Limits...
Viral Transmission Dynamics at Single-Cell Resolution Reveal Transiently Immune Subpopulations Caused by a Carrier State Association.

Cenens W, Makumi A, Govers SK, Lavigne R, Aertsen A - PLoS Genet. (2015)

Bottom Line: Monitoring the complex transmission dynamics of a bacterial virus (temperate phage P22) throughout a population of its host (Salmonella Typhimurium) at single cell resolution revealed the unexpected existence of a transiently immune subpopulation of host cells that emerged from peculiarities preceding the process of lysogenization.Upon subsequent division, the daughter cell inheriting this episome became lysogenized by an integration event yielding a prophage, while the other daughter cell became P22-free.The iterative emergence and infection of transiently resistant subpopulations suggests a new bet-hedging strategy by which viruses could manage to sustain both vertical and horizontal transmission routes throughout an infected population without compromising a stable co-existence with their host.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Food Microbiology, Department of Microbial and Molecular Systems (M²S), Faculty of Bioscience Engineering, KU Leuven, Leuven, Belgium.

ABSTRACT
Monitoring the complex transmission dynamics of a bacterial virus (temperate phage P22) throughout a population of its host (Salmonella Typhimurium) at single cell resolution revealed the unexpected existence of a transiently immune subpopulation of host cells that emerged from peculiarities preceding the process of lysogenization. More specifically, an infection event ultimately leading to a lysogen first yielded a phage carrier cell harboring a polarly tethered P22 episome. Upon subsequent division, the daughter cell inheriting this episome became lysogenized by an integration event yielding a prophage, while the other daughter cell became P22-free. However, since the phage carrier cell was shown to overproduce immunity factors that are cytoplasmically inherited by the P22-free daughter cell and further passed down to its siblings, a transiently resistant subpopulation was generated that upon dilution of these immunity factors again became susceptible to P22 infection. The iterative emergence and infection of transiently resistant subpopulations suggests a new bet-hedging strategy by which viruses could manage to sustain both vertical and horizontal transmission routes throughout an infected population without compromising a stable co-existence with their host.

Show MeSH

Related in: MedlinePlus

Categorizing single cell infection dynamics in the P22–S. Typhimurium model system.In the LT2ΔpSLT/pALA2705 reporter, presence of a P22 chromosome is revealed by the formation of a fluorescent GFP focus originating from GFP-ParB molecules bound to the parS sequence engineered in P22 parS. (A) The central picture shows an initial snapshot of an exponential phase population of LT2ΔpSLT/pALA2705 infected with P22 parS (MOI = 20) and further grown for 4 hours post infection in a semi-continuous culture. Time-lapse recordings of specific events are presented as a zoom in on the original snapshot as indicated by the black lines (box A1–A4). Four types of phage–host associations are seen in panel A: lysogenized cells in which the stably integrated P22 parS prophage yields a discrete GFP focus that replicates and segregates together with the host chromosome (box A1); lytically infected cells in which the replicating P22 parS chromosome yields a more diffuse and randomly dispersed GFP cloud throughout the cell prior to cell lysis (box A2); P22-free cells in which the absence of a P22 parS chromosome yields a diffuse cytoplasmic GFP fluorescence (box A3); phage carrier cells in which a polarly tethered P22 parS episome yields a coherent GFP cloud in one of the cell poles (box A4). Please note that the bright fluorescent cell at the bottom of panel A is a rare artifact. (B-D) Time-lapse series of (B) cells in the absence of P22 parS, (C) of growing cells from a P22 parS lysogen in LT2ΔpSLT/pALA2705, and (D) of LT2ΔpSLT/pALA2705 cells infected with P22 c2 parS (an obligate lytic derivative of P22 parS). (E) Snapshots from the lineages emerging from two phage carrier cells within a P22 parS infected LT2ΔpSLT/pALA2705 population, exhibiting either direct (left panel) or delayed (right panel) integration of the P22 parS prophage, resulting either in a homogeneous population of lysogens (left panel) or a heterogeneous population of both lysogens and P22-free cells (right panel). Analysis of 114 such lineages revealed the segregation of P22-free siblings in ca. 41% of cases. Phase contrast images (showing the cells) and GFP signal (reporting the P22 parS chromosomes) are merged. A 5 μm scale bar is shown at the bottom right of each panel. Timestamps are shown in the top left corners of time-lapse images. In panel D the timestamp is set at 0 min from the moment a ParB-GFP foci became visible. In all other panels the timestamp was started when first image was taken.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4697819&req=5

