 to p44-19 in Table I).
Estimation of Copy Numbers of p44-homologous Genes--
Two
primer pairs, pnf12/pnr12 and pcf12/pcr12, (Table I) were designed
based on both N- and C-terminal regions that had no homology each other
but were conserved among the deduced amino acid sequences of
p44-homologous genes. A pHGE 3.0 plasmid carrying a
p44-12 gene (see Fig. 4) was used as a template for PCR.
After PCR amplification with the primer pairs and the template, the amplicons were labeled with [ Western Immunoblotting and Indirect Fluorescent Antibody (IFA)
Labeling--
To examine the existence of p44-homologous
gene products in the HGE agent organisms, Western immunoblot analysis
and IFA were performed by using antisera against synthetic
oligopeptides specific to each P44-homologous proteins. Briefly, by
using DNASTAR program (DNASTAR Inc., Madison, WI), we analyzed the
antigenic index, surface probability, and hydrophilicity and selected
the unique amino acid sequence from the hypervariable region of P44-2
and P44-18 proteins. Two oligopeptides with sequences of
GHSSGVTQNPKLFST and KNQKSSDTDTGVEKA were synthesized (Alpha Diagnostic,
San Antonio, TX) and named Pep2 and Pep18, respectively (Fig.
2). Antisera against these synthetic
oligopeptides were generated by immunization of a rabbit and a mouse
with keyhole limpet hemacyanin (KLH) (Pierce)-conjugated synthetic
oligopeptides Pep2 and Pep18, respectively. Western immunoblot analysis
and IFA test with the above sera were performed as described elsewhere
(14, 13). The mouse anti-recombinant P44 serum was used as a positive
control. For double IFA staining, a lissamine rhodamine-labeled goat
anti-rabbit IgG and fluorescein isothiocyanate-labeled goat anti-mouse
IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) were
used as secondary antibodies.
Dot Immunoblot Assay--
The assay was carried out as described
elsewhere (13). The KLH-conjugated oligopeptides Pep2 and Pep18 were
used as antigens. Convalescent sera tested were from two patients
(patients 7 and 11) with clinical signs of HGE; diagnosis was confirmed
by IFA and PCR (12).
Sequence Analysis--
DNA and amino acid sequence analyses and
phylogenetic studies were performed as described previously (19).
mRNAs Transcribed from p44-homologous Genes--
By RT-PCR,
approximately 550-bp cDNAs were amplified from the total RNA of the
HGE agent in HL-60 cells. The amplicon was observed as a single band in
the gel by ethidium bromide, but no amplicon was detected without a
reverse transcriptase (data not shown), indicating the absence of
contamination of genomic DNA in the RNA preparation. After cloning the
amplicons, only five different nucleotide sequences were found in the
25 randomly selected cDNA clones, and the deduced amino acid
sequences are shown in Fig. 2 (boxed area). The five
sequences of the cDNAs were represented as P44-2, P44-18, P44-12,
P44-15, and P44-19. The numbers of cDNA clones with nucleotide
sequences identical to P44-2, P44-18, P44-12, P44-15, and P44-19 were
12, 5, 3, 3, and 2, respectively (Table
II). One or two substitutions were found
at the 3'-end of three nucleotide sequences in a set of 12 cDNA
clones represented by P44-2. We assume that these substitutions were
generated by nucleotide misincorporation with reverse transcriptase or
Taq polymerase. No cDNA sequence identical to the
p44 gene was found in these 25 cDNA clones. This
indicates that at least five different mRNAs from
p44-homologous genes are transcribed by the HGE agent
cultivated in HL-60 cell.
A comparison of the deduced amino acid sequences among the five
different cDNAs and p44 gene previously cloned (13)
revealed that a central region of approximately 94 amino acid residues corresponding to the 175th to 269th amino acid sequence of a protein (P44) encoded by the p44 gene was hypervariable, and the
flanking regions of approximately 30 residues each were highly
conserved (Fig. 2). Within the hypervariable region, the highest amino
acid sequence similarity was 32.8%, between the P44-2 and P44-18
proteins, and the lowest similarity was 19.9%, between P44-15 and P44
proteins. In comparison with flanking regions, the hypervariable region had higher hydrophilicity and antigenic index.
