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A GLOBAL TRANSCRIPTIONAL ANALYSIS OF STREPTOCOCCUS PNEUMONIAE IN RESPONSE
TO LOW-SHEAR MODELED MICROGRAVITY
Christopher Allen1, Cristi Galindo1, Nina Williams1, Utpal Pandya1, Ashok Chopra1, and David Niesel1
1
University of Texas Medical Branch, Galveston, TX 77555 USA
Long-term habitation in space exposes humans to a
variety of environmental stresses not encountered on earth
such as ionizing radiation and microgravity. Decreases in
immune function and increases in microbial growth and
antibiotic resistance during space flight could create an
increased health risk to crewmembers (Sonnenfeld et al.,
1990; Mishra et al., 1992). Streptococcus pneumoniae is
an upper respiratory pathogen which causes pneumonia,
meningitis, and bacteremia. S. pneumoniae has
previously been isolated from crewmembers and could
present a potential health hazard during extended
spaceflight (Cioletti et al., 1991). To facilitate groundbased microgravity analogue studies, NASA developed
high aspect ratio vessels (HARVs) which model lowshear microgravity conditions (LSMMG). The LSMMG
environment is generated through the continual rotation of
cell cultures in fluid-filled vessels which minimize
prolonged exposure to gravity through the randomization
of gravity vectors. Recently, these ground-based models
have been used to study the effects of LSMMG on
bacteria to understand the physiological and molecular
changes they undergo in the presence of microgravity as
encountered during spaceflight.
This study utilized S. pneumoniae as a Gram-positive
bacterial model to investigate how LSMMG affects
environmental stress resistance and global transcriptional
activity in opportunistic bacterial pathogens. DNA
microarray analysis was carried out on S. pneumoniae
strain TIGR4 after cultivation in HARVs under LSMMG
and compared to 1 x g and static conditions (Figure 1).
Significant decreases (p ≤ 0.05) in both thermal and acid
stress resistance were found in LSMMG-grown cultures
after exposure for 30 minutes using a Student’s t test.
Similar results were found after exposure when stress
conditions were increased up to 1 hour (data not shown).
These results were in contrast to previous reports which
have shown increases in environmental stress resistance
by Salmonella enterica serovar Typhimurium and
Escherichia coli (aWilson et al., 2002; Lynch et al., 2004)
in response to LSMMG. These results may be indicative
of the different responses by Gram-positive and Gramnegative bacteria to LSMMG. Alternatively, the results
may reflect the different phenotypes exhibited by enteric
and respiratory pathogens which colonize and persist in
distinct niches within the host.
Figure 1. HARV Vessel Orientations for Growth.
Figure 2. Thermal and Acid Stress Resistance in Response to
LSMMG.
Microarrays were acquired from The Institute for
Genomic Research (TIGR) Pathogen Functional
Genomics Resource Center (PFGRC).
They were
designed based on the genomic sequence of the TIGR4
strain and contain amplicons representing 2131 openreading frames.
Stress analysis was carried out on TIGR4 cultures
grown to mid-logarithmic phase under LSMMG and
controls (1 x g rotating) followed by exposure to thermal
(55°C) and acid (pH 3.5) stress conditions (Figure 2).
Thermal Stress Resistance
Acid Stress Resistance
In order to understand what genes may be involved in
bacterial LSMMG-mediated responses at the
transcriptional level, global transcriptional analysis was
carried out using mid-logarithmic phase cultures grown
under LSMMG and control cultures (1 x g rotating, 1 x g
static). A stringent analysis of the array data was carried
out using multiple analysis methods (Genepix Pro 6.0,
Spotfire 7.3, SAM, and ANOVA) and a threshold of ≥
1.5-fold was used to identify differentially expressed
genes. Results were confirmed by quantitative real time
Gravitational and Space Biology 19(2) August 2006
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C. Allen et al. – A Global Transcriptional Analysis of Streptococcus pneumoniae in Response to Low-Shear
Modeled Microgravity
RT-PCR of six representative genes differentially
expressed under LSMMG. Genes were chosen which
were differentially expressed ≥ 1.5-fold in response to
LSMMG, represented different functional categories, and
located within different regions within the genome.
