Space flight alters bacterial gene expression and
virulence and reveals a role for global regulator Hfq
Proc Natl Acad Sci U S A. 2007 Oct 9;104(41):16299-304.
“Space sick: What bugs us on Earth gets worse in
orbit”
“Germs sent to space come back meaner,
scientist reports”
“A New Reason to Fear Space Germs”
“Bacteria Grown in Space Become More Deadly”
“Lethal Bacteria Turn Deadlier After
Space Travel”
Why Study Bacteria In Space?
•Manned Missions
•Spacecraft
•Decontamination
•Insight to physiology on
Earth
Bacteria Make Spacecraft “Sick”
•Bacterial Contamination
•Window Gaskets
•Cable Insulations
Mir Space Station
•Wire insulations
•Space suits
•Possible Complication
•Corrosion of insulation
•Electrical failure
Fungal contamination in Mir
•Structural failure
Bacteria Make Astronauts Sick
Astronauts have impaired immune system in
space
PKA Pathway Disrupted
91 genes non-induced
8 genes inactivated
Reduces activation of T-Cells
Level of compromised T-Cell function only
known in 2 situations
1. Weightlessness
2. HIV Infection
Antibiotics in Space
Not-well tested
Limitations on drug studies
•Expensive $$$
•Experimental parameters differ between missions
•Immune Response factors hard to replicate
•Stress, radiation exposure, reduced gravity
•Drug stability in space
Shuttle does carry aide kits
Health maintenance
Emergency situations
Space flight alters bacterial gene
expression and virulence and reveals
a role for global regulator Hfq
•This Paper focuses on experiments
from Shuttle Mission STS-115
•Based of previous studies by
Nickerson and colleagues at ASU and
Tulane
•Salmonella subjected to LSMMG is
more virulent
•Acid/Thermal Stress
•Survival in Macrophages
Evidence for Microgravity
Affecting Salmonella Virulence
Microgravity as a Novel Environmental Signal Affecting
Salmonella enterica Typhimurium Virulence
Infection and Immunity, June 2000, p. 3147-3152
Microarray analysis identifies Salmonella genes belonging to the
low-shear modeled microgravity regulon
PNAS. October 15, 2002, vol 99, no. 21, p. 13807-13812
Salmonella enterica Serovar
Typhimurium
• Gram negative rod bacterium
• Causes gastroenteritis in humans
• Causes systemic disease in mice
– Similar to Typhoid fever in humans
X
Design of HARV
High-aspect-ratio rotating well vessel bioreactor
Bacterial cell
suspension
Growth Conditions
• Cells grown overnight in Lennox broth at 37ºC
• Diluted 1:200 into 50 ml LB and added to
HARV ensuring entire vessel was full
• Cells were grown to mid-log (~10 h)
– 1 x g conditions
– LSMMG conditions
Virulence in BALB/c Mice
Gravity
LSMMG
Condition
LD50
1xg
2.2 x 107
LSMMG
4.3 x 106
5 mice per dose
Trial carried out over 10 days
Oral inoculation 1.9 x 106 CFU
Acid Stress Survival
Gravity
LSMMG
Cells Subjected to Citrate buffer pH 3.5
Dilutions performed at time intervals to determine CFU/ml
Survival in Macrophages
1xg
• Salmonella added to well
plates with monolayer of
J774 macrophage cells
LSMMG
• Cells were incubated with
macrophages for 1h
• Cells washed with PBS &
incubated with MEM
media with gentamicin
• Macrophages were lysed
and viable cells were
counted
LSMMG Cells are prepared for initial stresses within macrophages
LSMMG Increases Virulence
• Cells grown under LSMMG have increased
virulence
– Increased resistance to acid stress
– Increased survivability on macrophages
– Increased virulence in mice
• What genes are causing this change?
Microarray analysis revels genes
regulated by LSMMG
• 163 genes found to be regulated by LSMMG
–
–
–
–
–
Transcriptional regulators
Virulence factors
Iron-utilization enzymes
LPS biosynthetic enzymes
Unknown proteins
• Looked and RpoS
– Primary σ-factor for stress response in Salmonella
– Essential for virulence in mice
RpoS NOT Involved in LSMMG
Stress Response
• An RpoS mutant shows the same response to
stresses as WT in LSMMG conditions
–
–
–
–
–
Resistance to acid stress
Resistance to high salt
Resistance to high temperatures
Increased survival in macrophages
Increased virulence in mice
• If RpoS involved in LSMMG response would
expect to see a decrease in stress resistance and
virulence
MISSION STS-115
Shuttle Mission STS-115
Have shown Salmonella to have increased
virulence in mimicked LSMMG on Earth
Is this the same in real space conditions?
