Molecular Characterization by Multilocus Sequence Typing and Diversity Analysis of Rickettsia asembonensis in Peru

Abstract

Despite several reports worldwide documenting the presence of Rickettsia asembonensis in samples derived from ectoparasites, animals and more recently humans, genomic information of these specimens remains scarce, and when available, is usually limited to small genomic fragments of limited value. We generated complete sequences for two conserved (17-kDa antigen gene and gltA) and three variable (sca4, ompB and ompA) genes in five R. asembonensis DNA samples detected in cat and dog fleas in Peru. Complete gene sequences were used to conduct multi-locus sequence typing and phylogenetic analyses to assess diversity and infer relationships among strains and other reference sequences. The 17-kDa antigen gene was highly conserved across Rickettsia species. Of the variable genes ompB was the most variable, but this diversity was not captured through phylogenetics alone even when efforts were made to maximize potential diversity in terms of flea species, animal host and location. Through a combination of de novo and reference-based genome assembly we identified a 75 bp insertion in ompA that encodes a 25 aa repetitive motif found in other Rickettsia species, but not present in the original prototype strain from Kenya. R. asembonensis has only recently been shown to be a bona-fide human pathogen. As such, and compounded by a lack of available genomic information, it remains understudied. Our work directly addresses the lack of genomic information available worldwide for the study of these novel Rickettsia species and specifically contributes to our understanding of the diversity and molecular epidemiology of R. asembonensis in Peru.

Keywords: Multilocus sequence typing; Peru; Rickettsia asembonensis.

FIG. 1.

Schematic representation of the ompA gene, including the insertion/deletion, found in the FhaB domain of the protein at amino acid positions 309 to 333. (a) Zoom-in view of an alignment of five sequences over the FhaB domain region of ompA. The Rickettsia asembonensis consensus sequence represents strains 8294D3, 8556D1, VGC2, LER197, and LER205 described in this work, which share 100% identity at nucleotide and amino acid levels over the region represented. The insertion is not present in the NMRCii reference strain from Kenya, and it was missed in the Peruvian strain VGD7 when it was first reported. However, the reported insertion is present as a motif in the R. helvetica and R. asiatica ompA gene with a high degree of conservation (conserved amino acids shown in blue and variable amino acids shown in red). (b) Zoom-out view of the complete ompA gene, with various functional domains color coded according to the legend.

FIG. 2.

Phylogenetic analysis of R. asembonensis specimens using the variable ompB gene. The analysis included 4263 nucleotides (82.8%) of the complete open reading frame of the ompB gene. It was carried out using the maximum likelihood method based on the General Time Reversible Gamma distributed model. Scale bars represent the number of substitutions per site. R. hoogstraalii and R. helvetica strains were included as outgroups.