Pneumococcal genes (81 total) which were found to be
responsive to LSMMG represented different functional
groups located throughout the genome (Table 1). The
statistical analysis program, Significance Analysis of
Microarrays (SAM), confirmed the statistical significance
of these differentially expressed genes based on three
independent experiments.
external forces such as shear and rotation exert individual
effects on cultured cells independent of LSMMG.
Overall, these results indicate that LSMMG represents
a unique environment that can alter a bacterium’s
transcriptional profile and physiological state.
Investigating how this unique environment can lead to
new properties will allow us to better understand the
impact of LSMMG on microbial virulence and the health
risks these changes may create during long-term space
habitation.
Functional Category
Sonnenfeld, G., Mandel A.D., Konstantinova I.V., Taylor
G.R., Berry W.D., Wellhausen S.R., Lesnyak A.T., Fuchs
B.B. 1990. Effects of Space Flight on Levels and Activity
of Immune Cells. Aviat. Space Env. Med. 61: 648.
LSMMG-Responsive Genes
Antibiotic Resistance
Cell Envelope
DNA Repair/Recombination
Metabolism
Signaling
Stress Response
Transcriptional Regulation
Transporters/Ion Channels
Hypothetical/Unknown
1
11
4
13
3
1
4
8
36
Table 1. Functional Gene Groups Significantly Altered by
LSMMG.
REFERENCES
Mishra, S., Pierson, D. 1992. Space Flight, Effects on
Microorgansims. In: Encyclopedia of Microbiology.Vol.
4. Academic Press, Inc. pp. 53-60.
Cioletti, L., Pierson, D., Mishra, S. 1991. Microbial
Growth and Physiology in Space: A Review. SAE
Technical Paper Series No.911512.
a
Interestingly, LSMMG-responsive genes were found to be
predominantly down regulated in response to LSMMG.
Previous studies have shown that global transcriptional
profiles of Gram-negative enteric bacteria contain both
up- and down-regulated genes after growth under
LSMMG (bWilson et al., 2002). This consistant pattern of
down-regulation seen in S. pneumoniae represents a
distinct response by this Gram-positive respiratory
pathogen compared with the more extensively
characterized Gram-negative enteric pathogens.
Hierarchical clustering analysis (Cluster/Treeview,
CLUSFAVOR 6.0, and Spotfire 7.3) revealed several
gene clusters displaying similar expression patterns.
These clusters consisted of different genes from diverse
functional groups. Currently, how these genes function in
response to LSMMG remains unknown. Likewise, a
common mechanism to explain these regulatory changes
in response to LSMMG remains uncharacterized. Further
studies will need to be carried out to identify LSMMGresponsive regulators.
Controls for the LSMMG experiments included both a
1 x g rotating control and a 1 x g static control (Figure 1)
as a secondary filter to identify LSMMG-responsive
genes. SAM analysis of genes differentially expressed
between static and rotating controls revealed 147
differentially expressed genes. Among these genes, 46
were also altered between 1 x g and LSMMG conditions
while 9 were altered between static and LSMMG
conditions. The results from this analysis clearly indicate
that the choice of control has a significant impact on the
assignment of LSMMG-responsive genes and possibly
physiological properties. Further, the presence of other
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Gravitational and Space Biology 19(2) August 2006
Wilson, J., Ott, C.M., Ramamurthy, R., Porwollik, S.,
McClelland, M., Pierson, D.L., Nickerson, C.A. 2002.
Low-Shear Modeled Microgravity Alters the Salmonella
enterica Serovar Typhimurium Stress Response in an
RpoS-Independent Manner. Appl Environ Microbiol
68(11): 5408-5416.
Lynch, S.V., Brodie, E.L., Matin, A. 2004. Role and
Regulation of sigma S in General Resistance Conferred
by Low-Shear Simulated Microgravity in Escherichia
coli. J Bacteriol 186(24): 8207-8212.
b
Wilson, J.W., Ramamurthy, R., Porwollik, S.,
McClelland, M., Hammond, T., Allen, P., Ott, C.M.,
Pierson, D.L., Nickerson, C.A. 2002. Microarray Analysis
Identifies Salmonella Genes Belonging to the Low-Shear
Modeled Microgravity Regulon. Proc Natl Acad Sci USA
99(21): 13807-13812.