scholarly journals Two Monoclonal Antibodies with Defined Epitopes of P44 Major Surface Proteins Neutralize Anaplasma phagocytophilum by Distinct Mechanisms

2006 ◽  
Vol 74 (3) ◽  
pp. 1873-1882 ◽  
Author(s):  
Xueqi Wang ◽  
Takane Kikuchi ◽  
Yasuko Rikihisa
Keyword(s):  
Monoclonal Antibodies ◽  
Anaplasma Phagocytophilum ◽  
Outer Membrane Proteins ◽  
Critical Role ◽  
Hypervariable Region ◽  
Passive Immunization ◽  
Amino Acid Sequences ◽  
Surface Proteins ◽  
Host Cells ◽  
Bacterial Surface

ABSTRACT Anaplasma phagocytophilum is an obligatory intracellular bacterium that causes human granulocytic anaplasmosis. The polymorphic 44-kDa major outer membrane proteins of A. phagocytophilum are dominant antigens recognized by patients and infected animals. However, the ability of anti-P44 antibody to neutralize the infection has been unclear due to a mixture of P44 proteins with diverse hypervariable region amino acid sequences expressed by a given bacterial population and lack of epitope-defined antibodies. Monoclonal antibodies (MAbs) 5C11 and 3E65 are directed to different domains of P44 proteins, the N-terminal conserved region and P44-18 central hypervariable region, respectively. Passive immunization with either MAb 5C11 or 3E65 partially protects mice from infection with A. phagocytophilum. In the present study, we demonstrated that the two monoclonal antibodies recognize bacterial surface-exposed epitopes of naturally folded P44 proteins and mapped these epitopes to specific peptide sequences. The two MAbs almost completely blocked the infection of the A. phagocytophilum population that predominantly expressed P44-18 in HL-60 cells by distinct mechanisms: MAb 5C11 blocked the binding, but MAb 3E65 did not block binding or internalization. Instead, MAb 3E65 inhibited internalized A. phagocytophilum to develop into microcolonies called morulae. Some plasma from experimentally infected horses and mice reacted with these two epitopes. Taken together, these data indicate the presence of at least two distinct bacterial surface-exposed neutralization epitopes in P44 proteins. The results indicate that antibodies directed to certain epitopes of P44 proteins have a critical role in inhibiting A. phagocytophilum infection of host cells.

scholarly journals Characterization of Monoclonal Antibodies to the 44-Kilodalton Major Outer Membrane Protein of the Human Granulocytic Ehrlichiosis Agent

1998 ◽  
Vol 36 (11) ◽  
pp. 3278-3284 ◽  
Author(s):  
Hyung-Yong Kim ◽  
Yasuko Rikihisa
Keyword(s):  
Monoclonal Antibodies ◽  
Western Blot ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Passive Immunization ◽  
Human Infection ◽  
Host Cells ◽  
Granulocytic Ehrlichiosis ◽  
Human Granulocytic Ehrlichiosis ◽  
Molecular Sizes

The major outer membrane proteins (OMPs) of the human granulocytic ehrlichiosis (HGE) agent, with molecular sizes of 44 to 47 kDa, are immunodominant antigens in human infection. Monoclonal antibodies (MAbs) to the OMPs were made by immunizing BALB/c mice with the purified HGE agent and then by fusing spleen cells with myeloma cells. The immunologic specificities of three MAbs (3E65, 5C11, and 5D13) were examined with five human HGE agent isolates and one tick isolate. By Western blot analysis, all three MAbs recognized the HGE agent but notEhrlichia chaffeensis, Ehrlichia sennetsu,Ehrlichia canis, or their host cells. MAb 3E65 reacted with a 44-kDa protein in the homologous human isolate but not in the remaining five isolates. The two remaining MAbs recognized proteins with molecular sizes of 44 to 47 kDa in all six isolates. Western blot results with the OMP fraction of the six isolates were consistent with results with the whole HGE agent. Immunofluorescent-antibody staining and immunogold labeling with these MAbs showed that these antigens were primarily present on the membrane of the HGE agent. MAbs 5C11 and 5D13 recognized the recombinant 44-kDa protein by Western immunoblot analysis, but MAb 3E65 did not. Passive immunization with MAb 3E65 was more effective in protecting mice from HGE agent infection than with MAbs 5C11 and 5D13. These MAbs would be useful for analyzing the role of the major OMP antigens in HGE agent infection and for serodiagnosis.

