This is a presentation given to the American Association for Aerosol Research, Fall 2010 in Portland, OR. It details some of our work examining the gene level response of B. pertussis to aerosolization.
Potential for dsRNA-based management of plant diseases - Karl-Heinz Kogel, Ju...
Pertussis Aaar Talk
1. Viability of Aerosolized Bordetella pertussis and Indications of Gene Level Control of Putative Environmental Survival Factors Kevin M. McCabe, Ph.D. University of Colorado at Boulder Department of Civil, Environmental, and Architectural Engineering
2. Virulence Gene Expression in B. pertussis is Controlled by Environmental Signals If B. pertussis is transmitted directly from one airway to the next as an aerosol, why would the virulence factor genes be turned off? Why are other genes turned on instead?
3. Survival of B. pertussis as a Function of Relative Humidity
4. Hypothetical role for ris -activated genes in B. pertussis infectious life-cycle Bvg-Activated Genes (Virulence Genes On - vrg s Off) Virulence Repressed Genes ( vrg s On - Virulence Genes Off) Survival outside the host Initial interaction with host Survival and replication in the host Bvg+ Bvg-
5. B. pertussis in Virulent State Bvg+ Adenylate Cyclase Toxin Pertussis Toxin Pertactin Fimbrea FHA BrkA Tracheal Colonization Factor Ptl Tracheal Cytotoxic Toxin
6. Modulation of Virulent vs. Environmental Survival States Virulent Environmental Survival? Vrg6 Vrg73 Vrg18 Bvg- Vrg24 Bvg+ Low Temperature [Nicotinic Acid] [MgSO 4 ] (modulation)
7. Hypothetical role for ris -activated genes in B. pertussis infectious life-cycle Bvg-Activated Genes (Virulence Genes On- vrg s Off) Virulence Repressed Genes ( vrg s On- Virulence Genes Off) Bvg+ Bvg- Survival outside the host Initial interaction with host Survival and replication in the host Vrg6 Vrg73 Vrg18 Bvg- Vrg24 Bvg+
16. Contribution of ris -activated genes to survival in aerosols
17. Contribution of ris -activated genes to survival in aerosols
18. Role for ris -activated genes in B. pertussis infectious life-cycle Bvg-Activated Genes (Virulence Genes On- Env. Survival Off) Virulence Repressed Genes ( ENVIRONMENTAL SURVIVAL GENES ON Virulence Genes Off) Bvg+ Bvg- Survival outside the host Initial interaction with host Survival and replication in the host Vrg6 Vrg73 Vrg18 Bvg- Vrg24 Bvg+
19. Thanks! FDA: Tod Merkel Vanessa Kelly Gloria Lee USDA: Tracy Nicholson Sarah Shore CU Boulder: Mark Hernandez Jane Turner Alina Handorean Alison Ling Other past lab members I will be presenting a poster on another aspect of our B. pertussis work and would be happy to answer any additional questions we do not have time for here. Poster 2L.3 Today 1-2:50pm Abstract #427 Tod Jane Mark
Hinweis der Redaktion
Hello and good morning (Title if not read by Jordan) Today I will be presenting: Background information regarding gene regulation of B. pertussis virulence genes, possible Environmental Survival factors, and how they affect aerosol based transmission of whooping cough. I will outline our methods using mutant and wild-type strains to isolate aerosol environmental stress as a process variable. Finally I will present data supporting our hypothesis that pertussis responds to aerosolization, or being coughed out of its host, at a gene expression level. CLICK
Read slide. CLICK
We know from our early characterization studies that wild-type B. pertussis is extremely sensitive to aerosolization, particularly at reduced relative humidity. This extreme sensitivity to aerosol environmental stress could be the reason B. pertussis needs this other set of genes. What if these genes, rather than aiding replication and growth in the host like the virulence genes, aid environmental stability and survival outside the host? CLICK
We also know that virulence gene expression is sensitive to environmental signals, and that the virulence genes and the virulence repressed genes or vrg s are oppositely regulated. Meaning that when the virulence genes are on, vrg s are off and vice versa. In order to survive outside the host, we believe pertussis responds environmental signals. These signals turn off the virulence genes and turn on the vrgs, a set of genes of unknown function. Bvg stands for Bordetella virulence gene and a Bvg plus state indicates the virulence genes are expressed. A Bvg minus state indicates the virulence genes are off and the vrgs are on. CLICK
In the Bvg+ state, with the virulence genes expressed, pertussis is a immune cell inhibiting, toxin producing, tissue necrosing machine. To date this year Whooping Cough has killed 10 infants in the California outbreak alone. CLICK
However, CLICK in response to low temperature and increases into the milimolar range for nicotinic acid or magnesium sulfate, expression of these toxins and other virulence factors are shut off CLICK and the vrg CLICK or virulence repressed genes are turned on. These are extracellular and secreted proteins that as I have said, have an as yet unknown function. We believe they aid in CLICK environmental survival. So with these environmental signals we can transition from a Bvg+ to a Bvg- state. CLICK
Essentially pertussis can sense it has been coughed out of the host (in the virulent state) CLICK and into the environment. There it will alter gene expression CLICK to address aerosol environmental stress. CLICK
