B. pseudomallei genes differentially expressed during host cell infection. B. pseudomallei genes differentially expressed during host cell infection

Prokaryotic cell transcriptomics have been limited to mixed or sub-population dynamics and individuality of cells within heterogeneous populations. This significantly hampers further knowledge into spatiotemporal and stage-specific processes of prokaryotic cells within complex environments. Herein, we developed a ‘TRANSITomic’ approach to profile transcriptomes of single Burkholderia pseudomallei (Bp) cells as they transit through host cell infection at defined stages, yielding pathophysiological insights. Bp transits through host cells during infection in three observable stages: i) vacuole entry; ii) cytoplasmic escape and replication; and iii) membrane protrusion, promoting cell-to-cell spread. The Bp ‘TRANSITome’ revealed dynamic gene-expression flux during transit in host cells. Some genes undergoing changes are required for pathogenesis. We discovered several hypothetical proteins and assigned them to virulence mechanisms such as attachment, cytoskeletal modulation, and autophagy evasion. The Bp ‘TRANSITome’ has provided high-resolution understanding of host-pathogen interactions and opens the door for future single-cell transcriptomic analysis of other prokaryotic processes. Overall design: Single B. pseudomallei cell isolation at different stages during macrophage infection. RFP-YFP-tagged B. pseudomallei strain was used to infect macrophage RAW 264.7 cell line. After infection and fixation, the macrophage plasma membrane and endo-vesicular membrane were stained far red and the actin was stained green. Nine single B. pseudomallei cells were observed and isolated at different stages during macrophage infection, including inside of macrophage vacuoles, in the cytoplasm during actin polymerization, and during protrusion. The in vitro control (nine B. pseudomallei cells) was obtained by incubating B. pseudomallei in the DMEM medium in the absence of macrophages. Single B. pseudomallei cells and pooled control B. pesudomallei cells were processed using previously described methods for single cell transcriptomic analysis.