scholarly journals Anaplasma phagocytophilum p44 mRNA Expression Is Differentially Regulated in Mammalian and Tick Host Cells: Involvement of the DNA Binding Protein ApxR

2007 ◽  
Vol 189 (23) ◽  
pp. 8651-8659 ◽  
Author(s):  
Xueqi Wang ◽  
Zhihui Cheng ◽  
Chunbin Zhang ◽  
Takane Kikuchi ◽  
Yasuko Rikihisa
Keyword(s):  
Dna Binding ◽  
Anaplasma Phagocytophilum ◽  
Binding Protein ◽  
Critical Role ◽  
Dna Binding Protein ◽  
Surface Proteins ◽  
Host Cells ◽  
Mammalian Host ◽  
Promoter Regions ◽  
Cells Cultured

ABSTRACT The natural life cycle of Anaplasma phagocytophilum, an obligatory intracellular bacterium that causes human granulocytic anaplasmosis, consists of alternate infection of two distinct hosts, ticks and mammals, in which bacterial surface proteins are expected to have a critical role. The present study investigated regulation of A. phagocytophilum p44 genes, which encode the P44 major surface proteins. Quantitative real-time reverse transcription-PCR analysis revealed that the amount of p44 mRNA obtained from spleens of A. phagocytophilum-infected SCID mice was approximately 10-fold greater than the amount obtained from salivary glands of A. phagocytophilum-infected Ixodes scapularis nymphs. Similarly, the amount of p44 mRNA obtained from A. phagocytophilum-infected HL-60 cells per bacterium was significantly greater than the amount obtained from infected ISE6 tick cells. The relative amount of p44 mRNA was approximately threefold higher in A. phagocytophilum-infected HL-60 cells cultured at 37°C than in A. phagocytophilum-infected HL-60 cells cultured at 28°C. Although there are more than 100 p44 paralogs, we observed expression mainly from the p44 expression locus (p44E) in various host environments. Interestingly, transcription of the A. phagocytophilum gene encoding the DNA binding protein ApxR was also significantly greater in A. phagocytophilum-infected HL-60 cells than in infected ISE6 tick cells. Gel mobility shift and DNase I protection assays revealed recombinant ApxR binding to the promoter regions of p44E and apxR. ApxR also transactivated the p44E and apxR promoter regions in a lacZ reporter assay. These results indicate that p44 genes and apxR are specifically up-regulated in the mammalian host environment and suggest that ApxR not only is positively autoregulated but also acts as a transcriptional regulator of p44E.

scholarly journals Invasion mechanisms of Gram-positive pathogenic cocci

2007 ◽  
Vol 98 (09) ◽  
pp. 488-496 ◽  
Author(s):  
Patric Nitsche-Schmitz ◽  
Manfred Rohde ◽  
Gursharan Chhatwal
Keyword(s):  
Surface Proteins ◽  
Host Cells ◽  
Human Pathogens ◽  
Gram Positive ◽  
Bacterial Surface ◽  
Surface Receptors ◽  
Major Threat ◽  
Invasion Mechanisms ◽  
Invasive Infections ◽  
Gram Positive Cocci

SummaryGram-positive cocci are important human pathogens. Streptococci and staphylococci in particular are a major threat to human health,since they cause a variety of serious invasive infections. Their invasion into normally sterile sites of the host depends on elaborated bacterial mechanisms that involve adhesion to the host tissue, its degradation, internalisation by host cells, and passage through epithelia and endothelia. Interactions of bacterial surface proteins with proteins of the host’s extracellular matrix as well as with cell surface receptors are crucial factors in these processes, and some of the key mechanisms are similar in many pathogenic Gram-positive cocci.Therapies that interfere with these mechanisms may become efficient alternatives to today’s antibiotic treatments.

