[show abstract][hide abstract] ABSTRACT: Despite accumulating data implicating Propionibacterium acnes in a variety of diseases, its precise role in infection remains to be determined. P. acnes antigen-specific CD4(+) T cells are present in early inflamed acne lesions and may be involved in the inflammatory response; however, little is known about the specific antigens involved. In this study, B cell and T cell antigens from P. acnes expression libraries were cloned and evaluated and the four predominant proteins identified were investigated. Two of these antigens share some homology with an M-like protein of Streptococcus equi and have dermatan-sulphate-binding activity (PA-25957 and 5541). The remaining two antigens, PA-21693 and 4687, are similar to the product of the Corynebacterium diphtheriae htaA gene from the hmu ABC transport locus, although only one of these (PA-21693) is encoded within an hmu-like operon and conserved amongst a range of clinical isolates. All four proteins contain an LPXTG motif, although only PA-21693 contains a characteristic sortase-sorting signal. Variation in the expression of PA-4687, 25957 and 5541 is evident amongst clinical isolates and is generated both by frameshifts associated with the putative signal peptide and by variable numbers of repeat regions toward the carboxy-terminus, potentially generating heterogeneity of molecular mass and antigenic variation. In addition, in the case of PA-25957, a frameshift in a C-rich region at the extreme carboxy-terminus eliminates the LPXTG motif in some isolates. For the dermatan-sulphate-binding PA-25957, IgG1 antibody in serum from acne-positive donors was shown to be specific for the amino-terminal region of the protein, which also contains a CD4(+) T cell epitope. In contrast, serum from acne-negative donors shows an IgG2 and IgG3 antibody subclass response to the carboxy-terminal region. These data have implications for the potential role of P. acnes in inflammatory acne and other diseases.
[show abstract][hide abstract] ABSTRACT: Analysis of the draft genome sequence of the opportunistic pathogen Propionibacterium acnes type strain NCTC 737 (=ATCC 6919) revealed five genes with sequence identity to the co-haemolytic Christie-Atkins-Munch-Peterson (CAMP) factor of Streptococcus agalactiae. The predicted molecular masses for the expressed proteins ranged from 28 to 30 kDa. The genes were present in each of the three recently identified recA-based phylogenetic groupings of P. acnes (IA, IB and II), as assessed by PCR amplification. Conserved differences in CAMP factor gene sequences between these three groups were also consistent with their previous phylogenetic designations. All type IA, IB and II isolates were positive for the co-haemolytic reaction on sheep blood agar. Immunoblotting and silver staining of SDS-PAGE gels, however, revealed differential protein expression of CAMP factors amongst the different groups. Type IB and II isolates produced an abundance of CAMP factor 1, detectable by specific antibody labelling and silver staining of SDS-PAGE gels. In contrast, abundant CAMP factor production was lacking in type IA isolates, although larger amounts of CAMP factor 2 were detectable by immunoblotting compared with type II isolates. While the potential role of the abundant CAMP factor 1 in host colonization or virulence remains to be determined, it should be noted that the type strain of P. acnes used in much of the published literature is a type IA isolate and is, therefore, lacking in this attribute.
[show abstract][hide abstract] ABSTRACT: Although two phenotypes of the opportunistic pathogen Propionibacterium acnes (types I and II) have been described, epidemiological investigations of their roles in different infections have not been widely reported. Using immunofluorescence microscopy with monoclonal antibodies (MAbs) QUBPa1 and QUBPa2, specific for types I and II, respectively, we investigated the prevalences of the two types among 132 P. acnes isolates. Analysis of isolates from failed prosthetic hip implants (n = 40) revealed approximately equal numbers of type I and II organisms. Isolates from failed prosthetic hip-associated bone (n = 6) and tissue (n = 38) samples, as well as isolates from acne (n = 22), dental infections (n = 8), and skin removed during surgical incision (n = 18) were predominately of type I. A total of 11 (8%) isolates showed atypical MAb labeling and could not be conclusively identified. Phylogenetic analysis of P. acnes by nucleotide sequencing revealed the 16S rRNA gene to be highly conserved between types I and II. In contrast, sequence analysis of recA and a putative hemolysin gene (tly) revealed significantly greater type-specific polymorphisms that corresponded to phylogenetically distinct cluster groups. All 11 isolates with atypical MAb labeling were identified as type I by sequencing. Within the recA and tly phylogenetic trees, nine of these isolates formed a cluster distinct from other type I organisms, suggesting a further phylogenetic subdivision within type I. Our study therefore demonstrates that the phenotypic differences between P. acnes types I and II reflect deeper differences in their phylogeny. Furthermore, nucleotide sequencing provides an accurate method for identifying the type status of P. acnes isolates.
