The Burkholderia pseudomallei intracellular 'TRANSITome'

Abstract

Prokaryotic cell transcriptomics has been limited to mixed or sub-population dynamics and individual cells within heterogeneous populations, which has hampered further understanding of spatiotemporal and stage-specific processes of prokaryotic cells within complex environments. Here we develop a 'TRANSITomic' approach to profile transcriptomes of single Burkholderia pseudomallei cells as they transit through host cell infection at defined stages, yielding pathophysiological insights. We find that B. pseudomallei transits through host cells during infection in three observable stages: vacuole entry; cytoplasmic escape and replication; and membrane protrusion, promoting cell-to-cell spread. The B. pseudomallei 'TRANSITome' reveals dynamic gene-expression flux during transit in host cells and identifies genes that are required for pathogenesis. We find several hypothetical proteins and assign them to virulence mechanisms, including attachment, cytoskeletal modulation, and autophagy evasion. The B. pseudomallei 'TRANSITome' provides prokaryotic single-cell transcriptomics information enabling high-resolution understanding of host-pathogen interactions.

Fig. 1. Single Bp cell isolation and the TRANSITome.

a–d YFP-RFP-tagged Bp infected RAW264.7 cells were fixed and stained red (plasma and vacuolar membranes) and green (actin). Single Bp cells were isolated from macrophage vacuoles (a), cytoplasm (b), and during membrane protrusion (c). Bp control cells were grown in DMEM medium and isolated as the baseline for gene expression analysis (d). Scale bars = 5 μm. Single bacterial cells were cut with the focused laser (white circle) and catapulted using the low-intensity unfocused laser (blue dot) by LCM. e Bp undergoes dynamic gene expression changes relative to the control cells as it transits through the host cell. Each line represents a single gene with log₂FC represented in a rainbow color scheme (red lines are upregulated, black lines are downregulated). f Genes upregulated (log₂FC > 1) and downregulated (log₂FC <−1) showed high reproducibility among biological triplicates. Each color circle represents a biological replicate with gray representing overlap in all biological replicates. Numbers represent the number of genes within each overlapping section of the Venn diagram.

Fig. 2. Characterizations of BPSL0097 and BPSS1860 mutants.

a The BPSL0097 gene is upregulated within the macrophage vacuole. First three boxes of each infection stage represent three biological replicates, the fourth represents the mean. b Immunofluorescence to a BPSL0097-HA fusion strain suggests that BPSL0097 is located on the surface of Bp while the BPSL0097 mutant strain showed no signal. Scale bar = 1 μm. c Numbers of plaques formed (PFU) by the BPSL0097 mutant in HEK293T monolayer (n = 3) is comparable to wild-type Bp indicating no defect in host cell invasion; however, reduced plaque diameters indicate overall infection defects. d The BPSL0097 mutant showed reduced attachment efficiencies compared to wild-type Bp, suggesting a role in attachment to multiple host cell lines (n = 3). e TEM confirms the location of BPSL0097 to be the surface of Bp and further isolates its location to the poles of the cell. Scale bar = 100 nm. f The BPSL0097 mutant is attenuated in BALB/c mice (n = 5) when infected via the intranasal route. Bacterial burdens of surviving mice suggest that the BPSL0097 mutant persists in vivo. g BPSS1860 gene is upregulated during cell infection relative to in vitro condition. First three boxes of each infection stage represent three biological replicates, the fourth represents the mean. h Immunofluorescence to a BPSS1860-HA fusion strain suggests that BPSS1860 is located on the surface of Bp while the BPSS1860 mutant strain showed no signal. Scale bar = 1 μm. i Numbers of plaques formed (PFU) by the BPSS1860 mutant in HEK293T monolayer (n = 3) is comparable to wild-type Bp but show reduced plaque diameters indicating an overall infection defect. j The BPSS1860 mutant showed reduced attachment efficiencies compared to wild-type Bp, suggesting a role in attachment (n = 3). k TEM confirms that BPSS1860 is located on the surface of Bp and that it is distributed throughout the entire cell length. Scale bar = 100 nm. l Antibodies against purified BPSS1860 were detected via immunoblot (IB) in pooled melioidosis patients’ serum samples (n = 7). M, Precision Plus Protein Standard (Bio-Rad). m The BPSS1860 mutant is attenuated in BALB/c mice (n = 5) when infected via the intranasal route. The BPSS1860 mutant was completely cleared in all surviving mice. Data in bar graphs represent means ± s.e.m. and analyzed via two-sided unpaired t-test. P values presented above relevant comparisons.

