Polymorphic Outer Membrane Protein Research Articles

Expression Level of the mip, pmp18D, and ompA Genes in Chlamydia abortus Isolated from Aborted Ewes

In this manuscript, we report the proteins macrophage infectivity potentiator (mip, CAB080), major outer membrane protein (momp, CAB048), and polymorphic outer membrane protein (pmp18D, CAB776) that are expressed in different times of pregnancy in mice infected with Chlamydia abortus. Enzootic abortion of ewes (EAE) by C. abortus, an obligate intracellular pathogen, is a critical zoonotic disease-causing significant economic loss to livestock farming globally. This study was carried out for the detection and characterization of macrophage infectivity potentiator (mip, CAB080), major outer membrane protein (momp, CAB048), and polymorphic outer membrane protein (pmp18D, CAB776) using RT-qPCR. These proteins are believed to be expressed as virulence factors in C. abortus isolated from aborted ewes. BALB/c mice (pregnant and nonpregnant) were used as an animal model to be injected intraperitoneally with C. abortus culture in Vero cells since the endometrial lymphoid tissues of these animals resembles that of ewes. Also, the short duration of pregnancy in mice makes them a suitable animal model for obstetric studies. Tissue samples were taken from the mice after 10, 15, and 20 days of pregnancy to compare the expression of the genes mip, pmp18D, and ompA. Transcription level was quantified using RT-qPCR, the GAPDH transcription quantification, as a normalization signal. Abortion occurred in pregnant mice, and apparent differences between the transcriptional levels of the mip, pmp18D, and ompA genes in the samples taken during different time intervals of pregnancy were not observed (p > 0.05). The result indicated that the three bacterial genes, mip, pmp18D, and ompA, play a role as virulence factors in abortion and are differentially expressed in pregnant and nonpregnant animals. Inactivation of the genes is suggested to confirm the hypothesis.

Polymorphic membrane protein 20G: A promising diagnostic biomarker for specific detection of Chlamydia psittaci infection

Psittacosis is a zoonotic disease caused by Chlamydia psittaci (C. psittaci), leading to high risk for animal industry and human health. Lack of reliable commercial kits and effective vaccines is hampering control of C. psittaci infection. Polymorphic outer membrane protein Gs (PmpGs) are enriched in diverse C. psittaci, and its role are unclear during C. psittaci infection. In the present study, pmp20G gene was cloned into pET-28a vector and then the constructed plasmid was transferred into Escherichia coli Rossetta (DE3). After denaturation and renaturation, the recombinant Pmp20G-N was identified by SDS-PAGE and Western blot. Afterwards Pmp20G-N was used as the coating antigen to develop an indirect ELISA (I-ELISA) assay. Both the specificity and sensitivity of Pmp20G-N ELISA were 100%, while the MOMP-ELISA had 93.65% sensitivity and 98.94% specificity, respectively. The concordance between MOMP-ELISA and Pmp20G-N ELISA assay was 98.1%. Hence, Pmp20G-N ELISA has the potential to be a diagnostic antigen for detection C. psittaci antibody. However, further studies are needed to be done for differentiating C. psittaci from Chlamydia spp. and other C.psittaci-specific serovars using Pmp20G-N ELISA.

Development of Transposon Mutagenesis for Chlamydia muridarum.

