Identification of novel surface proteins of Anaplasma phagocytophilum by affinity purification and proteomics

Anaplasma phagocytophilum is the etiologic agent of human granulocytic anaplasmosis (HGA), one of the major tick-borne zoonoses in the United States. The surface of A. phagocytophilum plays a crucial role in subverting the hostile host cell environment. However, except for the P44/Msp2 outer membrane protein family, the surface components of A. phagocytophilum are largely unknown. To identify the major surface proteins of A. phagocytophilum, a membrane-impermeable, cleavable biotin reagent, sulfosuccinimidyl-2-[biotinamido]ethyl-1,3-dithiopropionate (Sulfo-NHS-SS-Biotin), was used to label intact bacteria. The biotinylated bacterial surface proteins were isolated by streptavidin agarose affinity purification and then separated by electrophoresis, followed by capillary liquid chromatography-nanospray tandem mass spectrometry analysis. Among the major proteins captured by affinity purification were five A. phagocytophilum proteins, Omp85, hypothetical proteins APH_0404 (designated Asp62) and APH_0405 (designated Asp55), P44 family proteins, and Omp-1A. The surface exposure of Asp62 and Asp55 was verified by immunofluorescence microscopy. Recombinant Asp62 and Asp55 proteins were recognized by an HGA patient serum. Anti-Asp62 and anti-Asp55 peptide sera partially neutralized A. phagocytophilum infection of HL-60 cells in vitro. We found that the Asp62 and Asp55 genes were cotranscribed and conserved among members of the family Anaplasmataceae. With the exception of P44-18, all of the proteins were newly revealed major surface-exposed proteins whose study should facilitate understanding the interaction between A. phagocytophilum and the host. These proteins may serve as targets for development of chemotherapy, diagnostics, and vaccines.     

Porin activity of Anaplasma phagocytophilum outer membrane fraction and purified P44

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 beta-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 beta-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.     

Anaplasma phagocytophilum major surface protein-2 (Msp2) forms multimeric complexes in the bacterial membrane

Anaplasma phagocytophilum 44-kDa major surface protein-2 (Msp2) mediates partial neutrophil adhesion and interactions. Since A. phagocytophilum 44-kDa monoclonal antibodies also react with 160- and 100-kDa bands, a putative adhesin complex was studied. After separate excision/immunoprecipitation of these three bands, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) resolved each into three bands again with increased 44-kDa protein under reducing conditions suggesting oligomerization of Msp2 44-kDa monomers. With 9 M urea, each separately excised band was resolved only into 44-kDa monomers with three different pIs. With protein cross-linking, immunoblots showed four additional bands and increased high molecular mass band intensity, suggesting homo- and hetero-polymerization with other A. phagocytophilum proteins. Recognition of Msp2 complexes facilitates understanding of A. phagocytophilum-neutrophil adhesion.     

Antibodies to whole-cell or recombinant antigens of Borrelia burgdorferi, Anaplasma phagocytophilum, and Babesia microti in white-footed mice

Serum samples were obtained from white-footed mice (Peromyscus leucopus) in tick-infested areas of Connecticut during the period 2001 through 2003 and analyzed for antibodies to Borrelia burgdorferi, Anaplasma phagocytophilum, and Babesia microti. Emphasis was placed on the evaluations of highly specific recombinant VlsE or protein (p) 44 antigens of B. burgdorferi and A. phagocytophilum, respectively, in a newly developed enzyme-linked immunosorbent assay (ELISA) as well as testing sera with whole-cell antigens by conventional ELISA or indirect fluorescent antibody staining methods. Of the 414 mouse sera analyzed, 310 (75%) had antibodies to whole-cell B. burgdorferi, whereas 157 (38%) were positive to the VlsE antigen. The latter nearly equaled the overall antibody prevalence rate (37%) computed when sera were tested separately with the p44 antigen. Mice were exposed to these pathogens and B. microti (antibody prevalence = 25%) in extreme northern Connecticut as well as the southern coastal areas of the state, thus indicating further geographic expansion of these infections. Fifty-three (13%) sera from widely separated sites had antibodies to all three pathogens. With expression and immunological recognition of VlsE and p44 antigens in P. leucopus, separate incorporation of these fusion proteins in an ELISA was very helpful in confirming past or current infections and in identifying specific foci for B. burgdorferi and A. phagocytophilum.     

