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SPRINGER BRIEFS IN MICROBIOLOGY
EXTREMOPHILIC BACTERIA
Prasanti Babu
Anuj K. Chandel
Om V. Singh
Extremophiles
and Their
Applications
in Medical
Processes
SpringerBriefs in Microbiology
Extremophilic Bacteria
Series editors
Sonia M. Tiquia-Arashiro, Dearborn, USA
Melanie Mormile, Rolla, USA
Prasanti Babu •Anuj K. Chandel
Om V. Singh
Extremophiles and Their
Applications in Medical
Processes
123
Prasanti Babu
Om V. Singh
Division of Biological and Health Sciences
University of Pittsburgh
Bradford, PA
USA
Anuj K. Chandel
Department of Chemical Engineering
University of Arkansas
Fayetteville, AR
USA
ISSN 2191-5385 ISSN 2191-5393 (electronic)
ISBN 978-3-319-12807-8 ISBN 978-3-319-12808-5 (eBook)
DOI 10.1007/978-3-319-12808-5
Library of Congress Control Number: 2014953259
Springer Cham Heidelberg New York Dordrecht London
©The Author(s) 2015
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Printed on acid-free paper
Springer is part of Springer Science+Business Media (www.springer.com)
OVS gratefully dedicates this book
to Daisaku Ikeda, Uday V. Singh, Indu
Bala, and Late Prof. Ben M.J. Pereira in
appreciation for their encouragement
Contents
1 Introduction ........................................ 1
2 Survival Mechanisms of Extremophiles .................... 9
2.1 Survival and Potential Therapeutic Strategies . . . . . . . . . . . . . 14
2.1.1 Ectoine-Mediated Mechanism . . . . . . . . . . . . . . . . . . 15
2.1.2 Evolutionary Diversity. . . . . . . . . . . . . . . . . . . . . . . 16
2.1.3 Increased Catalytic Activity . . . . . . . . . . . . . . . . . . . 17
2.1.4 Amino Acid Accumulation. . . . . . . . . . . . . . . . . . . . 18
2.1.5 Aggregation Resistance Strategies . . . . . . . . . . . . . . . 18
2.1.6 Activation of the Nuclear Factor . . . . . . . . . . . . . . . . 19
2.1.7 Resistance to Cell Death . . . . . . . . . . . . . . . . . . . . . 19
2.1.8 Cellular Compartmentalization . . . . . . . . . . . . . . . . . 20
2.1.9 Overexpression of Heat Shock Protein Genes . . . . . . . 22
3 Therapeutic Implications of Extremophiles ................. 25
3.1 Radiation-Resistant Organisms . . . . . . . . . . . . . . . . . . . . . . . 29
3.2 Thermophiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
3.3 Halophiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
3.4 Acidophiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3.5 Mesophiles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.6 Psychrophiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.7 Geophiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3.8 Barophiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
vii
4 Challenges in Advancing Extremophiles for Therapeutic
Applications ........................................ 37
4.1 Isolation and Purification of Extremolytes . . . . . . . . . . . . . . . 38
4.2 Systems Biology of Extremophiles . . . . . . . . . . . . . . . . . . . . 39
4.3 Extremophiles Like Other Organisms . . . . . . . . . . . . . . . . . . 40
5 Conclusion ......................................... 43
References............................................. 45
viii Contents
Abstract
Extremophiles are a large group of organisms with the ability to thrive under
extreme environmental conditions such as high and low temperatures, high salt
levels, radiation, and high antibiotic concentrations. They have been the center of
attention due to the remarkable benefits they may have for humanity. The ex-
tremophiles’survival mechanisms are being investigated in order to meet chal-
lenges associated with human health; understanding these mechanisms could result
in solutions to various medical problems humans face. This article discusses their
survival mechanisms and possible implications in medical processes, as well as the
major issues and challenges involved in advancing the commercial exploitation of
extremophiles.
Keywords Extremophiles Microorganisms Genes Proteins Metabolic
processes Medical applications Challenges
ix
Chapter 1
Introduction
Abstract Microorganisms that are able to thrive in extreme environment are
known as extremophiles. The metabolites and products that are secreted through
these extremophiles are of immense importance. The environments that these ex-
tremophiles thrive enable the study of the metabolites and carry them over with the
hope that they will help in medical and therapeutic applications. As more infor-
mation is processed about extremophiles, it is easier to identify them and classify
them for their metabolic properties. Extremozymes, the enzymes that are secreted
through extremophiles are used in the therapeutics of treating different medical
conditions. Studies have showed that these enzymes are important in the therapies,
yet a throughout understanding of these extremophiles will enable for more
research to be done to prove that these products and primary and secondary
metabolites will be of use in the medical field and help to treat diseases.
Keywords Extremophiles Metabolic processes Extremozymes Therapeutics
Survival strategies
Extremophiles are a group of organisms that survive in extreme and harsh envi-
ronments. Their identification, classification, and potential for commercial appli-
cations in medical and therapeutic fields are receiving increased interest and
attention. Primary and secondary metabolic products of commercial significance,
i.e., extremolytes and extremozymes, are among the substances that are currently
being studied for their potential benefits as medical solutions to several human
diseases (Copeland et al. 2013; Kumar and Singh 2013; Singh 2013; Chakravorty
and Patra 2013). Thriving in harsh environments make extremophiles good can-
didates for the exploration of bioprocesses and biotechnological applications. An
important characteristic of extremophiles is maintaining the stability of their exis-
tence in order to thrive in their environments. Identifying the essential genes
responsible for the activity of the organisms, the stability of their chemical prop-
erties, and their adaptability under harsh environmental conditions may have
medical and therapeutic applications (Margesin and Schinner 1994; Feller 2003;
Furusho et al. 2005; Ma et al. 2010).
©The Author(s) 2015
P. Babu et al., Extremophiles and Their Applications in Medical Processes,
Extremophilic Bacteria, DOI 10.1007/978-3-319-12808-5_1
1
Exploration of extremophiles and their biodiversity in order to make them useful
for developing processes and products to serve humanity begins with the catego-
rization of these organisms (MacElroy 1974; Rothschild 2007). Studies have not
even scratched the surface in identifying extremophiles from natural habitats: less
than 1 % of the organisms have been identified and even fewer have been
sequenced for their beneficial properties. Table 1.1 summarizes major known ex-
tremophiles and their living conditions. The identification of extremophiles thus far
has already provided opportunities for industrial and medical use, as reviewed by
several researchers (Adams 1993; Adams and Kelly 1998; Niehaus et al. 1999;
Blumer-Schuette et al. 2012; Gabani and Singh 2013).
Within the past two decades, a number of studies have explored beneficial roles
of extremophilic bacteria and their usage in medicinal practices (Corry et al. 2014;
Gabani and Singh 2013; Ksouri et al. 2012). The exploration of extremophilic
organisms in various ecological and biodiversity settings has increased through
identification of the various environmental conditions under which the microor-
ganisms live and thrive (Ksouri et al. 2012; Cavicchioli et al. 2011). The genome
sequences of these extremophilic microorganisms have helped provide a better
understanding of their potential roles in industrial and medical applications (Lin and
Xu 2013; Majhi et al. 2013; Jaubert et al. 2013; Wemheuer et al. 2013; Shin et al.
2014). Considerable progress has been made in genome sequencing methods and
processes for identifying by-products from extremophiles (Ksouri et al. 2012; Liu
et al. 2013, 2014). Applications of extremophiles in processes, such as biocatalysis
and biotransformation, show great promise for benefits to human welfare (Hough
and Danson 1999; Singh 2013).
The application of beneficial characteristics of extremophiles for industrial and
medical purposes is becoming more possible now that the processes by which they
contribute such benefits are better understood than ever before. Table 1.2 introduces
a few major therapeutic roles for extremophiles. Copeland et al. (2013) provide a
schematic illustration to demonstrate one of these contributions. Figure 1.1 sum-
marizes the conversion of starch and cellulose into glucose and fructose that can be
fermented into ethanol with the use of extremozymes found in thermophiles.
Understanding how extremozymes can be used and applied in medical and thera-
peutic processes requires a fuller understanding of the systems biology (i.e.,
genomics, proteomics, and metabolomics) of these microorganisms. The scientific
community has recognized “Extremophiles”as an economically and ecologically
important group of microorganisms (Baker-Austin and Dopson 2007; Liu et al.
