John F. Kearney

Identification of the Immunodominant Protein and Other Proteins of the Bacillus anthracis Exosporium

Spores of Bacillus anthracis, the causative agent of anthrax, are enclosed by a prominent loose-fitting, balloon-like layer called the exosporium. Although the exosporium serves as the source of surface antigens and a primary permeability barrier of the spore, its molecular structure and function are not well characterized. In this study, we identified five major proteins in purified B. anthracis (Sterne strain) exosporia. One protein was the recently identified collagen-like glycoprotein BclA, which appears to be a structural component of the exosporium hair-like nap. Using a large panel of unique antispore monoclonal antibodies, we demonstrated that BclA is the immunodominant antigen on the B. anthracis spore surface. We also showed that the BclA protein and not a carbohydrate constituent directs the dominant immune response. In addition, the length of the central (GXX)(n) repeat region of BclA appears to be strain specific. Two other unique proteins, BxpA and BxpB, were identified. BxpA is unusually rich in Gln and Pro residues and contains several different tandem repeats, which also exhibit strain-specific variation. In addition, BxpA was found to be cleaved approximately in half. BxpB appears to be glycosylated or associated with glycosylated material and is encoded by a gene that (along with bclA) may be part of an exosporium genomic island. The other two proteins identified were alanine racemase and superoxide dismutase, both of which were reported to be associated with the surface of other Bacillus spores. Possible functions of the newly identified proteins are discussed.

Characterization of the Exosporium Basal Layer Protein BxpB of Bacillus anthracis

Bacillus anthracis spores, the cause of anthrax, are enclosed by a prominent loose-fitting structure called the exosporium. The exosporium is composed of a basal layer and an external hair-like nap. The filaments of the hair-like nap are apparently formed by a single collagen-like glycoprotein called BclA, whereas several different proteins form or are tightly associated with the basal layer. In this study, we used immunogold electron microscopy to demonstrate that BxpB (also called ExsF) is a component of the exosporium basal layer. Binding to the basal layer by an anti-BxpB monoclonal antibody was greatly increased by the loss of BclA. We found that BxpB and BclA are part of a stable complex that appears to include the putative basal layer protein ExsY and possibly other proteins. Previous results suggested that BxpB was glycosylated; however, our results indicate that it is not a glycoprotein. We showed that DeltabxpB spores, which lack BxpB, contain an exosporium devoid of hair-like nap even though the DeltabxpB strain produces normal levels of BclA. These results indicated that BxpB is required for the attachment of BclA to the exosporium. Finally, we found that the efficiency of production of DeltabxpB spores and their resistance properties were similar to those of wild-type spores. However, DeltabxpB spores germinate faster than wild-type spores, indicating that BxpB suppresses germination. This effect did not appear to be related to the absence from DeltabxpB spores of a hair-like nap or of enzymes that degrade germinants.

Isolation and purification of CD14-negative mucosal macrophages from normal human small intestine.

Mucosal macrophages play a fundamental role in the regulation of immunological events and inflammation in the small intestine. Because no information is available on normal small intestinal macrophages, we developed a technique for the isolation and purification of jejunal lamina propria macrophages in order to study their phenotype and activity. From sections of normal human jejunum, lamina propria mononuclear cells were isolated by neutral protease digestion and then subjected to counterflow centrifugal elutriation to purify the macrophages. The cells isolated by this procedure contained < 1% CD3+ lymphocytes and displayed the size distribution, morphological features, ultrastructure and phagocytic activity of mononuclear phagocytes. In contrast to blood monocytes, however, mucosal macrophages from the jejunum did not exhibit adherence properties or express CD14, a receptor for the lipopolysaccharide-binding protein. The purification of large numbers of lamina propria macrophages by this procedure offers the opportunity to define the role of this cell in the physiological inflammation characteristic of normal intestinal mucosa and the pathological inflammation associated with small intestinal diseases.

The ExsY Protein Is Required for Complete Formation of the Exosporium of Bacillus anthracis

The outermost layer of the Bacillus anthracis spore is the exosporium, which is composed of a paracrystalline basal layer and an external hair-like nap. The filaments of the nap are formed by a collagen-like glycoprotein called BclA, while the basal layer contains several different proteins. One of the putative basal layer proteins is ExsY. In this study, we constructed a DeltaexsY mutant of B. anthracis, which is devoid of ExsY, and examined the assembly of the exosporium on spores produced by this strain. Our results show that exosporium assembly on DeltaexsY spores is aberrant, with assembly arrested after the formation of a cap-like fragment that covers one end of the forespore-always the end near the middle of the mother cell. The cap contains a normal hair-like nap but an irregular basal layer. The cap is retained on spores prepared on solid medium, even after spore purification, but it is lost from spores prepared in liquid medium. Microscopic inspection of DeltaexsY spores prepared on solid medium revealed a fragile sac-like sublayer of the exosporium basal layer, to which caps were attached. Examination of purified DeltaexsY spores devoid of exosporium showed that they lacked detectable levels of BclA and the basal layer proteins BxpB, BxpC, CotY, and inosine-uridine-preferring nucleoside hydrolase; however, these spores retained half the amount of alanine racemase presumed to be associated with the exosporium of wild-type spores. The DeltaexsY mutation did not affect spore production and germination efficiencies or spore resistance but did influence the course of spore outgrowth.

Immunoglobulin isotype expression.

Many different kinds of observations suggest that during development of the B cell line, individual V H genes determining the antibody specificity of an immunoglobulin molecule can be expressed with multiple C H genes. Thus each clone of B cells, defined by expression of a variable-region gene for the light and heavy chains of the immunoglobulin molecule, ultimately contains plasma cells synthesizing immunoglobulins of all classes. The most convincing observations to support this concept include (1) shared light chains and V-region sequences by biclonal myeloma proteins of different heavy-chain isotypes (Wang et al., 1969, 1970), and (2) production of antibodies of different heavy-chain classes but with identical idiotype by the progeny of single precursor cells in clonal assays (Press and Klinman, 1973).

Accelerated Systemic Autoimmunity in the Absence of Somatic Hypermutation in 564Igi: A Mouse Model of Systemic Lupus with Knocked-In Heavy and Light Chain Genes

564Igi mice have knocked-in immunoglobulin (Ig) heavy (H) and light (L) chain genes that encode an autoantibody recognizing RNA. Previously, we showed that these mice produce pathogenic IgG autoantibodies when activation-induced deaminase (AID) is expressed in pre-B and immature B cells but not when it is expressed only in mature B cells. AID has two functions; it is necessary for somatic hypermutation (SHM) and class switch recombination (CSR). To determine the role of each of these functions in the generation of pathogenic autoantibodies, we generated 564Igi mice that carry a mutant AID-encoding gene, Aicda (AicdaG23S), which is capable of promoting CSR but not SHM. We found that 564Igi AicdaG23S mice secreted class-switched antibodies (Abs) at levels approximately equal to 564Igi mice. However, compared to 564Igi mice, 564Igi AicdaG23S mice had increased pathogenic IgG Abs and severe systemic lupus erythematosus-like disease, including, glomerulonephritis, and early death. We suggest that in 564Igi mice SHM by AID changes Ig receptors away from self reactivity, thereby mitigating the production of autoantibody, providing a novel mechanism of tolerance.