Functional genetic analysis of Chlamydia has been a challenge due to the historical genetic intractability of Chlamydia, although recent advances in chlamydial genetic manipulation have begun to remove these barriers. Here, we report the development of the Himar C9 transposon system for Chlamydia muridarum, a mouse-adapted Chlamydia species that is widely used in Chlamydia infection models. We demonstrate the generation and characterization of an initial library of 33 chloramphenicol (Cam)-resistant, green fluorescent protein (GFP)-expressing C. muridarum transposon mutants. The majority of the mutants contained single transposon insertions spread throughout the C. muridarum chromosome. In all, the library contained 31 transposon insertions in coding open reading frames (ORFs) and 7 insertions in intergenic regions. Whole-genome sequencing analysis of 17 mutant clones confirmed the chromosomal locations of the insertions. Four mutants with transposon insertions in glgB, pmpI, pmpA, and pmpD were investigated further for in vitro and in vivo phenotypes, including growth, inclusion morphology, and attachment to host cells. The glgB mutant was shown to be incapable of complete glycogen biosynthesis and accumulation in the lumen of mutant inclusions. Of the 3 pmp mutants, pmpI was shown to have the most pronounced growth attenuation defect. This initial library demonstrates the utility and efficacy of stable, isogenic transposon mutants for C. muridarum The generation of a complete library of C. muridarum mutants will ultimately enable comprehensive identification of the functional genetic requirements for Chlamydia infection in vivoIMPORTANCE Historical issues with genetic manipulation of Chlamydia have prevented rigorous functional genetic characterization of the ∼1,000 genes in chlamydial genomes. Here, we report the development of a transposon mutagenesis system for C. muridarum, a mouse-adapted Chlamydia species that is widely used for in vivo investigations of chlamydial pathogenesis. This advance builds on the pioneering development of this system for C. trachomatis We demonstrate the generation of an initial library of 33 mutants containing stable single or double transposon insertions. Using these mutant clones, we characterized in vitro phenotypes associated with genetic disruptions in glycogen biosynthesis and three polymorphic outer membrane proteins.

Analysis of Salmonella enterica Serovar Typhi by Outer Membrane Protein (OMP) Profiling, Random Amplification of Polymorphic DNA (RAPD) and Pulsed Field Gel Electrophoresis (PFGE)

A number of countries, including developed countries, still have typhoid fever as a major problem resulting in frequent outbreaks. The importance of controlling spread of typhoid fever is well known and necessitates periodic studies to delineate epidemiological relationships. Although phage typing remains to be the preferred conventional method for characterisation of typhoid bacilli, it is of limited use due to prevalence of few predominant phage types in the country like India. Therefore, an effort has been made to assess three molecular methods [Outer Membrane Protein (OMP) Profiling, Random Amplification of Polymorphic DNA (RAPD) and Pulsed Field Gel Electrophoresis (PFGE)] for typing of Salmonella enterica serovar Typhi. 128 Salmonella enterica serovar Typhi isolates were identified using biotyping and serotyping followed by antimicrobial susceptibility testing. These isolates were further subjected to OMP analysis, RAPD and PFGE. PFGE (114 unique clusters) was found to be the most discriminatory method followed by RAPD (94 unique clusters) and OMP profiling (50 unique clusters). Multidrug resistant strains were well discriminated by all three methods used in the study. PFGE still remains the most preferred method for detailed epidemiological investigations. However, random amplification of polymorphic DNA and outer membrane protein profiling can also be considered for molecular discrimination of the isolates in the laboratories lacking high-end facilities.

Development of a Novel PmpD-N ELISA for Chlamydia psittaci Infection

ObjectiveChlamydia psittaci is an avian respiratory pathogen and zoonotic agent. The wide prevalence of C. psittaci poses a threat to the poultry industry and its employees. However, few commercial kits are available for detecting avian antibodies excluding the in-house ELISA kit. In this study, we developed a novel ELISA kit for detecting antibodies against C. psittaci based on the N-terminal fragment of polymorphic outer membrane protein D (PmpD-N) as the coating antigen. MethodsThe antigen concentrations, primary antibody, and cut-off value were determined and optimized. The ELISA, designated PmpD-N ELISA, was assessed for sensitivity, specificity, and concordance using sera samples from 48 experimentally infected and 168 uninfected SPF chickens. ResultsThe sensitivity and specificity of PmpD-N ELISA were 97.9%, 100%, respectively, while the concordance was 98.1% as compared to that of MOMP-ELISA. No cross-reaction with positive sera for other avian pathogens was found. Using PmpD-N ELISA, 799/836 clinical samples were positive, including 93.0% and 98.1% positivity in layers and broilers, respectively. ConclusionThese data indicate that indirect ELISA with PmpD-N as the antigen candidate is a promising approach for the surveillance of C. psittaci infection.

A characteristic of polymorphic membrane protein F of Chlamydia trachomatis isolated from male urogenital tracts in Japan.

