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

Abstract

Ehrlichia chaffeensis, a tick-transmitted obligate intracellular rickettsial agent, causes human monocytic ehrlichiosis. In recent studies, we described substantial advances in developing random and targeted gene disruption methods to investigate the functions of E. chaffeensis genes. We reported earlier that the Himar1 transposon-based random mutagenesis is a valuable tool in defining E. chaffeensis genes critical for its persistent growth in vivo in reservoir and incidental hosts. The method also aided in extending studies focused on vaccine development and immunity. In this study, we describe the generation and mapping of 55 new mutations. To define the critical nature of the bacterial genes, infection experiments were carried out in the canine host with pools of mutant organisms. Infection evaluation in the physiologically relevant host by molecular assays and by xenodiagnoses allowed the identification of many proteins critical for the pathogen's persistent in vivo growth. Genes encoding proteins involved in biotin biosynthesis, protein synthesis and fatty acid biosynthesis, DNA repair, electron transfer, and a component of an multidrug resistence (MDR) efflux pump were concluded to be essential for the pathogen's in vivo growth. Three known immunodominant membrane proteins, i.e., two 28 kDa outer membrane proteins (P28/OMP) and a 120 kDa surface protein, are also recognized as necessary for the pathogen's obligate intracellular lifecycle. The discovery of many E. chaffeensis proteins crucial for its continuous in vivo growth will serve as a major resource for investigations aimed at defining pathogenesis and developing novel therapeutics for this and related pathogens of the rickettsial family Anaplasmataceae.

Comparative Analysis of Genome of Ehrlichia sp. HF, a Model Bacterium to Study Fatal Human Ehrlichiosis

“…7 c, Table S 4 ). Previous studies have indicated that E. chaffeensis TRP proteins are highly immunogenic in infected patients and animals [ 144 ], and could play important roles in host–pathogen interactions [ 143 , 145 – 152 ]. Our recent study using Himar1 transposon mutagenesis of Ehrlichia sp.…”

“…During the review process of the present paper, Wang et al . [ 152 ] reported (published online on August 3, 2020) that an Himar1 transposon insertion mutant in TRP120 gene of E. chaffeensis was recovered in DH82 cells. This mutant had an initial lag phase but recovered afterwards in DH82 cell culture; however, it could not infect dogs when mixtures of E. chaffeensis transposon mutants were inoculated into dogs.…”

Mutations in Ehrlichia chaffeensis Genes ECH_0660 and ECH_0665 Cause Transcriptional Changes in Response to Zinc or Iron Limitation

“…3C ), although the mutant tested positive for the remaining six genes (data not shown). We recently generated another mutant with functional disruption in ECH_0665, the last gene of the gene cluster ( 39 ). Unlike the ECH_0660 mutation, this mutation has no impact on the pathogen’s in vivo growth, as it progressed very similarly to that of the wild type when assessed in a vertebrate host ( 39 ).…”

“…In contrast, transcript levels were similar to wild-type or slightly downregulated for the ECH_0665 mutant. We recently reported that this mutant has a minimal impact on its in vivo growth (39).…”

“…In earlier studies, we reported that a mutation in the E. chaffeensis ECH_0660 gene encoding a phage head-to-tail connector protein results in the rapid clearance of the pathogen from vertebrate hosts (white-tailed deer and in dogs). In contrast, persistent infection occurs with wild-type E. chaffeensis and ECH_0665 mutant (34,35,39,40). Pathogenic intracellular on April 22, 2021 by guest http://jb.asm.org/ Downloaded from bacteria are often challenged due to the restricted availability of metal ions such as iron and zinc in the host cells (20).…”