Porin Activity of Anaplasma phagocytophilum Outer Membrane Fraction and Purified P44

ABSTRACT Anaplasma phagocytophilum, an obligatory intracellular bacterium that causes human granulocytic anaplasmosis, has significantly less coding capacity for biosynthesis and central intermediary metabolism than do free-living bacteria. Thus, A. phagocytophilum needs to usurp and acquire various compounds from its host. Here we demonstrate that the isolated outer membrane of A. phagocytophilum has porin activity, as measured by a liposome swelling assay. The activity allows the diffusion of l-glutamine, the monosaccharides arabinose and glucose, the disaccharide sucrose, and even the tetrasaccharide stachyose, and this diffusion could be inhibited with an anti-P44 monoclonal antibody. P44s are the most abundant outer membrane proteins and neutralizing targets of A. phagocytophilum. The P44 protein demonstrates characteristics consistent with porins of gram-negative bacteria, including detergent solubility, heat modifiability, a predicted structure of amphipathic and antiparallel β-strands, an abundance of polar residues, and a C-terminal phenylalanine. We purified native P44s under two different nondenaturing conditions. When reconstituted into proteoliposomes, both purified P44s exhibited porin activity. P44s are encoded by approximately 100 p44 paralogs and go through extensive antigenic variation. The 16-transmembrane-domain β-strands consist of conserved P44 N- and C-terminal regions. By looping out the hypervariable region, the porin structure is conserved among diverse P44 proteins yet enables antigenic variation for immunoevasion. The tricarboxylic acid (TCA) cycle of A. phagocytophilum is incomplete and requires the exogenous acquisition of l-glutamine or l-glutamate for function. Efficient diffusion of l-glutamine across the outer membrane suggests that the porin feeds the Anaplasma TCA cycle and that the relatively large pore size provides Anaplasma with the necessary metabolic intermediates from the host cytoplasm.

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Anaplasma phagocytophilum Has a Functional msp2 Gene That Is Distinct from p44

Infection and Immunity ◽

10.1128/iai.72.7.3883-3889.2004 ◽

2004 ◽

Vol 72 (7) ◽

pp. 3883-3889 ◽

Cited By ~ 31

Author(s):

Quan Lin ◽

Yasuko Rikihisa ◽

Suleyman Felek ◽

Xueqi Wang ◽

Robert F. Massung ◽

...

Keyword(s):

Outer Membrane ◽

Anaplasma Phagocytophilum ◽

Ixodes Scapularis ◽

Outer Membrane Proteins ◽

Gene Families ◽

Single Copy ◽

The United States ◽

Granulocytic Anaplasmosis ◽

The United Kingdom ◽

Membrane Protein Gene

ABSTRACT The msp2 and p44 genes encode polymorphic major outer membrane proteins that are considered unique to the intraerythrocytic agent of Anaplasma marginale and the intragranulocytic agent of Anaplasma phagocytophilum, respectively. In the present study, however, we found an msp2 gene in A. phagocytophilum that was remarkably conserved among A. phagocytophilum strains from human granulocytic anaplasmosis (HGA) patients, ticks, and a horse from various regions in the United States, but the gene was different in a sheep isolate from the United Kingdom. The msp2 gene in the A. phagocytophilum strain HZ genome was a single-copy gene and was located downstream of two Ehrlichia chaffeensis omp-1 homologs and a decarboxylase gene (ubiD). The msp2 gene was expressed by A. phagocytophilum in the blood from HGA patients NY36 and NY37 and by A. phagocytophilum isolates from these patients cultured in HL-60 cells at 37°C. The msp2 gene was also expressed in a DBA/2 mouse infected by attaching ticks infected with strain NTN-1 and in a horse experimentally infected by attaching strain HZ-infected ticks. However, the transcript of the msp2 gene was undetectable in A. phagocytophilum strain HZ in SCID mice and Ixodes scapularis ticks infected with strain NTN-1. These results indicate that msp2 is functional in various strains of A. phagocytophilum, and relative expression ratios of msp2 to p44 vary in different infected hosts. These findings may be important in understanding roles that Msp2 proteins play in granulocytic ehrlichia infection and evolution of the polymorphic major outer membrane protein gene families in Anaplasma species.