Although sexually transmitted disease due to Chlamydia trachomatis occurs similarly in both men and women, the female urogenital tract differs from that of males anatomically and physiologically, possibly leading to specific polymorphisms of the bacterial surface molecules. In the present study, we therefore characterized polymorphic features in a high-definition phylogenetic marker, polymorphic outer membrane protein (Pmp) F of C. trachomatis strains isolated from male urogenital tracts in Japan (Category: Japan-males, n = 12), when compared with those isolated from female cervical ducts in Japan (Category: Japan-females, n = 11), female cervical ducts in the other country (Category: Ref-females, n = 12) or homosexual male rectums in the other country (Category: Ref-males, n = 7), by general bioinformatics analysis tool with MAFFT software. As a result, phylogenetic reconstruction of the PmpF amino acid sequences showing three distinct clusters revealed that the Japan-males were limited into cluster 1 and 2, although there were only four clusters even though including an outgroup. Meanwhile, the phylogenetic distance values of PmpF passenger domain without hinge region, but not its full-length sequence, showed that the Japan-males were more stable and displayed less diversity when compared with the other categories, supported by the sequence conservation features. Thus, PmpF passenger domain is a useful phylogenetic maker, and the phylogenic features indicate that C. trachomatis strains isolated from male urogenital tracts in Japan may be unique, suggesting an adaptation depending on selective pressure, such as the presence or absence of microbial flora, furthermore possibly connecting to sexual differentiation.

OmpA family proteins and Pmp-like autotransporter: new adhesins of Waddlia chondrophila

Waddlia chondrophila is a obligate intracellular bacterium belonging to the Chlamydiales order, a clade that also includes the well-known classical Chlamydia responsible for a number of severe human and animal diseases. Waddlia is an emerging pathogen associated with adverse pregnancy outcomes in humans and abortion in ruminants. Adhesion to the host cell is an essential prerequisite for survival of every strict intracellular bacteria and, in classical Chlamydia, this step is partially mediated by polymorphic outer membrane proteins (Pmps), a family of highly diverse autotransporters that represent about 15% of the bacterial coding capacity. Waddlia chondrophila genome however only encodes one putative Pmp-like protein. Using a proteomic approach, we identified several bacterial proteins potentially implicated in the adhesion process and we characterized their expression during the replication cycle of the bacteria. In addition, we demonstrated that the Waddlia Pmp-like autotransporter as well as OmpA2 and OmpA3, two members of the extended Waddlia OmpA protein family, exhibit adhesive properties on epithelial cells. We hypothesize that the large diversity of the OmpA protein family is linked to the wide host range of these bacteria that are able to enter and multiply in various host cells ranging from protozoa to mammalian and fish cells.

Evaluation of a multisubunit recombinant polymorphic membrane protein and major outer membrane protein T cell vaccine against Chlamydia muridarum genital infection in three strains of mice

An efficacious vaccine is needed to control Chlamydia trachomatis infection. In the murine model of Chlamydia muridarum genital infection, multifunctional mucosal CD4 T cells are the foundation for protective immunity, with antibody playing a secondary role. We previously identified four Chlamydia outer membrane proteins (PmpE, PmpF, PmpG and PmpH) as CD4 T cell vaccine candidates using a dendritic cell-based immunoproteomic approach. We also demonstrated that these four polymorphic membrane proteins (Pmps) individually conferred protection as measured by accelerated clearance of Chlamydia infection in the C57BL/6 murine genital tract model. The major outer membrane protein, MOMP is also a well-studied protective vaccine antigen in this system. In the current study, we tested immunogenicity and protection of a multisubunit recombinant protein vaccine consisting of the four Pmps (PmpEFGH) with or without the major outer membrane protein (MOMP) formulated with a Th1 polarizing adjuvant in C57BL/6, Balb/c and C3H mice. We found that C57BL/6 mice vaccinated with PmpEFGH+MOMP elicited more robust cellular immune responses than mice immunized with individual protein antigens. Pmps elicited more variable cellular immune responses than MOMP among the three strains of mice. The combination vaccine accelerated clearance in the three strains of mice although at different rates. We conclude that the recombinant outer membrane protein combination constitutes a promising first generation Chlamydia vaccine construct that should provide broad immunogenicity in an outbred population.