scholarly journals Production and Characterization of Monoclonal Antibodies Specific for a Conserved Epitope within Hepatitis C Virus Hypervariable Region 1

2001 ◽  
Vol 75 (24) ◽  
pp. 12412-12420 ◽  
Author(s):  
Chengyao Li ◽  
Daniel Candotti ◽  
Jean-Pierre Allain
Keyword(s):  
Monoclonal Antibodies ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Neutralizing Antibodies ◽  
Hypervariable Region ◽  
Passive Immunization ◽  
Immune Recognition ◽  
Hypervariable Region 1 ◽  
Neutralizing Capacity ◽  
Conserved Epitope

ABSTRACT Frequent mutations in hypervariable region 1 (HVR1) of the main envelope protein of hepatitis C virus (HCV) is a major mechanism of persistence by escaping the host immune recognition. HVR1 contains an epitope eliciting neutralizing antibodies. This study was aimed to prepare broadly cross-reacting, high-affinity, monoclonal antibodies (MAb) to the HVR1 C terminus of HCV with potential therapeutic neutralizing capacity. A conserved amino residue group of glycine (G) at position 23 and glutamic acid (Q) at position 26 in HVR1 was confirmed as a key epitope against which two MAbs were selected and characterized. MAbs 2P24 and 15H4 were immunoglobulin G1 kappa chain [IgG1(κ)], cross-reacted with 32 and 30 of 39 random C-terminal HVR1 peptides, respectively, and did not react with other HCV peptides. The VH of 2P24 and 15H4 heavy chains originated from Igh germ line v gene family 1 and 8, respectively. In contrast, the VL κ sequences were highly homologous. The affinity (K d ) of 2P24 and 15H4 (10−9 or 10−8 M with two immunizing peptides and 10−8 M with two nonimmunizing HVR1 peptides) paralleled the reactivity obtained with peptide enzyme immunoassay. MAbs 2P24 and 15H4 captured 25 of 31 (81%) HCV in unselected patients' plasmas. These antibodies also blocked HCV binding to Molt-4 cells in a dose-dependent fashion. The data presented suggest that broadly cross-reactive MAbs to a conserved epitope within HCV HVR1 can be produced. Clinical application for passive immunization in HCV-related chronic liver disease and after liver transplantation is considered.

scholarly journals Surfaceome of Leptospira spp.

2005 ◽  
Vol 73 (8) ◽  
pp. 4853-4863 ◽  
Author(s):  
Paul A. Cullen ◽  
Xiaoyi Xu ◽  
James Matsunaga ◽  
Yolanda Sanchez ◽  
Albert I. Ko ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Surface Expression ◽  
Surface Proteins ◽  
Enzyme Linked Immunosorbent Assay ◽  
Two Dimensional Gel Electrophoresis ◽  
Affinity Capture ◽  
Bacterial Surface ◽  
Protective Antigens ◽  
Cell Enzyme

