This is my project proposal (Sarah Hu) for Quiz #3

*work in progress, feedback highly appreciated!

Is there differential gene expression in Vibrio tubiashii in ocean acidified water with a host (oyster) present?

A. Introduction / Rationale for Research (with references)
B. Proposed methods
C. Budget
D. Possible Pitfalls


Introduction

Vibrio tubiashii is a rod-shaped gram-negative bacterium. It is one of the leading causes of vibriosis in larval shellfish. Later the pathogenic bacterium was identified as vibriosis and named Vibrio tubiashii. Its importance in the shellfish production industry was established (Brown and Losee 1978, Elston 1993). With recent larval shellfish mortalities due to Vibrio tubiashii it is important to examine the virulence factors that contribute to these outbreaks.
Elston et al. 2008 reported the reappearance of Vibrio tubiashii along the West Coast and high mortality among shellfish larvae and juveniles. The nearshore outbreaks of vibriosis are associated with environmental conditions such as cold upwelled waters meeting with the warm sea surface (Elston et al. 2008). The re-emergence of virulent Vibrio tubiashii requires attention and further investigation due to potentially large economic losses in the shellfish hatchery.

V. tubiashii outbreaks as a result of environmental change, elevated ocean temperature, suggests that pathogenicity may be dependent on environmental cues or conditions. The environmental changes associated with global warming have recently caused research to focus on the biological impacts of climate change. A review (Marcogliese 2001) stressed the importance of evaluating responses of parasites in aquatic organisms. Parasites in these systems respond directly to climatic changes and can provide needed insight for host-pathogen relationship and possibly modeling future climate change responses (Marcogliese 2001).

In a similar comparison, it is important to consider the higher implications of ocean acidification and bacteria virulence. Evaluating the response of bacterium is essential for assessment of future biological and ecological changes to global warming. Climatic changes have the potential to alter host-pathogen relationship and pathogenicity. Determination of the virulence genes of pathogens is essential for understanding pathogen responses to climate change.

The recently isolated virulent strains of Vibrio tubiashii have shown that metalloprotease (vtpA) and a transcriptional regular for metalloprotease (vtpR) is expressed at higher levels (Hasegawa et al. 2008, Hasegawa and Hase 2009). The metalloprotease gene is potentially one of the virulence factors in Vibrio tubiashii. The use of metalloprotease inhibitors results in reduced infection in larval oysters. Metalloprotease gene was also transformed into Vibrio cholerae, which produced a high mortality of larval oysters. The metalloprotease gene was also removed from the V. tubiashii, which reduced infection (Hasegawa et al. 2008).
VtpR gene was associated with vtpA as a transcriptional regulator (Hasegawa and Hase 2009). This study...

Based on this evidence, I will be examining differential gene expression of Vibrio tubiashii virulence (vtpA and vtpR). My objectives are to compare gene expression between V. tubiashii with and without a host to colonize, the Pacific oyster larvae (Crassotrea gigas). Furthermore I will investigating these under two environmental conditions, ambient CO2 (380ppm) and elevated CO2 (840ppm). These findings will provide information on the virulence genes of V. tubiashii in two environmental conditions.

The prospect of differential gene expression between V. tubiashii with and without a host present, suggests that quorum sensing may be related to virulence factors in vibriosis. Quorum signaling in marine bacterial species has been observed with elevated population densities (Miller and Bassler). An accumulation of virulent V. tubiashii in conjunction with cell-to-cell communication may present an opportunity


Proposed methods

Vibrio tubiahii
Need help with where Vt is from, which strain??
Inoculation of 30uL of Vt (concentration??)


Experimental design
With help from Emma and Moose the ocean acidification set-up allowed me to have two ocean acidified conditions. One treatment is held at 380 ppm (current CO2) and the other is held at about 900 ppm (projected). There are six containers for each CO2 level, two controls with inoculated sea water, two controls of oyster larvae with no V. tubiashii and two treatment containers with inoculated oyster larvae. The containers are a static system with a constant in flow of air from both elevated CO2 conditions. The water is changed daily, at which time oyster larvae are counted and water samples are taken for V. tubiashii analysis.

Water is collected in 50mL falcon tubes and spun down for 30 minutes. The water is then transferred to 1.5mL microcentrifuge tubes and spun down at a higher speed. V. tubiashii will theoretically remain at the bottom as a "pellet". The pellet is then saved in RNA later and stored at 4C.