Evaluation of a Pmp18D vibro cholerae ghost vaccine against chlamydia abortus in mice (VET2P.1046)

Abstract Chlamydia abortus (C. abortus) is an obligate intracellular pathogen that causes abortion in pigs and poses a zoonotic risk in pregnant women. Currently available attenuated and inactivated vaccines show marginal protective efficacy underlining the need for the development of effective vaccines. Recombinant proteins expressed on gram negative bacterial envelopes are known to induce protective immunity (Th1) without the need of any additional adjuvant. Pmp18D is a member of polymorphic outer membrane protein family of C.abortus. The PmpD protein of C. abortus is expressed throughout the chlamydial developmental cycle. Pmp protein family members are known to exhibit higher immunogenicity. In this study an evaluation of immune responses to Vibrio cholerae ghosts expressing Pmp18D(rVCG) was done in comparison to rPmp18D codelivered with either Flt3L or CpG. Mice were immunized 3 times intranasally 2 weeks apart .By day 14 post last immunization, the rVCG vaccine elicited significantly higher levels of antigen-specific IgG2a and IgA antibodies both in serum and in vaginal washes. Proliferative responses of mucosal (genital tract) as well as splenic (systemic) T cells were significantly higher in mice receiving rVCG vaccine.These results indicate that the rVCG-Pmp18D vaccine is capable of inducing antigen-specific cellular and humoral immune responses that may provide protective immunity against a live challenge infection with C.abortus.

In vitro assessment of halobacterial gas vesicles as a Chlamydia vaccine display and delivery system

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted disease worldwide and while antibiotic treatment is effective in eliminating the pathogen, up to 70% of all infections are asymptomatic. Despite sustained efforts over the past 2 decades, an effective chlamydial vaccine remains elusive, due in large part to the lack of an effective delivery system. We explored the use of gas vesicles derived from Halobacterium salinarium as a potential display and delivery vehicle for chlamydial antigens of vaccine interest. Various size gene fragments coding for the major outer membrane protein (MOMP), outer membrane complex B (OmcB) and polymorphic outer membrane protein D (PompD) were integrated into and expressed as part of the gas vesicle protein C (gvpC) on the surface of these stable structures. The presence of the recombinant proteins was confirmed by Western blots probed using anti-gvpC and anti-Chlamydia antibodies as well as sera from Chlamydia-positive patients. Tissue culture evaluation revealed stability and a time-dependent degradation of recombinant gas vesicles (r-Gv) in human and animal cell lines. In vitro assessment using human foreskin fibroblasts (HFF) confirmed Toll-like receptor (TLR) 4 and 5 engagement by wild type and r-Gv, leading to MyD88 activation, TNF-α, IL-6 and IL-12 production. The data suggest that r-GV could be an effective, naturally adjuvanting, time-release antigen delivery system for immunologically relevant Chlamydia vaccine antigens which are readily recognized by human immune sera.

Amniotic and allantoic fluids from experimentally infected sheep contain immunoglobulin specific for Chlamydophila abortus

Chlamydophila abortus, the aetiological agent of enzootic abortion of ewes (EAE), replicates in trophoblast cells leading to their destruction and dissemination of the bacterium to foetal organs. To further understand the pathogenesis of EAE, amniotic and allantoic fluids were collected from experimentally infected pregnant ewes at 30 (7 samples from each fluid), 35 (8 samples from each fluid), 40 (10 samples from each fluid) and 43 (6 amniotic fluids and 7 allantoic fluids) days post-infection to determine pathogen numbers and other markers of infection. Whilst experimentally infected ewes had characteristic placental lesions, only two amniotic and seven allantoic fluid samples were positive for C. abortus by real-time PCR. In contrast, all amniotic and allantoic fluids were positive for immunoglobulin. Immunoglobulins were generally detected earlier in allantoic fluid than in amniotic fluid and the numbers of samples containing immunoglobulins increased as infection progressed. IgG in amniotic and allantoic fluids was shown to be specific for C. abortus, and reacted with the major outer membrane proteins, polymorphic outer membrane protein and macrophage infectivity potentiator protein. A comparison of two-dimensional immunoblots using purified IgG from the allantoic fluid, amniotic fluid, ewe serum and foetal serum of a C. abortus infected animal at 40 days post infection indicated a pattern of reactivity intermediate between that of the ewe serum and the foetal serum. Results suggest that a maternal source of immunoglobulin is predominant at 30 days post-infection but that foetal derived antibodies may be contributed at a later stage.