ABSTRACT The identification of the subset of outer membrane proteins exposed on the surface of a bacterial cell (the surfaceome) is critical to understanding the interactions of bacteria with their environments and greatly narrows the search for protective antigens of extracellular pathogens. The surfaceome of Leptospira was investigated by biotin labeling of viable leptospires, affinity capture of the biotinylated proteins, two-dimensional gel electrophoresis, and mass spectrometry (MS). The leptospiral surfaceome was found to be predominantly made up of a small number of already characterized proteins, being in order of relative abundance on the cell surface: LipL32 > LipL21 > LipL41. Of these proteins, only LipL32 had not been previously identified as surface exposed. LipL32 surface exposure was subsequently verified by three independent approaches: surface immunofluorescence, whole-cell enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy. Three other proteins, Q8F8Q0 (a putative transmembrane outer membrane protein) and two proteins of 20 kDa and 55 kDa that could not be identified by MS, one of which demonstrated a high degree of labeling potentially representing an additional, as-yet-uncharacterized, surface-exposed protein. Minor labeling of p31LipL45, GroEL, and FlaB1 was also observed. Expression of the surfaceome constituents remained unchanged under a range of conditions investigated, including temperature and the presence of serum or urine. Immunization of mice with affinity-captured surface components stimulated the production of antibodies that bound surface proteins from heterologous leptospiral strains. The surfaceomics approach is particularly amenable to protein expression profiling using small amounts of sample (<107 cells) offering the potential to analyze bacterial surface expression during infection.

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

2004 ◽  
Vol 72 (10) ◽  
pp. 5574-5581 ◽  
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.

scholarly journals Mechanisms of Variable p44 Expression by Anaplasma phagocytophilum

2003 ◽  
Vol 71 (10) ◽  
pp. 5650-5661 ◽  
Author(s):  
Quan Lin ◽  
Yasuko Rikihisa ◽  
Norio Ohashi ◽  
Ning Zhi
Keyword(s):  
Gene Expression ◽  
Amino Acid ◽  
Anaplasma Phagocytophilum ◽  
Hypervariable Region ◽  
Amino Acid Sequences ◽  
Hypervariable Regions ◽  
Tandem Genes ◽  
Flanking Regions ◽  
Passage Cell ◽  
Expression Status

ABSTRACT The human intragranulocytic bacterium Anaplasma phagocytophilum promotes variation of P44s, which are surface-exposed proteins encoded by a p44 multigene family. In the present study, the specific p44 gene expression loci in four strains of A. phagocytophilum were identified and it was determined that each consisted of four tandem genes, tr1, omp-1X, omp-1N, and p44. A putative σ70-type promoter was found upstream of tr1. The p44 genes include a central hypervariable region flanked by conserved regions. The hypervariable region sequence in the p44 expression locus was duplicated and, regardless of the expression status, conserved at another locus in both low- and high-passage cell cultures of strain NY-37. No significant differences in the hypervariable region were found when we compared p44 sequences, at the level of cDNA, within the expression locus and within other loci in the genomes of strains NY-37 and HZ. Similarly, in cDNA isolated from patients and from assorted cultures of strains NY-31, NY-36, and NY-37, hypervariable regions of 450 deduced amino acid sequences of various p44s within each strain were found to be identical, as were those of p44 sequences in the genome of strain HZ. These data suggest that variations in p44 sequences at the level of the p44 expression locus occur through unidirectional conversion of the entire (nonsegmental) p44 hypervariable region including flanking regions with a corresponding sequence copied from one of the conserved donor p44 genomic loci. The data suggest that the P44 antigenic repertoire within the hypervariable region is restricted.

scholarly journals Cyclic Dimeric GMP Signaling Regulates Intracellular Aggregation, Sessility, and Growth of Ehrlichia chaffeensis

2011 ◽  
Vol 79 (10) ◽  
pp. 3905-3912 ◽  
Author(s):  
Yumi Kumagai ◽  
Junji Matsuo ◽  
Zhihui Cheng ◽  
Yoshihiro Hayakawa ◽  
Yasuko Rikihisa
Keyword(s):  
Serine Protease ◽  
Serine Proteases ◽  
Matrix Protein ◽  
Response Regulator ◽  
Bacterial Biofilm ◽  
Surface Proteins ◽  
Host Cells ◽  
Ehrlichia Chaffeensis ◽  
Content Type ◽  
Bacterial Surface