Southern Blotting and Genomic Cloning of the p44-homologous Genes
Expressed by the HGE Agent--
By using a DNA probe specific to each
of cDNAs of P44-18, P44-12, P44-19, and the p44 gene, a
single DNA band was detected in all restriction digestions tested (Fig.
3). Because restriction enzymes used do
not cut within a p44 gene or any cDNA clones, this
suggests that each gene corresponding to the four probes is a single
copy in the HGE agent genome. However, the probes specific to the
cDNAs of P44-2 and P44-15 generated two or three bands (Fig. 3),
showing that two or three gene copies with sequences identical or
highly homologous to those of the cDNAs of P44-2 or P44-15 exist in
the HGE agent genome. Hybridization patterns with the probes specific
to P44-18 cDNA and the p44 gene were identical to each
other in the four restriction digestions. This is consistent with the
result of our sequence analysis data that the p44 gene and a
p44-homologous gene (p44-18) without a universal start codon (AUG) were overlapped by 21 bp in an XbaI
fragment in pHGE1221 plasmid (Fig. 4).
Overall, at least nine copies of p44-homologous genes (a
total of the copies of genes identified in Table II) either expressed
and/or highly homologous were detected in the genome by using cDNA-
specific probes.
Three DNA fragments of 3.0, 3.4, and 3.9 kb were cloned from the
XbaI-digested genomic DNA of the HGE agent with the probes specific to the cDNAs of P44-2, P44-12, and P44-15 (Fig. 4).
Sequencing of the 3.9- and 3.0-kb fragments revealed two complete ORFs
of 1275 and 1173 bp encoding 425- and 391-amino acid proteins with molecular weights of 44,969 and 41,179, respectively. As expected, these ORFs (p44-2 and p44-12) contained the
sequences identical to the cDNAs of P44-2 and P44-12, respectively
(Figs. 2 and 4). In the 3.9-kb DNA, an additional small ORF
(p44-c), which had a DNA sequence corresponding to 82 amino
acid residues (9062 Da) at the C terminus of the P44-2 protein, was
found at 111 nucleotides downstream from the p44-2 gene. The
3.4-kb DNA fragment contained an ORF of 834 bp encoding a 278-amino
acid protein with a molecular weight of 29,387. This ORF
(p44-15) included a sequence identical to P44-15 cDNA.
The p44-15 did not have a universal start codon and lacked
DNA sequence corresponding to 82 amino acid residues at the N terminus
of the P44 protein. Consensus sequences of
The N-terminal amino acid sequence (HDDVSALETG) of the native 44-kDa
protein previously determined (13) was also found in P44-2 and P44-12
proteins as shown in Fig. 2. A comparison of deduced amino acid
sequences of these cloned genes suggests that the identical amino acid
sequence consisting of 15 residues at the N terminus of P44, P44-2, or
P44-12 protein is a signal peptide. The alignment also showed that the
N- and C-terminal portions are highly conserved among three P44
homologs (P44-2, P44-12, and P44), except for the existence of
additional 34 amino acid residues at C terminus of P44-2 (Fig. 2). At N
and C termini of both P44-15 and P44-18, proteins that are encoded by
the ORFs without universal start codons had a short stretch of an amino acid sequence consisting of 8-28 residues without any homology to
other P44 homologs, including P44, P44-2, and P44-12 (Fig. 2).
Northern Blot Analysis--
To confirm whether the relative
proportion of the cDNA clones represented different levels of
transcription of the p44-homologous genes and to determine
the transcript sizes, the Northern blotting was performed with three
RNA probes. The probe specific to P44-2 and P44-18 cDNAs strongly
hybridized with the respective 1.5-kb mRNA transcript (Fig.
5). Using same amounts of total RNA and each probe, the density of band hybridized with P44-2-specific probe
was at least 2-fold greater than that with P44-18-specific probe. No
hybridized band was observed when p44-specific probe was
used. These findings support the cDNA cloning results that, among
the p44-homologous genes, p44-2 was more
abundantly expressed in the HGE agent than p44-18, and the
p44 gene was silent.