2014).
Despite recent progress, practical applications for extremophiles are still in their
infancy in many areas, including therapeutic and medical applications. A large
number of potentially beneficial extremophiles have yet to be investigated, and
development of large-scale cultivation processes for extremophile organisms is still
in its early stages. In addition, further efforts are needed to improve our under-
standing of the stability of the substances derived from extremophiles (Fornbacke
and Clarsund 2013). The progress made in scientific methods such as random
mutagenesis, protein engineering, direct evolution, and DNA shuffling has shown
2 1 Introduction
Table 1.1 Types of extremophiles, major species, and their living extremities in the environment
Types of
extremophile
Selective species Living extremities References
Radiation-
resistant
Deinococcus depolym-
erans;D. guangrien-
sis;D. radiodurans;D.
wulumuqiensis;D.
xibeiensis;D. gobien-
sis;D. gradis;D. mi-
sasensis;Cellulosi-
microbium cellulans
(UVP1); Bacillus
pumilus (UVP4); B.
stratosphericus
(UVR3); Enterobacter
sp. (UVP3); Roultella
planticola (UVR1);
Aeromonas eucreno-
phila (UVR4); Arthro-
bacter mysorens
(UVR5a); Micrococ-
cus yunnanensis
(UV20HR); Steno-
trophomonas sp.
(YLP1); Brevundi-
monas olei (BR2)
Resistance to survive
under ionizing radia-
tion; UVR
resistance >600 J m
−2
Gabani et al. (2012),
Copeland et al. (2013),
Gabani et al. (2014),
Asker et al. (2011),
Sun et al. (2009),
Wang et al. (2009),
Yun et al. (2009a, b),
Yun and Lee (2009)
Thermophiles Geobacillus thermo-
denitrificans;Thermus
aquaticus (YT-1)
High temperature
above 45 °C and up to
121 °C
Arena et al. (2009),
Lin et al. (2011)
Halophiles Halobacterium spp;
Haloferax spp; Hal-
oarcula spp; Halo-
monas stenophilia B-
100
High salt concentra-
tion such as salter
pond brines and natu-
ral salt lakes
Ma et al. (2010),
Buommino et al.
(2005), Oren (2002),
Llamas et al. (2011)
Acidophiles Ferroplasma
acidarmanus
Extreme pH levels;
pH level 0
Edwards et al. (2000),
Baker-Ausin and
Dopson (2007)
Alkaliphiles Alkaliphilus
transvaalensis
pH level 12.5
Takai et al. (2001),
Kobayashi et al.
(2007)
Mesophiles Phyla included: prote-
obacteria, firmicutes,
and actinobacteria
+11 to +45 °C Zheng and Wu (2010),
Widdel and Bak
(1992)
Habitats include
yogurt, cheese, and
moderate
environments
Psychrophiles Himalayan midge −18 °CD’Amico et al. (2006),
De Maayer et al.
(2014), Irgens et al.
(1996)
Polaromonas
vacuolata
<15 °C
(continued)
1 Introduction 3
Table 1.1 (continued)
Types of
extremophile
Selective species Living extremities References
Geophiles Geobacillus thermo-
glucosidasius;G. ste-
arothermophilus;G.
thermodenitrificans;
G.
thermopakistaniensis
Habitats include rich
soils as well as kera-
tin-enriched
environments
Espina et al. (2014),
Lin et al. (2014),
Siddiqui et al. (2014),
Pennacchia et al.
(2014), Yao et al.
(2013)
Barophiles Moritella spp;
alphaproteobacterium
High hydrostatic
pressure; 80 Mpa
Kato et al. (1998),
Emiley et al. (2011)
Table 1.2 Influensive roles of extremozymes/extremolytes in therapeutics from major
extremophiles
Type of
extremophile
Influensive role of extremolytes/
extremozymes in therapeutics
References
Radiation-resistant Extremolytes, ecotines, and other
natural compounds used to cope
with the stress of different
environments
Rastogi et al. (2010), Singh and
Gabani (2011, 2013), Copeland
et al. (2013)
Decreases the risks of skin dam-
age and skin- related cancers
Thermophiles “Thermozymes”showed
increased resistance to denaturing
chemical agents; “ability to crys-
talize”; stowed away the aggre-
gate prone regions (ARPs)
Sælensminde et al. (2008), Cava
et al. (2009), Irwin and Baird
(2004), Thangakani and Kumar
(2012)
Prevent misfolding of proteins of
Alzheimer’s and Parkinson’s
diseases; certain genes found in
thermophiles are used for the heat
precipitation from the host cell is
found to isolate and purify the
expressed protein
Halophiles Ex:
Halomonas steno-
phila (B-100 and
N-12)
Metabolism of carbohydrates;
oxygenic and an oxygenic
phototrophs, aerobic hetero-
trophs, fermenters, de-nitrifiers,
sulfate reducers, and
methanogens
Tomlinson et al. (1978), Oren
(2002), Llamas et al. (2011),
Ruiz-Ruiz et al. (2011), Molina
et al. (2013)
Prevent cancer, chronic inflam-
mation, cardiovascular disorder,
aging process; promote antican-
cer activity through synthetic
lipids
Blocked the growth of human T-
lymphocyte tumors
Exopolysaccharides (EPSs) as
antitumoral agents
(continued)
4 1 Introduction
Table 1.2 (continued)
Type of
extremophile
Influensive role of extremolytes/
extremozymes in therapeutics
References
Acidophiles Amylases, proteases, ligases,
cellulases, xylanases, a-glucosi-
dases, endoglucanases, and
esterases; plasmids, rusticynin,
and maltose-binding protein
Sharma et al. (2012)
Used in evolutionary medicine;
preventive measures for ulcer
disease and gastric cancer;
enzymes are used toward poly-
mer degradation
Mesophiles Cellular components, including
their membranes, energy gener-
ating systems, protein synthesis
machinery, biodegradative
enzymes, and the components
responsible for nutrient uptake;
biochemical substrate storage
capability
Metpally and Reddy (2009), Insel
et al. (2007)
Used throughout in proteases, for
developments and advancements
in extremozymes/extremolytes
Psychrophiles Energy transduction; regulation
of intracellular environment and
metabolism; functioning of
enzymes; and protein conforma-
tion; synthesis of the gene prod-
ucts are not prevented by cold
shock in cold-adapted microor-
ganisms; secrete special “anti-
freeze”, also known as
cryoprotecant molecules that aid
in decreasing the water point
within a cell
Margesin and Schinner (1993),
Irwin and Baird (2004),
Zecchinon et al. (2001)
Membrane fluidity; aid in main-
taining viral vaccinations stabil-
ity; also provide less tension for a
cell to maintain a certain tem-
perature and atmosphere in order
to survive; help cells to survive in
harsh winters
Geophiles Enriched in proteins including
keratin proteins, fungal growth
diminishing with the aid of
proteins
Weitzman and Summerbell
(1995), Achterman and White
(2011), Lakshmipathy and
Kannabiran (2010), Arena et al.
(2009), Barbara et al. (2013)
Prevent stomach ulcers, as well
as gastric cancers that can
develop with bacterial infections
(continued)
1 Introduction 5
Table 1.2 (continued)
Type of
extremophile
Influensive role of extremolytes/
extremozymes in therapeutics
References
from parasitic worms; prevent
chronic inflammation; adjuvant
agents in equilibrating the
immune response in viral
diseases
Barophiles Mechanotransduction; proteases;
gene expression is regulated by
the functions of barophiles; gene
encoding RNA polymerase
activity; gene ompH is found
regulated in barophilic organism
Tan et al. (2006), Irwin and Baird
(2004), Nakasone et al. (1996),
Bartlett et al. (1993)
Used in coping mechanisms for
cells in order to avoid detrimental
changes to the environmental
pressure changes of the cell;
ensure cells are viable in all
situations
Fig. 1.1 Extremozymes and their production of sugars and ethanol from sources found in
thermophiles (adopted and modified from Copeland et al. 2013)
6 1 Introduction
promise for improving the stability and modifying the specificity of chemical
substances derived from extremophiles (Danson and Hough 1998; Sellek and
Chouduri 1999).