ABSTRACTCyclic dimeric GMP (c-di-GMP), a bacterial second messenger, is known to regulate bacterial biofilm and sessility. Replication of an obligatory intracellular pathogen,Ehrlichia chaffeensis, is characterized by formation of bacterial aggregates called morulae inside membrane-bound inclusions. WhenE. chaffeensismatures into an infectious form, morulae become loose to allow bacteria to exit from host cells to infect adjacent cells.E. chaffeensisexpresses a sensor kinase, PleC, and a cognate response regulator, PleD, which can produce c-di-GMP. A hydrophobic c-di-GMP antagonist, 2′-O-di(tert-butyldimethysilyl)-c-di-GMP (CDGA) inhibitsE. chaffeensisinternalization into host cells by facilitating degradation of some bacterial surface proteins via endogenous serine proteases. In the present study, we found that PleC and PleD were upregulated synchronously during exponential growth of bacteria, concomitant with increased morula size. While CDGA did not affect host cells, when infected cells were treated with CDGA, bacterial proliferation was inhibited, morulae became less compact, and the intracellular movement of bacteria was enhanced. Concurrently, CDGA treatment facilitated the extracellular release of bacteria with lower infectivity than those spontaneously released from sham-treated cells. Addition of CDGA to isolated inclusions induced dispersion of the morulae, degradation of an inclusion matrix protein TRP120, and bacterial intrainclusion movement, all of which were blocked by a serine protease inhibitor. These results suggest that c-di-GMP signaling regulates aggregation and sessility ofE. chaffeensiswithin the inclusion through stabilization of matrix proteins by preventing the serine protease activity, which is associated with bacterial intracellular proliferation and maturation.

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

2004 ◽  
Vol 72 (2) ◽  
pp. 659-666 ◽  
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.

scholarly journals Anaplasma phagocytophilum Asp14 Is an Invasin That Interacts with Mammalian Host Cells via Its C Terminus To Facilitate Infection

2012 ◽  
Vol 81 (1) ◽  
pp. 65-79 ◽  
Author(s):  
Amandeep Kahlon ◽  
Nore Ojogun ◽  
Stephanie A. Ragland ◽  
David Seidman ◽  
Matthew J. Troese ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Anaplasma Phagocytophilum ◽  
Affinity Purification ◽  
Surface Protein ◽  
Surface Proteins ◽  
Host Cells ◽  
Mammalian Host ◽  
Content Type

Anaplasma phagocytophilum, a member of the familyAnaplasmataceae, is the tick-transmitted obligate intracellular bacterium that causes human granulocytic anaplasmosis. The life cycle ofA. phagocytophilumis biphasic, transitioning between the noninfectious reticulate cell (RC) and infectious dense-cored (DC) forms. We analyzed the bacterium's DC surface proteome by selective biotinylation of surface proteins, NeutrAvidin affinity purification, and mass spectrometry. Transcriptional profiling of selected outer membrane protein candidates over the course of infection revealed thataph_0248(designatedasp14[14-kDaA. phagocytophilumsurface protein]) expression was upregulated the most duringA. phagocytophilumcellular invasion.asp14transcription was induced during transmission feeding ofA. phagocytophilum-infected ticks on mice and was upregulated when the bacterium engaged its receptor, P-selectin glycoprotein ligand 1. Asp14 localized to theA. phagocytophilumsurface and was expressed duringin vivoinfection. Treating DC organisms with Asp14 antiserum or preincubating mammalian host cells with glutathioneS-transferase (GST)–Asp14 significantly inhibited infection of host cells. Moreover, preincubating host cells with GST-tagged forms of both Asp14 and outer membrane protein A, anotherA. phagocytophiluminvasin, pronouncedly reduced infection relative to treatment with either protein alone. The Asp14 domain that is sufficient for cellular adherence and invasion lies within the C-terminal 12 to 24 amino acids and is conserved among otherAnaplasmaandEhrlichiaspecies. These results identify Asp14 as anA. phagocytophilumsurface protein that is critical for infection, delineate its invasion domain, and demonstrate the potential of targeting Asp14 in concert with OmpA for protecting against infection byA. phagocytophilumand otherAnaplasmataceaepathogens.

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