Protein and DNA synthesis demonstrated in cell-free Ehrlichia chaffeensis organisms in axenic medium

Vijay K Eedunuri,Yuntao Zhang,Chuanmin Cheng,Huitao Liu,Anders Omsland,Dan Boyle,Roman R Ganta,Li Chen

doi:10.1038/s41598-018-27574-z

Abstract

Ehrlichia chaffeensis, a tick-transmitted rickettsial bacterium, is the causative agent of human monocytic ehrlichiosis. Biochemical characterization of this and other related Rickettsiales remains a major challenge, as they require a host cell for their replication. We investigated the use of an axenic medium for E. chaffeensis growth, assessed by protein and DNA synthesis, in the absence of a host cell. E. chaffeensis organisms harvested from in vitro cultures grown in a vertebrate cell line were fractionated into infectious dense-core cells (DC) and the non-infectious replicating form, known as reticulate cells (RC) by renografin density gradient centrifugation and incubated in the axenic medium containing amino acids, nucleotides, and different energy sources. Bacterial protein and DNA synthesis were observed in RCs in response to glucose-6-phosphate, although adenosine triphosphate, alpha-ketoglutarate or sodium acetate supported protein synthesis. The biosynthetic activity could not be detected in DCs in the axenic medium. While the data demonstrate de novo protein and DNA synthesis under axenic conditions for E. chaffeensis RCs, additional modifications are required in order to establish conditions that support bacterial replication, and transition to DCs.

Highlights

Ehrlichia chaffeensis is an obligate intracellular, tick-transmitted bacterium that is maintained in nature in a cycle involving at least one and perhaps several vertebrate reservoir hosts[1,2]
E. chaffeensis dense-core cells (DCs) and reticulate cells (RCs) were purified from the infected Vero cells or DH82 cells by renografin gradient centrifugation which fractionated at the interface between PBS/25% renografin and between 25–35% renografin fraction, respectively
Two major limitations of carrying out research on obligate intracellular bacterial pathogens, including the study of Rickettsiales belonging to the Anaplasmataceae family, are the lack of fully established methods for targeted mutagenesis and the inability to grow the bacteria in the absence of a host cell

Summary

Introduction

Ehrlichia chaffeensis is an obligate intracellular, tick-transmitted bacterium that is maintained in nature in a cycle involving at least one and perhaps several vertebrate reservoir hosts[1,2]. Recent advances with Coxiella burnetii demonstrate that axenic media aids greatly in studies focused on biochemical and genetic analysis of the pathogen[14,15,16,17,18,19]. Has been reported for Chlamydia trachomatis, bacterial replication is yet to be demonstrated[20,21] These studies suggest that it is possible to take advantage of the cell-free growth medium for other important obligates belonging to Rickettsiales, such as E. chaffeensis. The ability to grow obligate intracellular bacteria under axenic conditions represents a major advancement, as it will enable new paths of investigation, such as aiding the manipulation of the pathogenic organisms in the absence of host cells, clonal purification of bacterial mutants, and detailed biochemical and genetic studies. We describe the use of CIP-1 in supporting both protein and DNA biosynthesis in DC and RC forms of E. chaffeensis in the absence of host cells

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