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. 2015 Feb 3:4:186.
doi: 10.3389/fcimb.2014.00186. eCollection 2014.

Orientia tsutsugamushi ankyrin repeat-containing protein family members are Type 1 secretion system substrates that traffic to the host cell endoplasmic reticulum

Lauren VieBrock  1 , Sean M Evans  1 , Andrea R Beyer  1 , Charles L Larson  2 , Paul A Beare  2 , Hong Ge  3 , Smita Singh  1 , Kyle G Rodino  1 , Robert A Heinzen  2 , Allen L Richards  3 , Jason A Carlyon  1
Affiliations

Orientia tsutsugamushi ankyrin repeat-containing protein family members are Type 1 secretion system substrates that traffic to the host cell endoplasmic reticulum

Lauren VieBrock et al. Front Cell Infect Microbiol. .

Abstract

Scrub typhus is an understudied, potentially fatal infection that threatens one billion persons in the Asia-Pacific region. How the causative obligate intracellular bacterium, Orientia tsutsugamushi, facilitates its intracellular survival and pathogenesis is poorly understood. Many intracellular bacterial pathogens utilize the Type 1 (T1SS) or Type 4 secretion system (T4SS) to translocate ankyrin repeat-containing proteins (Anks) that traffic to distinct subcellular locations and modulate host cell processes. The O. tsutsugamushi genome encodes one of the largest known bacterial Ank repertoires plus T1SS and T4SS components. Whether these potential virulence factors are expressed during infection, how the Anks are potentially secreted, and to where they localize in the host cell are not known. We determined that O. tsutsugamushi transcriptionally expresses 20 unique ank genes as well as genes for both T1SS and T4SS during infection of mammalian host cells. Examination of the Anks' C-termini revealed that the majority of them resemble T1SS substrates. Escherichia coli expressing a functional T1SS was able to secrete chimeric hemolysin proteins bearing the C-termini of 19 of 20 O. tsutsugamushi Anks in an HlyBD-dependent manner. Thus, O. tsutsugamushi Anks C-termini are T1SS-compatible. Conversely, Coxiella burnetii could not secrete heterologously expressed Anks in a T4SS-dependent manner. Analysis of the subcellular distribution patterns of 20 ectopically expressed Anks revealed that, while 6 remained cytosolic or trafficked to the nucleus, 14 localized to, and in some cases, altered the morphology of the endoplasmic reticulum. This study identifies O. tsutsugamushi Anks as T1SS substrates and indicates that many display a tropism for the host cell secretory pathway.

Keywords: ER-tropic effector; Rickettsia; ankyrin repeat; bacterial effector; bacterial secretion; intracellular bacteria; scrub typhus; secretory pathway.

