Analysis of Orientia tsutsugamushi promoter activity

Abstract

Orientia tsutsugamushi is an obligate intracellular bacterium that causes scrub typhus, a potentially fatal rickettsiosis, and for which no genetic tools exist. Critical to addressing this technical gap is to identify promoters for driving expression of antibiotic resistance and fluorescence reporter genes in O. tsutsugamushi. Such promoters would need to be highly conserved among strains, expressed throughout infection, and exhibit strong activity. We examined the untranslated regions upstream of O. tsutsugamushi genes encoding outer membrane protein A (ompA), 22-kDa type-specific antigen (tsa22) and tsa56. The bacterium transcribed all three during infection of monocytic, endothelial and epithelial cells. Examination of the upstream noncoding regions revealed putative ribosome binding sites, one set of predicted -10 and -35 sequences for ompA and two sets of -10 and -35 sequences for tsa22 and tsa56. Comparison of these regions among geographically diverse O. tsutsugamushi patient isolates revealed nucleotide identities ranging from 84.8 to 100.0%. Upon examination of the candidates for the ability to drive green fluorescence protein expression in Escherichia coli, varying activities were observed with one of the tsa22 promoters being the strongest. Identification and validation of O. tsutsugamushi promoters is an initial key step toward genetically manipulating this important pathogen.

Keywords: Orientia tsutsugamushi; Rickettsia; Rickettsiales; promoter; scrub typhus.

Figure 3.

Comparison of −10 and −35 regions of O. tsutsugamushi ompA, tsa22 and tsa56 with those of other Rickettsiales species and E. coli. The −10 and −35 sequences are indicated by boldface text. Dashes were introduced to enable alignment with the portion of the O. tsutsugamushi ompA UTR sequence depicted.

Figure 1.

Orientia tsutsugamushi transcriptionally expresses ompA, tsa22 and tsa56 during infection of mammalian host cells. HeLa, RF/6A or THP-1 cells were synchronously infected with O. tsutsugamushi (Ot) followed by collection of total RNA at 24, 48 or 72 h. RT-qPCR was performed using gene-specific primers. Relative ompA-, tsa22- and tsa56-to-Ot 16S rRNA gene (ott16S) expression was determined using the 2^−ΔΔCT method. Data are mean values ± SD from three experiments performed in triplicate.

Figure 2.

The UTR of ompA, tsa22 and tsa56 each contains a predicted RBS and −10 and −35 sequences. Upstream sequences of ompA(A), tsa22(B) and tsa56(C) were evaluated for the presence of RBS and −10 and −35 sequences. Initiation codons are indicated by white boldface text and purple highlighting. Putative RBS sequences are underlined. (A) The ompA UTR −10 and −35 sequences are denoted by boldface text. The two sets of −10 and −35 sequences in the UTRs of tsa22 (B) and tsa56 (C), which are designated ‘down’ and ‘up’ based on proximity to the start codon, are indicated by blue and red boldface text, respectively. Nucleotides cloned into pPROBE-NT for assessing promoter activity of tsa22-down and tsa56-down are highlighted gray, while those for evaluating promoter activity of tsa22-up and tsa56-up are highlighted yellow.

Figure 4.

Promoter activity evaluation of ompA, tsa22 and tsa56 upstream noncoding regions. Putative promoter regions of tsa56 (Ptsa56-fl [full length], Ptsa56-down [dn], Ptsa56-up, ompA [PompA] and tsa22 [Ptsa22-fl, Ptsa22-down and Ptsa22-up]) were cloned upstream of promoter-less gfp in pPROBE-NT. The recombinant plasmids or empty pPROBE-NT (control) were transformed into E. coli. (A) LB agar plate streaked with E. coli transformed with the indicated constructs imaged under blue light illumination to detect GFP fluorescence. (B) Western-blotted whole cell lysates of E. coli transformed with the indicated vector were screened with GFP antibody. (C) Mean ± GFP signal intensity of triplicate samples each normalized to total protein signal of duplicate Coomassie brilliant blue stained gels. Data in panels (B) and (C) are representative of three experiments with similar results. Statistically significant (*P < 0.05; ***P < 0.003; ^****P < 0.0001) values are indicated. Black asterisks denote GFP signal values that are statistically significant versus control, whereas green asterisks indicate statistical significance of Ptsa22-up GFP signal compared with those produced from other O. tsutsugamushi promoter sequences.