Expression and characterization of recombinant interferon gamma (IFN-gamma) from the nine-banded armadillo (Dasypus novemcinctus) and its effect on Mycobacterium leprae-infected macrophages

Abstract

Armadillos (Dasypus novemcinctus) manifest the full histopathological spectrum of leprosy, and are hosts of choice for in vivo propagation of Mycobacterium leprae. Though potentially useful as a model of leprosy pathogenesis, few armadillo-specific reagents exist. We have identified a region of high homology to the interferon gamma (IFN-gamma) of other mammals within the recently published armadillo whole genomic sequence. cDNA was made from ConA-stimulated armadillo peripheral blood mononuclear cells (PBMC), amplified, and cloned into a pET expression vector for transformation and over-expression in Escherichia coli. The recombinant protein (rDnIFN-gamma) was characterized by western blot and its biological function confirmed with bioassays including intracellular killing of Toxoplasma gondii and induction of indoleamine 2, 3-dioxygenase activity. In using rIFN-gamma to activate macrophages from mice, humans or armadillos, similar to humans, rIFN-gamma-activated armadillo MPhi did not produce nitrite and or inhibit the viability of M. leprae in vitro. Conversely, murine rIFN-gamma-activated mouse MPhi produced high levels of nitrite and killed intracellular M. leprae in vitro. These data indicate that the response of armadillo MPhi to rDnIFN-gamma is similar to that which occurs in humans, and demonstrates a potentially important value of the armadillo as a model in leprosy research.

Figure 1

Putative DnIFN-γ cDNA (GI: DQ094083) and predicted protein. Italicized characters indicate the putative signal peptide.

Figure 2

Clustal W alignment of DnIFN-γ-IFN-γ Dasypus (Dasypus novemcinctus) with Equus (Equus caballus), Camelus ( Camelus bactrianus), Lama (Lama glama), Canis (Canis familiaris), Sus (Sus scrofa), Homo (Homo sapiens), and Mus (Mus musculus) IFN-γs. Epitopes used to generate polyclonal antibodies =Anti-DnIFN-γ #1 and Anti-DnIFN-γ #2 are indicated by bold text. The signal peptide is indicated in italics. “*” indicates positions which have a single, fully conserved residue. “:” indicates that one of the following “strong” groups is fully conserved: STA NEQK NHQK NDEQ QHRK MILV MILF HY FYW. “.” indicates that one of the following “weaker” groups is fully conserved: CSA ATV SAG STNK STPA SGND SNDEQK NDEQHK NEQHRK FVLIH FYM.

Figure 3

(A and C). Purified rDnIFN-γ (21.22 kDa) separated on a 4% to 20% gradient polyacrylamide gel and stained with Coomassie Blue Brilliant; (B and D) Western blots with polyclonal antibodies Anti-DnIFN-g #1 and Anti-DnIFN-g #2, respectively. E. Semi-quantitative RT-PCR analysis of DnIFN-γ gene expression in armadillo PBMC: Control, non-stimulated armadillo PBMC cDNA; ConA, ConA-stimulated armadillo PBMC cDNA; Negative, buffer control; DnIFN-γ, DnIFN-γ PCR products; DnG₃PDH, DnG₃PDH housekeeping gene PCR products.

Figure 4

Inhibition of intracellular growth of T. gondii, viability of M. leprae, and Nitrite production in mouse, human and armadillo ṂΦ. MΦ were unstimulated (Control) or activated for 24 h with the appropriate rIFNγ + 5 ng/ml LPS (ACT) prior to challenge with T. gondii or M. leprae. Growth of T. gondii indexed by enumerated intracellular rosettes, and viability of M. leprae indexed with radiorespirometry of ¹⁴C-palmitate. Nitrites were assessed on supernatants using the Griess reagent system. Results are representative of three independent experiments in mouse, 8 different human donors, and 4 different armadillos.