Predominant outer membrane antigens of Bartonella henselae

Abstract

A hallmark of Bartonella henselae is persistent bacteremia in cats despite the presence of a vigorous host immune response. To understand better the long-term survival of B. henselae in cats, we examined the feline humoral immune response to B. henselae outer membrane (OM) proteins in naturally and experimentally infected cats. Initially, a panel of sera (n = 42) collected throughout North America from naturally infected cats was used to probe B. henselae total membranes to detect commonly recognized antigens. Twelve antigens reacted with sera from at least 85% of cats, and five were recognized by sera from all cats. To localize these antigens further, OMs were purified on discontinuous sucrose density step gradients. Each membrane fraction (OM, hybrid or inner membrane [IM]) contained less than 1% of the total malate dehydrogenase activity (soluble marker), indicating very little contamination by cytoplasmic proteins. FtsI, an integral IM cell division protein, was used to identify the low-density fraction (rho = 1.13 g/cm3) as putative IM (<5% of the total FtsI localized to the high-density fraction) while lipopolysaccharide (LPS) and Pap31, a homolog of the Bartonella quintana heme-binding protein A (HbpA), defined the high-density fraction (rho = 1.20 g/cm3) as putative OM. Additionally, little evidence of cross-contamination between the IM and OM was evident by two-dimensional gel electrophoresis. When purified OMs were probed with feline sera, antigenic proteins profiles were very similar to those observed with total membranes, indicating that many, but not all, of the immunoreactive proteins detected in the initial immunoblots were OM components. Interestingly, two-dimensional immunoblots indicated that B. henselae LPS and members of the Hbp family of proteins did not appear to stimulate an humoral response in any infected cats. Seven proteins were recognized by at least 70% of sera tested, but only three were recognized by all sera. Nanospray-tandem mass spectrometry was used to identify OM components, including the immunodominant OM proteins. Recognition of the nonimmunogenic nature of the major OM components, such as LPS, and identification of the predominant immunogens should elucidate the mechanisms by which B. henselae establishes persistent bacteremic infections within cats. Additionally, the common antigens may serve as potential feline vaccine candidates to eliminate the pathogen from its animal reservoir.

FIG. 1.

Western immunoblot demonstrating recognition of B. henselae membrane antigens by sera from naturally infected cats. Total membrane proteins were separated by SDS-PAGE, transferred to nitrocellulose, and screened with sera. Lane 1 was incubated with only secondary goat anti-cat antibodies, and lane 2 was incubated with serum from a specific-pathogen-free cat. Lanes 3 to 9 were incubated with sera from infected cats. Molecular mass standards (std) are indicated to the left. Antigens recognized by >85% of sera screened are marked with asterisks, and antigens recognized by 100% of sera are marked with arrows. Ctrl, control.

FIG. 2.

Protein profiles of B. henselae subcellular fractions and localization of membrane marker proteins. Lanes: I, cell lysate; II, high-density membrane fraction; III, intermediate-density membrane fraction; IV, low-density membrane fraction. (A) Membrane fractions were isolated by sucrose gradient centrifugation, separated by SDS-PAGE, and visualized by Coomassie blue staining. Molecular mass standards (std) are indicated to the left. (B) Following separation by SDS-PAGE, membrane markers were visualized in membrane fractions by either modified silver staining for LPS or immunoblotting with antibodies raised against HbpA, FtsI, and IalB.

FIG. 3.

Two-dimensional SDS-PAGE profiles of B. henselae OM (A) and IM (B). Isolated OM and IM fractions were separated first by pI and then by molecular mass. Proteins were visualized by silver staining with the PlusOne kit. LPS is labeled in red in panel A. The pH gradient is indicated at the top, and molecular mass standards (std) are indicated to the left.

FIG. 4.

Western immunoblot of B. henselae OM proteins screened with sera from infected cats. After SDS-PAGE separation, OM proteins were transferred to nitrocellulose and incubated with sera from naturally infected cats (lanes 2 to 8), sera from experimentally infected cats (designated EICS; lanes 9 and 10), and sera from naive cats (lanes 1 and 11). OM antigens recognized by all sera from naturally infected cats are marked with asterisks on the right. The arrow on the right indicates the dye front, where LPS migrates. Molecular mass standards (std) are indicated to the left. Ctrl, control.

FIG. 5.

Two-dimensional characterization of B. henselae OM proteins and their recognition by feline immune serum. Following separation by two-dimensional SDS-PAGE, OM proteins were either visualized by silver staining (A) or transferred to nitrocelluose and probed with sera from infected cats (B) (representative immunoblot) or with specific antibodies raised against IalB (C). LPS and IalB are labeled on the panels. Proteins marked with arrows are discussed in the text. Molecular mass standards (std) are indicated to the left.