Identification, characterization, and application of a recombinant antigen for the serological investigation of feline hemotropic Mycoplasma infections

Abstract

In felids, three hemotropic mycoplasma species (hemoplasmas) have been described: Mycoplasma haemofelis, "Candidatus Mycoplasma haemominutum," and "Candidatus Mycoplasma turicensis." In particular, M. haemofelis may cause severe, potentially life-threatening hemolytic anemia. No routine serological assays for feline hemoplasma infections are available. Thus, the goal of our project was to identify and characterize an M. haemofelis antigen (DnaK) that subsequently could be applied as a recombinant antigen in a serological assay. The gene sequence of this protein was determined using consensus primers and blood samples from two naturally M. haemofelis-infected Swiss pet cats, an experimentally M. haemofelis-infected specific-pathogen-free cat, and a naturally M. haemofelis-infected Iberian lynx (Lynx pardinus). The M. haemofelis DnaK gene sequence showed the highest identity to an analogous protein of a porcine hemoplasma (72%). M. haemofelis DnaK was expressed recombinantly in an Escherichia coli DnaK knockout strain and purified using Ni affinity, size-exclusion, and anion-exchange chromatography. It then was biochemically and functionally characterized and showed characteristics typical for DnaKs (secondary structure profile, thermal denaturation, ATPase activity, and DnaK complementation). Moreover, its immunogenicity was assessed using serum samples from experimentally hemoplasma-infected cats. In Western blotting or enzyme-linked immunosorbent assays, it was recognized by sera from cats infected with M. haemofelis, "Ca. Mycoplasma haemominutum," and "Ca. Mycoplasma turicensis," respectively, but not from uninfected cats. This is the first description of a full-length purified recombinant feline hemoplasma antigen that can readily be applied in future pathogenesis studies and may have potential for application in a diagnostic serological test.

FIG. 1.

Phylogenetic tree demonstrating the relationship of the deduced M. haemofelis DnaK protein sequences to mycoplasma DnaK protein sequences from the GenBank database. Phylogenetic relationships were calculated using the neighbor-joining algorithm. Evolutionary distances are shown to scale. The data set was resampled 1,000 times to generate bootstrap percentage values. Bootstrap values greater than 70% are given at the nodes of the tree. GenBank accession numbers are indicated in parentheses. Mycoplasma groups, which previously have been classified based on their 16S rRNA gene (23, 34) or on their RNase P RNA gene sequences (hemoplasma and haemofelis groups) (22), are indicated. The sequence of E. coli served as an outgroup, establishing the root of the tree.

FIG. 2.

(A) SDS-PAGE of recombinant M. haemofelis DnaK for purity assessment after the final purification step of anion-exchange chromatography. Lane M, low-molecular-weight marker (Amersham Pharmacia Biotech); lane 1, M. haemofelis rDnaK. (B) Western blot analyses of samples from felines taken before and after hemoplasma infection using 540 ng recombinant M. haemofelis DnaK per lane. Blots were reacted with serum from M. haemofelis-infected cat QLA5 preinfection (lane 2) and 21 dpi (lane 3), plasma from “Ca. Mycoplasma haemominutum”-infected cat 09NFR2 preinfection (lane 4) and 56 dpi (lane 5), and serum from “Ca. Mycoplasma turicensis”-infected cat Y preinfection (lane 6) and 109 dpi (lane 7). Lanes M, peqGold prestained protein marker IV (PEQLAB Biotechnologie; Erlangen, Germany).

FIG. 3.

Biochemical and functional characterization of M. haemofelis rDnaK. (A) Circular dichroism spectra of M. haemofelis rDnaK in the absence or presence of ATP and/or K⁺ and Mg²⁺ ions. (B) Thermal denaturation curves of M. haemofelis rDnaK recorded at a wavelength of 222 nm in the absence or presence of ATP and/or K⁺ and Mg²⁺ ions. T, temperature. (C) Michaelis-Menten plot of M. haemofelis rDnaK (400 nM) showing ATPase activity at indicated ATP concentrations. The kinetic constants k_cat and K_m of M. haemofelis rDnaK were determined from curve fitting to the Michaelis-Menten equation. (D) Evaluation of in vivo complementation of biological DnaK function by M. haemofelis rDnaK in the DnaK-deficient E. coli strain JW0013 at permissive (30°C; upper) and nonpermissive (43°C; lower) temperatures. JW0013 cells were expressing either the M. haemofelis rDnaK gene (M. haemofelis rDnaK) or the E. coli chorismate mutase gene (EcCM), the latter serving as a negative control. Overnight cultures of transformed cells were diluted sequentially 10-fold down to 10⁻⁶ before being spotted onto selective agar plates supplemented with 1 mM salicylate. Their ability to grow was evaluated by counting colonies.

FIG. 4.

Course of experimental M. haemofelis infection in cat QLA5: hematocrit, M. haemofelis load, and anti-M. haemofelis rDnaK antibodies. (A) Hematocrit and M. haemofelis blood copy numbers during the first year of experimental M. haemofelis infection. M. haemofelis blood copy numbers were determined using TaqMan real-time PCR (35). The dashed lines indicate the lower and the upper limit (5 and 95% quantiles) of the hematocrit reference range (33 to 45%). (B) Anti-M. haemofelis rDnaK serum antibodies and M. haemofelis blood copy numbers during 29 months of experimental M. haemofelis infection.