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

Obligate intracellular bacteria of the order Rickettsiales include important hu-man 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 alphaproteo-bacteria. Using a novel fluorescence microscopy approach, we quantified R. parkeri morphol-ogy 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.

Automated summary

The researchers developed a toolkit to study the biology of R. parkeri, an important human pathogen. They analyzed the cell wall composition and morphology of R. parkeri and found unique features. Using fluorescence microscopy, they quantified R. parkeri morphology in live host cells and demonstrated the localization of fluorescence fusions. They also developed an imaging-based assay to evaluate population growth kinetics and showed that the actin homologue MreB is required for R. parkeri growth and shape.