Expression Sites of p44-homologous Genes in the HGE Agent
Genome--
In a PFGE gel, EagI and BamHI
digestions produced 14 and 23 bands ranging from 9 to 145 kb and from 2 to 194 kb, respectively (data not shown). In the Southern blotting
(Fig. 6), the probes hybridized to larger
fragments of 100, 97, 49, and 12 kb in EagI digestion and
50, 20 and 18 kb in BamHI digestion. These results show that
these five different expressed genes did not form a cluster in the
genome of the HGE agent.
Copy Number of p44-homologous Genes--
In the genomic Southern
blot analysis (Fig. 7), the hybridization
patterns of major bands with p44-12N and p44-12C probes (no homology
each other) in two restriction digestions were almost identical to each
other, except that two restriction fragments of 2.2 and 1.5 kb in
XbaI and three restriction fragments of 2.0, 1.7, and 1.2 kb
in PstI hybridized only with either p44-12N or p44-12C
probes (Fig. 7, asterisks). This suggests that two and three
copies of p44-homologous genes lack the 3'- or 5'-end
conserved regions, respectively. The restriction sites of these enzymes were not found in the DNA sequences of nine p44-homologous
genes so far cloned (five in the present study, three in Ref. 15, and
one in Ref. 16). However, one or two p44-homologous genes that have not been cloned may be digested by the restriction enzymes. Because any sequence divergences between the probes and target p44-homologous genes reduce the hybridization signal, ratios
of densities of the bands to the 3.0-kb band approximately represent a
minimum copy number of p44-homologous genes in each of these hybridization bands. Therefore, we concluded that there are at least 18 of p44-homologous genes in the HGE agent genome. Because by
genomic Southern blotting with cDNA-specific probes, only nine copies of p44-homologous genes were detected (Fig. 3 and
Table II), at least nine more copies with sequences in their
hypervariable regions divergent from any of those expressed genes
appear to exist in the HGE agent genome.
Analysis of HGE Agent Organisms Expressing P44-homologous
Proteins--
The rabbit anti-Pep2 and the mouse anti-Pep18 sera
reacted with a single band of 44 and 43 kDa, respectively, in the HGE
agent lysate (Fig. 8). In agreement with
our RT-PCR and Northern blot results, the size of the protein
recognized by the anti-Pep18 serum was not 26.4 kDa, as was predicted
from the 759-bp ORF of the p44-18 gene. The result of
Southern blotting with p44-18-specific probe ruled out the
possibility of the existence of other expression loci for
p44-18 in the genome of the HGE agent. Therefore, all these
results suggest that transcriptional modification, such as splicing, is
involved in the expression of p44-18. Mouse antibody against
a recombinant P44 protein (rP44) that is expected to detect most of
p44-homologous gene products because the rP44 protein includes N-terminal region highly conserved among P44 homologs (13),
predominantly reacted with two proteins of 44 and 43 kDa (Fig. 8). The
anti-rP44 serum was used as a positive control to visualize most
organisms expressing P44 homologs by a double immunofluorescence labeling (Fig. 9). In host cells, the HGE
agent resides in membrane-bound inclusions that appear as clusters of
organisms that resemble mulberries and are therefore called morulae.
The morulae are considered as microcolonies derived from a single
organism. As shown in Fig. 9 (arrowheads), six morulae in an
HL-60 cell that reacted with the mouse anti-rP44 serum were also
recognized by the rabbit anti-Pep2 serum. All organisms were
double-labeled in 100 infected cells scored in three independent
labeling experiments. This means that a p44-2 gene is
probably expressed in all of the HGE agent organisms cultivated in
HL-60 cells. With a mouse anti-Pep18 serum, even at a low (1:5)
dilution, the immunofluorescence labeling of organisms or morulae was
extremely weak (data not shown), although Fig. 8 showed that a 43-kDa
protein of the HGE agent was strongly recognized with the serum at
1:400. The reason may be that the epitope(s) of this oligopeptide is
masked or sterically hidden in the HGE agent organism. Based on these
results, the existence of mRNA for five p44-homologous
genes, and the relative proportion of cDNA clones, we suggest that
multiple p44-homologous genes are differentially expressed
by each organism. Accordingly, HGE agents cultivated in HL-60 cells
probably consist of a heterogeneous population of organisms expressing
one, two, or multiple p44-homologous gene products.