This gene level environmental response is the focus of these studies. We examine pertussis mutant strains that control virulence gene expression and virulence repressed gene (vrg ) expression. This system relies on cyclic di-GMP (cyclic di-Guanosine Monophosphate) as an activator of vrg expression. CLICK RisX is a di-guanlyate cyclase that converts 2 GTP molecules to one cyclic di-GMP. CLICK BvgR is a phosphodiesterase that can cleave cyclic di-GMP into a third and inactive molecule. CLICK Cyclic di-GMP binds to and activates RisA turning on vrg expression, and we believe conferring environmental resistance. CLICK
Environmental cues are sensed by and transmitted through BvgS and BvgA, which reside here in the cytoplasmic membrane, to affect levels of BvgR. CLICK By inhibiting BvgAS these environmental cues reduce BvgR levels. (SAY VERY SLOWLY!!!) As BvgR inhibits vrg expression, these cues serve to reduce inhibition of vrg expression, or in more direct terms, these environmental signals increase vrg expression. CLICK, CLICK RisA is a required activator of vrg expression. CLICK (active transcription or gene expression is noted by this black arrow) CLICK
Putting together the cyclic diGMP and environmental signaling aspects of this pathway: RisX is a diguanylate cyclase that makes cyclic di-GMP. BvgR is a phosphodiesterase that can cleave cyclic di-GMP into another inactive compound. These two enzymes, thus, control the cyclic di-GMP pool in the cell. RisX makes cyclic di-GMP, permitting binding to, and activation of RisA. Active RisA triggers expression of the vrg genes. CLICK (again, noted by this black arrow) Increased BvgR reduces cyclic di-GMP levels, reducing active RisA levels and reducing vrg expression. CLICK CLICK
The first of the mutant strains studied is a delta-risA or deletion of the risA gene. RisA is a required activator of vrg expression. CLICK, CLICK, CLICK Deleting the risA gene eliminates the RisA protein. CLICK Regardless of cyclic diGMP levels there will be no vrg expression without RisA. CLICK This locks the pertussis in a virulent or Bvg+ state. CLICK
The next mutant strain we utilize is a delta-bvgR or deletion of the bvgR gene. CLICK Deleting the bvgR gene eliminates the BvgR protein. CLICK, CLICK BvgR is phosphodieseterase that reduces cyclic di-GMP levels which would normally serve to reduce levels of active RisA. CLICK, CLICK Cyclic diGMP levels therefore will increase CLICK , increasing levels of active RisA, increasing vrg expression. CLICK This locks the pertussis in the environmental survival state. CLICK PAUSE CLICK
If these vrg s do aid environmental survival, we would expect that delta-risA, locked in the virulent state and lacking the ability to produce the vrg proteins, would be very sensitive to environmental exposure relative to wild-type Bp536 which remains environmentally responsive. CLICK, CLICK Therefore CLICK, CLICK , delta-bvgR, with ongoing expression of the vrg genes and the constant presence of these “environmental stability factors” should be resistant to environmental stress relative to delta-risA and wild-type Bp536. CLICK
Read slide. CLICK
Once again, we expect the delta-risA to have reduced viability relative to the CLICK, CLICK wild-type. We also expect the delta-bvgR CLICK CLICK to have increased viability relative to the wild-type. CLICK
Relative Humidity is held near 82% throughout these experiments. Viability is normalized to the time zero sample which is collected as the nebulization is completed, and for the first minute following. Error bars represent one standard deviation. CLICK Wild type looses over 50% viability over the first 2 minutes and continues dropping over the intervening 8 minutes to 16%. CLICK As expected, delta-risA is more sensitive, loosing 75% viability in the first two minutes and falling to near the detection limit by 10 minutes. CLICK Further confirming our hypothesis, delta-bvgR, while loosing similar viability to wild-type early on, then maintains 50% viability for 10 minutes. CLICK
In a duplicate set of experiments, we see a similar pattern. CLICK Wild-type again looses about 50% and continues to loose viability over the next 8 minutes. CLICK Delta -risA is once again significantly more sensitive than wild-type. CLICK And delta- bvgR , after an initial 50% loss in the first 2 minutes, holds its viability through 10 minutes. CLICK
In conclusion, through the use of these mutant and wild-type Bordetella pertussis strains, we have isolated environmental aerosol exposure as a process variable. Further, we have demonstrated decreased viability of the delta-risA mutant and increased viability of the delta-bvgR mutant relative to wild-type pertussis . These results confirm our hypothesis that the vrgs or virulence repressed gene products confer environmental aerosol stress resistance. Thus, when the bacteria that cause whooping cough are coughed out into the air, they recognize the change in their environment. They turn off now the unneeded virulence genes, in favor of a set of environmental stability genes. When a new host is encountered, the bacteria again recognize the change in environment, turn off vrg expression and turn back on the virulence genes to effectively infect the new host.
Thank you for your attention. I would like to note I have a poster this afternoon regarding another aspect of our pertussis work and any questions or extended discussion we don’t have time for here, I can entertain at that time. That said, I would be happy to take any questions now.