Given the breadth of this field, the current article aims to take a critical look at
the survival strategies of the major extremophiles in order to investigate potential
therapeutic targets for multiple disease types. There is a need to understand the
molecular events in these organisms and their potential benefits in therapeutic
applications. This article also brings together major issues and challenges in
research areas related to extremophiles.
1 Introduction 7
Chapter 2
Survival Mechanisms of Extremophiles
Abstract It is vital for extremophiles to cope with their environments making them
viable to withstand under harsh environmental conditions. Extremophiles are
known to adapt to the changes in their environment and surroundings that enable
them to stabilize the changes in their homeostasis. The adaptability of extremo-
philes arrives from alteration of varying genes and proteins. Extremophiles produce
extremolytes, which helps them to maintain their homeostasis such as ectoine-
mediated mechanism, which is produced by halophiles and organisms alike. Evo-
lutionary diversity, increased catalytic activity, amino acid accumulation, aggre-
gation resistance strategies, resistance to cell death, activation of the nuclear factor,
the use of heat shock proteins, and cellular compartmentalization, are all vital tools
that extremophiles take on in order to conserve their genes.
Keywords Survival mechanisms Proteins Genes Evolution Diversity
Extremolytes Metabolites
The general mechanisms that are studied and exploited in all the therapeutic and
medical applications of extremophiles relate to how the extremophiles develop
defensive mechanisms to survive in harsh environments and how their metabolisms
are involved in these survival processes.
Extremophiles have been able to live in extreme and harsh conditions mainly
due to their adaptability (Mallik and Kundu 2014; van Wolferen et al. 2013; Singh
2013). The adaptation mechanisms of such extremophiles would help researchers to
understand their survival mechanisms, which in turn would help to figure out the
process by which their molecular elements (i.e., proteins and genes) could be
altered and used for therapeutic implications. Table 2.1 provides an overview of the
survival and defensive strategies of selected extremophiles.
Singh and Gabani (2011) reviewed and described one such survival pathway in
the radiation-resistant microorganism Deinococcus radiodurans. The microbial
resistance against ionizing radiation induces pathway-specific genes, modulated
proteins, and enzymes as part of the DNA repair mechanism. Figure 2.1 summa-
rizes the survival strategy of D. radiodurans. This mechanism operates in three
©The Author(s) 2015
P. Babu et al., Extremophiles and Their Applications in Medical Processes,
Extremophilic Bacteria, DOI 10.1007/978-3-319-12808-5_2
9
distinct steps, as shown in Fig. 2.2. Three different pathways of survival have been
identified through the process of homologous recombination, which is responsible
for gene induction. First, the UVR-induced gene uvrA reveals uvrABC system
protein A, representing a universal function in DNA repair and survival of
Table 2.1 Survival and defensive strategies in major extremophiles to thrive under extreme
environmental conditions
Extremophiles/
extremolytes
Survival and defensive strategies References
Thermophiles/car-
bohydrate extrem-
olytes/
hydroxyectoine
Stabilization of enzymes from
stress and freeze drying; protec-
tion of oxidative protein damage;
reduction of VLS in immunotoxin
therapy
Kumar et al. (2010)
Halophiles/ecotines Protection of skin immune cells
from UV radiation; enzyme sta-
bilization against heating, freez-
ing, and drying; protection of the
skin barrier against water loss and
drying out; block of UVA-
induced ceramide release in
human keratinocytes
Buommino et al. (2005), Singh
and Gabani (2011), Ortenberg
et al. (2000)
Acidophiles/
alkaliphiles
Maintaining a circumneutral
intracellular pH; constant pump-
ing of protons in and out of
cytoplasm; acidic polymers of the
cell membrane; passive regula-
tion of the cytoplasmic pools of
polyamines and low membrane
permeability
Baker-Austin and Dopson
(2007), Horikoshi (1999),
Bordenstein (2008)
Psychrophiles Translation of cold-evolved
enzymes; increased flexibility in
the portions of protein structure;
presence of cold shock proteins
and nucleic acid binding proteins;
reduction in the packing of acyl
chains in the cell membranes
Berger et al. (1996), Feller and
Gerdey (2003), D’Amico et al.
(2006), Chakravorty and Patra
(2013)
Geophiles/EPS-
V264; EPS-1,2,3
Mucoidal layer enveloping cell
colonies; biofilm formation as
stress response to extreme envi-
ronmental conditions
Arena et al. (2009), Kambourova
et al. (2009), Barbara et al.
(2013)
Barophiles Homeoviscous adaptation, tight
packing of their lipid membranes;
and increased levels of unsatu-
rated fatty acids; polyunsaturated
fatty acids maintain the mem-
brane fluidity; robust DNA repair
systems; highly conserved pres-
sure regulated operons; presence
of heat shock proteins
Lauro and Bartlett (2007), Yano
et al. (1998), Rothschild and
Mancinelli (2001), Kato et al.
(1995 1996a, b), Kato and
Bartlett (1997), Marteinsson et al.
(1999)
10 2 Survival Mechanisms of Extremophiles
D. radiodurans (Fig. 2.2). This process of induction helps D. radiodurans to thrive
in high-radiation conditions. UV induction in RecQ has been revealed to control
DNA helicase, which further helps in managing the nature and the quantity of the
DNA damage that is needed for RecQ functions in D. radiodurans. RecE pathway-
dependent modulation in recO and recF reveals a functional repertoire of DNA
repair protein, DNA metabolism, and replication and SOS inducibility. The mod-
ulation in recR could resist interstrand cross-links and DNA recombination acting
with recF and recO (Singh and Gabani 2011). Thus, studying the defensive and
survival mechanisms of the extremophiles in terms of their genome structure and
the chemical properties of the compounds derived from them will help in the
discovery of novel therapeutic and medical applications.
Fig. 2.1 A survival strategy of radiation-resistant microorganism D. radiodurans shows microbial
resistance against ionizing radiation that induces pathway-specific genes, proteins, and enzymes of
pathways in DNA repair mechanism (adopted with permission from Singh and Gabani 2011)
2 Survival Mechanisms of Extremophiles 11
Similar defensive mechanisms have been studied and described for other types
of extremophiles. For example, in the case of acidophiles, which survive in highly
acidic conditions, Baker-Austin and Dopson (2007) reviewed various survival
pathways and mechanisms that enable these organisms to thrive at low pH.
Impermeability of the cell membrane to protons is one such mechanism. Figure 2.2
summarizes various pH homeostasis mechanisms that have been identified (Booth
1985; Matin 1990). In general, the mechanism by which acidophiles use pH
homeostasis has not been fully understood. However, efforts in sequencing the
genomes of several acidophiles have shed light on several interrelated processes,
including pH homeostatic mechanisms, impermeable cell membrane, cytoplasmic
buffering, active proton extrusion, and organic acid degradation (Osorio et al. 2008;
Cardenas et al. 2010; Liljeqvist et al. 2013; Guo et al. 2014).
Konings et al. (2002) describes the role of the cell membrane in the survival
of bacteria and archaea found under extreme environmental conditions. The
acidophiles have a rigid and impermeable cell membrane, which can restrict the
cytoplasmic influx of protons. This helps to regulate the proton motive forces of the
cell by determining the rate at which protons flow inward and pump outward
Fig. 2.2 pH homeostasis processes of acidophiles (adopted and modified from Baker-Austin and
Dopson 2007)
12 2 Survival Mechanisms of Extremophiles
(Konings et al. 2002). Shimada et al. (2002) provided concrete evidence of this
phenomenon in Thermoplasma acidophilum, whose cell membranes are made of
tetraether lipids. Other examples of acidophiles include Picrophilus oshimae (van de
Vossenberg et al. 1998a), Sulfolobus solfataricus (van de Vossenberg et al. 1998b),
Ferroplasma acidarmanus (Macalady and Banfield 2003), and Ferroplasma acid-
iphilum (Golyshina et al. 2000; Batrakov et al. 2002; Pivovarova et al. 2002). Tyson
et al. (2004) reported reconstruction of near-complete genomes of Leptospirillum
group II and Ferroplasma type II and suggested that a wide variety of genes could be
responsible for the impermeability of the cell membrane and preventing the inflow of
protons to the cells. The above studies indicated that the genomes of organisms in
microbial communities may reveal pathways for carbon fixation and nitrogen fixa-
tions, including energy generation, which will help us learn more about the survival
strategies of microorganisms in extreme environments.