pgen.1005770.g001: Categorizing single cell infection dynamics in the P22–S. Typhimurium model system.In the LT2ΔpSLT/pALA2705 reporter, presence of a P22 chromosome is revealed by the formation of a fluorescent GFP focus originating from GFP-ParB molecules bound to the parS sequence engineered in P22 parS. (A) The central picture shows an initial snapshot of an exponential phase population of LT2ΔpSLT/pALA2705 infected with P22 parS (MOI = 20) and further grown for 4 hours post infection in a semi-continuous culture. Time-lapse recordings of specific events are presented as a zoom in on the original snapshot as indicated by the black lines (box A1–A4). Four types of phage–host associations are seen in panel A: lysogenized cells in which the stably integrated P22 parS prophage yields a discrete GFP focus that replicates and segregates together with the host chromosome (box A1); lytically infected cells in which the replicating P22 parS chromosome yields a more diffuse and randomly dispersed GFP cloud throughout the cell prior to cell lysis (box A2); P22-free cells in which the absence of a P22 parS chromosome yields a diffuse cytoplasmic GFP fluorescence (box A3); phage carrier cells in which a polarly tethered P22 parS episome yields a coherent GFP cloud in one of the cell poles (box A4). Please note that the bright fluorescent cell at the bottom of panel A is a rare artifact. (B-D) Time-lapse series of (B) cells in the absence of P22 parS, (C) of growing cells from a P22 parS lysogen in LT2ΔpSLT/pALA2705, and (D) of LT2ΔpSLT/pALA2705 cells infected with P22 c2 parS (an obligate lytic derivative of P22 parS). (E) Snapshots from the lineages emerging from two phage carrier cells within a P22 parS infected LT2ΔpSLT/pALA2705 population, exhibiting either direct (left panel) or delayed (right panel) integration of the P22 parS prophage, resulting either in a homogeneous population of lysogens (left panel) or a heterogeneous population of both lysogens and P22-free cells (right panel). Analysis of 114 such lineages revealed the segregation of P22-free siblings in ca. 41% of cases. Phase contrast images (showing the cells) and GFP signal (reporting the P22 parS chromosomes) are merged. A 5 μm scale bar is shown at the bottom right of each panel. Timestamps are shown in the top left corners of time-lapse images. In panel D the timestamp is set at 0 min from the moment a ParB-GFP foci became visible. In all other panels the timestamp was started when first image was taken.

Mentions: In order to be able to specifically track the intracellular whereabouts of the P22 chromosome during live infection of its S. Typhimurium LT2 host with time-lapse fluorescence microscopy, we (i) recombineered a parS sequence into the P22 chromosome (yielding P22 parS; [24]), and (ii) equipped its LT2 host with the pALA2705 plasmid ([25]; yielding LT2/pALA2705) expressing a cognate GFP-ParB fusion protein that binds to and multimerizes around this parS locus [26]. In the course of these experiments, however, we noticed that presence of the pSLT virulence plasmid in LT2 interfered with the proper localization of GFP-ParB (likely mediated by the presence of the pSLT specific parS/ParAB segregation system [27]; S1A Fig). Once the pSLT plasmid was cured from LT2 (resulting in LT2ΔpSLT/pALA2705), GFP-ParB assumed the proper diffuse cytoplasmic distribution indicative for the absence of a parS sequence in the cell (Figs 1 and S1B).


