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

Ying Wang,Ulrike G Munderloh,Kathleen Trinkl,Charan K Ganta,Huitao Liu,Arathy D S Nair,Nicole Burkhardt,Roman R Ganta,Andy Alhassan,Paidashe Hove,Deborah C Jaworski,Guy H Palmer

doi:10.1128/iai.00316-20

Abstract

Ehrlichia chaffeensis, a tick-transmitted obligate intracellular rickettsial agent, causes human monocytic ehrlichiosis. In recent reports, 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. Here, 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 a multidrug resistance (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, were also recognized as necessary for the pathogen's obligate intracellular life cycle. 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.

Highlights

During the past 3 decades, rickettsial diseases caused by Anaplasmataceae family pathogens in the genera Ehrlichia and Anaplasma have emerged as a growing public health concern [1,2,3,4,5,6,7,8], and they are considered the second leading human tick-borne diseases in the United States and many parts of the world
These diseases include human monocytic and granulocytic ehrlichiosis caused by Ehrlichia chaffeensis and Ehrlichia ewingii, respectively, which are transmitted by Amblyomma americanum, and human granulocytic anaplasmosis resulting from infection with Anaplasma phagocytophilum, which is transmitted by Ixodes species ticks
Random mutagenesis is described for other members of the alphaproteobacterial order Rickettsiales [19,20,21,22,23,24]

Summary

Introduction

During the past 3 decades, rickettsial diseases caused by Anaplasmataceae family pathogens in the genera Ehrlichia and Anaplasma have emerged as a growing public health concern [1,2,3,4,5,6,7,8], and they are considered the second leading human tick-borne diseases in the United States and many parts of the world These diseases include human monocytic and granulocytic ehrlichiosis caused by Ehrlichia chaffeensis and Ehrlichia ewingii, respectively, which are transmitted by Amblyomma americanum, and human granulocytic anaplasmosis resulting from infection with Anaplasma phagocytophilum, which is transmitted by Ixodes species ticks. The study aided in identifying many essential genes and genomic regions of E. chaffeensis

Methods

Results

Conclusion