Michels and Bakker (1985) reported that bacteria such as B. acidocaldarius and
T. acidophilum have exhibited the ability to actively pump protons out of their
cytoplasm to maintain pH homeostasis. Such proton removal systems have also been
reported in the Ferroplasma type II and Leptospirillum group II (L. ferriphilum)
sequenced genomes (Tyson et al. 2004). Another key mechanism that acidophile
cells use to maintain pH homeostasis is regulating the size and permeability of the
cell membrane. Reducing the pore size of the cell membrane channels has been
suggested as another mechanism to prevent protons from the acidic environment
from entering the cell, thus helping maintain pH homeostasis. Amaro et al. (1991)
characterized the outer membrane porin of the acidophile and revealed a large
external loop that could be responsible for controlling the size of the pores in the cell
as well as the ion selectivity. Guiliani and Jerez (2000) reported that at a pH level of
2.5, the external loop controlled the inflow of the protons across the outer membrane.
In the event of protons entering the cell membrane, acidophiles have a number of
intracellular mechanisms to reduce damage that might be caused by the entering
protons. The cells of acidophiles have a buffering mechanism to release the protons,
as summarized in Fig. 2.2. This is possible because of the presence of certain
cytoplasmic buffer molecules that contain basic amino acids such as lysine, histi-
dine, and arginine that help in the proton sequestering process. Studying the
cytoplasmic homeostasis of pH in the acidophilic bacterium Theobacillus aci-
dophilus, Zychlinsky and Matin (1983) proposed that the amino acid side chains
were primarily responsible for acidophile cytoplasmic buffering. Castenie-Cornet
et al. (1999) supported this by finding that decarboxylation of amino acids such as
arginine-induced cell buffering in Escherichia coli by consuming the protons and
transporting them outside the cell membrane.
Another mechanism acidophiles use to maintain homeostasis is uncoupling the
organic acids. This process is called cytoplasmic protonation, and is a result of the
dissociation of protons in the cytoplasm. Researchers studying this organic acid
degradation reported the authenticity of uncoupling reactions at low pH (Kishimoto
et al. 1990; Alexander et al. 1987; Ciaramell et al. 2005).
Heliobacter pylori are known for causing gastric ulcers, and are able to survive
in harsh acidic conditions. E. coli can survive in harsh acidic environments
2 Survival Mechanisms of Extremophiles 13
(pH 2–3) for shorter time spans, even though it prefers to be at neutral pH. The
mechanisms that these two different microorganisms use to withstand acid in the
stomach differ significantly. It is unclear how E. coli is able to survive the high acid
levels in the stomach; however, studies have suggested three systems that enable
microorganisms to resist high levels of acid for longer periods of time (Foster
2004). In the stationary phase, alternative sigma factor is what makes the cells
tolerant to the various levels of acid. Another part of this mechanism is the cAMP
receptor protein (CRP), which binds with the sigma factor to create a complex that
tolerates high levels of acidity (pH 1–2) in the stomach. Acidophiles also have
pumps that move protons in and out of the cell in order to neutralize the cyto-
plasmic membrane. This is required because when bacterial cells come into contact
with extreme acid stress, as is the case with acidophiles, there is an influx of protons
that decreases the internal pH of the cell (Foster 2004).
The defensive and survival mechanisms used by radiation-resistant and acido-
philic organisms, as well as the other specific mechanisms that enable extremo-
philes to adapt to various environments, make them excellent candidates for
exploring beneficial properties and therapeutic implications for multiple disease
types (Furusho et al. 2005; Buommino et al. 2005; Kumar and Singh 2013;
Copeland et al. 2013). However, the advantages the medical world can derive from
these extremophiles are only in the early stages of recognition and realization. Some
extremophiles may have the solutions, but the task at hand is to find what mech-
anisms can be effective in synthesizing potentially useful therapeutic products. To
advance our therapeutic uses of extremophiles toward treatments of specific dis-
eases in the future, it is necessary to have a better understanding of the physiology
of these extremophiles.
2.1 Survival and Potential Therapeutic Strategies
It has been recognized that the characteristics that help extremophiles to survive in
extreme environmental conditions could be effectively used in medical processes to
develop applications that have benefits to human health. Radiation-resistant ex-
tremophiles have been reviewed to reveal their implications for developing anti-
cancer drugs, antioxidants, and sunscreens (Singh and Gabani 2011; Gabani and
Singh 2013). Similarly, thermophilic bacteria have been known to help in DNA
processing, production of proteins and enzymes, and biotechnological processes
(Oost 1996). Acidophilic bacteria contribute to acid mine drainage and help to
neutralize the pH of certain cytoplasmic membranes by pumping protons into the
cellular space (Edwards et al. 2000).
To advance the role of extremophiles in the search for specific therapeutic
mechanisms and their implications, it is pertinent to ask what metabolic products
such as extremolytes and extremozymes are produced and how these primary and
secondary products can be effectively exploited for medical purposes. Here we
discuss some therapeutic mechanisms of the selected extremolytes.
14 2 Survival Mechanisms of Extremophiles
2.1.1 Ectoine-Mediated Mechanism
Aerobic, chemoheterotrophic, and halophilic organisms contain ectoine, which is
chemically identified as (5)-2-methyl-1, 4, 5, 6-tetrahydropyridine-4-carboxylic
acid. High levels of radiation can alter DNA structure and produce cancer unless the
structure is repaired by cellular machinery. Copeland et al. (2013) reviewed and
demonstrated usage of extremolytes in their setting, and proposed an ectoine-
mediated hypothetical survival mechanism (Fig. 2.3). The mechanism of ectoine
biosynthesis led to UV neutralization, revealing therapeutic implications of halo-
philes as summarized in Fig. 2.3. Halophilic extremophiles engage in a three-step
process to produce ectoine from aspartate semialdehyde (ASA) (Fig. 2.3).
Nakayama et al. (2000) reported that the gene cluster of EctA,EctB, and EctC
encodes the enzymes needed for the synthesis of ectoine (Fig. 2.3A). Ectoine was
produced through fermentation of Halobacter elongate in a continuous process and
microfiltration of the biomass, and the ectoine filtrates were purified through
electrodialysis, chromatography, and crystallization (Lentzen and Schwarz 2006).
Skin is protected from UVA irradiation when human keratinocyte cells are pre-
treated with ectoine (Bunger and Driller 2004). The ways in which ectoine may
Fig. 2.3 Extremolytes in halophile bacterium H. elongate and proposed hypothetical survival
mechanism. (ASA aspartate semialdehyde; DABA 1-2,4-diaminobutyrate; ADABA N-acetyl-1-2,4-
diaminobutyrate) (adopted and modifed from Copeland et al. 2013)
2.1 Survival and Potential Therapeutic Strategies 15
help prevent damage to cells are shown in Fig. 2.3B: the release of secondary
messengers (i.e., kinase), transcription factor AP-2 activation, intercellular adhesion
molecule-1 expression, and mitochondrial DNA mutation. Beyer et al. (2000)
demonstrated the immunoprotective effects of ectoine treatment through treating
Langerhans cells under UV stress with 1 % ectoine. All these studies provide ample
evidence of the protective properties of ectoine, which is hypothesized to provide
protection from DNA damage and hence from cancer (Fig. 2.3B).
The therapeutic implication of this defensive mechanism is that ectoine helps
stabilize the membrane structures, resulting in a higher level of resistance to UVA
damage. Further, ectoine-mediated neutralization has been found to reduce or
prevent dehydration of dry atopic skin and prevent skin aging (Singh and Gabani
2011). Similar roles of ectoine have been explored in research on apoptotic
cell deaths in the contexts of Machado–Joseph disease (Furusho et al. 2005) and
Alzheimer’s disease (Kanapathipillai et al. 2005).
2.1.2 Evolutionary Diversity
Despite the diversity in living world, microorganisms are yet to see the tip of the
iceberg. Most microorganisms existing in nature, particularly bacteria have yet to be
identified. There is very little known to the current microbiologists on how to grow
wide variety of microorganisms. In the sense of unknown growth medium for most
microbial life, metagenomics have been considered to extract the total nucleic acid
from environment with limited success to explore the hidden microbial life. The
challenges remain for microbiologists to isolate novel microbial species from a
variety of extreme environmental conditions.