Viral Transmission Dynamics at Single-Cell Resolution Reveal Transiently Immune Subpopulations Caused by a Carrier State Association.

Cenens W, Makumi A, Govers SK, Lavigne R, Aertsen A - PLoS Genet. (2015)

Categorizing single cell infection dynamics in the P22–S. Typhimurium model system.In the LT2ΔpSLT/pALA2705 reporter, presence of a P22 chromosome is revealed by the formation of a fluorescent GFP focus originating from GFP-ParB molecules bound to the parS sequence engineered in P22 parS. (A) The central picture shows an initial snapshot of an exponential phase population of LT2ΔpSLT/pALA2705 infected with P22 parS (MOI = 20) and further grown for 4 hours post infection in a semi-continuous culture. Time-lapse recordings of specific events are presented as a zoom in on the original snapshot as indicated by the black lines (box A1–A4). Four types of phage–host associations are seen in panel A: lysogenized cells in which the stably integrated P22 parS prophage yields a discrete GFP focus that replicates and segregates together with the host chromosome (box A1); lytically infected cells in which the replicating P22 parS chromosome yields a more diffuse and randomly dispersed GFP cloud throughout the cell prior to cell lysis (box A2); P22-free cells in which the absence of a P22 parS chromosome yields a diffuse cytoplasmic GFP fluorescence (box A3); phage carrier cells in which a polarly tethered P22 parS episome yields a coherent GFP cloud in one of the cell poles (box A4). Please note that the bright fluorescent cell at the bottom of panel A is a rare artifact. (B-D) Time-lapse series of (B) cells in the absence of P22 parS, (C) of growing cells from a P22 parS lysogen in LT2ΔpSLT/pALA2705, and (D) of LT2ΔpSLT/pALA2705 cells infected with P22 c2 parS (an obligate lytic derivative of P22 parS). (E) Snapshots from the lineages emerging from two phage carrier cells within a P22 parS infected LT2ΔpSLT/pALA2705 population, exhibiting either direct (left panel) or delayed (right panel) integration of the P22 parS prophage, resulting either in a homogeneous population of lysogens (left panel) or a heterogeneous population of both lysogens and P22-free cells (right panel). Analysis of 114 such lineages revealed the segregation of P22-free siblings in ca. 41% of cases. Phase contrast images (showing the cells) and GFP signal (reporting the P22 parS chromosomes) are merged. A 5 μm scale bar is shown at the bottom right of each panel. Timestamps are shown in the top left corners of time-lapse images. In panel D the timestamp is set at 0 min from the moment a ParB-GFP foci became visible. In all other panels the timestamp was started when first image was taken.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4697819&req=5