Detection of Antibodies Specific to P44-2 and P44-18 Proteins in
the Sera of Patients with HGE Infection--
Whether antibodies
against P44-2 and P44-18 proteins were present in convalescent sera
from patients with HGE was examined by dot immunoblot assay with the
oligopeptides of Pep2 and Pep18 as antigens (Fig.
10). Serum 7 reacted strongly to both
Pep2 and Pep18. Serum 11 also reacted strongly to Pep18 but weakly to
Pep2. This result indicates that p44-2 and p44-18
genes are actually expressed by the HGE agent in infected patients;
thus, antibodies specific to these P44 homologs were developed.
Phylogenetic Analysis--
A phylogenetic analysis revealed nine
P44 homologs had identities of 58.3-99.5% (Table
III) and made a cluster separated from two major surface protein 2 (MSP2) (2-11.2 and 2-DF (20, 21)) and a
MSP4 (22) of Anaplasma marginale (Fig.
11). The result is in agreement with
phylogenetic analysis based on 16 S rRNA sequence comparison. This also
indicates that P44 homologs of HGE and USG3 isolates were not
segregated either by geography (e.g. New York
versus Massachusetts) or host origin (e.g. human versus tick).
This study demonstrated that multiple p44 genes are
expressed by the HGE agent in HL-60 cells and probably in patients,
which may be the reason why multiple bands of 38-49 kDa were found in our previous Western blot analysis studies with patients' sera (12) or
with monoclonal antibodies (14). Our data set the framework for a
better understanding of the mechanism(s) underpinning the expression of
p44 multigenes in the HGE agent. Four of expressed p44-homologous genes (p44-2, p44-12, p44-15, and
p44-18) and one silent gene (p44) from the
genomic DNA were cloned. The proteins encoded by these
p44-homologous genes were found to consist of a single
central hypervariable region of approximately 94 amino acid residues
and N- and C-terminal regions highly conserved among the homologs. It
appears that two kinds of mechanisms may involve in the expressions of
p44-homologous genes: (i) the normal expression of the
genes, such as p44-2 and p44-12, with complete
ORFs from the respective expression sites, and (ii) the unique
expression with a specific event, probably transcriptional modification
such as splicing, of the genes, such as p44-18 and
p44-15, which lack a universal start codon. The latter
mechanism may come to existence to overcome the deficiency in creating
antigenic diversity by a common mechanism, such as recombination.
p44 multigenes are homologous to two A. marginale
msp2 genes that had been cloned (~ 45% amino acid similarity
(13, 15, 16)). Recently, the study on A. marginale revealed
that multiple (at least 4) msp2 genes were expressed in each
peak of rickettsemia that occurred at 6-8-week intervals in two cattle
(20). The distribution of the genes in the genome (broad distribution
throughout the genome) and the protein structure (e.g. a
central hypervariable region flanked with conserved regions) are
similar between msp2 genes and p44 multigenes.
Because the copy number of msp2 multigenes in A. marginale was estimated as 10 (21), the relative ratio (4 in 10)
of expressed genes against the total copy number of msp2
multigenes at a given stage of infection is higher than that (5 in 18)
of p44 multigenes of the HGE agent. In other words, the HGE
agent may have more potential genetic capacity to generate the
diversity of the P44-homologous proteins. Because for A. marginale, the msp2 genes corresponding to the
transcripts were not identified, the expression mechanism is unknown.
Among rickettsia closely related to HGE agent, persistent infection and
recurrence after recovery from the clinical disease are known for
Ehrlichia canis, Ehrlichia platys, E. phagocytophila, Cowdria ruminantium, and A. marginale (20, 23). The HGE
agent was detected in the serum of one untreated patient by PCR at 30 day after onset of illness (24), suggesting that the HGE agent may also
cause persistent infection in humans. In addition, persistence of the
HGE agent in reservoir rodent host would be an important adaptation
that allows greater access of uninfected tick populations to an
infectious blood meal. French et al. (20) proposed that the
expression of distinct sets of msp2 genes at each
rickettsemia peak in A. marginale infection of cattle may
allow immunoevation of anaplasma to persist in immunocompetent hosts.
Multiple expression of p44 multigenes may be also related to
immunoavoidance in human and rodent hosts and potential persistence.