Due to their biochemical properties, extremophiles are of high interest to both
basic and applied microbiologists. Thermophiles contain DNA binding proteins,
which have a potential role in maintaining DNA in a double-stranded form at high
temperatures (Pereira and Reeve 1998). In order to diversify microbial community
in the thermal environment, the heat-mediated alteration was reported to affect the
membrane stability by opposing hydrophobic residues from each layer of the “lipid
bilayer”membrane together forming the lipid monolayer instead of a bilayer that
prevents the cell membrane to melt at high temperature (van de Vossenberg et al.
1998a). This diversifies the microbial survival at specific niche. However, at low
temperature, proteins are being revealed to be more polar and less hydrophobic than
proteins in thermophiles. In addition, psychrophiles regulate chemical composition
of their membranes by maintaining the length and degree of unsaturation of fatty
acids. This regulation keeps the membrane structure in sufficiently fluid form
allowing transport process to occur, even below freezing temperatures (Horikoshi
and Grant 1998).
Extremophiles have been reported to carry a set of essential genes that are
evolutionarily conserved (Duplantis et al. 2010). These essential genes play an
important role in translating the useful products that enable their survival under
16 2 Survival Mechanisms of Extremophiles
harsh environmental conditions. The variations in extreme environmental charac-
teristics exert pressure on essential genes (ligASf,ligACp,ligAPh,hemCCp,pyr-
GCp,dnaKCp,murGCp,dnaKSf,fmtCp,ftsZCp,cmkCp, and tyrSCp) that help
extremophiles adapt to the environment. This phenomenon is referred to as “evo-
lutionary diversity”and the properties of the essential genes could be used to
engineer bacterial pathogens that are stable and temperature sensitive which further
could be used as vaccines. Figure 2.4 summarizes the involvement of essential
genes from psychrophilic bacteria transformed into temperature-sensitive meso-
philic host organisms. Studies have also substituted essential genes of bacteria
found in arctic environments for the genes in pathogenic organisms (Duplantis et al.
2010; Shanmugam and Parasuraman 2012).
2.1.3 Increased Catalytic Activity
Another extremophilic mechanism with the potential for therapeutic applications is
the metabolic fluxes among psychrophilic microorganisms (Georlette et al. 2003).
Psychrophilic organisms thrive in extreme cold habitats, produce enzymes that are
Fig. 2.4 Essential genes from a psychrophilic bacterium are transformed into temperature-
sensitive mesophilic host organism (adopted and modified from Shanmugam and Parasuraman
2012)
2.1 Survival and Potential Therapeutic Strategies 17
active in cold temperatures, and can cope with the low-temperature-induced
reduction in chemical reaction rates. The enzymes produced by psychrophilic
organisms will have high catalytic efficiency at low temperatures.
It has been suggested that at the active sites, cold-adapted DNA ligase has
specific characteristics such as high conformational flexibility, increased activity at
low and moderate temperatures and overall destabilization of the molecular edifice
(Georlette et al. 2003), revealing potential implications for biotechnology appli-
cations. These characteristics are reversed in mesophiles and thermophiles, which
show reduced activity at low temperatures, high stability, and reduced flexibility.
Because of the complexity involved in understanding these properties, large entropy
changes are involved in the denaturation process of these microorganisms. The
results of this study, conducted by adapting the different thermal habitats, indicated
functional links between activity, flexibility, and stability. Studies have also been
conducted on amylases and xylanases derived from extremophiles (Elleuche et al.
2011; Qin and Huang 2014; Liu et al. 2014). The therapeutic implications of
increased catalytic activity in psychrophilic organisms due to the tradeoff between
the low temperature and lower thermal energy resulting in specific changes in the
molecular structure need to be further exploited.
2.1.4 Amino Acid Accumulation
Some extremophilic adaptation mechanisms that produce substances useful to
humans are explained (Hendry 2006). While the enzymes produced by acidophiles
and alkaliphiles can be useful in extreme conditions, the organisms themselves can
also regulate their cytoplasmic activities at neutral pH conditions. Halophiles,
however, adapt by regulating the salt concentration in their cytoplasm; the cyto-
plasmic proteins of the halophiles adapt to the environment by accumulating
anionic amino acids on the cell surfaces. This property is also useful in improving
their stability and activity in nonaqueous solvents. Halophiles also tend to reduce
their osmotic pressure by gathering high levels of low-molecular-weight neutral
organic species (Hendry 2006).
2.1.5 Aggregation Resistance Strategies
Maintenance of metabolic flux and cellular mechanisms relies upon the organisms’
ability to keep their functional states when they are under extreme stress. By
understanding the aggregation resistance strategies of thermophilic proteins, it is
possible to resolve the response of the aggregation-prone regions in proteins.
Thermophiles produce proteins that help in addressing the protein aggregation that
18 2 Survival Mechanisms of Extremophiles
reduces the functional state of the organisms (Merkley et al. 2011; Kufner and
Lipps 2013). Thangakani et al. (2012) compared the aggregation resistance strat-
egies adapted by thermophilic proteins and their mesophilic homologs using a
dataset of 373 protein families and found that the thermophilic proteins had better
utilization of the aggregation resistance strategies. Thermophiles tend to accumulate
osmolyte molecules that can stabilize their proteins and macromolecules, which
could help in the design and formulation of proteins and antibodies with therapeutic
applications.
2.1.6 Activation of the Nuclear Factor
The ability of heat shock proteins (HSPs) to inhibit the genetic expression of
proinflammatory cytokines has been explored as another mechanism by which
extremophiles survive under harsh environmental conditions. Buommino et al.
(2005) reported that the transcription of proinflammatory cytokines is dependent on
the activation of the nuclear factor kappa-B (NF-kappaB). Studies indicate that
ectoine, a biomolecule produced by halophiles, activates certain heat shock pro-
teins. The authors used reverse transcriptase-polymerase chain reaction (RT-PCR)
and immunoblot analysis to determine the increased levels of gene expression of
HSPs in human keratinocytes that were treated with ectoine and heat stress. The
findings had important implications for the development of additives that can be
used as protective tools for treating human skin infections or inflammation.
2.1.7 Resistance to Cell Death
Other recent investigations have examined extremophiles’resistance to cell death
and the pathways by which this process occurs, as shown in Fig. 2.5. One major
hypothesis that has been supported by several studies involves the role of mito-
chondria in the death of brain cells: a set of protein components affects mito-
chondria and begins their destruction, leading to cell death under various
conditions. Thus, further research on extremophiles and the proteins in their
mitochondria may provide clues for identifying compounds that do not destroy
mitochondria. Biochemical assays and protein sequencing will assist in identifying
the mechanisms of molecular mediation in cell death. This can lead to the devel-
opment of drugs to target proteins that cause cell degeneration and reduce the
development of neurodegenerative diseases. This mechanism for using extremo-
philes in the development of therapeutic applications is summarized in Fig. 2.5.
2.1 Survival and Potential Therapeutic Strategies 19
2.1.8 Cellular Compartmentalization
A UVR neutralization model using cellular compartmentalization of scytonemin
biosynthesis in cyanobacteria was studied (Soule et al. 2009). Here, we attempt to
summarize the possible therapeutic implications of this model (Fig. 2.6). The outer
membrane of the cyanobacterium absorbs the UVA irradiation (Fig. 2.6, left),
which further stimulates a cluster of genes such as Tyrp. This activates production
of tryptophan and p-hydroxyphenyl pyruvate monomers from chorismate. In
addition, it is proposed that certain precursors are processed by ScyA, ScyB, and
ScyC and NpR1259 in the cytoplasm. Using these precursors, reduced forms of
scytonemins are produced by perisplasmic enzymes (ScyD, ScyE, ScyF, DsbA, and
TyrP). These reduced forms of scytonemin autooxidize from the extracellular slime
layer in sufficient quantity to block the incoming UVR (Soule et al. 2009).
Singh and Gabani (2011) conceptualized this model for the eukaryotic cell.
