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

Abstract

Ehrlichia chaffeensis causes human monocytic ehrlichiosis by replicating within phagosomes of monocytes/macrophages. A function disruption mutation within the pathogen’s ECH_0660 gene encoding a phage head-to-tail connector protein resulted in the rapid clearance of the pathogen in vivo, while aiding to induce sufficient immunity in a host to protect against wild-type infection challenge. In this study, we describe the characterization of a cluster of seven genes spanning from ECH_0659 to ECH_0665, which contained four genes encoding bacterial phage proteins, including the ECH_0660 gene. Assessment of the promoter region upstream to the first gene of the seven genes (ECH_0659) in Escherichia coli demonstrated transcriptional enhancement under zinc and iron starvation. Further, transcription of the seven genes was significantly higher for E. chaffeensis having a mutation in the ECH_0660 gene compared to the wild-type pathogen under zinc and iron starving conditions. In contrast, transcription from the genes was mostly similar to wild-type or moderately downregulated for the ECH_0665 gene mutant with the function disruption. Recently, we reported that this mutation caused a minimal impact on the pathogen’s in vivo growth, as it persisted similar to wild-type. The current study is the first in describing how zinc and iron contribute to E. chaffeensis biology. Specifically, we demonstrated that the functional disruption in the gene encoding the predicted head-to-tail connector protein in E. chaffeensis results in the enhanced transcription of seven genes including those encoding phage proteins during zinc and iron limitation. IMPORTANCE Ehrlichia chaffeensis, a tick-transmitted bacterium, causes human monocytic ehrlichiosis by replicating within phagosomes of monocytes/macrophages. A function disruption mutation within the pathogen’s gene encoding a phage head-to-tail connector protein resulted in the rapid clearance of the pathogen in vivo, while aiding to induce sufficient immunity in a host to protect against wild-type infection challenge. In the current study, we investigated if the functional disruption in the predicted head-to-tail connector protein gene caused transcriptional changes resulting from metal ion limitations. This is the first study describing how zinc and iron may contribute to E. chaffeensis replication.

Persistence of obligate intracellular pathogens: alternative strategies to overcome host-specific stresses

“…The severity of infections seems to vary depending on the geographical area with more severe infections in the USA than in European countries ( Rar et al., 2021 ). Symptoms of HME are not specific and range from influenza-like symptoms to life-threatening disease ( Torres-Escobar et al., 2021 ). Chronic infections were observed for A. phagocytophilum in infected animals like horses, sheep, dogs, or lambs.…”

“…For example, the gene ECH_0660 was required for Ehrlichia growth in deer ( Cheng et al., 2013 ). Additional studies revealed ECH_0660-dependent upregulation of a cluster of seven genes during iron or zinc deprivation ( Torres-Escobar et al., 2021 ). A transposon mutagenesis library of E. chaffeensis by Wang and colleagues identified mutants for the genes ECH_0837 (coding for the metal ion binding protein MiaB) and ECH_1144 (coding for an outer membrane protein) with a significantly slower growth than wild-type E. chaffeensis in the macrophage cell line DH82 ( Wang et al., 2020 ).…”

Targeted mutagenesis in Anaplasma marginale to define virulence and vaccine development against bovine anaplasmosis

“…chaffeensis phage head to tail connector protein ( phtcp ) gene (gene tag # ECH_0660) has minimal impact for its in vitro growth, while inducing attenuated growth in two different vertebrate hosts [ 12 , 13 ]. As the phtcp gene orthologs are broadly conserved in Anaplasma and Ehrlichia species [ 18 ], we reasoned that it serves as an ideal target for defining targeted mutagenesis in diverse Anaplasmataceae organisms. Indeed, the current study demonstrated such feasibility in A .…”

“…Earlier, we reported that the phtcp gene disruption of E . chaffeensis causes an in vivo growth defect, possibly resulting from limiting the bacterial zinc and iron acquisition [ 18 ]. The data presented in the current study extends our prior data reporting that the functional phtcp protein is also critical for A .…”

“…Our recent data also suggest that the functional disruption in the E . chaffeensis phtcp gene is likely detrimental in altering the pathogen’s ability to obtain metal ions to support its growth within a phagosome of infected host macrophages [ 18 ]. Since the phtcp gene orthologs are well-conserved in all known Anaplasma and Ehrlichia species [ 18 ], we reasoned that targeted mutagenesis disrupting the function of phtcp orthologs in other related Anaplasmataceae pathogens could facilitate establishing mutagenesis methods for application in developing live attenuated vaccines.…”