pgen.1005770.g001: Categorizing single cell infection dynamics in the P22–S. Typhimurium model system.In the LT2ΔpSLT/pALA2705 reporter, presence of a P22 chromosome is revealed by the formation of a fluorescent GFP focus originating from GFP-ParB molecules bound to the parS sequence engineered in P22 parS. (A) The central picture shows an initial snapshot of an exponential phase population of LT2ΔpSLT/pALA2705 infected with P22 parS (MOI = 20) and further grown for 4 hours post infection in a semi-continuous culture. Time-lapse recordings of specific events are presented as a zoom in on the original snapshot as indicated by the black lines (box A1–A4). Four types of phage–host associations are seen in panel A: lysogenized cells in which the stably integrated P22 parS prophage yields a discrete GFP focus that replicates and segregates together with the host chromosome (box A1); lytically infected cells in which the replicating P22 parS chromosome yields a more diffuse and randomly dispersed GFP cloud throughout the cell prior to cell lysis (box A2); P22-free cells in which the absence of a P22 parS chromosome yields a diffuse cytoplasmic GFP fluorescence (box A3); phage carrier cells in which a polarly tethered P22 parS episome yields a coherent GFP cloud in one of the cell poles (box A4). Please note that the bright fluorescent cell at the bottom of panel A is a rare artifact. (B-D) Time-lapse series of (B) cells in the absence of P22 parS, (C) of growing cells from a P22 parS lysogen in LT2ΔpSLT/pALA2705, and (D) of LT2ΔpSLT/pALA2705 cells infected with P22 c2 parS (an obligate lytic derivative of P22 parS). (E) Snapshots from the lineages emerging from two phage carrier cells within a P22 parS infected LT2ΔpSLT/pALA2705 population, exhibiting either direct (left panel) or delayed (right panel) integration of the P22 parS prophage, resulting either in a homogeneous population of lysogens (left panel) or a heterogeneous population of both lysogens and P22-free cells (right panel). Analysis of 114 such lineages revealed the segregation of P22-free siblings in ca. 41% of cases. Phase contrast images (showing the cells) and GFP signal (reporting the P22 parS chromosomes) are merged. A 5 μm scale bar is shown at the bottom right of each panel. Timestamps are shown in the top left corners of time-lapse images. In panel D the timestamp is set at 0 min from the moment a ParB-GFP foci became visible. In all other panels the timestamp was started when first image was taken.
Mentions: In order to be able to specifically track the intracellular whereabouts of the P22 chromosome during live infection of its S. Typhimurium LT2 host with time-lapse fluorescence microscopy, we (i) recombineered a parS sequence into the P22 chromosome (yielding P22 parS; [24]), and (ii) equipped its LT2 host with the pALA2705 plasmid ([25]; yielding LT2/pALA2705) expressing a cognate GFP-ParB fusion protein that binds to and multimerizes around this parS locus [26]. In the course of these experiments, however, we noticed that presence of the pSLT virulence plasmid in LT2 interfered with the proper localization of GFP-ParB (likely mediated by the presence of the pSLT specific parS/ParAB segregation system [27]; S1A Fig). Once the pSLT plasmid was cured from LT2 (resulting in LT2ΔpSLT/pALA2705), GFP-ParB assumed the proper diffuse cytoplasmic distribution indicative for the absence of a parS sequence in the cell (Figs 1 and S1B).

Bottom Line: Monitoring the complex transmission dynamics of a bacterial virus (temperate phage P22) throughout a population of its host (Salmonella Typhimurium) at single cell resolution revealed the unexpected existence of a transiently immune subpopulation of host cells that emerged from peculiarities preceding the process of lysogenization.Upon subsequent division, the daughter cell inheriting this episome became lysogenized by an integration event yielding a prophage, while the other daughter cell became P22-free.The iterative emergence and infection of transiently resistant subpopulations suggests a new bet-hedging strategy by which viruses could manage to sustain both vertical and horizontal transmission routes throughout an infected population without compromising a stable co-existence with their host.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Food Microbiology, Department of Microbial and Molecular Systems (M²S), Faculty of Bioscience Engineering, KU Leuven, Leuven, Belgium.

ABSTRACT
Monitoring the complex transmission dynamics of a bacterial virus (temperate phage P22) throughout a population of its host (Salmonella Typhimurium) at single cell resolution revealed the unexpected existence of a transiently immune subpopulation of host cells that emerged from peculiarities preceding the process of lysogenization. More specifically, an infection event ultimately leading to a lysogen first yielded a phage carrier cell harboring a polarly tethered P22 episome. Upon subsequent division, the daughter cell inheriting this episome became lysogenized by an integration event yielding a prophage, while the other daughter cell became P22-free. However, since the phage carrier cell was shown to overproduce immunity factors that are cytoplasmically inherited by the P22-free daughter cell and further passed down to its siblings, a transiently resistant subpopulation was generated that upon dilution of these immunity factors again became susceptible to P22 infection. The iterative emergence and infection of transiently resistant subpopulations suggests a new bet-hedging strategy by which viruses could manage to sustain both vertical and horizontal transmission routes throughout an infected population without compromising a stable co-existence with their host.

Show MeSH
Related in: MedlinePlus