The expression mechanisms of the several multigene families in the
human pathogens Neisseria gonorrhoeae (pil (25)
and opa (26)), Borrelia hermsii (vmp
(27)), Borrelia burgdorferi (vls (28)), African trypanosomes (vsg (29)), and Plasmodium
falciparum (var (30)), have been well studied. These
multigene families are involved in pathogenesis, e.g.
antigenic variation (or phase variation) and cytoadhesin. The switching
of gene expression can be divided into two major mechanisms: one
depends on DNA rearrangement, and another occurs at transcriptional
level. In pil, vmp, vls, and
vsg, switching of expression between members of the
corresponding gene families occurs through programmed DNA
rearrangements (gene conversion), moving a transcriptionally silent
gene into an active expression site. In opa, expression is
regulated by a reversible frameshift mutation of DNA, i.e. a
slipped-strand mispairing of the number of pentanucleotide coding
repeat units in the single peptides (26), whereas in the var
gene of P. falciparum, each parasite expresses only a single
and distinct var gene product. Each var gene is
an independent transcription unit in which promoter activity determines
the expression status. p44 multigene expression appears to
be different from those of pil, vmp,
vls, and vsg in that multiple expressed
p44 genes are located in several large DNA fragments in the
HGE agent genome, i.e. the transcription of the genes
apparently does not occur from a unique expression site. The expression
p44 multigene, therefore, may not involve DNA rearrangement.
Expressions of p44 multigenes, as well as msp2 genes (21), are also different from that of var genes,
because a single organism appear to express more than two gene products.
In ehrlichiae, recently, additional multigene families encoding major
outer membrane proteins have been discovered. We identified the
omp-1 gene family of Ehrlichia
chaffeensis (31) and the p30 gene family of
E. canis (19). More than a dozen copies of the
omp-1 and p30 gene families are tandemly arranged
in the genome, in contrast to p44 multigenes and
msp2 genes. Furthermore the protein structures encoded by
the omp-1 and p30 gene families is distinct from
those of p44 and msp2, consisting of three short, hypervariable segments interposed with relatively conserved segments (19, 31). This suggests that the expression mechanism of
omp-1 and p30 gene families is probably different
from those of p44 and msp2. Immunization with a
recombinant P28 protein (one of the omp-1 multigene
products) of E. chaffeensis and native MSP2 of A. marginale has been demonstrated to induce almost complete and
partial protections against the infection in mice and cattle, respectively (31, 32). We previously observed that passive immunization
with monoclonal antibodies specific to P44-homologous proteins of the
HGE agent induced partial protection against challenge with the HGE
agent in mice (14). Active immunization with the recombinant P44
protein also partially protected mice from HGE infection.2 This indicates
that the p44 multigene family encodes a potential protective
antigen. However, because only a fraction of genes is differentially
expressed as shown in this study, it seems to be essential to determine
which genes are expressed in the host for identification of the most
protective antigen.
By a GenBankTM data base homology search, the
P44-homologous proteins of the HGE agent were found to possess a
similarity (~64%) with the N-terminal region of Hsf protein (surface
fibrils) involved in binding of Hemophilus influenzae type b
to human epithelial cells (33). The similar sequence identified
was located in the center of all P44 homologs, including the
hypervariable domain (span of approximately 200 amino acid
residues). This finding suggests that P44s may have a similar function,
such as cytoadhesion to human granulocytes.
The basic information obtained in this study would facilitate the
understanding of the role of the p44 multigene family in causing disease in humans, in the persistence of HGE agent in white-footed mice, and in transmission of this agent from tick to
human. Further analysis of expression mechanism of these genes and the
genes encoding more protective antigens would assist in designing an
effective vaccine candidate against the human ehrlichiosis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Primer positions in the p44
gene and the truncated ORF homologous to p44 in
the recombinant plasmid pHGE1221 (13). Primers p3708 and p4257
were designed for RT-PCR to amplify the cDNA of
p44-homologous genes. Primers hvf and hvr were designed to
prepare the cDNA-specific probes by PCR for Southern and Northern
blot analysis.
Oligodeoxynucleotides used in RT-PCR and
PCR
-32P]dATP by the random primer method
with a kit (Amersham Pharmacia Biotech) and used as a probe.
Hybridization was performed in rapid hybridization buffer (Amersham
Pharmacia Biotech) as described elsewhere (13). The membrane was
exposed to a Hyperfilm (Amersham Pharmacia Biotech).