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Outer Membrane Protein Sequence Variation in Lambs Experimentally Infected with Anaplasma phagocytophilum

Infection and Immunity ◽

10.1128/iai.01206-07 ◽

2007 ◽

Vol 76 (1) ◽

pp. 120-126 ◽

Cited By ~ 24

Author(s):

Erik G. Granquist ◽

Snorre Stuen ◽

Anna M. Lundgren ◽

Margrethe Bråten ◽

Anthony F. Barbet

Keyword(s):

Outer Membrane ◽

Anaplasma Phagocytophilum ◽

Sequence Variation ◽

Antigenic Variation ◽

Outer Membrane Proteins ◽

Model Systems ◽

Mammalian Host ◽

Host Immune System ◽

Expression Site ◽

Disease Persistence

ABSTRACT Anaplasma phagocytophilum has long been known to cause tick-borne fever in ruminants and has been identified more recently as the causative agent of the emerging disease human granulocytic anaplasmosis. The related organism Anaplasma marginale uses gene conversion of the expression site for two major outer membrane proteins (OMPs) to generate extensive sequence and antigenic variation in these OMPs. This is thought to present a continuously varying repertoire of epitopes to the mammalian host and allow disease persistence. Recent genomic and structural data on human strains of A. phagocytophilum, together with animal studies in model systems, have implicated an orthologous OMP of A. phagocytophilum in a similar mechanism of variation. However, to date there has been little investigation of the mechanisms of antigenic variation or disease persistence in hosts naturally infected with field strains of A. phagocytophilum. Approximately 300,000 lambs in Norway suffer severe disease caused by A. phagocytophilum annually. We show here the persistent and cyclic nature of infection in these animals that is accompanied by loosely programmed sequence variation of the major OMP expression site in each rickettsemic peak. These data will allow analysis of interactions between A. phagocytophilum and the host immune system in naturally occurring persistent infections and provide an important comparison with enduring infections of cattle caused by A. marginale.

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Structural analysis of a p44/msp2 expression site of Anaplasma phagocytophilum in naturally infected ticks in Japan

Journal of Medical Microbiology ◽

10.1099/jmm.0.011775-0 ◽

2009 ◽

Vol 58 (12) ◽

pp. 1638-1644 ◽

Cited By ~ 17

Author(s):

Wuritu ◽

Yutaka Ozawa ◽

Gaowa ◽

Fumihiko Kawamori ◽

Takashi Masuda ◽

...

Keyword(s):

Amino Acid ◽

Anaplasma Phagocytophilum ◽

Outer Membrane Proteins ◽

Hypervariable Region ◽

Molecular Characteristics ◽

Ixodes Persulcatus ◽

Significant Information ◽

Genomic Expression ◽

Granulocytic Anaplasmosis ◽

Expression Site

Anaplasma phagocytophilum, an agent of human granulocytic anaplasmosis, infects neutrophils and causes an emerging tickborne febrile disease. The genome of this bacterium contains a large number of p44/msp2-related genes encoding 44 kDa major outer-membrane proteins, and it is known that a specific p44/msp2 gene is predominantly transcribed from a single expression locus. This study successfully characterized the genomic expression site for p44/msp2 (3.8 kb) in uncultured A. phagocytophilum from Ixodes persulcatus ticks inhabiting a northern part of Japan. Comparative analysis of the sequences revealed that the structures of the expression sites in Japanese A. phagocytophilum were similar to those of US strains from human patients and European strains from a dog and sheep, but omp-1N (upstream from p44/msp2) and a truncated recA (downstream from p44/msp2) in the p44/msp2 expression site seemed to share similarities with those of US and European strains. The central hypervariable region sequences of Japanese p44/msp2 were found to be quite diverse (24.4–100 % amino acid similarities) and distinct from their closest relatives from US human patients or animal host origins (56.3–97.6 % amino acid similarities) with some exceptions. Thus, this study provides significant information about the molecular characteristics of A. phagocytophilum in East Asia, as well as the global diversity of p44/msp2.

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Functional Activity of Antibodies against the Recombinant OpaJ Protein from Neisseria meningitidis

Infection and Immunity ◽

10.1128/iai.71.5.2331-2340.2003 ◽

2003 ◽

Vol 71 (5) ◽

pp. 2331-2340 ◽

Cited By ~ 18

Author(s):

M. I. de Jonge ◽

G. Vidarsson ◽

H. H. van Dijken ◽

P. Hoogerhout ◽

L. van Alphen ◽

...

