Quantitative analysis of morphogenesis and growth dynamics in an obligate intracellular bacterium

Abstract

Obligate intracellular bacteria of the order Rickettsiales include important human pathogens. However, our understanding of the biology of Rickettsia species is limited by challenges imposed by their obligate intracellular lifestyle. To overcome this roadblock, we developed methods to assess cell wall composition, growth, and morphology of Rickettsia parkeri, a human pathogen in the spotted fever group of the Rickettsia genus. Analysis of the cell wall of R. parkeri revealed unique features that distinguish it from free-living alphaproteobacteria. Using a novel fluorescence microscopy approach, we quantified R. parkeri morphology in live host cells and found that the fraction of the population undergoing cell division decreased over the course of infection. We further demonstrated the feasibility of localizing fluorescence fusions, for example, to the cell division protein ZapA, in live R. parkeri for the first time. To evaluate population growth kinetics, we developed an imaging-based assay that improves on the throughput and resolution of other methods. Finally, we applied these tools to quantitatively demonstrate that the actin homologue MreB is required for R. parkeri growth and rod shape. Collectively, a toolkit was developed of high-throughput, quantitative tools to understand growth and morphogenesis of R. parkeri that is translatable to other obligate intracellular bacteria.

FIGURE 1:

Characterization of R. parkeri PG by UPLC and mass spectrometry. (A) Representative chromatogram of R. parkeri PG. The most abundant muropeptides are indicated with arrows, and structures are drawn. *Control indicates uninfected Vero cell. (B, C) Relative muropeptide molar abundance and percentage of each muropeptide detected in R. parkeri PG for two biological replicates (n = 2; 20 T175 cm² flasks per “n” per condition). For chain length, values (not percentages) are plotted.

FIGURE 2:

Quantitative evaluation of the morphology of R. parkeri in human lung epithelial (A549) cells. (A) Strategy for the quantitative evaluation of the morphology of R. parkeri producing GFPuv (Rp-GFPuv) in A549 cells. Briefly, human lung epithelial cells (A549) were grown in 35 mm MatTek dishes (step I), infected with Rp-GFPuv (step II), and imaged at 6, 12, 24, and 48 hpi (step III). Images were analyzed using MicrobeJ (step IV). (B) Composite of raw Rp-GFPuv images obtained via epifluorescence microscopy (12 hpi) used to detect cell outlines (“Outline”) using MicrobeJ software. Representative cells (bounded by white boxes) were selected from different images to make a composite. Scale bar = 1 µm. (C) Cell length distributions of live Rp-GFPuv imaged at the indicated hpi, and the middle line represents the global median cell length (Mdn_CL). (D) Cell width distributions of live Rp-GFPuv imaged at the indicated hpi, and the middle line represents the global average cell width (A_CW). Colored dots indicate single cell measurements from three independent biological replicates, whereas the colored boxes represent independent biological replicate Mdn_CL or A_CW. Error bars represent the interquartile range (IQR). Data represent three biological replicates (n = 3, 50 bacterial cells per “n” per time point). A Kruskal–Wallis with Dunn’s posttest was performed to compare the Mdn_CL for all the time points compared with each other, whereas a Welch and Brown–Forsythe ANOVA test with a Dunnett’s posttest was used to compare the A_CW for all the time points to each other. No statistical significance was found comparing cell length or cell width across time points.

FIGURE 3:

Image-based evaluation of R. parkeri cell division. (A) Composite image of actively dividing intracellular Rp-GFPuv in A549. Representative cells (bounded by white boxes) were selected from different images to make a composite. Scale bar = 2 µm. (B) Demograph of R. parkeri cells producing cytoplasmic GFPuv revealed predivisional cells (boxed area). (C) Percentage of constricting cells in the population at 6, 12, 24, and 48 hpi. Data represent three biological replicates (n = 3) per time point. (D) Spatiotemporal localization of ZapA-mNG on live R. parkeri cells expressing cytoplasmic BFP at 24 hpi. Scale bar = 2 µm. Data represent three biological replicates (n = 3).

FIGURE 4:

Fluorescence-based quantification of intracellular growth dynamics of R. parkeri in A549 cells (A) Strategy for the quantitative evaluation of the intracellular growth of R. parkeri in A549 cells. (B) Phase-contrast and GFP images taken with the BioTek Cytation1 at 6, 24, and 48 h postimaging. Scale bar = 100 µm. (C) Semi-log plot of GFP intensity over time for A549 cells infected with Rp-GFPuv or Rp-GFPAa. t_D indicates the doubling time of Rp-GFPuv or Rp-GFPAa. Data represent three biological replicates (n = 3; nine images per “n” per time point).

FIGURE 5:

MreB is required to maintain cell width and support robust growth in R. parkeri. (A) Epifluorescence micrographs of fixed A549 cells infected with Rp-GFPuv and treated with 25 or 50 µM MP265 for 24 h. *Control = untreated. Scale bar = 2 µm. (B) Median cell length (Mdn_CL) and (C) average (A_CW) cell width distributions of fixed Rp-GFPuv imaged at 48 hpi. Colored dots indicate three independent biological replicates (n = 3). A Kruskal–Wallis with Dunn’s posttest was performed to compare the cell lengths for all the groups (time points) compared with each other, whereas a Welch and Brown–Forsythe ANOVA test with a Dunnett’s posttest was used to compare the cell widths for all the groups (time points) compared with each other. * < P 0.1; *** < P 0.001. (D) Semi-log plot of sum GFP intensity over time for A549 cells infected with Rp-GFPuv and treated with the indicated concentration of MP265. Data represent two biological replicates (n = 2; nine images per “n” per time point). Doubling times with the indicated treatments. t_D indicates the doubling time of Rp-GFPuv or Rp-GFPAa in 24-well plates.