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, which encodes a phage head-to-tail connector protein, resulted in the rapid clearance of the pathogen in vivo, while aiding in induction of 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 of the first gene of the seven genes (ECH_0659) in Escherichia coli demonstrated transcriptional enhancement under zinc and iron starvation conditions. Furthermore, transcription of the seven genes was significantly higher under zinc and iron starvation conditions for E. chaffeensis carrying a mutation in the ECH_0660 gene compared to the wild-type pathogen. In contrast, for the ECH_0665 gene mutant with the function disruption, transcription from the genes was mostly similar to that of the wild type or was moderately downregulated. Recently, we reported that this mutation caused a minimal impact on the pathogen's in vivo growth, as it persisted similarly to the wild type. The current study is the first to describe how zinc and iron contribute to E. chaffeensis biology. Specifically, we demonstrated that the functional disruption in the gene encoding the phage head-to-tail connector protein in E. chaffeensis results in the enhanced transcription of seven genes, including those encoding phage proteins, under 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 in induction of sufficient immunity in a host to protect against wild-type infection challenge. In the current study, we investigated if the functional disruption in the phage 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.

Keywords: Anaplasmataceae pathogens; Ehrlichia chaffeensis; Rickettsiales; iron; metal ion deficiency; metal ions; tick-borne diseases; zinc.

FIG 1

The putative promoter region upstream of the ECH_0659 coding region contains DNA binding motifs that likely contribute to metal ion regulation and transcription. (A) Putative promoter region upstream of the ECH_0659 coding region. Regions that resemble the consensus for the Fur-binding site are indicated by magenta shading and in boldface type and are labeled Fur-(1), Fur-(2), and Fur-(3). The region that resembles the consensus for the Zur-binding site is indicated in boldface type in magenta color and labeled “partial Zur box.” Predicted −35 and −10 sequences are shown in blue and cyan-blue text, respectively, and are labeled P1-35, P2-35, P2b-35, P3-35, P4-35, P2-10, P2b-10, P3-10, and P4-10. (B) Inverted repeat sequences that are separated are a-a′, b, b′, c-c′, d-d′-d″, e-e′ and f-f′; inverted repeats that are located at close proximity are identified with underlines and listed as h, i, and j. Direct repeats are shown after the inverted repeats. (C and D) Zur (C) and Fur (D) partial consensus binding site sequences from other Gram-positive and Gram-negative bacteria are indicated by gray shading. Bt, Burkholderia thailandensis; Bs, Bacillus subtilis; Bj, Bradyrhizobium japonicum; Ec, E. coli; Ech, E. chaffeensis.

FIG 2

A promoter region upstream of ECH_0659 assessed in an Escherichia coli surrogate system using zinc- and iron-sufficient or depletion conditions. (A) Endogenous alkaline phosphatase activity (PhoA) of the E. coli XL1-Blue MRF′ strain harboring the plasmid vector pJT3. Bacterial cells were grown in chemically defined medium (Tris-glucose) with zinc or phosphate restriction or supplemented with 15 μM ZnSO₄ or 64 mM buffer phosphate. For iron response, bacterial cells were grown in Tris-glucose medium, iron depleted or supplemented with 100 μM FeCl₂ or 100 μM FeCl₃, as described in Materials and Methods. (B) Schematic diagram of the ECH_0659-promoter region and transcriptional fusion constructed pJT294, pJT189, pJT145, pJT130, pJT96, pJT50, and the control pJT3 plasmid, showing the putative binding regions for Zur and Fur (colored boxes), the −10 and −35 promoter elements (bent arrows), and the secondary structures (lines with open circles [j, i, and h]). Inverted repeat sequences are indicated with black boxes. The numbering of the nucleotides is relative to the ECH_0659 translational start codon. (C, D, E, and F) β-Gal activity for each construct is expressed as Miller units and was measured in Escherichia coli as described in Materials and Methods. The values are results from three independent experiments. Statistical significance was determined by one-way analysis of variance (ANOVA) followed by Tukey’s multiple-comparison test (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

FIG 3

Transcripts from the seven genes assessed for the E. chaffeensis wild type and the ECH_0660 and ECH_0665 mutants. (A) Schematic representation of the seven genes on E. chaffeensis wild-type chromosome, and ECH_0660 and ECH_0665 mutants. (B) Reverse transcription-PCR (RT-PCR) targets identified. The primer pairs and their estimated products are represented with arrows and bars, respectively. (C) RT-PCR data presented for the amplicons generated targeting the 7 genes. “D” refers to a positive control with genomic DNA as the template; + and − refer to the RT-PCR assays performed with or without reverse transcriptase, respectively. Molecular weight markers (MWM) were included when resolving the PCR products to help locating specific amplicons. Expected amplicons for the internal coding regions of all seven genes were detected for the wild type when reverse transcriptase was added but were absent for the mutation insertion regions of ECH_0660 and ECH_0665 mutants (PCRs 2 and 11, respectively). Also, amplicons from overlapping coding regions of genes were detected as shown. For both ECH_0660 and ECH_0665 mutants, the transcription of the aadA gene from the Himar1 transposon (Tn) insertion was also observed (PCR 12).

FIG 4

Effects of zinc and iron starvation on the E. chaffeensis wild type and ECH_0660 and ECH_0665 mutants. The genes ECH_0659 to ECH_0665 from E. chaffeensis are upregulated under zinc and iron depletion conditions. The expression of ECH_0659 (A), ECH_0660 (B), ECH_0661 (C), ECH_0662 (D), ECH_0663 (E), ECH_0664 (F), and ECH_0665 (G) from wild-type, ECH_0660 mutant, and ECH_0665 mutant organisms was measured during the stationary phase of infection under zinc repletion or depletion conditions, and similarly under iron repletion or depletion conditions, by quantitative real-time RT-PCR. The data represent the mean ± standard deviation (SD) from 2 biological replicas, each of which comprises 3 technical replicas. Fold expression changes are normalized using gyrB as an endogenous reference gene (ΔC_T). Statistical significance was determined by one-way analysis of variance (ANOVA) followed by Tukey’s multiple-comparison test (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).