Scytonemin was anticipated to provide a novel pharmacophore for the development
of protein kinase inhibitors as antiproliferative and antiinflammatory drugs. It was
Fig. 2.5 A process of studying the mechanism for using extremophiles in the development of
therapeutic applications for neurodegenerative diseases (based on: http://www.projectsmagazine.
eu.com/randd_projects/mitochondrial_mechanisms_of_disease_lessons_from_extremophiles)
20 2 Survival Mechanisms of Extremophiles
also hypothesized that scytonemin derivatives may be involved in the survival of
healthy cells through mediated activation of stress-activated protein kinases
(SAPKs), shown with dotted arrows on the right in Fig. 2.6.
Singh and Gabani (2011) reviewed the ATP-competitive inhibitors of polo-like
kinases (PLKs), which have been theorized to control oncogenes in human cells,
since they have the ability to switch off the activity by binding to ATP-binding
sites. PLK1 has been highly regarded as a mitotic cancer target, and can be inhibited
by scytonemin, which is recognized as a nonspecific ATP competitor. Further,
scytonemin has the property to treat hyperproliferative disorders (Stevenson et al.
2002b). Luo et al. (2009) demonstrated that due to mitotic stress, cells become
highly sensitive to PLK1 inhibition when they have mutant Ras acting as an
oncogene. The scytonemin-mediated inhibition of PLK1 expression has been
shown to induce apoptosis in osteosarcoma cells and other cancer cell types
(Stevenson et al. 2002a; Duan et al. 2010).
These studies suggested that scytonemin could function as a novel pharmaco-
phore for the development of protein kinase inhibitors and antiproliferative and
antiinflammatory drugs (Fig. 2.6). In addition, SAPKs such as p38⁄RK⁄CSBP kinase
Fig. 2.6 Cellular protection of biosynthesized scytonemin in prokaryotes and hypothesized
proposed mechanism of cellular protection in eukaryotic cell (adopted and modified with
permission from Singh and Gabani 2011)
2.1 Survival and Potential Therapeutic Strategies 21
and c-Jun N-terminal kinase (JNK) could help in the development of therapeutic
responses to shock and UV-radiation-related stress. It is known that SAPKs when
activated can further activate transcription factors (c-Jun, ATF2, and Elk-1)
responsible for gene expression responses to external environmental stresses.
Alternatively, Singh and Gabani (2011) anticipated that scytonemin-mediated
activation of SAPKs could help in eukaryotic cell survival. There also exists some
complementarity of the scytonemin activity responsible for the UV insensitivity of
photosynthesis in Nostoc flagelliforme and the UV absorption of mycosporine-like
amino acids (Ferroni et al. 2010). The therapeutic propositions of these studies
indicate that there is complementarity between biologically mediated UV protection
and the pharmaceutical compounds used for UV protection.
2.1.9 Overexpression of Heat Shock Protein Genes
HSPs have immunomodulatory properties that could have proinflammatory func-
tions that help in immune responses. The mechanism that HSPs use to regulate
autoimmunity can be effectively harnessed to develop therapeutic tools for the
treatment of autoimmune disorders and some forms of cancer. Welch (1993)
showed that high levels of HSPs could be achieved by exposing the cells to various
types of chemical agents including metabolic poisons, heavy metals, protein
modifiers, amino acid analogs, and ionophores. In another study, Zügel and
Kaufmann (1999) demonstrated that during periods of stress caused by infection or
inflammation, HSP synthesis could protect prokaryotic or eukaryotic cells.
Boummino et al. (2005) reported that ectoine from halophiles may help in pro-
tecting cells from stress and prevent cell damage at higher levels of HSP70. HSPs
are known for their role in the cytoprotection and repair of cells and tissues against
the stresses and trauma they might face in extreme conditions (Morimoto and
Santoro 1998).
HSPs involve overexpression of the single or collective HSP genes to help
protect the skin from various stresses such as high levels of heat, drug toxicity, UV
radiation, and other pollutants (Simon et al. 1995; Zhou et al. 1998). Among
modulated variations in HSPs during cellular stress, HSP70 was revealed to be a
major inducible and cytoprotective protein (Buommino et al. 2005). The overex-
pression of HSP70 significantly reduces the release of IL-6 induced by UVA, UVB
irradiation, and oxidative stress (Buommino et al. 2005). Halophilic organisms were
anticipated to be an effective source of small organic molecules that can be used to
treat skin diseases that originate from infections or inflammation by overexpression
of HSP70. Further, ectoine, a key extremozyme from the halophiles, could be
effectively used as a protective additive for skin defense and in protein synthesis by
increasing the basal levels of HSP70 (Buommino et al. 2005). Relatively similar
mechanisms have been identified and documented in other extremophiles, and can
be effectively applied in developing products that may have therapeutic values. Liu
et al. (2010) reported that CiHsp70, a molecular chaperone of the HSP family, may
22 2 Survival Mechanisms of Extremophiles
play a role in enabling Antarctic ice algae Chlamydomonas sp. ICE-L to acclimatize
to the polar environment. Yamauchi et al. (2012) studied the protein-folding
mechanism of the GroEL system in psychrophilic bacterium Colwellia psych-
rerythraea 34H, and found that the CpGroEL system has an energy-saving
mechanism that allows it to avoid excess utilization of ATP to ensure microbial
growth at low temperatures.
2.1 Survival and Potential Therapeutic Strategies 23
Chapter 3
Therapeutic Implications of Extremophiles
Abstract Extremophiles use specific mechanisms to alter the primary and/or sec-
ondary metabolites (i.e., extremolytes) to thrive under harsh environmental condi-
tions, which could be exploited in therapeutics. Extremolytes from thermophiles,
i.e., stable proteins, stable amylase, and thermozymes, have potential implications in
regulation of intracellular environment and metabolism, and in energy transduction.
Extremolytes mycosporine-like amino acids (MAAs), scytonemin, bacterioruberin,
ectoine from radiation-resistant extremophiles can help to protect from UVR and
gamma radiations. Extremolytes from acidophiles have been considered to use in
protein pumps, reduce the pH of the cells within the cell surfaces, and as probiotics.
Halophile carries unprecedented properties of carotenoids, high-antioxidant com-
position, and reductases. Further, investigations on extremolytes will pave the way
toward next-generation medical innovation.
Keywords Extremolytes Extremozymes Extracellular polymeric substances
Neurodegenerative disease Proteases Amylase
The interest in the study of extremophiles stems from the fact that they are a good
source of useful substances (i.e., extremolytes), including beneficial enzymes (i.e.,
extremozymes). These substances can be used as biocatalytic agents because of
their unique characteristics, including their stability under extreme environmental
conditions. Extremolytes are being applied in various ways in the medical field
today. For example, they have been used in cancer research, in the form of anti-
cancer drugs, antioxidants, cell-cycle blocking elements, and anticholesteric drugs.
Extremozymes can be stable and active under conditions that are thought to be
incompatible with normal biological organisms and materials (Hough and Danson
1999; Eichler 2001; Egorova and Antranikian 2005; Gabani and Singh 2013;
Elleuche et al. 2014). One major potential use for extremozymes is the development
of special industrial chemicals, agrochemicals, and intermediary products for the
pharmaceutical industries (Demirjian et al. 2001). Table 3.1 summarizes the ther-
apeutic implications of the substances that can be derived from extremophiles.
©The Author(s) 2015
P. Babu et al., Extremophiles and Their Applications in Medical Processes,
Extremophilic Bacteria, DOI 10.1007/978-3-319-12808-5_3
25
Table 3.1 Extremozymes found in extremophiles and their implicative products in therapeutics
Types of
extremophile
Role in disease types Extremolytes (proteins/
enzymes) from extremophiles
Implications References
Radiation-
resistant
Prevents Alzheimer’s and Par-
kinson’s diseases from
developing;
Prevents damage from bone
marrow and protects skin from
exterior radiations; prevent
skin damage due to from UVR;
Skin cancer, etc.
Mycosporine like amino acids;
scytonemin; bacterioruberin;
ectoine
Extremolytes; extremozymes,
used for their high stability;
color pigmentation, help to
protect from UVR and gamma
radiations; increase of con-
sumption of carbohydrates;
anticancer drugs: UV absorp-
tion and sun screen
Copeland et al. (2013), Rastogi
et al. (2012), Zhang et al.
(2007), Singh and Gabani
(2011), Yuan et al. (2009a, b),
Karsten et al. (2009), Gabani
and Singh (2013), Bunger and
Driller (2004), Asgarani et al.
(2000), Stevenson et al.