Use of recombinant antigens of Borrelia burgdorferi and Anaplasma phagocytophilum in enzyme-linked immunosorbent assays to detect antibodies in white-tailed deer

Serum samples obtained from white-tailed deer (Odocoileus virginianus) in Connecticut (n=218) and South Carolina (n=20) (USA) during the period 1992-2002 were analyzed for antibodies to whole-cell or recombinant antigens (i.e., fusion proteins) of Borrelia burgdorferi sensu stricto and Anaplasma phagocytophilum, etiologic agents of Lyme borreliosis and granulocytic ehrlichiosis, respectively. In enzyme-linked immunosorbent assays (ELISAs) with whole-cell B. burgdorferi, the overall seropositivity rate for Connecticut (53%) exceeded that for South Carolina (30%). In separate tests of seven recombinant antigens of B. burgdorferi by an ELISA, seroprevalence for the VlsE antigen was highest (48%) in Connecticut followed by outer surface protein (OspF) (21%), whereas serum reactivities to the protein (p) 41-G antigen (55%) and VlsE (25%) were most frequent for South Carolina sera. In analyses for antibodies to the recombinant protein (p) 44 antigen of A. phagocytophilum, seroprevalences of 52% and 25% were recorded for Connecticut and South Carolina samples, respectively. These findings paralleled those determined by indirect fluorescent antibody staining methods with whole cells (43% and 30%). Moreover, there was good agreement (74%) in results of Western blot analyses and an ELISA when a subset of 39 sera was screened with whole-cell or recombinant p44 antigens of A. phagocytophilum. An ELISA with highly specific recombinant VlsE or p44 antigens can be used in conjunction with other antibody tests to determine whether deer living in different regions of eastern United States were exposed to B. burgdorferi or A. phagocytophilum.     

Expression and porin activity of P28 and OMP-1F during intracellular Ehrlichia chaffeensis development

Ehrlichia chaffeensis, an obligatory intracellular gram-negative bacterium, must take up various nutrients and metabolic compounds because it lacks many genes involved in metabolism. Nutrient uptake by a gram-negative bacterium occurs primarily through pores or channels in the bacterial outer membrane. Here we demonstrate that isolated E. chaffeensis outer membranes have porin activities, as determined by a proteoliposome swelling assay. The activity was partially blocked by an antibody that recognizes the two most abundant outer membrane proteins, P28/OMP-19 and OMP-1F/OMP-18. Both proteins were predicted to have structural features characteristic of porins, including 12 transmembrane segments comprised of amphipathic and antiparallel beta-strands. The sodium dodecyl sulfate stability of the two proteins was consistent with a beta-barrel structure. Isolated native P28 and OMP-1F exhibited porin activities, with pore sizes similar to and larger than, respectively, that of OprF, which is the porin with the largest pore size known to date. E. chaffeensis experiences temperature changes during transmission by ticks. During the intracellular development of E. chaffeensis, both P28 and OMP-1F were expressed mostly in the mid-exponential growth phase at 37 degrees C and the late-exponential growth phase at 28 degrees C. The porin activity of proteoliposomes reconstituted with proteins from the outer membrane fractions derived from bacteria in the mid- and late-exponential growth phases at 28 degrees C and 37 degrees C correlated with the expression levels of P28 and OMP-1F. These results imply that P28 and OMP-1F function as porins with large pore sizes, suggesting that the differential expression of these two proteins might regulate nutrient uptake during intracellular E. chaffeensis development at both temperatures.     

Analysis of membrane protein genes in a Brazilian isolate of Anaplasma marginale

The sequencing of the complete genome of Anaplasma marginale has enabled the identification of several genes that encode membrane proteins, thereby increasing the chances of identifying candidate immunogens. Little is known regarding the genetic variability of genes that encode membrane proteins in A. marginale isolates. The aim of the present study was to determine the degree of conservation of the predicted amino acid sequences of OMP1, OMP4, OMP5, OMP7, OMP8, OMP10, OMP14, OMP15, SODb, OPAG1, OPAG3, VirB3, VirB9-1, PepA, EF-Tu and AM854 proteins in a Brazilian isolate of A. marginale compared to other isolates. Hence, primers were used to amplify these genes: omp1, omp4, omp5, omp7, omp8, omp10, omp14, omp15, sodb, opag1, opag3, virb3, VirB9-1, pepA, ef-tu and am854. After polimerase chain reaction amplification, the products were cloned and sequenced using the Sanger method and the predicted amino acid sequence were multi-aligned using the CLUSTALW and MEGA 4 programs, comparing the predicted sequences between the Brazilian, Saint Maries, Florida and A. marginale centrale isolates. With the exception of outer membrane protein (OMP) 7, all proteins exhibited 92-100% homology to the other A. marginale isolates. However, only OMP1, OMP5, EF-Tu, VirB3, SODb and VirB9-1 were selected as potential immunogens capable of promoting cross-protection between isolates due to the high degree of homology (over 72%) also found with A. (centrale) marginale.     