Fig. 3. Characterizations of Bp mutant BPSS1818.

a BPSS1818 gene is upregulated within the macrophage cytosol, then significantly downregulated during the protrusion stage of infection. First three boxes of each infection stage represent three biological replicates, the fourth represents the mean. b Number of plaques formed (PFU) by the BPSS1818 mutant is comparable to wild-type Bp, indicating no defect in host cell invasion; however, reduced plaque diameters were observed indicating defect in infection spread (n = 3). Data represent means ± s.e.m. and analyzed via two-sided unpaired t-test. P values presented above relevant comparisons. c Fused RAW264.7 cells infected with the BPSS1818 mutant show extended host cell cytoskeleton and distended MNGCs compared to wild-type Bp. Scale bars = 5 μm. d The BPSS1818 mutant is completely attenuated in BALB/c mice (n = 5) when compared to wild-type Bp. Bacterial burdens were only observed at low levels in lungs of three mice. e RAW264.7 cells infected with the BPSS1818 mutant showed elongated microtubules with pronounced fibers when magnified in contrast to wild-type Bp infected cells. Scale bars = 10 μm.

Fig. 4. Characterization of Bp mutant BPSS0015.

a BPSS0015 is upregulated at the protrusion stage during infection. First three boxes of each infection stage represent three biological replicates, the fourth represents the mean. b Number of plaques formed (PFU) by the BPSS0015 mutant is similar to wild-type Bp, indicating no defects in host cell invasion; however, reduced plaque diameters were observed, suggesting defects during the progression of infection (n = 3). c Rapamycin-induced (RAP) autophagy reduced the survival of both wild-type Bp and the BPSS0015 mutant (n = 3), whereas the inhibition of autophagy with 3-methyladenine (3-MA) significantly restored intracellular replication of the BPSS0015 mutant to wild-type level (n = 6). d Representative TEM images of macrophages infected with the BPSS0015 mutant show each bacterium surrounded by membrane structures. Scale bars = 1 μm. e Infected HEK293T cells stably expressing GFP-LC3 show co-localization of the autophagy marker, LC3 (green), with the BPSS0015 mutant (red), indicated by the white arrow, and no co-localization with wild-type Bp (red). Scale bars = 10 μm. f The BPSS0015 mutant is completely attenuated in BALB/c mice (n = 5) but bacterial burdens of surviving mice suggest that this mutant persists in vivo. Data in bar graphs represent means ± s.e.m. and analyzed via two-sided unpaired t-test. P values presented above relevant comparisons.

Fig. 5. Model of Bp intracellular pathogenesis.

Bp attaches unknown receptors (orange) on host cells, using surface attachment proteins BPSL0097 and BPSS1860. After internalization, Bp escapes the vacuole utilizing the Burkholderia secretion apparatus (T3SS_Bsa) and moves within the host cell using its secondary flagella locus or by polymerizing host cell actin (green) with bacterial BimA (red boxes on the bacterial cell pole). BPSS1818 facilitates changes in host cell cytoskeletal structure through modulation of tubulin (maroon), while BPSS0015 contributes to autophagy evasion. PAS: pre-autophagosomal structure. Bacteria replicate intracellularly and spread to neighboring cells by actin-based membrane protrusion and by promoting cell fusion to form multinucleated giant cells (MNGC) using a Type VI Secretion System (T6SS)^,,.