Identification of Immunologically Relevant Proteins ofChlamydophila abortusUsing Sera from Experimentally Infected Pregnant Ewes

Chlamydophila abortus is an intracellular pathogen and the etiological agent of enzootic abortion of ewes (EAE). C. abortus has a biphasic development cycle; extracellular infectious elementary bodies (EB) attach and penetrate host cells, where they give rise to intracellular, metabolically active reticulate bodies (RB). RB divide by binary fission and subsequently mature to EB, which, on rupture of infected cells, are released to infect new host cells. Pregnant ewes were challenged with 2 x 10(6) inclusion forming units (IFU) of C. abortus cultured in yolk sac (comprising both EB and RB). Serum samples were collected at 0, 7, 14, 21, 27, 30, 35, 40, and 43 days postinfection (dpi) and used to identify antigens of C. abortus expressed during disease. Additionally, sera from fetal lambs were collected at 30, 35, 40, and 43 dpi. All serum samples collected from experimentally infected pregnant ewes reacted specifically with several antigens of EB as determined by one-dimensional (1-D) and 2-D gel electrophoresis; reactive antigens identified by mass spectrometry included the major outer membrane protein (MOMP), polymorphic outer membrane protein (POMP), and macrophage infectivity potentiator (MIP) lipoprotein.

Transcriptional analysis of in vitro expression patterns ofChlamydophila abortuspolymorphic outer membrane proteins during the chlamydial developmental cycle

Chlamydophila abortus is the aetiological agent of ovine enzootic abortion. Sequencing, annotation and comparative analysis of the genome of C. abortus strain S26/3 has revealed variation in the loci encoding the polymorphic membrane proteins (Pmps). These Pmps resemble autotransporter proteins of the type V secretion system, suggesting an important role in chlamydial pathogenesis. The purpose of this study was to characterise the transcriptional expression patterns of this family during the developmental cycle of C. abortus. McCoy cells were infected with C. abortus and analysed for pmp mRNA expression over a 72 h period. Few pmp transcripts were detected in the early stages of the developmental cycle. Peak expression occurred at 48 h post-infection (p.i.) other than for pmp5E, where it was observed at 24 h p.i. Overall, expression of pmps 5E, 18D and 10G were found to be 40 to 100-fold higher than the lowest expressing pmps (6H, 13G and 15G) at 24 h p.i., while pmps 18D and 17G were 14 to 16-fold higher than the lowest (11G, 14G and 15G) at 48 h. Levels of expression for all the other pmp genes were below one copy per genome at any time point. The expression of all the pmps reduced to near base-line levels by 60 h p.i. These results demonstrate that pmp expression in C. abortus is mid to late cycle, consistent with conversion of the reticulate body to the elementary body. The low level of pmp transcription may be indicative of heterogeneity in expression, suggesting a possible role for some of the Pmps in antigenic variation and chlamydial pathogenesis.

High prevalence of antibodies against Chlamydiaceae and Chlamydophila abortus in wild ungulates using two “in house” blocking-ELISA tests