我国部分地区禽源性大肠杆菌的外膜蛋白型

测定了从我国１８个省、市、自治区分离到的２０４个禽病原性大肠杆菌优势血清型分离株的外膜蛋白型（ＯｕｔｅｒＭｅｍｂｒａｎｅＰｒｏｔｅｉｎＰａｔｅｒｎｓ,ＯＭＰ型）。这些分离株共产生了４个ＯＭＰ型,５６个Ｏ１８分离株可分为３个ＯＭＰ型,５４个Ｏ７８分离株、２８个Ｏ２分离株、２６个Ｏ８８分离株、２２个Ｏ１１分离株和１８个Ｏ２６分离株,分别出现了４、２、１、３和１个ＯＭＰ型。其中,ＯＭＰ１型为６个血清型所共有,ＯＭＰ３型则同时存在于Ｏ１８、Ｏ７８、Ｏ２和Ｏ１１分离株中。结果表明,优势血清型中,Ｏ１８、Ｏ７８、Ｏ２和Ｏ１１分离株具有多样性的ＯＭＰ型,而Ｏ８８、Ｏ２６分离株的ＯＭＰ型则高度一致,所测６个优势血清型的分离株间存在共同的ＯＭＰ型     

Immunogenicity of iron-regulated outer membrane proteins of Pasteurella multocida B:2 in mice model

The present study was conducted to investigate the role of iron-regulated outer membrane proteins (IROMP) of Pasteurella multocida B:2 in mice as potential immunogens. Outer membrane proteins extracted from P. multocida B:2 grown under normal (OMP) and iron-deficient (IROMP) conditions were subjected to discontinuous SDS-PAGE. Nine polypeptides of MW ranging from 85.1 to 16.7 kDa from OMP preparations and two additional polypeptides of MW 95.4 and 89.1 kDa from IROMP preparations were observed with bands of MW 37.2 and 34.7 kDa as major proteins. Mice were immunized twice with OMP, IROMP-enriched fractions and whole cell lysate (WCL) via subcutaneous route at day 0 and 21. Antibody titers were determined from sera collected at weekly interval and protection was studied against challenge using 10(2) cfu of P. multocida two weeks after secondary immunization via intranasal and subcutaneous routes. IROMP and OMP immunized mice provoked significant antibody responses and IROMP induced higher antibody responses. IROMP and OMP immunized mice showed protection (100%) upon intranasal challenge and a protection (84%) following subcutaneous challenge as compared to high mortality (84%) in control mice. These results indicate that OMP enriched with IROMP fractions can be superior means of immunization.     

Seroprevalence of Anaplasma phagocytophilum in domestic and wild animals from central Italy

From January 2004 to July 2007, 2455 sera were collected from domestic and wild animals living in central Italy and tested by indirect immunofluorescence assay to detect antibodies against Anaplasma phagocytophilum. Considering sera with 1:40 antibody titers as positive, 336 (13.68%) animals scored positive. The percentages of seropositivity were: 46.26% (31/67) in fallow deer, 46.15% (24/52) in red deer, 16.89% (134/793) in horses, 16.78% (23/137) in cattle, 12.74% (13/102) in sheep, 8.76% (108/1232) in dogs, 4.16% (3/72) in goats. These data confirm the presence of A. phagocytophilum in wild ruminants and domestic animals, including pets, in central Italy.     

Tick-borne rickettsial pathogens in ticks and small mammals in Korea

In order to investigate the prevalence of tick-borne infectious agents among ticks, ticks comprising five species from two genera (Hemaphysalis spp. and Ixodes spp.) were screened using molecular techniques. Ticks (3,135) were collected from small wild-caught mammals or by dragging/flagging in the Republic of Korea (ROK) and were pooled into a total of 1,638 samples (1 to 27 ticks per pool). From the 1,638 tick samples, species-specific fragments of Anaplasma phagocytophilum (1 sample), Anaplasma platys (52 samples), Ehrlichia chaffeensis (29 samples), Ehrlichia ewingii (2 samples), Ehrlichia canis (18 samples), and Rickettsia rickettsii (28 samples) were amplified by PCR assay. Twenty-one pooled and individual tick samples had mixed infections of two (15 samples) or three (6 samples) pathogens. In addition, 424 spleen samples from small captured mammals (389 rodents, 33 insectivores, and 2 weasels) were screened for selected zoonotic pathogens. Species-specific DNA fragments of A. phagocytophilum (110 samples), A. platys (68 samples), E. chaffeensis (8 samples), E. ewingii (26 samples), E. canis (51 samples), and Rickettsia sp. (22 samples) were amplified by PCR assay. One hundred thirty small mammals had single infections, while 4, 14, and 21 striped field mice (Apodemus agrarius) had mixed infections of four, three, and two pathogens, respectively. Phylogenetic analysis based on nucleotide sequence comparison also revealed that Korean strains of E. chaffeensis clustered closely with those from China and the United States, while the Rickettsia (rOmpA) sequences clustered within a clade together with a Chinese strain. These results suggest that these agents should be considered in differential diagnosis while examining cases of acute febrile illnesses in humans as well as animals in the ROK.     