Journal of Clinical Microbiology 02/2005; 43(1):326-34. · 4.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: Expression cloning involves the selection of specific polypeptides, generated from a cDNA or genomic DNA library, based on certain characteristics of the expressed proteins, such as antibody or ligand binding, recognition by T-cells, function, or complementation of cell defects. Here we describe the detailed construction of a genomic, random shear lambda expression library, adsorption of anti Escherichia coli antibody from antiserum, the screening of an expression library with specific antisera, and the cloning of genes with potential use in the diagnosis of infectious disease. This approach has been used successfully by our laboratory for the discovery of antigenic components of diagnostics and vaccines for several infectious agents including: Mycobacterium tuberculosis, Anaplasma phagocytophila (formerly Ehrlichia spp. or E. phagocytophila), Babesia microti, Trypanosoma cruzi, Leishmania chagasi, and Chlamydia spp.
[show abstract][hide abstract] ABSTRACT: The need for improved diagnostic reagents to identify human long-term carriers of the zoonotic parasite Babesia microti is evidenced by numerous reported cases of transfusion-acquired infections. This report describes the identification and initial characterization of 27 clones representing seven genes or gene families that were isolated through serological expression cloning by using a technique that we specifically designed to screen for shed antigens. In this screen, sera from B. microti-infected SCID mice, putatively containing secreted or shed antigens from the parasites, were harvested and used to immunize syngeneic immunocompetent mice (BALB/c). After boosting, the sera from the BALB/c mice, containing antibodies against the immunodominant secreted antigens, were used to screen a B. microti genomic expression library. Analyses of the putative peptides encoded by the novel DNA sequences revealed characteristics indicating that these peptides might be secreted. Initial serological data obtained with recombinant proteins and a patient serum panel demonstrated that several of the proteins could be useful in developing diagnostic tests for detection of B. microti antibodies and antigens in serum.
Journal of Clinical Microbiology 03/2003; 41(2):723-9. · 4.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: Tick-borne diseases, particularly babesiosis and ehrlichiosis, represent recently emerging infections. Despite an increased recognition of the threat tick-borne agents pose to blood safety, our understanding of the prevalence and transmissibility of these agents in blood donors is limited.
Babesia microti and Anaplasma phagocytophila (previously Ehrlichia sp.) seroprevalence was determined in random Connecticut and Wisconsin donors, and subsequently in Connecticut donors reporting tick bites. In the interim, a postcard survey regarding tick bites during the previous 6 months was sent to 6,000 random donors in six geographically distinct collection regions.
In total, 3 of 999 Wisconsin donors (0.3%) and 6 of 1,007 Connecticut donors (0.6%) had antibodies to B. microti. Of 992 donors tested for A. phagocytophila, 5 Wisconsin donors (0.5%) and 35 Connecticut donors (3.5%) were seropositive. A total of 2,482 donors (41.4%) completed the survey; 103 (4.1%) reported a tick bite. Of 848 Connecticut donors (0.4%) reporting tick bites, 3 had B. microti antibodies, while 8 (0.9%) had A. phagocytophila antibodies. These rates were not significantly different from control donors.
Blood donors seropositive for B. microti and A. phagocytophila are present in Connecticut and Wisconsin. Donors readily recall previous tick bites, but self-reported bites are not reliable indicators of serologic status. The exposure of blood donors to tick-borne pathogens does suggest a need to better understand the transfusion transmission potential of these agents.
[show abstract][hide abstract] ABSTRACT: Babesia microti is a tick-borne agent that is increasingly implicated in transfusion-acquired infection, especially in immunocompromised and elderly recipients. To develop a test that can detect antibody responses to B. microti, peptide epitopes identified in two serocomplementary B. microti-specific antigens were used in a prototype EIA.
A prototype peptide EIA was used to detect B. microti-specific antibodies in 15 sera taken before infection and 107 taken after infection from 59 individuals with known tick-borne infections previously confirmed by other methods. Three additional groups of samples were also tested: a proficiency panel of 18 sera positive for B. microti by IFA, 38 sera from blood donors confirmed positive by IFA, and 30 sera from random blood donors.
The combination peptide detected 98 out of 107 sera taken after infection that were IgG blot positive (4 equivocal). This included all 12 samples that were PCR positive and six sera from smear-negative patients that were confirmed positive by PCR, immunoblot, or IFA. Of the IgG blot-positive specimens that were equivocal (four specimens) or did not react (nine specimens) by EIA, most had low IFA titers consistent with previous exposure. In a second evaluation, 15 out of 15 Babesia IFA-positive sera and 3 out of 3 Babesia-Ehrlichia IFA-positive sera were positive, whereas sera from 30 random donors were negative. Finally, of 38 IFA-positive blood-donor samples, 35 were positive by peptide EIA. The three EIA-negative sera were Western blot negative.