Few data are available on the prevalence and relevance of chlamydiae in wild mammals, and even fewer studies have been conducted to determine the prevalence of Chlamydophila abortus in wildlife hosts, most probably due to the absence of suitable species-specific serological assays for testing sera from wild animals. In light of this, we have developed two in-house blocking-ELISA tests for detection of antibodies against Chlamydiaceae and C. abortus in wild ungulates, and analyzed the relationship between geographical and biological factors and the prevalence of antibodies against Chlamydiaceae and C. abortus in 434 wild ungulates from Spain, including sera from European wild boar, Red deer, Fallow deer, Roe deer, Mouflon, Barbary sheep, Southern chamois, and Iberian ibex. Serology revealed that 41.7 ± 4% of the sera were positive for the b-ELISA-LPS ( Chlamydiaceae-specific) and 18.9 ± 3% for the b-ELISA-rPOMP ( C. abortus-specific). Antibodies against Chlamydiaceae lipopolysaccharide (LPS) were detected in sera from all eight ungulate species, the prevalence ranging from 23 to 60%. Iberian ibex was the only wild ungulate not showing seropositivity to the C. abortus specific polymorphic outer membrane protein (POMP). The prevalence of anti-POMP antibodies in the other seven wild ungulate species ranged from 7 to 40%. While significant seroprevalence differences were detected among species and among sampling regions, no effect of age and sex was observed. The high prevalence levels found should be considered with regards to livestock and human health, and warrant further research.

Comparative evaluation of eight serological assays for diagnosing Chlamydophila abortus infection in sheep

Chlamydophila abortus is one of the principal causes of late-term abortion (enzootic abortion of ewes or EAE) in sheep across Europe. Serological diagnosis of EAE is routinely carried out by the complement fixation test, although the interpretation of results can often be difficult because of cross reaction with Chlamydophila pecorum, which also commonly infects sheep. The purpose of this study was to evaluate and compare four ELISAs developed at Moredun Research Institute and based on whole C. abortus elementary bodies (EBs), an outer membrane preparation of the whole organism (SolPr) and two recombinant polymorphic outer membrane protein fragments (rOMP90-3 and rOMP90-4), with 3 commercial tests, the CHEKIT ® Chlamydophila Abortus, Pourquier ® ELISA Chlamydophila abortus and ImmunoComb Ovine Chlamydophila Antibody tests. The tests were evaluated using a panel of 202 sera from experimentally and naturally infected animals, as well as from EAE-free flocks. The EB, SolPr and CHEKIT ELISAs performed similarly to the CFT, all lacking in specificity by cross reacting with sera from C. pecorum infected animals. The ImmunoComb also lacked specificity with C. pecorum sera, but also badly cross reacted with sera from EAE-free flocks. The rOMP90-3, rOMP90-4 and Pourquier ELISAs were the most specific, although the Pourquier test appeared less sensitive with sera from naturally infected animals. Overall, the rOMP90-3 ELISA performed the best, with high sensitivity (96.8%) and no cross reaction with sera from C. pecorum infected animals or from EAE-free flocks (100% specificity) and so would be a suitable alternative to the CFT for the serological diagnosis of EAE.

Comparison of ELISA and CFT assays for Chlamydophila abortus antibodies in ovine sera

To compare the sensitivity and specificity of Chlamydophila abortus antibody assays, to find a suitable serological assay for testing sheep for export. Comparison of results from known positive and negative sheep populations. Fifty-five positive and fifty negative sera were analysed by four enzyme linked immunosorbent assays (ELISA), three using recombinant antigens based on the chlamydial polymorphic outer membrane proteins (POMP90-3, POMP90-4, POMP80-90) and one using a synthetic peptide based on chlamydial major outer membrane proteins (MOMP-P). They were also analysed by complement fixation tests (CFT) using crude antigens from chlamydia isolated from an Australian sheep, a Californian parakeet and a Texan turkey. Assay sensitivity and specificity were expressed as point estimates and 95% confidence intervals. Results were compared using McNemar's test for paired samples. ELISA sensitivity ranged from 70 to 98% and complement fixation test sensitivity from 60 to 96%; with POMP90-3 > POMP90-4 > CFT (parakeet) > CFT (turkey) > POMP80-90 > MOMP-P > CFT (sheep). There was no significant difference from POMP90-3 to POMP80-90 (P > 0.05). ELISA specificity ranged from 88 to 100% and CFT specificity was 100% for all three antigens; with CFT and POMP90-4 > MOMP-P > POMP80-90 > POMP90-3. There was no significant difference from CFT to POMP80-90 (P > 0.05). Changing the CFT cut-off from 1:32 to 1:4 substantially reduced the specificity with little improvement in sensitivity. Assays using POMP90-4, POMP80-90, CFT (parakeet) and CFT (turkey) had equivalent sensitivity and specificity; none of the ELISAs were more specific than any CFT. The POMP80-90 ELISA is recommended as an alternative to CFT (parakeet) but as its specificity is not ideal the search for a more specific assay should continue.

