Transcription of the Rickettsia felis ompA gene in naturally infected fleas

Abstract

Rickettsia felis is maintained transovarially in Ctenocephalides felis fleas in a widespread geographic distribution and is transmitted to humans and animals, including opossums. This rickettsia is phylogenetically a member of the spotted fever group, most closely related to Rickettsia akari and R. australis. An unusual feature of this rickettsia is that the gene for the outer membrane protein A (OmpA) is interrupted by stop codons. To determine if this putatively dying gene is expressed, mRNA was extracted from laboratory-maintained, R. felis-infected cat fleas. Reverse transcriptase-polymerase chain reaction amplification of three segments of the ompA gene indicated that mRNA of ompA is actively transcribed in fleas. The cDNA sequences expressed represented mRNA of the first 1860-basepair segment of ompA, which includes domains I and II, part of domain III, the region from site 1836 to site 2180, despite the presence of several stop codons, and the open reading frame from site 2788 to site 3837. The detected sequences showed several differences in the amino acid composition when compared with the previously reported sequence.

FIGURE 1

Non-scale schematic representation of the Rickettsia felis outer membrane protein A (ompA) gene. The R. felis ompA sequence (GenBank accession no. AF191026) is represented by the open rectangle. The double head arrows at the top indicate the domains, and the filled circles indicate where the stop codons are present. The filled arrows below indicate the positions of the primers used for the reverse transcriptase— polymerase chain reaction (RT-PCR). ORF = open reading frame; nt = nucleotides.

FIGURE 2

Reverse transcriptase—polymerase chain reaction (RTPCR) assay of the outer membrane protein A (ompA) gene of Rickettsia felis. A, RT/PCR assay with total mRNA and ompA primers set 1 (nucleotides 1—1860). Molecular sizes were determined by a 1-kb DNA ladder, Lane 1, genomic PCR; lane 2, RT/PCR product of R. felis RNA; lane 3, water; lane 4 PCR of R. felis RNA without RT (control for potential DNA contamination). Kbp = kilobasepairs. B, RT/PCR with total mRNA using primer set 2 (nucleotides 2788— 3837). Lane 1, RT/PCR product.

FIGURE 3

Reverse transcriptase—polymerase chain reaction (RTPCR) assay of the stop codon region of the outer membrane protein A (ompA) gene of Rickettsia felis using primer set 3 (nucleotides 1836—2180). Molecular sizes were determined by a 100-basepair DNA ladder. Lane1, RT/PCR product of R. felis RNA; lane 2, genomic PCR; lane 3, water; lane 4, PCR of R. felis RNA without RT (control for DNA contamination). Kbp = kilobasepairs.