Transposon mutagenesis of Rickettsia felis sca1 confers a distinct phenotype during flea infection

Abstract

Since its recognition in 1994 as the causative agent of human flea-borne spotted fever, Rickettsia felis, has been detected worldwide in over 40 different arthropod species. The cat flea, Ctenocephalides felis, is a well-described biological vector of R. felis. Unique to insect-borne rickettsiae, R. felis can employ multiple routes of infection including inoculation via salivary secretions and potentially infectious flea feces into the skin of vertebrate hosts. Yet, little is known of the molecular interactions governing flea infection and subsequent transmission of R. felis. While the obligate intracellular nature of rickettsiae has hampered the function of large-scale mutagenesis strategies, studies have shown the efficiency of mariner-based transposon systems in Rickettsiales. Thus, this study aimed to assess R. felis genetic mutants in a flea transmission model to elucidate genes involved in vector infection. A Himar1 transposase was used to generate R. felis transformants, in which subsequent genome sequencing revealed a transposon insertion near the 3’ end of sca1. Alterations in sca1 expression resulted in unique infection phenotypes. While the R. felis sca1::tn mutant portrayed enhanced growth kinetics compared to R. felis wild-type during in vitro culture, rickettsial loads were significantly reduced during flea infection. As a consequence of decreased rickettsial loads within infected donor fleas, R. felis sca1::tn exhibited limited transmission potential. Thus, the use of a biologically relevant model provides evidence of a defective phenotype associated with R. felis sca1::tn during flea infection.

Cat Flea Coinfection with Rickettsia felis and Rickettsia typhi

“…Alternatively, R. felis can quickly disseminate to distal tissues from the midgut ( e.g ., the hindgut, reproductive tissues, and salivary glands) as early as 24 hpi (Danchenko et al, 2021 ; Laukaitis et al, 2022 ; Thepparit et al, 2013 ). Although also detected in flea feces, in similar levels to that of R. typhi , R. felis exhibited increased loads within its flea host over a 21-day period (Hirunkanokpun et al, 2011 ; Laukaitis et al, 2022 ; Reif et al, 2011 ; Reif et al, 2008 ; Thepparit et al, 2013 ), suggesting bacterial replication in other tissues compensate for those rickettsiae expelled in feces. The contrasting infection phenotypes presented in this study pose an intriguing opportunity to investigate the implications of distinct interspecies interactions between Rickettsia spp.…”

“…Cat fleas were purchased from Elward II Laboratory (Soquel, CA) and maintained using an artificial dog system (Wade and Georgi, 1988 ). Before use in each bioassay, a subset of fleas was confirmed to be pathogen-specific ( R. felis and R. typhi ) free by quantitative PCR (qPCR) (Danchenko et al, 2021 ; Laukaitis et al, 2022 ).…”

“…For in vitro infection bioassays, the Ixodes scapularis -derived cell line (ISE6), and the rickettsial isolate, R. felis strain LSU passage 3 was maintained as previously described (Brown et al, 2015 ; Danchenko et al, 2021 ; Laukaitis et al, 2022 ; Pornwiroon et al, 2006 ). R. typhi strain Wilmington passage <5 was originally cultured in Vero cells using Dulbecco's Modified Eagle Media (DMEM) supplemented with 5% fetal bovine serum (FBS) at 34°C with 5% CO 2 .…”

“…For flea infections, R. typhi was grown in ISE6 cells. Rickettsial infection was monitored by Diff-Quik staining (Brown et al, 2015 ; Danchenko et al, 2021 ; Laukaitis et al, 2022 ; Pornwiroon et al, 2006 ).…”

“…Fleas were surface sterilized (Danchenko et al, 2021 ; Laukaitis et al, 2022 ) and homogenized using 2- and 3-mm stainless steel beads in a Bead Ruptor 96 (Omni International). Flea lysates were incubated overnight at 56°C, and gDNA was extracted from the sample using the DNeasy Blood and Tissue Kit (Qiagen) following the manufacturer's blood extraction protocol.…”