Reactivity of the B. microti-specific peptide EIA shows a high correlation with IFA, PCR, and B. microti immunoblot in confirmed B. microti cases. The peptide EIA may be the most suitable B. microti infection test for adaptation to the blood bank environment if testing for B. microti is required in the future.
[show abstract][hide abstract] ABSTRACT: BACKGROUND : Tick-borne diseases, particularly babesiosis and ehrlichiosis, represent recently emerging infections. Despite an increased recognition of the threat tick-borne agents pose to blood safety, our understanding of the prevalence and transmissibility of these agents in blood donors is limited.STUDY DESIGN AND METHODS : Babesia microti and Anaplasma phagocytophila (previously Ehrlichia sp.) seroprevalence was determined in random Connecticut and Wisconsin donors, and subsequently in Connecticut donors reporting tick bites. In the interim, a postcard survey regarding tick bites during the previous 6 months was sent to 6000 random donors in six geographically distinct collection regions.RESULTS : In total, 3 of 999 Wisconsin donors (0.3%) and 6 of 1007 Connecticut donors (0.6%) had antibodies to B. microti. Of 992 donors tested for A. phagocytophila, 5 Wisconsin donors (0.5%) and 35 Connecticut donors (3.5%) were seropositive. A total of 2482 donors (41.4%) completed the survey; 103 (4.1%) reported a tick bite. Of 848 Connecticut donors (0.4%) reporting tick bites, 3 had B. microti antibodies, while 8 (0.9%) had A. phagocytophila antibodies. These rates were not significantly different from control donors.CONCLUSION : Blood donors seropositive for B. microti and A. phagocytophila are present in Connecticut and Wisconsin. Donors readily recall previous tick bites, but self-reported bites are not reliable indicators of serologic status. The exposure of blood donors to tick-borne pathogens does suggest a need to better understand the transfusion transmission potential of these agents.
[show abstract][hide abstract] ABSTRACT: Screening of genomic expression libraries from Mycobacterium tuberculosis with sera from tuberculosis (TB) patients or rabbit antiserum to M. tuberculosis led to the identification of novel antigens capable of detecting specific antibodies to M. tuberculosis. Three antigens, Mtb11 (also known as CFP-10), Mtb8, and Mtb48, were tested together with the previously reported 38-kDa protein, in an enzyme-linked immunosorbent assay (ELISA) to detect antibodies in TB patients. These four proteins were also produced as a genetically fused polyprotein, which was tested with two additional antigens, DPEP (also known as MPT32) and Mtb81. Sera from individuals with pulmonary and extrapulmonary TB, human immunodeficiency virus (HIV)-TB coinfections, and purified protein derivative (PPD)-positive and PPD-negative status with no evidence of disease were tested. In samples from HIV-negative individuals, the ELISA detected antibodies in >80% of smear-positive individuals and >60% smear-negative individuals, with a specificity of approximately 98%. For this group, smears detected 81.6% but a combination of smear and ELISA had a sensitivity of approximately 93%. The antigen combination detected a significant number of HIV-TB coinfections as well as antibodies in patients with extrapulmonary infections. Improved reactivity in the HIV-TB group was observed by including the antigen Mtb81 that was identified by proteomics. The data indicate that the use of multiple antigens, some of which are in a single polyprotein, can be used to facilitate the development of a highly sensitive test for M. tuberculosis antibody detection.
[show abstract][hide abstract] ABSTRACT: A panel of seven recombinant antigens, derived from Ehrlichia phagocytophila (the agent of human granulocytic ehrlichiosis), was evaluated by class-specific enzyme-linked immunosorbent assays (ELISAs) for utility in the diagnosis of the infection. Fourteen genomic fragments, obtained by serologic expression screening, contained open reading frames (ORFs) encoding 16 immunodominant antigens. Eleven of these antigens were members of the major surface protein (MSP) multigene family. Alignment of their predicted protein sequences revealed a pattern of conserved sequences, which contained short direct repeats, flanking a variable region. In addition, two genomic clones contained two and three MSP ORFs, respectively, indicating that these genes are clustered in tandem copies. The implications for this pattern of both genomic and protein arrangements in antigenic variations of MSPs and in their utilities in a diagnostic assay are discussed. In addition to two MSP recombinant antigens (rHGE-1 and -3) and a fusion protein of these antigens (rErf-1), five further recombinants were evaluated by ELISA. Two of these antigens (rHGE-14 and -15) were novel, while a third (rHGE-2), with no known function, has been described. The final two recombinant antigens (rHGE-9 and -17) represent overlapping segments of the ankyrin gene (ank). The addition of rHGE-9 ELISA data resulted in the detection of 78% (21 of 27) of acute-phase sera. When serologic data for all recombinants are combined, 96.2% (26 of 27) of convalescent-phase patient serum samples and 85.2% (23 of 27) of acute-phase patient serum samples are detected, indicating the potential of these antigens for use in the development of a rapid serologic assay for the detection of E. phagocytophila infection.