RNA extractions
Samples were spun down to remove RNAlater and 1mL of Trizol was added. After mixing well and incubating, 200uL of chloroform was added and the samples were vortexed thoroughly. After incubate and spinning down the samples at 12,000 g for 15 minutes at 4C. The aqueous phase was transferred to a new tube, approximately 500uL of obtained. RNA was precipitated out by adding 500uL of isopropanol and mixing. After a 5 minute incubation and centrifugation for 8 minutes at 12,00 g, the supernatant was removed and the pellet was washed with 75% ethanol. The sample was mixed with ethanol and then spun down for 5 minutes at 7,500g. The ethanol was removed and the pellet was air dried and then resuspended in 20uL DEPC H2O.

Reverse transcription

qPCR:
For each reaction, used 2.5uL of Sybr seni mix, 0.5uL each of forward and reverse primers, rest with water for 13uL of each reaction. 2uL of cDNA was used for qPCR.

Thermal profile for qPCR:

  1. 7 minutes, 95C
  2. 10 seconds, 95C
  3. 30 seconds, 60C
  4. 30 seconds, 72C
  5. 1 minutes, 95C
  6. 30 seconds, 55C
  7. 30 seconds, 95C
  8. repeat for 40 cycles (from step 2)

Annealing temperatures differed (step 3)
Primers used:
VtpA: metalloprotease
VtpR: transcriptional regular for metalloprotease and hemolysin secretion
16S: for normalizing


qPCR plate layout?

Analysis:


Budget


 Cost per unit
RNAlater
 $1.00/mL
RNA extraction
 $1.25/sample
cDNA
 $3.00/sample
qPCR reagents
 $0.50/sample





Pitfalls:

Inoculation of water in a static system may not be the best approach for investigation of Vibrio tubiashii. The system does allow us to extract water samples with knowledge that V. tubiashii is present, but with exchanges of clean water, the V. tubiashii densities may not have been constant. After spinning down the water samples, there was no visible pellet, but care was taken to preserve the bacteria.
There is no way to quantify RNA at FHL, but there was amplification from qPCR. Based on these issues, the data may only suggest results. The water temperature and CO2 input also may not have been consistent, but alterations were small.
Another pitfall is that there are multiple virulence factors that come into play in the host-pathogen relationship, thus these results are only a small look at a very complex system. Environmental cues and elements have not been incorporated into the environment, such as the upwelling events (Elston et al. 2008) that potentially have a huge impact on how infectious V. tubiashii can be.
The pathogenesis of vibriosis particularly to larval bivalves, also involves multiple virulence factors (Nottage and Birkbeck 1987). This study is only a basic look at two virulence genes.


Sources:

Brown, Carolyn and Losee, Ellen. 1977. Natural and Induced Epizootics of Vibriosis in Crassostrea virginica Larvae. Jour. of Invertebrate Path. 31: 41-47.

Elston, Ralph A. 1993. Infectious Diseases of Pacific Oyster, Crassostrea gigas. Annual Rev. of Fish Diseases. 3:259-276.

Elston, Ralph A., Hasegawa, Hiroaki, Humphrey, Karen L., Polyak, Ildiko K., and Hase, Claudia C. 2008. Re-emergence of Vibrio tubiashii in bivalve shellfish aquaculture: severity, environmental drivers, geographic extent and management. Dis Aquat Org. 82: 119-134.

Hasegawa, Hiroaki and Hase, Claudia C. 2009. TetR-Type Transcriptional Regulator VtpR Function as a Global Regulator in Vibrio tubiashii. Appl. Environ. Microbiol. 75 (24):7602-7609.

Hasegawa, Hiroaki, Lind, Erin J., Boin, Markus A., and Hase, Claudia C. 2008. The Extracellular Metalloprotease of Vibrio tubiashii Is a major Virulence Factor of Pacific Oyster (Crassostrea gigas) Larvae. Appl. Environ. Microbiol. 74 (13): 4101-4110.

Hasegawa, Hiroaki, Gharaibeh, Dima N., Lind, Erin J., Hase, Claudia C. 2009. Virulence of metalloproteases produced by Vibrio species on Pacific oyster Crassostrea gigas larvae. Dis Aquat Org. 85: 123-131.