Cloning and Expression of the 44-Kilodalton Major Outer Membrane Protein Gene of the Human Granulocytic Ehrlichiosis Agent and Application of the Recombinant Protein to Serodiagnosis

Abstract

A 44-kDa major outer membrane protein of the human granulocytic ehrlichiosis (HGE) agent is an immunodominant antigen in human infection. A gene encoding this protein was cloned and sequenced. Southern blot results revealed the existence of multigenes homologous to the P44 gene in the genome of the HGE agent. The recombinant 44-kDa protein (rP44) was expressed by using expression vector pET30a. The reactivity of the affinity-purified rP44 was evaluated by Western immunoblot analysis and dot blot immunoassay. Western immunoblot analysis showed that mouse anti-rP44 serum reacted with 44- to 42-kDa proteins in six different HGE agent strains tested except strain 2, in which three proteins of 42, 40, and 38 kDa were recognized. Eleven HGE patient serum samples, a horse anti-HGE serum, and a horse anti-Ehrlichia equi serum recognized the rP44 protein. This suggests that rP44 is an HGE-E. equi group-specific antigen. Neither human anti-Ehrlichia chaffeensis serum nor rabbit anti-Borrelia burgdorferi serum reacted with rP44. Sera from two patients coinfected with the HGE agent and B. burgdorferi reacted positively with rP44 and the HGE agent. Sera from 20 HGE patients with indirect fluorescent-antibody (IFA) titers ranging from 1:20 to 1:2,560 gave distinct positive reactions in a dot immunoblot assay. There was a positive correlation between the color densities of the dot reactions and the IFA titers when greater than 50 ng of recombinant antigen per dot was used. The use of the affinity-purified rP44 protein as antigen would provide a more specific, consistent, and simpler serodiagnosis for HGE than the use of whole infected cells or purified HGE agents.

Outer Membrane Protein-Specific Monoclonal Antibodies Protect SCID Mice from Fatal Infection by the Obligate Intracellular Bacterial Pathogen Ehrlichia chaffeensis

“…Thus, the OMPs are major targets of the protective immune response in the mouse, and are also likely to be important in human immunity (26). The OMPs in E. chaffeensis and related ehrlichiae are encoded by multigene families (10,21,22,(27)(28)(29)(30), so it is likely that variation in the expression of different OMPs contribute to immune evasion in the natural host for E. chaffeensis, the white tailed deer (31). Indeed, cyclic rickettsemia has been reported to occur in cattle infected with the related erythrocyte tropic ehrlichial pathogen Anaplasma marginale (32,33).…”

Activation of a p44 pseudogene in Anaplasma phagocytophila by bacterial RNA splicing: a novel mechanism for post‐transcriptional regulation of a multigene family encoding immunodominant major outer membrane proteins

“…We further examined the ability of self‐splicing of p44–1 IVS under the optimal conditions that are generally used in in vitro splicing experiments for group I and group II introns respectively. To prepare a radiolabelled transcript including the A. phagocytophila intron, we first amplified the DNA fragment (1197 bp) consisting of p44–1 IVS flanked by 5′‐region of 189 bp and 3′‐region of 201 bp by PCR with a primer pair of p3457 and p4693 from a pHGE1221 plasmid (Zhi et al. , 1998) and then cloned the amplicon into pBluescript‐II KS+ vector (Fig.…”

“…Therefore, the change of the bacterial phenotype is required for their adaptation in different host environments. Because the p44 multigene family consisting of more than 20‐genome‐wide distributed paralogous genes encodes polymorphic immunodominant major outer membrane proteins (Zhi et al ., 1998; 1999; 2002), it is one of the candidates enabling such adaptation of the human granulocytic ehrlichiosis agent, A. phagocytophila . In our experimental tick transmission study (Zhi et al ., 2002), one of p44 paralogues, p44–18 , is predominantly transcribed by A. phagocytophila in mammalian hosts at the early stage of infection, regardless of animal species (mouse or horse), inoculation routes (intraperitoneal inoculation, intravenous inoculation or inoculation by tick feeding), or organism sources (culture or tick), whereas in the organisms transmitted from mammals to ticks, the expression of p44–18 is extremely downregulated and the several other species of p44 transcripts are detectable.…”

“…The open reading frames (ORFs) of a p44–1 paralogue (1173 bp encoding a 44 kDa protein) and a p44–18 paralogue (759 bp encoding a 26‐kDa protein) are tandemly arranged and overlapped by 21 basepairs (bp) (a p44–1/p44–18 gene locus) in the A . phagocytophila genome (Zhi et al ., 1998; 1999). The downstream p44–18 gene lacks an AUG start codon and is −1 out of frame from the upstream p44–1 paralogue.…”

Antigenic Variation of Anaplasma marginale by Expression of MSP2 Mosaics

“…The hypervariable msp2 region, characterized by nucleotide insertions, deletions, and substitutions, is flanked by highly conserved 5Ј and 3Ј ends. This structure is shared by the MSP2 homolog (Blastp E value of 10 Ϫ102 ) in a causative agent of human granulocytic ehrlichiosis (13,18,32). Using cDNA clones derived from A. marginale during acute rickettsemia, we identified transcripts that extended 5Ј to the ATG initiation codon for MSP2 (7).…”