Journal of Clinical Microbiology 08/2001; 39(7):2466-76. · 4.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: Improved diagnostics are needed for the detection of Mycobacterium tuberculosis, especially for patients with smear-negative disease. To address this problem, we have screened M. tuberculosis (H37Rv and Erdman strains) genomic expression libraries with pooled sera from patients with extrapulmonary disease and with sera from patients with elevated reactivity with M. tuberculosis lysate. Both serum pools were reactive with clones expressing a recombinant protein referred to here as MTB48. The genomic sequence of the resulting clones was identical to that of the M. tuberculosis H37Rv isolate and showed 99% identity to the Mycobacterium bovis and M. bovis BCG isolate sequences. The genomic location of this sequence is 826 bp upstream of a region containing the esat-6 gene that is deleted in the M. bovis BCG isolate. The mtb48 1,380-bp open reading frame encodes a predicted 47.6-kDa polypeptide with no known function. Southern and Western blot analyses indicate that this sequence is present in a single copy and is conserved in the M. tuberculosis and M. bovis isolates tested but not in other mycobacterial species tested, including Mycobacterium leprae and Mycobacterium avium. In addition, the native protein was detected in the cytoplasm, as was a processed form that was also shed into the medium during culture. Serological analysis of recombinant MTB48 and the M. tuberculosis 38-kDa antigen with a panel of patient and control sera indicates that the inclusion of recombinant MTB48 in a prototype serodiagnostic test increases assay sensitivity for M. tuberculosis infection when it is combined with other known immunodominant antigens, such as the 38-kDa antigen.
Journal of Clinical Microbiology 08/2001; 39(7):2485-93. · 4.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: Increased recognition of the prevalence of human babesiosis in the United States, together with rising concern about the potential for transmission of this infection by blood transfusion, has provided motivation to develop definitive serologic and molecular tests for the causative agent, Babesia microti. To develop more sensitive and specific assays for B. microti, we screened a genomic expression library with patient serum pools. This screening resulted in the identification of three classes of novel genes and an additional two novel, unrelated genes, which together encode a total of 17 unique B. microti antigens. The first class (BMN1-2 family) of genes encodes seven closely related antigens with a degenerate six-amino-acid repeat that shows limited homology to Plasmodium sp. merozoite and sporozoite surface antigens. A second class (BMN1-8 family) of genes encodes six related antigens, and the third class (BMN1-17 family) of genes encodes two related antigens. The two remaining genes code for novel and unrelated sequences. Among the three classes of antigens and remaining novel sequences, five were chosen to code for the most immunodominant antigens (BMN1-2, -9, -15, and -17 and MN-10). Western blot analysis with the resulting recombinant proteins indicated that these antigens were targets of humoral immune responses during B. microti infection in humans.
Infection and Immunity 06/2000; 68(5):2783-90. · 4.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: A tetrapeptide and a recombinant protein, each representing 4 immunodominant epitopes of Trypanosoma cruzi, were tested by use of ELISA for the detection of serum antibodies. Sera from individuals with Chagas' disease, including persons untreated and successfully or unsuccessfully treated, were tested. These assays detected antibody in 100% of the parasitemias. The antibody reactivity decreased based on the success of treatment. Higher sensitivity was observed for tetrapeptide/recombinant protein assays than for lysate-based ELISA, and specificity was improved, particularly with Leishmania sera. The results indicate that multiepitope antigens provide a more sensitive and specific alternative to lysate for detection of anti-T. cruzi antibodies, as required for developing blood screening assays.