Designed an experiment flown on Atlantis
Mission STS-115 on September 9, 2006
STS-115
• Primary goal was to build onto
the international space station
• Added a solar panel truss which
doubled the power generating
capacity of the station
• Required 3 space walks over 4
days
“MICROBE”
• Tested microgravity environments on three
different organisms
– Salmonella typhimurium
– Pseudomonas aeruginosa
– Candida albicans
• Experiments carried out in a fluid processing
apparatus
• Operated by crew member using a hand crank
• Ground experiments carried out simultaneously
Fluid Processing Apparatus
FPA At Work
Fixative
or LB
Bacteria
LB
Media
1st Crank
2nd Crank
Bacteria
dispensed
into LB
Fixative/LB
dispensed
into
LB/Bacteria
FPA At Work
Fixative
or LB
Bacteria
LB
Media
1st Crank
2nd Crank
Bacteria
dispensed
into LB
Fixative/LB
dispensed
into
LB/Bacteria
MICROBE
• On day 9 crew cranked FPA
releasing Salmonella into LB
medium
• 24h post inoculation crew
cranked FPA releasing Fixative
or fresh Broth
• When the shuttle landed fixed
samples were frozen and fresh
cultures were subjected to animal
testing
Increased Virulence in Space Flight
Microarray Reveals hfq
Involvement in LSMMG
• hfq expression decreased in flight
• Expression of 64 genes altered in flight that
belong to hfq regulon
• Interesting Down Regulated Genes
–
–
–
–
dps (Ferritin and stress response protein)
fnr (Transcriptional regulator)
Bfr (Bacerioferritin, iron storage)
ALL found to be required for virulence
• What is Hfq?
Hfq
• First identified as requirement for Q_
RNA phage replication
• Crystal structure shows homohexamer
• Regulates translation by:
– masking Shine-Dalgarno (ex: hfq)
– matchmaking of small untranslated
RNAs to target transcripts (ex:
ryhB – sodB)
– mRNA stability (ex: RNase E, and
other mRNA decay enzymes)
• Sm-like (Lsm) protein
– Spliceosome in eukaryotes
Sauter, C. et al. Nucleic Acids Res 31:4091-4098
Hfq and Eukaryotic Similarities
• Sm proteins involved in splicing of mRNA
– Sm autoantibodies discovered in lupus patients
• More than 16 Lsm proteins discovered in
eukaryotes
– processing mRNAs, snoRNAs, tRNAs, rRNAs,
snRNPs of histone mRNA
– present in bacteria and archaea
Comparison of Hfq and Lsm Cellular
Interactions
Wilusz, CJ and Wilusz J. 2005. Nat Struct Mol Bio 12:1031-1036
Organization of Hfq Gene Region
E. coli K12
S. typhimurium LT2
P2Hfq
P3Hfq
P1Hfq
PHfq
• Expression of Hfq dependent on growth
- evidence from pulse chase experiments
Kajatani M. et al. 1994. J Bacteriol176:531-534
_
Hfq
__’
Involvement of Hfq in LSMMG
Stress Response
• Created three mutant strains
– Deletion of hfq with CMr cassette
– Fusion of CMr cassette downstream of hfq
– Deletion of invA (Unrelated to stress resistance)
• Tested effects of LSMGG on
– Acid resistance
– Survival in macrophages
Acid Resistance
Salmonella cells incubated with citrate buffer (pH3.5) 60min
Calculated % survival
Graph represents fold difference in survival between gravity and
microgravity
Survival in Macrophages
• Cells incubated with J774 macrophages for 2h and 24h
• # of viable bacteria counted by CFU/ml
• Graph represents fold difference between time points
Conclusions
• First documented results showing altered gene expression
in space flight
• Microgravity is a environmental signal that causes changes
in cell behavior
• Hfq is a global regulator of responses under LSMMG
conditions
Questions
• What is the mechanism of Hfq regulation?
• How long does this increased virulence last?