(2002a, b)
Thermophiles Treat misfolded proteins: aid in
curing neurodegenerative
diseases;
thermolytes: used in protein
degradation; suppress
cancer activity: known to aid
in T cell proliferation of leu-
kocytes; antitumor suppression
Stable proteins; thermosomes,
(chaperone protein), act as a
facilitator and maintain the rest
of the chaperone proteins for
the cell; stable amylases; ther-
mozymes are being produce in
surrogate mesophilic hosts;
enzymes and proteins from the
genus Thermus, and thermo-
zymes in general are good
candidates for biocatalytical
processes as they often present
higher operational stability,
and enhanced co-solvent
compatibility
Phytosterols; cellulose produc-
tion; energy transduction; reg-
ulation of intracellular
environment and metabolism;
functioning of enzymes; and
protein conformation; citrate
synthase from P. furiosus; and
glucose dehydrogenase from
Thermoplasma acidophilum
Jorge et al. (2011), Acharya
and Chaudhary (2012),
Margesin and Schinner (1994),
Jorge et al. (2011), Kohama
et al. (1994), Sellek and
Chaudhuri (1999), Thangakani
and Kumar (2012), Irwin and
Baird (2004)
Halophiles Prevent multiple cancer types;
chronic inflammation; athero-
sclerosis; cardiovascular disor-
der; aging process;
Reductase from H. volcanii
and malate dehydrogenase
from Haloarcula marismortui;
dihydrofolate reductase and
Motility capacity; unfolding
and refolding of a halophilic
enzyme has been studied; 3-
hydroxy-3-methylglutaryl-
Ksouri et at. (2012), Agrawala
and Goel (2002), Le Bail et al.
(1998), Sellek and Chaudhuri
(continued)
26 3 Therapeutic Implications of Extremophiles
Table 3.1 (continued)
Types of
extremophile
Role in disease types Extremolytes (proteins/
enzymes) from extremophiles
Implications References
anticarcinogenic flavonoids
help to prevent cancer; pro-
mote antiproliferative cancer
activity
dihydrolipoamide dehydroge-
nase; polyunsaturated fatty
acids; carotenoids; vitamins;
sterols; essential oils; polysac-
charides; glycosides: phenyl
compounds; high- antioxidant
composition; proteins espe-
cially in halophiles require an
excess of negatively charged
amino acids on the protein
surface; contains antifreeze
proteins that can freeze body
organs for study
coenzyme; inhibition of insulin
and amyloid formation; reduc-
tion of apopotic cell death;
protection of mitochondrial
DNA in human dermal
fibroblasts
(1999), Buommino et al.
(2005), Arora et al. (2004)
Acidophiles Used in situ experiments; Fe
cycling; Used in evolutionary
medicine; preventive measures
for ulcer disease and gastric
cancer
Protein pumps are used exces-
sively; peptidoglycan is used
to reduce the pH of cells within
the cell surface; biocatalysts;
bio-enzymes, probiotics
Cultured media allows for
special growth in amino acids;
Helicobacter pylori: secretion
of urease; enzymes stable at
low pH such as amylases,
proteases, ligases, cellulases,
xylanases, α-glucosidases, en-
doglucanases, and esterases are
known from various acido-
philic microbes
Lu et al. (2010), Lopez De
Saro et al. (2013), Sachs et al.
(2011), Foster (2004), Irwin
and Baird (2004), Sellek and
Chaudhuri (1999), Sharma
et al. (2012)
Mesophiles Commonly used in proteases Glutamate dehydrogenase; a
protein that enables mesophiles
to prevent the formation of
protein aggregates. This
enzyme is used for protein
folding
Proteases; cold-adapted,
enabling the microorganisms
to adapt to changes in
temperature
Fornbacke and Clarsund
(2013), Singh et al. (2009),
Sellek and Chaudhuri (1999),
Thangakani et al. (2012)
(continued)
3 Therapeutic Implications of Extremophiles 27
Table 3.1 (continued)
Types of
extremophile
Role in disease types Extremolytes (proteins/
enzymes) from extremophiles
Implications References
Psychrophiles Membrane fluidity; frostbite;
hypothermia; enzyme activa-
tion at high temperatures;
marginal cellular growth
Flexible cellular proteins; anti-
freeze proteins; special pro-
teins and molecular chaperones
enable microbes to adapt and
survive in cold temperatures;
citrate synthase from an Ant-
arctic bacterium, and α-amy-
lase from Alteromonas
haloplanctis; DNA polymerase
from the uncultivated psy-
chrophilic Archaeon Crenar-
chaeum symbiosum
Able to sense changes in tem-
peratures: modulating mem-
brane fluidity; leading bacteria
to become temperature sensi-
tive; aiding in establishing
viral vaccinations; protecting
against diseases that require
cold-mediated immunity;
increased enzyme production;
lowering the temperature can
change the flexibility of human
proteins
Singh et al. (2009),
Shanmugam et al. (2012),
Margesin and Schinner (1994),
Garcia-Descalzo et al. (2013),
Selleck and Chaudhuri (1999),
Gerday et al. (2000),
Singh (2013), Irwin and
Baird (2004)
Geophiles Skin-related diseases; stomach
ulcers; gastric cancers; help in
finding treatments to prevent
stomach worms as such
Protease could be an evolu-
tionary adaptation in therapeu-
tics; may lead to the decreased
risk of derma-related diseases
Acid proteinases, elastase,
keratinases; antibiotic cells;
cell mediated immune reac-
tions: can rid the fungus from
the skin; leukocytes; antibodies
Weitzman and Summerbell
(1995), Dahl (1987), Singh
(2013)
Barophiles Used in coping mechanisms
for cells in order to avoid
detrimental changes due to the
environmental pressure; affect
the cells working ability to
adapt in different environments
(pressures)
P60 protein was discovered to
have pressures below standard
condition; analysis was done
on the protein and found heat
shock properties in the protein
and resembling archaic organ-
isms; stress proteins are mostly
expressed in barophilic
organisms
Changes in membrane fluidity;
amounts of fatty acids; changes
in biochemical processes;
Pressure-sensing mechanisms/
organs: pressure-regulated
operons
Marteinsson et al. (1999),
Yano et al. (1998), Kato and
Bartlett (1997), Marquis and
Keller (1975)
28 3 Therapeutic Implications of Extremophiles
3.1 Radiation-Resistant Organisms
Radiation-resistant organisms have been studied for the development of medical
applications that can help humans suffering from radiation consequences such as
skin cancer and premature aging. Mycosporine-like amino acids (MAAs) are found
in diverse extremophiles, including cyanobacteria and eukaryotic algae. MAAs
absorbs radiation with wavelengths of 310–365 nm, and are commercially used in
sunscreens and other cosmetic purposes.
MAAs are known to prevent the formation of DNA dimers, which damage DNA
when cells are exposed to UVR. MAAs have also been shown to protect fibroblast
cells in human skin from UVR exposure (Oyamada et al. 2008). MAAs such as
palythine, asterina, palythinol, and palythene have been suggested as prime can-
didates for UV protection. Some of these MAAs are found in several species of
microalgae such as G. galatheanum and G. venificum (Llewellyn and Airs 2010).
Torres et al. (2006) reported the isolation of mycosporine with UVB-absorbing
properties from Collemacristatum, a lichenized ascomycete. In cultured human
keratinocytes, MAAs exhibited protection from UVB-induced processes, such as
erythema, pyrimidine dimer formation, and other types of membrane destruction.
Researchers have suggested the use of MAAs to prevent UVR-induced cancers
such as melanoma (De la Coba et al. 2009).
Mukherjee et al. (2006) suggested that maristentorin found in Maristerntor
dinoferus could have properties to protect from UV irradiation. In addition to
MAAs, bacteriorubrins have also been shown to be highly resistant to ionizing
radiation. Bacteria such as Rubrobacter radiotoleransis,D. radiodurans, and
Halobacterium salinarium have been reported to have considerable resistance to
UVR and H
2
O
2
(Asgarnai et al. 2000). In a study using a halobacterium that
contained bacterioruberin, Shahmohammadi et al. (1998) found that the presence of
bacterioruberin was also found to have DNA repair mechanisms.