The Journal of Infectious Diseases 01/2000; 181(1):325-30. · 5.85 Impact Factor
[show abstract][hide abstract] ABSTRACT: Human babesiosis in the United States is caused predominantly by Babesia microti, a tick-transmitted blood parasite. Improved testing methods for the detection of infection with this parasite are needed, since asymptomatic B. microti infection represents a potential threat to the blood supply in areas where B. microti is endemic. We performed immunoscreening of an expression library of genomic DNA from a human isolate of B. microti (strain MN1). Among 17 unique immunoreactive clones, we identified 9 which represent a related family of genes with little sequence homology to other known sequences but with an architecture resembling that of several surface proteins of Plasmodium. Within this family, a tandem array of a degenerate six-amino-acid repeat (SEAGGP, SEAGWP, SGTGWP, SGTVGP) was found in various lengths between relatively well conserved segments at the N and C termini. In order to examine within-clone variation, we developed a PCR protocol for direct recovery of a specific bmn1-6 homologue directly from 30 human blood isolates, 4 corresponding hamster isolates, and 5 geographically corresponding Peromyscus leucopus (white-footed mouse) isolates. Isolates from the hamsters had the same sequences as those found in the corresponding human blood, suggesting that genetic variation of bmn1-6 does not occur during passage. However, clones from different patients were often substantially different from each other with regard to the number and location of the degenerate repeats within the bmn1-6 homologue. Moreover, we found that strains that were closely related geographically were also closely related at the sequence level; nine patients, all from Nantucket Island, Mass., harbored clones that were indistinguishable from each other but that were distinct from those found in other northeastern or upper midwestern strains. We conclude that considerable genetic and antigenic diversity exists among isolates of B. microti from the United States and that geographic clustering of subtypes may exist. The nature of the bmn1-6 gene family suggests a mechanism of antigenic variation in B. microti that may occur by recombination, differential expression, or a combination of both mechanisms.
Journal of Clinical Microbiology 01/2000; 38(1):362-8. · 4.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: Culture filtrate proteins (CFP) of Mycobacterium tuberculosis have been shown to contain immunogenic components that elicit at least partial protective immunity against Mycobacterium infection. To clone genes encoding some of the immunogenic proteins, we made a high-titer rabbit anti-CFP serum and used it to screen an M. tuberculosis genomic expression library in Escherichia coli. In this paper, we describe the molecular cloning of two new protein components of CFP and identified them as members of the serine protease gene family. Their open reading frames contain N-terminal hydrophobic secretory signals consistent with their detection in CFP. The predicted molecular masses of the mature, fully processed forms of both antigens are approximately 32 kDa, in agreement with their observed sizes on immunoblots of CFP probed with polyclonal rabbit antisera made to the recombinant proteins. Thus, these proteins have been designated MTB32A and MTB32B. Interestingly, and despite 66% amino acid sequence homology between the two proteins, polyclonal rabbit antisera made to each of the recombinant proteins were found to be specific for the respective immunizing antigens. The recombinant proteins were also evaluated in in vitro assays with donor peripheral blood mononuclear cells (PBMC) from healthy purified protein derivative (PPD)-positive individuals of diverse ethnic backgrounds. MTB32A but not MTB32B stimulated PBMC from healthy PPD-positive donors but not from PPD-negative donors to proliferate and secrete gamma interferon. MTB32A is encoded by a single-copy gene which is present in both virulent and avirulent strains of the M. tuberculosis complex and the BCG strain of Mycobacterium bovis but absent in the environmental mycobacterial species tested. In addition, nucleotide sequence comparison of mtb32a of the avirulent H37Ra strain and the virulent Erdman strain, as well as with the corresponding sequences (identified in the databases) of strain H37Rv and the clinical isolate CSU93, revealed 100% identity. MTB32A, therefore, represents a candidate for inclusion in subunit vaccine development. Finally, the possible role of MTB32 serine proteases as a virulence factor(s) during Mycobacterium spp. infection is discussed.
Infection and Immunity 09/1999; 67(8):3998-4007. · 4.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: Peptide epitopes of Trypanosoma cruzi have been identified through expression cloning. A tripeptide (2/D/E) containing three epitopes (TcD, TcE, PEP-2) was used in ELISA to detect antibodies to T. cruzi in 239 of 240 consensus-positive sera and 41 of 42 sera confirmed positive by radioimmunoprecipitation assay. The 1 discrepant consensus-positive serum was used to expression-clone a novel gene that contained a repeat sequence. A peptide corresponding to this sequence, TcLo1.2, was specific for T. cruzi. This antigen detected the discrepant consensus-positive serum and enhanced reactivity of low-positive sera in the tripeptide assay. A branched synthetic peptide, 2/D/E/Lo1.2, or a linear recombinant, r2/D/E/Lo1.2, realized all of the diagnostic features of the four epitopes, including the ability to boost reactivity of low-reactive sera. These studies show that peptides and recombinants containing multiple repeat epitopes are powerful tools for developing assays for T. cruzi antibody detection and have direct application in blood screening.
The Journal of Infectious Diseases 06/1999; 179(5):1226-34. · 5.85 Impact Factor