Keyword(s):

Outer Membrane ◽

Functional Activity ◽

Outer Membrane Proteins ◽

Hypervariable Region ◽

Surface Expression ◽

Cell Surface Expression ◽

Transfected Cells ◽

E Coli ◽

Human Polymorphonuclear Leukocytes ◽

Opacity Proteins

ABSTRACT The opacity proteins belong to the major outer membrane proteins of the pathogenic Neisseria and are involved in adhesion and invasion. We studied the functional activity of antibodies raised against the OpaJ protein from strain H44/76. Recombinant OpaJ protein was obtained from Escherichia coli in two different ways: cytoplasmic expression in the form of inclusion bodies followed by purification and refolding and cell surface expression followed by isolation of outer membrane complexes (OMCs). Immunization with purified protein and Quillaja saponin A (QuilA) induced high levels of Opa-specific antibodies, whereas the E. coli OMC preparations generally induced lower levels of antibodies. Two chimeric Opa proteins, hybrids between OpaB and OpaJ, were generated to demonstrate that the hypervariable region 2 is immunodominant. Denatured OpaJ with QuilA induced high levels of immunoglobulin G2a (IgG2a) in addition to IgG1, whereas refolded OpaJ with QuilA induced IgG1 exclusively. These sera did not induce significant complement-mediated killing. However, all sera blocked the interaction of OpaJ-expressing bacteria to CEACAM1-transfected cells. In addition, cross-reactive blocking of OpaB-expressing bacteria to both CEACAM1- and CEA-transfected cells was found for all sera. Sera raised against purified OpaJ and against OpaJ-containing meningococcal OMCs also blocked the nonopsonic interaction of Opa-expressing meningococci with human polymorphonuclear leukocytes.

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Polymorphism and Transcription at the p44-1/p44-18 Genomic Locus in Anaplasma phagocytophilum Strains from Diverse Geographic Regions

Infection and Immunity ◽

10.1128/iai.72.10.5574-5581.2004 ◽

2004 ◽

Vol 72 (10) ◽

pp. 5574-5581 ◽

Cited By ~ 14

Author(s):

Quan Lin ◽

Yasuko Rikihisa ◽

Robert F. Massung ◽

Zerai Woldehiwet ◽

Richard C. Falco

Keyword(s):

New York ◽

Anaplasma Phagocytophilum ◽

Functional Divergence ◽

Outer Membrane Proteins ◽

New York State ◽

Hypervariable Region ◽

Geographic Region ◽

Genomic Locus ◽

Specific Pcr ◽

Geographic Regions

ABSTRACT A polymorphic multigene family (p44) of Anaplasma phagocytophilum encodes the immunodominant 44-kDa major outer membrane proteins. With p44-specific PCR and gene-specific probes, p44-1 was found in all human isolates from New York State but not in isolates from Minnesota, whereas p44-18 and two other p44 species were found in isolates from both regions. We therefore sequenced the genomic locus corresponding to the p44-1/p44-18 tandem locus of A. phagocytophilum HZ in 14 other geographically divergent strains from various hosts. The locus was found in all 14 strains, and p44-18 was conserved among all 13 United States isolates studied. In all nine northeastern strains, p44-1 was conserved. However, in three of the Minnesota strains and in one California strain, p44-1 was replaced at this genomic locus by the novel gene p44-61 (p44-61/18), whose hypervariable region (hv) was a chimera of p44-20hv and p44-23hv. The conserved base sequence within the hv region linked the two segments. In contrast, in the Old Sourhope strain isolated from sheep in the United Kingdom, only a single and distinct p44, p44-OS, was found in this locus. This suggests different rates of evolution of p44-1 and p44-18 at this locus and conservation of the locus within strains isolated from the same geographic region. Locus-specific reverse transcription-PCR revealed expression of p44-1 by New York and p44-61 by Minnesota strains at this locus. These p44 loci provide insight into the molecular evolution and functional divergence of p44 paralogs and may serve as markers for typing strains from different geographic regions.

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Establishment of Cloned Anaplasma phagocytophilum and Analysis of p44 Gene Conversion within an Infected Horse and Infected SCID Mice

Infection and Immunity ◽

10.1128/iai.73.8.5106-5114.2005 ◽

2005 ◽

Vol 73 (8) ◽

pp. 5106-5114 ◽

Cited By ~ 26

Author(s):

Quan Lin ◽

Yasuko Rikihisa

Keyword(s):

