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. 2021 Feb 19;10(2):228.
doi: 10.3390/pathogens10020228.

Significant Growth by Rickettsia Species within Human Macrophage-Like Cells Is a Phenotype Correlated with the Ability to Cause Disease in Mammals

M Nathan Kristof  1 , Paige E Allen  1 , Lane D Yutzy  1 , Brandon Thibodaux  1 , Christopher D Paddock  2 , Juan J Martinez  1
Affiliations

Significant Growth by Rickettsia Species within Human Macrophage-Like Cells Is a Phenotype Correlated with the Ability to Cause Disease in Mammals

M Nathan Kristof et al. Pathogens. .

Abstract

Rickettsia are significant sources of tick-borne diseases in humans worldwide. In North America, two species in the spotted fever group of Rickettsia have been conclusively associated with disease of humans: Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, and Rickettsia parkeri, the cause of R. parkeri rickettsiosis. Previous work in our lab demonstrated non-endothelial parasitism by another pathogenic SFG Rickettsia species, Rickettsia conorii, within THP-1-derived macrophages, and we have hypothesized that this growth characteristic may be an underappreciated aspect of rickettsial pathogenesis in mammalian hosts. In this work, we demonstrated that multiple other recognized human pathogenic species of Rickettsia, including R. rickettsii, R. parkeri, Rickettsia africae, and Rickettsiaakari can grow within target endothelial cells as well as within PMA-differentiated THP-1 cells. In contrast, Rickettsia bellii, a Rickettsia species not associated with disease of humans, and R. rickettsii strain Iowa, an avirulent derivative of pathogenic R. rickettsii, could invade both cell types but proliferate only within endothelial cells. Further analysis revealed that similar to previous studies on R. conorii, other recognized pathogenic Rickettsia species could grow within the cytosol of THP-1-derived macrophages and avoided localization with two different markers of lysosomal compartments; LAMP-2 and cathepsin D. R. bellii, on the other hand, demonstrated significant co-localization with lysosomal compartments. Collectively, these findings suggest that the ability of pathogenic rickettsial species to establish a niche within macrophage-like cells could be an important factor in their ability to cause disease in mammals. These findings also suggest that analysis of growth within mammalian phagocytic cells may be useful to predict the pathogenic potential of newly isolated and identified Rickettsia species.

Keywords: Rickettsia; THP-1 cells; pathogenesis; proliferation.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
R. rickettsii st. Sheila Smith proliferates inside both endothelial cells (EA.hy926) and human derived macrophage cells (THP-1). (A,B) EA.hy926 cells and PMA-differentiated THP-1 cells were infected with R. rickettsii st. Sheila Smith (MOI = 2.5), and genomic DNA was extracted at each time point post-infection. Each time point represents the ratio of R. rickettsii st. Sheila Smith sca1 to the host cell actin gene amplified from genomic DNA and determined by quantitative PCR (qPCR). Immunofluorescence microscopy growth analyses in EA.hy926 cells at days 1 and 4 post-infection (C,D) in PMA-differentiated THP-1 cells at days 1 and 5 post-infection demonstrate significant intracellular proliferation. DAPI (blue) was used to visualize host cell nuclei; anti-Rickettsia antibody (RcPFA) followed by Alexa Fluor 488 (green) was utilized to reveal R. rickettsii st. Sheila Smith, and Alexa Fluor 546 Phalloidin (red) was used to indicate the host actin cytoskeleton in C and D. Scale bar = 10 μm. A logistic regression test was used to measure significance (p < 0.05) in growth over time in both mammalian cell lines in A and B.
Figure 2
Figure 2
R. parkeri proliferates inside both endothelial cells (EA.hy926) and human derived macrophage cells (THP-1). (A,B) EA.hy926 cells and PMA-differentiated THP-1 cells were infected with R. parkeri st. Portsmouth (MOI = 2.5) and genomic DNA was extracted at each time point post-infection. Increase in growth is represented by the ratio of R. parkeri st. Portsmouth sca1 to the host cell actin gene determined by quantitative PCR (qPCR). A logistic regression test was used to measure significance (p < 0.05) in growth over time in both mammalian cell lines. Immunofluorescence microscopy demonstrated growth in EA.hy926 cells on days 1 and 4 post-infection (C) and in PMA-differentiated THP-1 cells on days 1 and 3 post-infection (D). Cells were stained with the following antibodies: DAPI (blue) to stain host cell nuclei, anti-Rickettsia antibody (RcPFA) followed by Alexa Fluor 488 (green) to stain R. parkeri st. Portsmouth, and Phalloidin (red) to stain actin. Scale bar = 10 μm.
Figure 3
Figure 3
R. bellii proliferates inside endothelial cells (EA.hy926) but does not grow in human derived macrophage cells (THP-1). (A,B) EA.hy926 cells and PMA-differentiated THP-1 cells were infected with R. bellii st. Yolo (MOI = 2.5), genomic DNA was extracted at indicated time points and growth was determined by qPCR. A logistic regression test was used to measure significance (p < 0.05) in growth over time in both mammalian cell lines. Immunofluorescence microscopy analyses confirmed growth in EA.hy926 cells at days 1 and 4 post-infection (C) but not in PMA-differentiated THP-1 cells at days 1 and 4 post-infection (D). DAPI (blue) was used to stain host cell nuclei; anti-Rickettsia antibody (RcPFA) followed by Alexa Fluor 488 conjugated anti-rabbit IgG (green) was used to stain R. bellii st. Yolo, and AlexaFluor 546-Phalloidin (red) was used to stain actin. Scale bar = 10 μm.
Figure 4
Figure 4
Differences in the co-localization of SFG rickettsial species with the activated, mature form of the lysosomal marker Cathepsin D. PMA-differentiated THP-1 cells were infected with (A) R. rickettsii Sheila Smith, (B) R. parkeri Portsmouth, and (C) R. bellii Yolo at MOIs of 10 for 24 h and then processed for immunofluorescence confocal microscopy analyses. Representative slices from z stacks of infected THP-1-derived macrophage are shown. (D–F) A generated RGB profile plot documents the relative fluorescence intensity along the indicated white line. Putative co-localization events for R. rickettsii (D), R. parkeri (E), and R. bellii (F) were deemed positive when fluorescence intensities from the green and red channels overlap at a given point in the image. Areas of interest that were used for determination of co-localization are enlarged to show detail (inset). Scale bar = 25 μm.
Figure 5
Figure 5
Pathogenic SFG rickettsiae, but not R. bellii, avoid co-localization with the lysosomal marker, LAMP-2. PMA-differentiated THP-1 cells were infected with (A) R. rickettsii Sheila Smith, (B) R. parkeri Portsmouth, and (C) R. bellii Yolo at MOIs of 10 for 24 h and then processed for immunofluorescence confocal microscopy analyses. Representative slices from z stacks of infected THP-1-derived macrophage 24 h post-infection are shown. (D–F) A generated RGB profile plot documents the relative fluorescence intensity along the indicated white line. Co-localization events were deemed positive when fluorescence intensities from the green and red channels overlap at a given point in the image and negative when intensity peaks do not overlap. Areas of interest for R. rickettsii (D), R. parkeri (E), and R. bellii (F) that were used for determination of co-localization are enlarged to show detail (inset). Scale bar = 25 μm.
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