Transcription Analysis of the Major Antigenic Protein 1 Multigene Family of Three In Vitro-Cultured Ehrlichia ruminantium Isolates

Abstract

Ehrlichia ruminantium, an obligate intracellular bacterium transmitted by ticks of the genus Amblyomma, causes heartwater disease in ruminants. The gene coding for the major antigenic protein MAP1 is part of a multigene family consisting of a cluster containing 16 paralogs. In the search for differentially regulated genes between E. ruminantium grown in endothelial and tick cell lines that could be used in vaccine development and to determine if differences in the map1 gene cluster exist between different isolates of E. ruminantium, we analyzed the map1 gene cluster of the Senegal and Gardel isolates of E. ruminantium. Both isolates contained the same number of genes, and the same organization as found in the genome sequence of the Welgevonden isolate (H. Van Heerden, N. E. Collins, K. A. Brayton, C. Rademeyer, and B. A. Allsopp, Gene 330:159-168, 2004). However, comparison of two subpopulations of the Gardel isolate maintained in different laboratories demonstrated that recombination between map1-3 and map1-2 had occurred in one subpopulation with deletion of one entire gene. Reverse transcription-PCR on E. ruminantium derived mRNA from infected cells using gene-specific primers revealed that all 16 map1 paralogs were transcribed in endothelial cells. In one vector (Amblyomma variegatum) and several nonvector tick cell lines infected with E. ruminantium, transcripts were found for between 4 and 11 paralogs. In all these cases the transcript for the map1-1 gene was detected and was predominant. Our results indicate that the map1 gene cluster is relatively conserved but can be subject to recombination, and differences in the transcription of map1 multigenes in host and vector cell environments exist.Ehrlichia ruminantium (formerly Cowdria ruminantium [12]) is the causative agent of heartwater, a rickettsial disease transmitted by ticks of the genus Amblyomma which causes major economic losses in wild and domestic ruminants. The disease is endemic in sub-Saharan Africa and also is present on some Caribbean islands (33), where it poses a risk of spreading to the American mainland. Feeding ticks transmit E. ruminantium to vertebrate hosts in their saliva and/or by gut regurgitation (8,19). Phylogenetic studies have revealed a close relationship between E. ruminantium, Ehrlichia canis, and Ehrlichia chaffeensis (30,39).In infections with these ehrlichial agents, the serological response is mainly directed against outer-membrane proteins of approximately 30 kDa. The genes coding for these proteins have been designated the major antigenic protein 1 (map1) in E. ruminantium (32,40), the outer membrane protein p28 (omp-1) in E. chaffeensis, and the p30 outer membrane protein (p30) in E. canis (27,28,30,35,36,42,43). The OMP-1 and P30 protein families are each encoded by a multigene family consisting of 22 genes arranged in a cluster between a hypothetical transcriptional regulator (upstream) and the secA gene (downstream). The 5Ј end of the cluster contains paralogs with short intergenic spaces, whereas the paralogs at t...

Differential transcription of the major antigenic protein 1 multigene family of Ehrlichia ruminantium in Amblyomma variegatum ticks

“…The differentiation of E. ruminantium to mammalian-infective forms in the tick appears to be a complex process in which the differential transcription of genes coding for outer membrane proteins of E. ruminantium in unfed and feeding ticks as demonstrated here for two map1 gene family paralogs may be involved. However it will be necessary to determine whether the differential transcription of map1 paralogs observed in vitro (Bekker et al, 2005) and in vivo is reflected at the protein level.…”

“…All 16 paralogs were transcriptionally active when E. ruminantium was grown in bovine endothelial cells, whereas between 4 and 11 paralogs were found to be transcribed in E. ruminantium-infected tick cell lines (Bekker et al, 2005;van Heerden et al, 2004) and transcripts of only two out of three paralogs were detected in unfed infected adult Amblyomma variegatum ticks (Bekker et al, 2002). In the present study, we analysed transcription of the entire map1 cluster in midguts and salivary glands of E. ruminantium-infected Amblyomma variegatum ticks and assessed levels of transcription of map1 genes in relation to the numbers of organisms present in different tissues of unfed and feeding ticks.…”

Comparative Genomic Analysis of Three Strains of Ehrlichia ruminantium Reveals an Active Process of Genome Size Plasticity

“…Membrane proteins such as Cpg-and Map1-related proteins are well-studied antigenic proteins (9,34,65), and comparative genomics may allow for more accurate targeting. Cpg proteins, and more specifically Cpg1, are clearly good candidates.…”

“…This deletion accounts for 80% of the size difference between the map1 clusters of Erga and Erwe/Erwo. Interestingly, map1-2 was shown to have recombined with map1-3 in a subset strain (strain CTVM) of Erga (9). In Erwe/Erwo, the only known gene to be affected is ftsA, which is 63 bp shorter than the Erga ortholog.…”

Tick cell lines: tools for tick and tick-borne disease research

“…The p44 gene expression site was found to be polymorphic in human and tick cells, with sequence changes in p44 variants being influenced by host cell type and culture conditions [47]. Although all 16 members of the E. ruminantium major antigenic protein 1 (map1) multigene family were transcribed in vitro in mammalian cells, between 4 and 11 paralogs were transcribed in different tick cell lines [48]. Differential macrophage and tick cell-specific protein expression from the p28/p30 outer membrane protein multigene locus in Ehrlichia chaffeensis and E. canis has been described [49].…”