Identification and characterization of a novel outer membrane protein (OMP J) of Moraxella catarrhalis that exists in two major forms

Moraxella catarrhalis is a common commensal of the human respiratory tract that has been associated with a number of disease states, including acute otitis media in children and exacerbations of chronic obstructive pulmonary disease in adults. During studies to investigate the outer membrane proteins of this bacterium, two novel major proteins, of approximately 19 kDa and 16 kDa (named OMP J1 and OMP J2, respectively), were identified. Further analysis indicated that these two proteins possessed almost identical gene sequences, apart from two insertion/deletion events in predicted external loops present within the putative barrel-like structure of the proteins. The development of a PCR screening strategy found a 100% (96/96) incidence for the genes encoding the OMP J1 and OMP J2 proteins within a set of geographically diverse M. catarrhalis isolates, as well as a significant association of OMP J1/OMP J2 with both the genetic lineage and the complement resistance phenotype (Fisher's exact test; P < 0.01). Experiments using two DeltaompJ2 mutants (one complement resistant and the other complement sensitive) indicated that both were less easily cleared from the lungs of mice than were their isogenic wild-type counterparts, with a significant difference in bacterial clearance being observed for the complement-resistant isolate but not for its isogenic DeltaompJ2 mutant (unpaired Student's t test; P < 0.001 and P = 0.32). In this publication, we characterize a novel outer membrane protein of Moraxella catarrhalis which exists in two variant forms associated with particular genetic lineages, and both forms are suggested to contribute to bacterial clearance from the lungs.     

Comparison of meningococcal outer membrane protein vaccines solubilized with detergent or C polysaccharide

Outer membrane proteins (OMPs) were isolated from meningococcal strain H44/76 (B:15:P1.16) by detergent extraction of bacteria. A final product containing class 1 (P1.16), 3(15), 4 OMPs and 5% (w/w) lipooligosaccharide was obtained. Two experimental vaccines were prepared: OMP-detergent and OMP-C polysaccharide. The OMP-detergent vaccine tended to show a better bactericidal: ELISA ratio for the antibodies induced as compared to the OMP-C polysaccharide vaccine. The vaccine induced bactericidal antibodies appeared for the greater part to be directed against the class 1 OMP (P1.16). By comparison of cultures grown in Mueller Hinton Broth with and without 0,25% (w/v) glucose, it was found that monoclonal antibodies against the serotype OMP (class 2 or 3) were not bactericidal against meningococci grown in MHB without glucose. Antibodies against class 1 OMP and lipooligosaccharide were not influenced by this. A new major outer membrane protein (appr. 40kd) is described that may function as a cation-specific porin.     

Anaplasma phagocytophilum in small mammals and ticks in northeast Florida

Human anaplasmosis is an emerging tick-borne disease in the United States, but few studies of the causative agent, Anaplasma phagocytophilum, have been conducted in southeastern states. The aim of this study was to determine if A. phagocytophilum is present in small mammals and ticks in northeast Florida. Polymerase chain reaction assays designed to amplify portions of the major surface protein 2 gene (p44), 16S rDNA, and groESL operons were used to test rodent blood and tick DNA samples for the presence of A. phagocytophilum. Positive samples were confirmed by DNA sequence analysis. Anaplasma phagocytophilum DNA was detected in less than 5% of cotton mice and 45% of cotton rats from two sites in northeast Florida. Anaplasma phagocytophilum DNA was also confirmed in 1.3% of host-seeking adult Ixodes scapularis tested and 2.7% of host-seeking adult Amblyomma americanum. This report describes the first DNA sequence data confirming strains of A. phagocytophilum in rodents and ticks in Florida. The DNA sequences of the msp2, 16S rDNA, and groESL gene fragments obtained in this study were highly similar to reference strains of human pathogenic strains of A. phagocytophilum. These findings suggest that A. phagocytophilum is present and established among some small mammal species in northeast Florida. Although the infection prevalence was low in the total number of ticks tested, the presence of A. phagocytophilum in two human biting tick species, one of which is a known competent vector, suggests that humans in this region may be at risk of granulocytic anaplasmosis caused by this pathogen.     