Anaplasma Phagocytophilum ◽

Antigenic Variation ◽

Severe Combined Immunodeficiency ◽

Hypervariable Region ◽

Scid Mice ◽

Infection Period ◽

Immune Pressure ◽

Precise Analysis ◽

Cloning Method ◽

Novel Method

ABSTRACT Diverse p44 alleles at the p44 expression locus (p44Es) encoding surface-exposed major membrane proteins, P44s, of Anaplasma phagocytophilum were hypothesized to be garnered by recombination to enact antigenic variation. However, this hypothesis has not been proven so far, due to inability to clone this obligate intragranulocytic rickettsia. To define the p44E recombination, we developed a novel method to clone A. phagocytophilum. This isogenic cloned population containing a defined p44E was used to infect a naive horse and severe combined immunodeficiency (SCID) mice. During a 58-day infection period in the blood of the horse, p44E conversion was evident in a total of 11 new p44Es, 48% (115/242) of the sequenced p44E population. During a 50-day infection period in the blood of SCID mice, p44E conversion was manifested in a total of 13 new p44Es, 42% (192/460) of the p44E population. Thus, similar levels of p44E convertants were detected in either the presence or absence of an acquired immune system, suggesting that T- and B-cell immune pressure was not essential for recombination and/or selection of the p44E variants. Analysis of sequentially changed p44Es revealed that the entire central hypervariable region of donor p44 pseudogenes or of donor full-length p44s replaced the same region of the resident p44E as a cassette. Putative recombination points were detected within p44 conserved regions flanking the central hypervariable region by the TOPALi analysis. Our results unambiguously demonstrated p44E recombination. The cloning method developed would facilitate precise analysis of the recombination process and the extent of diversity which the recombination creates in the antigenic repertoire.

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Sequence Analysis of p44 Homologs Expressed by Anaplasma phagocytophilum in Infected Ticks Feeding on Naive Hosts and in Mice Infected by Tick Attachment

Infection and Immunity ◽

10.1128/iai.72.2.659-666.2004 ◽

2004 ◽

Vol 72 (2) ◽

pp. 659-666 ◽

Cited By ~ 21

Author(s):

Suleyman Felek ◽

Sam Telford ◽

Richard C. Falco ◽

Yasuko Rikihisa

Keyword(s):

Sequence Analysis ◽

Salivary Glands ◽

Anaplasma Phagocytophilum ◽

Outer Membrane Proteins ◽

Hypervariable Region ◽

Mouse Blood ◽

Cdna Sequences ◽

Tick Attachment ◽

Different Strains ◽

Strain Genome

ABSTRACT The 44-kDa immunodominant outer membrane proteins (P44 proteins) of Anaplasma phagocytophilum are encoded by the p44 polymorphic multigene family. The present study examined p44 expression and analyzed the cDNA sequences of various p44 transcripts from the spleens and blood of mice infected by the bites of ticks infected with the A. phagocytophilum NTN-1 strain or of naturally infected nymphal ticks and in the salivary glands and midgut tissues of these ticks. A total of 300 p44 cDNAs were subjected to sequence analysis. Of these, 40 distinct p44 species were found, and all of these had orthologs in the A. phagocytophilum HZ strain genome that shared 95 to 100% base sequence identity. The number of unique p44 species expressed in mouse blood was greater than that for mouse spleens. Higher numbers of different p44 transcripts were also expressed in the salivary glands of ticks than in the midgut tissues. Variations in the sequences of the same p44 cDNA species within a single A. phagocytophilum strain and among different strains were concentrated in the conserved regions flanking the central hypervariable region of p44 genes. No mosaic sequences derived from two or more p44 species were found within the p44 hypervariable region. The conservation of the hypervariable region of each p44 cDNA species of A. phagocytophilum in naturally infected ticks and in different geographic isolates suggests that each A. phagocytophilum genome carries a set of p44 paralogs to be expressed. Thus, a large but restricted repertoire of p44 hypervariable sequences exists in A. phagocytophilum strains in the Northeastern United States.

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Proteomic Identification of a Novel Anaplasma phagocytophilum DNA Binding Protein That Regulates a Putative Transcription Factor

Journal of Bacteriology ◽

10.1128/jb.00318-07 ◽

2007 ◽

Vol 189 (13) ◽

pp. 4880-4886 ◽

Cited By ~ 20

Author(s):

Xueqi Wang ◽

Takane Kikuchi ◽

Yasuko Rikihisa

Keyword(s):

