Single-cell analysis: Understanding infected cell heterogeneity

Biological processes occasionally present a stochastic behavior. Variable performances can be observed for genetically identical organisms under the same environment conditions, which are invisible if phenomena are analyzed for the population as a whole, thus limiting a complete biological understanding. The technological developments in the field of fluorescence microscopy combined with increasingly automated analytical approaches, can now allow the study of extensive populations at the individual cell level. Over the last number of years, the number of publications reporting such an approach has increased with applications in a wide variety of fields including bacterial replication,^1,2 bacterial cell morphology,³ and deformation of red blood cells.⁴ In this issue of Virulence Ramos-Marquès et al.⁵ combine up-to-date technologies and single cell analysis to obtain an insight of how bacterial pathogens interact with host cells.

They take advantage of fluorescent proteins (FP) expressed in bacteria and in eukaryotic cells to follow over time the host-bacteria/pathogen interaction.⁶ FPs are usually not toxic and allow the long-term observation of organisms. Moreover, in most cases fluorescent tagging does not impact protein localization and function when compared to their native counterpart. These features, when applied in experiemental settings, therefore create great confidence regarding the meaningfulness of collected data. The team uses eukaryotic cells and Salmonella Typhimurium, expressing p65-GFP and DsRed respectively, to follow NF-κB activity during the infection process. From the initial stages in the gut through to chronic infection in the gallbladder, Salmonella modulates gene expression to colonize and survive in a large variety of cells and tissues. These bacteria initially activate the immune response to facilitate their spreading to different organs⁷ but for a long-lasting chronic infection, they must hide from the immune system and dampen inflammation. NF-κB is a protein complex that controls the transcription of genes involved in inflammation and Salmonella targets the NF-κB signaling pathway in order to facilitate a more favorable adaptive reponse.⁸

A large number of studies have reported the modulation of the NF-κB pathway in response to a Salmonella infection.⁸ However they generally use techniques that do not consider spatial and temporal parameters simultaneously. Microarrays, Northern- and Western-blotting are (so far) limited to the analysis of populations. By flow cytometry or PCR, it is not possible to track the same cells over time. Electron or classical fluorescence microscopy require the use of fixed samples that preclude an observation in a dynamic mode. This recent study⁵ analyses at the single cell level the dynamics of the NF-κB pathway during an infection. Using a software that automatically defines cell segmentation based on the Hoechst nucleus staining while tracking the p65-GFP signal, they were able to calculate the nuclear to cytoplasmic ratio of the GFP intensity, which was then exploited as a read-out of NF-κB activation. Additionally DsRed in the cytoplasmic region was used to identify and compare responses in infected or uninfected fibroblasts.

Thanks to the implementation of this strategy, they found that when compared to naïve cells, infected and uninfected cells exhibit specific oscillatory NF-κB signaling dynamics and a variable capacity to respond to external signals, such as TNF-α challenge or secondary infection. These differences may be due to bacterial effector proteins that alter signaling in infected cells while these cells may keep the capacity to transmit a proinflammatory signal to adjacent cells, possibly via gap junctions as it was shown in HeLa cells.⁹ These observation uncover the considerable benefit of performing a single cell study as compared to using average values from whole cell populations where such insights would be buried.

While the ability to survive and replicate in macrophages is essential for virulence, Salmonella also targets other cell types. In the mouse model of infection, bacteria reside in various cells of the immune system¹⁰ or epithelial cells of the intestinal tract and gallbladder.¹¹ Low numbers of Salmonella have also been found in non-phagocytic stromal cells of the intestinal lamina propria that are probably fibroblasts.¹² In this cell type Salmonella actively restrain their proliferation. Carrier state infections are commonly observed in human and domestic fowl following an acute phase. Fibroblasts infected at low level with dormant Salmonella may be a source of relapse and shedding. From this point of view and considering the different scenarios that can occur during a Salmonella infection, the development of this atypical fibroblast model is another valuable addition to the field.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Sean Hannify for the critical reading of the manuscript.