Novel genetic variants of Anaplasma phagocytophilum, Anaplasma bovis, Anaplasma centrale, and a novel Ehrlichia sp. in wild deer and ticks on two major islands in Japan

Wild deer are one of the important natural reservoir hosts of several species of Ehrlichia and Anaplasma that cause human ehrlichiosis or anaplasmosis in the United States and Europe. The primary aim of the present study was to determine whether and what species of Ehrlichia and Anaplasma naturally infect deer in Japan. Blood samples obtained from wild deer on two major Japanese islands, Hokkaido and Honshu, were tested for the presence of Ehrlichia and Anaplasma by PCR assays and sequencing of the 16S rRNA genes, major outer membrane protein p44 genes, and groESL. DNA representing four species and two genera of Ehrlichia and Anaplasma was identified in 33 of 126 wild deer (26%). DNA sequence analysis revealed novel strains of Anaplasma phagocytophilum, a novel Ehrlichia sp., Anaplasma centrale, and Anaplasma bovis in the blood samples from deer. None of these have been found previously in deer. The new Ehrlichia sp., A. bovis, and A. centrale were also detected in Hemaphysalis longicornis ticks from Honshu Island. These results suggest that enzootic cycles of Ehrlichia and Anaplasma species distinct from those found in the United States or Europe have been established in wild deer and ticks in Japan.     

Outer membrane protein-specific monoclonal antibodies protect SCID mice from fatal infection by the obligate intracellular bacterial pathogen Ehrlichia chaffeensis

Previous studies of Ehrlichia chaffeensis infection in the mouse have demonstrated that passive transfer of polyclonal Abs from resistant immunocompetent mice to susceptible SCID mice ameliorated infection and disease, even when Abs were administered during established infection. To identify particular Abs that could mediate bacterial clearance in vivo, E. chaffeensis-specific mAbs were generated and administered to infected SCID mice. Bacterial infection in the livers was significantly lowered after administration of either of two Abs of different isotypes (IgG2a and IgG3). Moreover, repeated administration of one Ab (Ec56.5; IgG2a) rescued mice from an otherwise lethal infection for at least 5 wk. Both protective Abs recognized the E. chaffeensis major outer membrane protein (OMP)-1g. Further studies revealed that both Abs recognized closely related epitopes within the amino terminus of the first hypervariable region of OMP-1g. Analyses of human sera showed that E. chaffeensis-infected patients also generated serological responses to OMP-1g hypervariable region 1, indicating that humans and mice recognize identical or closely related epitopes. These studies demonstrate that OMP-specific mAbs can mediate bacterial elimination in SCID mice, and indicate that Abs, in the absence of cell-mediated immunity, can play a significant role in host defense during infection by this obligate intracellular bacterium.     

Anaplasma phagocytophilum AnkA binds to granulocyte DNA and nuclear proteins

Human granulocytic anaplasmosis (HGA) is caused by the obligate intracellular bacterium Anaplasma phagocytophilum. The bacterium infects, survives, propagates in, and alters neutrophil phenotype, indicating unique survival mechanisms. AnkA is the only known A. phagocytophilum component that gains access beyond neutrophil vacuoles and is transported to the infected host cell nucleus. The ability of native and recombinant AnkA to bind DNA and nuclear proteins from host HL-60 cells was assessed by the use of immunoprecipitation after cis-diamminedichloroplatinum (cis-DDP) DNA-protein crosslinking, by probing uninfected HL-60 cell nuclear lysates for AnkA binding, and by recovery and sequence analysis of immunoprecipitated DNA. AnkA binds HL-60 cell DNA as well as nuclear proteins of approximately 86, 53 and 25 kDa, whereas recombinant A. phagocytophilum Msp2 or control proteins do not. DNA immunoprecipitation reveals AnkA binding to a variety of target genes in the human genome, including genes that encode proteins with ATPase, tyrosine phosphatase and NADH dehydrogenase-like functions. These data indicate that AnkA could exert some effect on cells through binding to protein:DNA complexes in neutrophil nuclei. Whether AnkA binding leads to neutrophil functional alterations, and how such alterations might occur will depend upon definitive identification of binding partners and associated metabolic and biochemical pathways.