Discovery of in vivo Virulence Genes of Obligatory Intracellular Bacteria by Random Mutagenesis

Abstract

Ehrlichia spp. are emerging tick-borne obligatory intracellular bacteria that cause febrile and sometimes fatal diseases with abnormal blood cell counts and signs of hepatitis. Ehrlichia HF strain provides an excellent mouse disease model of fatal human ehrlichiosis. We recently obtained and established stable culture of Ehrlichia HF strain in DH82 canine macrophage cell line, and obtained its whole genome sequence and annotation. To identify genes required for in vivo virulence of Ehrlichia, we constructed random insertional HF strain mutants by using Himar1 transposon-based mutagenesis procedure. Of total 158 insertional mutants isolated via antibiotic selection in DH82 cells, 74 insertions were in the coding regions of 55 distinct protein-coding genes, including TRP120 and multicopy genes, such as p28/omp-1, virB2, and virB6. Among 84 insertions mapped within the non-coding regions, seven are located in the putative promoter region since they were within 50 bp upstream of the seven distinct genes. Using limited dilution methods, nine stable clonal mutants that had no apparent defect for multiplication in DH82 cells, were obtained. Mouse virulence of seven mutant clones was similar to that of wild-type HF strain, whereas two mutant clones showed significantly retarded growth in blood, livers, and spleens, and the mice inoculated with them lived longer than mice inoculated with wild-type. The two clones contained mutations in genes encoding a conserved hypothetical protein and a staphylococcal superantigen-like domain protein, respectively, and both genes are conserved among Ehrlichia spp., but lack homology to other bacterial genes. Inflammatory cytokine mRNA levels in the liver of mice infected with the two mutants were significantly diminished than those infected with HF strain wild-type, except IL-1β and IL-12 p40 in one clone. Thus, we identified two Ehrlichia virulence genes responsible for in vivo infection, but not for infection and growth in macrophages.Keywords: Ehrlichia HF strain, Himar1 transposon mutagenesis, mouse virulence, inflammatory cytokines, staphylococcal superantigen-like domain, obligatory intracellular bacteria IMPORTANCE Ehrlichiosis, a sometimes deadly febrile disease in man and animal, is caused by infection with Gram-negative obligatory intracellular bacteria, Ehrlichia. Ehrlichia species lack typical pathogen-associate molecular patterns (PAMPs), such as lipopolysaccharide, peptidoglycan, pili, and flagella, yet they induce acute and/or chronic inflammatory cytokines in infected animals.Bekebrede et al. In vivo Virulence Genes of EhrlichiaStudies to identify virulence factors and PAMPs of Ehrlichia species are hampered by the limitation to applicable genetic tools and small laboratory animal models. Mouse infection with Ehrlichia HF strain provides an excellent model for fatal human ehrlichiosis, as the HF strain is highly virulent in laboratory mice. However, due to inability to culture the HF stain, the use of this model has been limited. As we have succeeded in culturing and who...

Multiple Ehrlichia chaffeensis Genes Critical for Its Persistent Infection in a Vertebrate Host Are Identified by Random Mutagenesis Coupled with In Vivo Infection Assessment

“…E. chaffeensis is among several tick-transmitted rickettsial bacteria responsible for causing zoonosis in people. Despite several recent advances in performing molecular genetic studies, only limited progress is documented in understanding pathogenesis and in identifying rickettsial proteins posited to contribute to bacterial virulence and host-pathogen interactions and those involved in supporting the immune evasion mechanisms ( 14 , 16 , 20 , 23 , 24 , 32 – 34 ). One of the challenges is that the pathogenic organisms are difficult to grow in axenic culture media ( 35 ).…”

“…Furthermore, the mutational success rate remains extremely limited even with Himar1 mutagenesis plasmids, possibly because Ehrlichia and Anaplasma species are not known to naturally harbor extrachromosomal plasmids. For example, several attempts are required to generate a moderately sized mutant library for Ehrlichia and Anaplasma species, and often none to only a few mutant organisms are recovered following a typical mutational experiment ( 14 , 19 , 23 , 24 ). We previously reported 9 random mutations within the E. chaffeensis genome in our prior study ( 14 ).…”

“…There were significantly more rapidly cleared mutant organisms compared to those that persisted (Table 4). The rapidly cleared mutants included 23 with gene disruption mutations (Table 4A) and 15 with insertion mutations in noncoding regions (Table 4B) (14,19,23,24). We previously reported 9 random mutations within the E. chaffeensis genome in our prior study (14).…”

“…We demonstrated that Himar1 mutagenesis is a valuable tool in elucidating host-pathogen interactions and in developing attenuated mutant vaccines ( 16 – 18 ). Similarly, random mutagenesis is described for other members of the alphaproteobacterial order Rickettsiales ( 19 – 24 ).…”

“…random mutagenesis is described in other members of the α-proteobacterial order Rickettsiales (19)(20)(21)(22)(23)(24).…”