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. pseudomall… Show more
“…To understand how intracellular pathogenesis of Bp is controlled, we first have to identify transcriptional regulators that control virulence mechanisms during intracellular infection. We identified BP1026B_II1198, a PadR-like transcriptional regulator that is differentially regulated during the Bp intracellular ‘transitome’ 7 . BP1026B_II1198 shows similarities to PadR transcriptional regulators with an N-terminal winged helix-like DNA binding domain (Pfam: PF03551) and a C-terminal virulence activator alpha domain (Pfam: PF10400) 18 , 19 .…”
Section: Resultsmentioning
confidence: 99%
“… Figure 1 BP1026B_II1198 is differentially regulated during cell infection and critical for intracellular survival. ( a) Single cell ‘transitome’ analysis 7 shows that BP1026B_II1198 is up-regulated in the vacuole and protrusion while down-regulated in the cytoplasm during infection of RAW264.7 cells. Boxes represent three biological replicates and an average (Ave) 7 .…”
Section: Resultsmentioning
confidence: 99%
“…( a) Single cell ‘transitome’ analysis 7 shows that BP1026B_II1198 is up-regulated in the vacuole and protrusion while down-regulated in the cytoplasm during infection of RAW264.7 cells. Boxes represent three biological replicates and an average (Ave) 7 . ( b ) A mutant in BP1026B_II1198 shows a reduced ability to form wildtype level MNGCs.…”
Section: Resultsmentioning
confidence: 99%
“…To cause such a wide array of clinical manifestations and be able to transit through the intracellular environment with such precision, Bp requires many virulence factors, some of which are described above. As recently demonstrated, Bp goes through drastic transcriptional changes, with 1,953 genes showing unique patterns of expression during the intracellular infection process 7 . This finding suggests that Bp has a highly complex regulatory network that is required to coordinate the differential expression of these genes during the progression of infection, from the initial stage of attachment to eventual protrusion towards neighboring cells.…”
Section: Introductionmentioning
confidence: 94%
“…Bp encounters many different environments as it infects most tissues of the human body, leading to the formation of localized abscesses, bacteremia, septic shock, and sometimes death 5 , 6 . While infecting a cell, Bp has a dynamic lifecycle that can be broken down into multiple stages: attachment to the host cell, host cell entry, vacuole escape, cytoplasmic replication, and protrusion towards neighboring cells culminating with the spread of infection 7 . In the vacuole, the Burkholderia secretion apparatus (T3SS Bsa ), a type III secretion system, must be expressed to allow entry into the host cell cytoplasm 8 .…”
Burkholderia pseudomallei (Bp) is the causative agent of melioidosis, a disease endemic to the tropics. Melioidosis manifests in various ways ranging from acute skin lesions to pneumonia and, in rare cases, infection of the central nervous system. Bp is a facultative intracellular pathogen and it can infect various cell types. The Bp intracellular lifecycle has been partially elucidated and is highly complex. Herein, we have identified a transcriptional regulator, BP1026B_II1198, that is differentially expressed as Bp transits through host cells. A deletion mutant of BP1026B_II1198 was attenuated in RAW264.7 cell and BALB/c mouse infection. To further characterize the function of this transcriptional regulator, we endeavored to determine the regulon of BP1026B_II1198. RNA-seq analysis showed the global picture of genes regulated while ChIP-seq analysis identified two specific BP1026B_II1198 binding regions on chromosome II. We investigated the transposon mutants of these genes controlled by BP1026B_II1198 and confirmed that these genes contribute to pathogenesis in RAW264.7 murine macrophage cells. Taken together, the data presented here shed light on the regulon of BP1026B_II1198 and its role during intracellular infection and highlights an integral portion of the highly complex regulation network of Bp during host infection.
“…To understand how intracellular pathogenesis of Bp is controlled, we first have to identify transcriptional regulators that control virulence mechanisms during intracellular infection. We identified BP1026B_II1198, a PadR-like transcriptional regulator that is differentially regulated during the Bp intracellular ‘transitome’ 7 . BP1026B_II1198 shows similarities to PadR transcriptional regulators with an N-terminal winged helix-like DNA binding domain (Pfam: PF03551) and a C-terminal virulence activator alpha domain (Pfam: PF10400) 18 , 19 .…”
Section: Resultsmentioning
confidence: 99%
“… Figure 1 BP1026B_II1198 is differentially regulated during cell infection and critical for intracellular survival. ( a) Single cell ‘transitome’ analysis 7 shows that BP1026B_II1198 is up-regulated in the vacuole and protrusion while down-regulated in the cytoplasm during infection of RAW264.7 cells. Boxes represent three biological replicates and an average (Ave) 7 .…”
Section: Resultsmentioning
confidence: 99%
“…( a) Single cell ‘transitome’ analysis 7 shows that BP1026B_II1198 is up-regulated in the vacuole and protrusion while down-regulated in the cytoplasm during infection of RAW264.7 cells. Boxes represent three biological replicates and an average (Ave) 7 . ( b ) A mutant in BP1026B_II1198 shows a reduced ability to form wildtype level MNGCs.…”
Section: Resultsmentioning
confidence: 99%
“…To cause such a wide array of clinical manifestations and be able to transit through the intracellular environment with such precision, Bp requires many virulence factors, some of which are described above. As recently demonstrated, Bp goes through drastic transcriptional changes, with 1,953 genes showing unique patterns of expression during the intracellular infection process 7 . This finding suggests that Bp has a highly complex regulatory network that is required to coordinate the differential expression of these genes during the progression of infection, from the initial stage of attachment to eventual protrusion towards neighboring cells.…”
Section: Introductionmentioning
confidence: 94%
“…Bp encounters many different environments as it infects most tissues of the human body, leading to the formation of localized abscesses, bacteremia, septic shock, and sometimes death 5 , 6 . While infecting a cell, Bp has a dynamic lifecycle that can be broken down into multiple stages: attachment to the host cell, host cell entry, vacuole escape, cytoplasmic replication, and protrusion towards neighboring cells culminating with the spread of infection 7 . In the vacuole, the Burkholderia secretion apparatus (T3SS Bsa ), a type III secretion system, must be expressed to allow entry into the host cell cytoplasm 8 .…”
Burkholderia pseudomallei (Bp) is the causative agent of melioidosis, a disease endemic to the tropics. Melioidosis manifests in various ways ranging from acute skin lesions to pneumonia and, in rare cases, infection of the central nervous system. Bp is a facultative intracellular pathogen and it can infect various cell types. The Bp intracellular lifecycle has been partially elucidated and is highly complex. Herein, we have identified a transcriptional regulator, BP1026B_II1198, that is differentially expressed as Bp transits through host cells. A deletion mutant of BP1026B_II1198 was attenuated in RAW264.7 cell and BALB/c mouse infection. To further characterize the function of this transcriptional regulator, we endeavored to determine the regulon of BP1026B_II1198. RNA-seq analysis showed the global picture of genes regulated while ChIP-seq analysis identified two specific BP1026B_II1198 binding regions on chromosome II. We investigated the transposon mutants of these genes controlled by BP1026B_II1198 and confirmed that these genes contribute to pathogenesis in RAW264.7 murine macrophage cells. Taken together, the data presented here shed light on the regulon of BP1026B_II1198 and its role during intracellular infection and highlights an integral portion of the highly complex regulation network of Bp during host infection.
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