. By using the colony
hybridization method (18) with the specific DNA probes same as those
used for Southern blot analysis, three positive clones were isolated,
and the DNA inserts were sequenced. The clones were designated pHGE3.0,
pHGE3.4, and pHGE3.9, containing ehrlichial DNA fragments of 3.0, 3.4, and 3.9 kb, respectively.
-32P]CTP-labeled single-stranded RNA probes (Table I)
that were specific to each of cDNAs derived from the recombinant
plasmids used for Southern blotting were prepared by using the
riboprobe in vitro transcription system (Promega Corp.,
Madison, WI). The hybridization was performed separately with 1 × 107cpm/ml of each 32P-labeled RNA probe (a
specific activity of 2 × 108 cpm/µg) in Prehyb/Hyb
solution (Ambion, Inc., Austin, TX) at 65 °C for 16 h. After
being washed twice each with low stringency solution and high
stringency solution (Ambion) at 65 °C for 15 min, the membranes were
exposed to a Hyperfilm. The densities of hybridized bands were analyzed
with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
-32P]dATP and used as DNA
probes. The probes were designated p44-12N (pnf12/pnr12) and p44-12C
(pcf12/pcr12) as shown in Table I and Fig. 4. For Southern blot
analysis, the genomic DNA of the HGE agent was digested with
XbaI and PstI. The hybridization was done separately with the two 32P-labeled probes as described
above. The densities of hybridized bands were analyzed by
PhosphorImager. Two 3.0-kb XbaI fragments of the HGE agent
that comigrated in a gel contained three p44-homologous gene
copies (p44 and p44-18 in pHGE1221 and
p44-12 in pHGE3.0 (see Fig. 4)). Consequently, the density
of this 3.0-kb hybridized band in the Southern blot analysis
represented three p44-homologous gene copies and was used as
a standard to estimate the copy number.
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Fig. 2.
Alignment of amino acid sequences deduced
from cDNA clones and the corresponding
p44-homologous genes of the HGE agent. Aligned
positions of identical amino acids with P44 of the HGE agent are shown
with dots. Gaps indicated by dashed lines were
introduced for an optimal alignment of all proteins. A boxed
area in the middle indicates the amino acid sequences deduced from
nucleotide sequences of cDNAs. Hypervariable regions are shown in
boldface. A bar indicates the N-terminal amino
acid sequence of the native P44 protein, and an arrowhead
shows the cleavage site of the putative signal peptide. The amino acid
sequences underlined in the hypervariable regions of P44-2 and P44-18
indicate the sequences that were used to prepare synthetic
oligopeptides, Pep2 and Pep18, respectively. The arrows
point out the positions of the primers used in RT-PCR. The numbers on
the right indicate the positions of amino acid residues in
P44-homologous proteins from the N terminus to C terminus.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Properties of p44-homologous genes of the HGE
agent
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Fig. 3.
Genomic Southern blot analysis of the HGE
agent with the DNA probes specific to each of five cDNAs and
p44 gene. Probes are shown at bottom
of each panel. Hybridization was performed at 65 °C for 16 h,
and the membrane was washed twice with 0.1× SSC (1× SSC, 0.15 M sodium chloride and 0.015 M sodium citrate)
and 0.1% SDS at 60 °C for 30 min. Numbers on the left
indicate molecular sizes in kb.
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Fig. 4.
Schematic diagram of the DNA inserts of
recombinant plasmids pHGE1221, pHGE3.9, pHGE3.0, and pHGE3.4. The
arrow indicates the orientation of ORF from 5' to 3'
end.
70 promoter
(AT-rich region about 10 base pairs upstream of the transcription start
site; the
10 sequence) and Shine-Dalgarno sequence were found in the
regions upstream from the start codon of p44-2,
p44-12, and p44-c. The properties of the
p44-homologous genes identified in this study were
summarized in Table II.
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Fig. 5.
Northern blot analysis of the mRNA
expression of the p44-homologous genes. The
single-stranded RNA probes specific to cDNAs of P44-2 and P44-18
and the p44 gene are shown at bottom of the
lanes. Numbers on the left indicate molecular size in
kb.
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Fig. 6.
Distribution of expressed
p44-homologous genes in the HGE agent genome.