Two Monoclonal Antibodies with Defined Epitopes of P44 Major Surface Proteins NeutralizeAnaplasma phagocytophilumby Distinct Mechanisms

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.

Antibody Responses to Recombinant Protein Fragments of the Major Outer Membrane Protein and Polymorphic Outer Membrane Protein POMP90 inChlamydophila abortus-Infected Pregnant Sheep

Chlamydophila abortus is one of the major causes of infectious abortion in pregnant sheep (enzootic abortion of ewes or EAE) worldwide. Organisms shed in infected placentas and uterine discharges at lambing time are the main sources of environmental contamination, responsible for transmission to susceptible animals and possible human contacts. In the present study, a recently developed test, based on a recombinant fragment of the polymorphic outer membrane protein POMP90 (rOMP90-4 indirect enzyme-linked immunosorbent assay [iELISA]) and one based on the variable segment 2 (VS2) region of the major outer membrane protein (MOMP) (MOMP VS2 iELISA) were compared using sera from C. abortus-infected ewes at different stages throughout pregnancy. The rOMP90 iELISA detected antibody much earlier in pregnancy than the MOMP iELISA, which, like the complement fixation test, detected antibody only at the time of abortion or lambing. No anti-MOMP antibody response could be detected in three of seven experimentally infected ewes. Furthermore, the rOMP90 iELISA detected antibody in an animal that seroconverted during the course of the study, which the MOMP iELISA failed to detect. Overall, the results show that the rOMP90-4 iELISA is considerably more sensitive than the MOMP VS2 iELISA for identifying animals infected with C. abortus. Earlier detection of infection will allow appropriate control measures to be taken to reduce environmental contamination, thus limiting the spread of infection, financial losses, and the possible risks of zoonotic transmission to humans.

Anaplasma phagocytophilum has a functional msp2 gene that is distinct from p44.

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 degrees 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.

Antigenic organization of the N-terminal part of the polymorphic outer membrane proteins 90, 91A, and 91B of Chlamydophila abortus.

A series of overlapping recombinant antigens, 61 to 74 residues in length, representing polymorphic outer membrane protein 90 (POMP90) of Chlamydophila abortus and two recombinant peptides spanning gene fragment p91Bf99 of POMP91B were assessed by immunoblotting to determine the antigen-binding sites of 20 monoclonal antibodies to POMP90, -91A, and -91B. The epitopes were further restricted by scanning 52 overlapping synthetic 12-mer peptides representing the N-terminal part of POMP90, and the 12-mer epitopes were then analyzed by using hexapeptides to the resolution of a single amino acid. Ten epitopes were defined: 1, TSEEFQVKETSSGT; 2, SGAIYTCEGNVCISYAGKDSPL; 3, SLVFHKNCSTAE; 4, AIYADKLTIVSGGPTLFS; 5, SPKGGAISIKDS; 6, ITFDGNKIIKTS; 7, LRAKDGFGIFFY; 7a, DGFGIF; 7b, GIFFYD; 8, IFFYDPITGGGS; 8a, FFYDPIT; 9, GKIVFSGE; and 10, DLGTTL. The 20-mer peptide LRAKDGFGIFFYDPITGGGS was a major epitope that was recognized by seven antibodies. Epitopes 7 to 10 were conserved in reference strains of the former species C. psittaci, whereas the strong antigenic peptides FYDPIT and IVFSGE were conserved among members of the genus CHLAMYDOPHILA: Epitopes 3 to 8 were located within the best-scoring beta-helical wrap (residues 148 to 293) predicted for POMP91B by the program BETAWRAP. Other studies have suggested an association of the POMPs with type V secretory autotransporter proteins. The results presented in this study provide some evidence for a passenger domain that is folded as a beta-helix pyramid with compact antigenic organization.