Transcription Factor ◽

Outer Membrane ◽

Anaplasma Phagocytophilum ◽

Promoter Region ◽

Outer Membrane Proteins ◽

Hypothetical Protein ◽

Etiologic Agent ◽

Major Protein ◽

Mobility Shift ◽

Putative Transcription Factor

ABSTRACT Anaplasma phagocytophilum, the etiologic agent of human granulocytic anaplasmosis, is an obligatory intracellular bacterium. Little is known about the gene regulatory mechanisms for this bacterium. A gene encoding a putative transcription factor, tr1, upstream of three tandem genes encoding outer membrane proteins, including the major outer membrane protein P44, is driven by a strong promoter. In the present study, gel mobility shift assays revealed the presence of A. phagocytophilum proteins that interact with the promoter region of tr1. These proteins interacting with the tr1 promoter region were purified by biotin-labeled DNA affinity chromatography from a large amount of host cell-free bacteria. Mass spectrometry identified the major protein as an A. phagocytophilum 12.5-kDa hypothetical protein, which was named ApxR. In a DNase I protection assay, recombinant ApxR (rApxR) bound cooperatively to four 24- or 25-bp sites within 235 bp upstream of tr1: regions III and IV proximal to tr1 had higher affinity than regions I and II did. Deletion assays showed that regions III and IV were essential for rApxR binding, whereas regions I and II upstream of regions III and IV were not. The primary cis-acting region was region IV, since region IV alone was sufficient for rApxR to strongly transactivate the downstream gene in a lacZ reporter assay. Addition of regions I, II, and III did not enhance transactivation. These results show that ApxR is a novel transcriptional regulator that directly regulates tr1.

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Analysis of Involvement of the RecF Pathway in p44 Recombination in Anaplasma phagocytophilum and in Escherichia coli by Using a Plasmid Carrying the p44 Expression and p44 Donor Loci

Infection and Immunity ◽

10.1128/iai.74.4.2052-2062.2006 ◽

2006 ◽

Vol 74 (4) ◽

pp. 2052-2062 ◽

Cited By ~ 25

Author(s):

Quan Lin ◽

Chunbin Zhang ◽

Yasuko Rikihisa

Keyword(s):

Escherichia Coli ◽

Gene Conversion ◽

Anaplasma Phagocytophilum ◽

Antigenic Variation ◽

Outer Membrane Proteins ◽

Etiologic Agent ◽

Intermediate Structure ◽

E Coli ◽

Recf Pathway ◽

Recombination Pathway

ABSTRACT Anaplasma phagocytophilum, the etiologic agent of human granulocytic anaplasmosis, has a large paralog cluster (approximate 90 members) that encodes the 44-kDa major outer membrane proteins (P44s). Gene conversion at a single p44 expression locus leads to P44 antigenic variation. Homologs of genes for the RecA-dependent RecF pathway, but not the RecBCD or RecE pathways, of recombination were detected in the A. phagocytophilum genome. In the present study, we examined whether the RecF pathway is involved in p44 gene conversion. The recombination intermediate structure between a donor p44 and the p44 expression locus of A. phagocytophilum was detected in an HL-60 cell culture by Southern blot analysis followed by sequencing the band and in blood samples from infected SCID mice by PCR, followed by sequencing. The sequences were consistent with the RecF pathway recombination: a half-crossover structure, consisting of the donor p44 locus connected to the 3′ conserved region of the recipient p44 in the p44 expression locus in direct orientation. To determine whether the p44 recombination intermediate structure can be generated in a RecF-active Escherichia coli strain, we constructed a double-origin plasmid carrying the p44 expression locus and a donor p44 locus and introduced the plasmid into various E. coli strains. The recombination intermediate was recovered in an E. coli strain with active RecF recombination pathway but not in strains with deficient RecF pathway. Our results support the view that the p44 gene conversion in A. phagocytophilum occurs through the RecF pathway.

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Antigenic Variation of Anaplasma marginale by Expression of MSP2 Mosaics

Infection and Immunity ◽

10.1128/iai.68.11.6133-6138.2000 ◽

2000 ◽

Vol 68 (11) ◽

pp. 6133-6138 ◽

Cited By ~ 87

Author(s):

Anthony F. Barbet ◽

Anna Lundgren ◽

Jooyoung Yi ◽

Fred R. Rurangirwa ◽

Guy H. Palmer

Keyword(s):

Outer Membrane ◽

Antigenic Variation ◽

Outer Membrane Proteins ◽

Anaplasma Marginale ◽

Life Cycles ◽

Reservoir Host ◽

Complex Life Cycles ◽

Genomic Expression ◽

Polycistronic Mrna ◽

Expression Site

ABSTRACT Anaplasma marginale is a tick-borne pathogen, one of several closely related ehrlichial organisms that cause disease in animals and humans. These Ehrlichia species have complex life cycles that require, in addition to replication and development within the tick vector, evasion of the immune system in order to persist in the mammalian reservoir host. This complexity requires efficient use of the small ehrlichial genome. A. marginaleand related ehrlichiae express immunoprotective, variable outer membrane proteins that have similar structures and are encoded by polymorphic multigene families. We show here that the major outer membrane protein of A. marginale, MSP2, is encoded on a polycistronic mRNA. The genomic expression site for this mRNA is polymorphic and encodes numerous amino acid sequence variants in bloodstream populations of A. marginale. A potential mechanism for persistence is segmental gene conversion of the expression site to link hypervariable msp2 sequences to the promoter and polycistron.

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