Southern blot analysis after PFGE was performed by using a mixture of
DNA probes specific to the cDNAs of P44-2, P44-12, P44-15, P44-18,
and P44-19. Hybridization was performed at 65 °C for 16 h, and
the membrane was washed twice with 0.1× SSC and 0.1% SDS at 60 °C
for 30 min. Numbers on the right indicate molecular sizes in
kb.
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Fig. 7.
Estimation of minimum copy numbers of
p44-homologous genes in the genome of the HGE
agent. A, Southern blotting profiles by using the DNA
probe specific to P44-12 cDNA and the DNA probes of p44-12N and
p44-12C that were derived from N-terminal and C-terminal regions of
p44-12, respectively. Each probe is shown at
bottom of the respective lane. Hybridization was performed
at 60 °C for 16 h, and the membrane was washed twice with 0.1×
SSC at 60 °C for 30 min. The asterisks indicate that the
unique restriction fragments recognized only by either p44-12N or
p44-12C but not by both. Numbers on the left indicate
molecular sizes in kb. B, schematic illustration of blotting
patterns for the copy number analysis. The numbers on the
right indicate a ratio of the band density against that of
the 3.0-kb band in XbaI digestion.
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Fig. 8.
Western blot analysis of rabbit anti-Pep2,
mouse anti-Pep18, and mouse anti-rP44 sera using the purified HGE
agent. Ten µg of proteins of the purified organisms were used as
an antigen in each lane. The three primary antisera were used at 1:400
dilution. The numbers on the left indicate molecular masses
in kDa based on the broad-range prestained standards (Bio-Rad).
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Fig. 9.
Double immunofluorescence labeling of the HGE
agent with rabbit anti-Pep2 and mouse anti-rP44 sera. The binding
of each antibody was detected by using lissamine rhodamine-labeled goat
anti-rabbit IgG and fluorescein isothiocyanate-labeled goat anti-mouse
IgG. Fluorescein isothiocyanate-labeled (right) and
lissamine rhodamine-labeled (left) morulae containing
multiple HGE agent organisms are shown by arrows in the
paired photomicrographs. Magnification, × 750.
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Fig. 10.
Dot immunoblot assay of HGE patient sera
(patients 7 and 11) with oligopeptide Pep2 and Pep18.
KLH-conjugated Pep2 and Pep18, and KLH as a negative control, were
blotted onto the nitrocellulose membrane at 200 ng of protein per dot.
The patient sera were preabsorbed with KLH and used at a 1:300
dilution.
Identities and evolutionary distances among amino acid sequences
predicted from entire genes of HGE agent P44 and its
homologs
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Fig. 11.
Phylogenetic classification among the
proteins of P44 family and other major outer membrane proteins of the
closely related rickettsiae based on the amino acid sequence
similarities. Scale bar shows 10% divergence in amino
acid sequences. The GenBankTM accession numbers of the
published sequences are as follows: P44 (HGE agent, HZ strain),
AF059181; HGE-44 (HGE agent, NCH-1 strain), AF037599; MSP-2A (GE sp.,
USG3 strain), AF029322; MSP-2B and -2C (GE sp., USG3 strain) and MSP4
(A. marginale, Florida strain), Q07408; MSP2-11.2 (A. marginale, Florida strain), U07862; and MSP2-DF (A. marginale, Florida strain), U36193.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant RO1 AI40934.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF135254, AF135255, AF135256, AF135257, AF135258, AF135259, AF132260, AF132261, AF132262, and AF132263.
To whom correspondence should be addressed: Dept. of Veterinary
Biosciences, College of Veterinary Medicine, The Ohio State University,
1925 Coffey Rd., Columbus, OH 43210-1093. Tel.: 614-292-9677; Fax:
614-292-6473; E-mail: rikihisa.1{at}osu.edu.
2 N. Zhi, N. Ohashi, and Y. Rikihisa, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: HGE, human granulocytic ehrlichiosis; RT, reverse transcription; PCR, polymerase chain reaction; IFA, indirect fluorescent antibody; KLH, keyhole limpet hemacyanin; PFGE, pulsed-field gel electophoresis; ORF, open reading frame; kb, kilobase(s); bp, base pair(s); rP44, recombinant P44.
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