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Figures

Figure 1
Figure 1
O. tsutsugamushi transcriptionally expresses Ank, T1SS, and T4SS genes during infection of mammalian host cells. RT-PCR using primers targeting genes encoding O. tsutsugamushi Anks (A), T1SS components (B), T4SS components (C), and the 16S rRNA gene (infection control) (A) was performed on DNA-free total RNA isolated from infected L929 cells (RT lanes). O. tsutsugamushi genomic DNA and water served as positive (+) and negative (−) controls, respectively. Results are representative of two experiments with similar results.
Figure 2
Figure 2
Experimental approach for assaying E. coli secretion of HlyA chimeric proteins bearing the C-termini of putative T1SS substrates in an HlyBD-dependent manner. E. coli BL21 (DE3) cells, which express TolC but not HlyB or HlyD, were transformed with pLG575 to constitutively express HlyB and HlyD and thereby functionally reconstitute the T1SS. E. coli with or without pLG575 were transformed with plasmids that express IPTG-inducible chimeric HlyA proteins having their 60 C-terminal residues (amino acids 964–1024) replaced with the 60–70 C-terminal residues of each O. tsutsugamushi Ank or control protein. The E. coli cultures were induced and centrifuged, and the resulting cell pellets and filter-sterilized supernatants were analyzed by Western blot using HlyA antibody to detect HlyA-fusion proteins that the bacteria expressed and secreted into the medium.
Figure 3
Figure 3
Validation of the T1SS assay: E. coli secretion of HlyA and a chimeric HlyA-LktA protein depends on the substrates' C-terminal T1SS recognition signals. E. coli BL21 (DE3) cells constitutively expressing HlyB and HlyD (+ HlyBD), or not (− HlyBD) were transformed with plasmids encoding full-length HlyA (A) HlyA lacking its 60 C-terminal amino acids (HlyAΔ965–1024) (A) or a chimeric HlyA-LktA protein corresponding to HlyAΔ965–1024 fused to the 70 C-terminal amino acids of M. haemolytica LktA (B). Following IPTG induction of HlyA or HlyA chimeric protein expression, the cultures were centrifuged. The resulting E. coli cell pellets and filter-sterilized supernatants (SN) were analyzed by Western blot using HlyA antibody for the presence of HlyA proteins that had been expressed (Pellet) and secreted into the medium by the bacteria (SN), respectively. Arrows denote bands corresponding to the expected sizes for full-length and naturally occurring break down products of HlyA, HlyAΔ965–1024, and HlyA-LktA. Results are representative of at least three independent experiments.
Figure 4
Figure 4
Extracellular secretion of HlyA-Ank proteins from E. coli in an HlyBD-dependent manner. E. coli BL21 (DE3) constitutively expressing HlyB and HlyD (+ HlyBD), or not (− HlyBD) were transformed with plasmids encoding the indicated chimeric HlyA-Ank protein or the negative control chimera, HlyA-OmpA. Following IPTG induction of HlyA chimeric protein expression, the cultures were centrifuged. The resulting E. coli cell pellets and filter-sterilized supernatants (SN) were analyzed by Western blot using HlyA antibody for the presence of HlyA chimeric proteins that had been expressed (pellet) and secreted into the medium by the bacteria (SN), respectively. Results per each HlyA-chimeric protein are representative of at least three independent experiments.
Figure 5
Figure 5
The C. burnetii Dot/Icm T4SS cannot secrete O. tsutsugamushi Anks. THP-1 macrophage-like cells were infected with C. burnetii transformants expressing CyaA-tagged O. tsutsugamushi Anks, CvpA (+), or CyaA alone (−). Bars represent the fold change in intracellular cAMP concentration for host cells infected with wild type C. burnetii transformants expressing CyaA-fusions relative to control cells infected with transformants expressing CyaA alone. Results are representative of two independent experiments.
Figure 6
Figure 6
Ectopically expressed O. tsutsugamushi Anks exhibit diverse subcellular localization patterns. (A) HeLa cells expressing GFP alone or the indicated Ank proteins N-terminally fused to GFP were screened with GFP antibody, and visualized using confocal microscopy. (B) Subcellular localization patterns of ectopically expressed Anks are not affected by the fusion tag itself or tag placement. HeLa cells expressing the indicated Anks as N-terminally (Flag-Ank) or C-terminally Flag-tagged (Ank-Flag) fusion proteins or Flag-BAP were screened with Flag tag antibody and examined by confocal microscopy. (A,B) HeLa cell nuclei were stained with DAPI (blue). Representative images from 2 to 4 experiments performed per each ectopically expressed Ank are presented.
Figure 7
Figure 7
Multiple ectopically expressed O. tsutsugamushi Anks localize to the endoplasmic reticulum. HeLa cells expressing GFP alone or GFP-Anks were screened with GFP antibody, and cells expressing Flag-BAP or Flag-Anks were screened with Flag antibody. Additionally, cells were stained with antibody against either of the ER lumenal markers calreticulin (A) or protein disulfide isomerase (C), or the ER transmembrane protein, calnexin (B) prior to examination by confocal microscopy. Representative fluorescence images of cells viewed for GFP (green), ER marker (red), and merged images plus DAPI (blue) are presented for each Ank from 2 to 4 independent experiments. Arrows denote representative areas of GFP and ER marker signal colocalization.
Figure 8
Figure 8
O. tsutsugamushi expresses Ank4 during infection of mammalian host cells. (A) Ank4 antiserum specifically recognizes its target. Western blots of Flag-tagged Ank4, Ank9, or BAP that had been immunoprecipitated from transfected HeLa cells were screened with Ank4 antiserum and Flag tag antibody. Data presented are representative of three experiments with similar results. (B) Ank4 immunofluorescent signal colocalizes with O. tsutsugamushi. L929 cells infected with O. tsutsugamushi were screened with antisera specific for the bacterium (Ot; green) and Ank4 (red) were examined by laser-scanning confocal microscopy. Host cell nuclei were stained with DAPI (blue). Data presented in (A) and (B) are each representative of three experiments with similar results.
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