Sphaerophorin and pannarin derived from lichens have been shown to have
antioxidant activity, which is useful in sunscreen products for UV protection
(Muller 2001). Russo et al. (2008) evaluated the effect of sphaerophorin and
pannarin on chemically induced DNA cleavage and found that both compounds
showed a protective effect on plasmid DNA. These compounds inhibited the growth
of human melanoma cells (M14 cell line) by inducing apoptosis in cell culture
experiments (Russo et al. 2008).
Studies indicate that substances from extremophiles such as bacterioruberin,
sphaerophorin, and pannarin play a significant role in the development of potential
treatments for diseases caused by excessive radiation, as reviewed by Singh and
Gabani (2011) and Copeland et al. (2013). The therapeutic implications of these
compounds for repairing DNA damage and thereby treating the diseases are yet to
be explored.
3.1 Radiation-Resistant Organisms 29
3.2 Thermophiles
Thermophiles require high temperatures to thrive, and have several medical
applications because their cell membranes have evolved to endure intense heat. As a
result, these extremophiles are in demand for treating types of cancers involving
intense heat. Since global warming has and will continue to have an impact on the
environmental temperature, there has to be ways to reduce the effects of high
temperature on human lives. Thermophiles have a mechanism in which they are
able to convert carbohydrate-rich substances into hydrogen, and because they are
able to withstand and grow at extremely high temperatures, this makes them
applicable for use in the medicinal and therapeutic formulations. One major
advantage that thermophiles may have is that they prevent the growth of pathogenic
organisms from entering into the human society, since they survive at high tem-
peratures at which most pathogens would not be able to proliferate (Suzuki et al.
2013).
Major neurodegenerative diseases such as Huntington’s and Parkinson’s are
characterized by protein folding malfunctions and the inclusion of formations inside
neurons in the brain (Jorge et al. 2011). Thermophiles may have extremozymes
with capabilities such as the inhibition of conformational changes of native or
mutant proteins, which may be useful to develop cures for neurodegenerative
diseases (Jorge et al. 2011). Jorge et al. (2011) suggested that novel organic solutes
known as thermolytes could serve to protect proteins’native structures. Thermo-
lytes have been revealed to affect the growth curves of HEK293 cells, which have
been cultured to aid in the treatment of Parkinson’s disease. The results of this
research showed a decrease in cell density in the cells that were treated with
thermolytes compared to untreated control cells (Jorge et al. 2011). Studies were
conducted to test the similar effects of thermolytes in Huntington’s disease using
transfected HEK293 cells with Htt-103QEGFP, and revealed an Mg- and DGP-
mediated significant effect on the formation of Huntington aggregates (Jorge et al.
2011).
Identifying the missing components in cytoplasmic disulfide bond formation in
hyperthermophiles where protein folding occurs may provide understanding of this
new property that may lead to the discovery of antiviral drugs and large-scale
production of therapeutics (Saaranen and Ruddock 2013). The interesting part of
this study is the role of the cytoplasmic machinery of specific extremophiles, as that
is where the protein folding takes place, and it is an important part of antiviral drug
production (Saaranen and Ruddock 2013). Another emerging global challenge that
is a result of increased temperatures is the scarcity of water, because the heat
evaporates the water in reservoirs. This has become an increasing challenge for the
water deprived communities of the world including in the North American state of
California. Conditions that help mesophilic organisms to survive at high tempera-
tures and water deprived environments need to be understood further to develop
outcomes that may be useful to keep animals and humans less dependent on water.
30 3 Therapeutic Implications of Extremophiles
3.3 Halophiles
Halophile archaea and bacteria are found in environments with high salt concen-
trations. The extremolytes from halophiles are categorized by their low-molecular
mass and accumulation to stress, usually in response to salt and temperature
(Chakravorty and Patra 2013). However, a major setback in using these extremo-
lytes is that they have not been cultured in cells with higher cellular density. The
biodiversity of the halophiles has been better understood and their therapeutic
implications have improved due to knowledge of the role that halophilic organisms
inhabit in the saltern crystallizer ponds (Oren 2002). Improved availability of
chemotaxonomic studies and the effective use of advanced culture techniques and
molecular biological methods have led to more effective exploitation of halophilic
microorganisms.
Lipids derived from halophilic bacteria have found uses as deliverers of drugs
and vaccines (Abrevaya 2013). Other compounds found in halophilic archaea, such
as siderophores, offer iron-chelating agents that can be used to treat iron deficiency
diseases or to increase antibiotic activity against bacteria (Abrevaya 2013). Oren
(2002) reported that halophiles’metabolic activities decrease in diversity when the
level of salinity they live in decreases. The level of energy generated by halophiles
and the energetic cost involved in their osmotic adaptation are increasingly directly
connected to high levels of salinity.
In general, extremophiles have a special characteristic in that they contain
extracellular polymeric substances (EPS). Although the therapeutic mechanisms
involved in these substances are not yet fully understood, they are known to help
extremophiles and other prokaryotic organisms adapt to changes in their environ-
ments and compensate for the deleterious effects of harsh environments (Barbara
et al. 2013). This is true for thermophiles and halophiles, as well as mesophiles that
may have high industrial value.
Neurodegenerative diseases such as Machado-Joseph disease are categorized by
mass protein misfolding as a key event in the pathogenesis. The overexpression of
chaperone proteins recognizes misfolding as a typical target for effective therapy.
Furusho et al. (2005) explored molecules from extremophiles that can potentially
influence protein folding. Ectoine, which was originally found in halophiles, is an
organic molecule of low-molecular mass and serves as an osmoprotectant; it is good
at preserving enzymatic activity against freeze-thawing treatments of protein sta-
bilization (Furusho et al. 2005). Ectoine was observed to reduce large cytoplasmic
inclusions and increase the frequency of nuclear inclusions, although the integrity
of the nuclei appeared to be maintained. Further, ectoine was shown to protect cells
from polyglutamine-induced toxicity (Furusho et al. 2005).
Ectoines are also helpful in treating Alzheimer’s disease. They play an important
role in inhibiting the formation of amyloid, which is a protein aggregation factor
involved in the misfolding of proteins (Kumar and Singh 2013). A majority of
extremolytes comes from marine organisms, including mannosylglycerate (firoin)
and mannoslyglyceramide (firoin A). Found abundantly in thermophilic bacterium
3.3 Halophiles 31
Rhodothermus marinus, these extremolytes are used against certain cancers and
related diseases (Kumar and Singh 2013).
Buommino et al. (2005) showed that by the induction of HSP70 protein at
elevated levels, ectoine exhibited a cytoprotection effect through bacterial lipo-
polysaccharide. Overexpressions in HSP70 and HSP70B9 were observed in
keratinocyte cells treated with ectoine along with the heat shocks. However, this
study was based on earlier findings, which showed that exposure to chemical
inducers lead to both HSP induction and inhibition of NF-kB (Thanoas and
Maniatis 1995). NF-kB is a eukaryotic transcription factor that can be induced by
bacterial and viral infections, inflammation, and UV radiation. Its activation is
a result of phosphorylation and degradation of the inhibitory protein IkB-a.
Buommino et al. (2005) evaluated the degradation of IkB-a and showed that ectoine
did not activate NF-kB in treated keratinocyte cells. Yoo et al. (2000) demonstrated
that the induction of HSPs inhibits proinflammatory cytokine expressions. Further,
the same studies indicated that the induction of HSPs can block the nuclear
translocation of NF-kB by inhibiting the degradation of IkB-a. Thus, it can be
interpreted that keratinocytes exhibiting cytoprotection mechanism could be a result
of the ability of ectoine to induce HSPs and downregulate proinflammatory signals.
The therapeutic implication could possibly be to prevent water loss in dry skin and
aging of the skin through ectoine-mediated neutralization of UVR.
3.4 Acidophiles
Acidophiles are widely used for therapeutic purposes, and have even been involved
in the evolution of medicine. They are mostly used in preventing gastric cancers
and stomach ulcers for those prone to infections (Foster 2004). However, they have
also been found useful for iron cycling and conducting in situ experiments. The
acidophilus organisms are collectively known as probiotics and intestinal inhabit-
ants. Some of these internal inhabitants are more helpful than others. Acidophilus
aids digestive tract function and reduces the presence of harmful organisms. For this
reason, use of probiotics can help prevent infectious diarrhea. Lactobacillus aci-
dophilus has been used to prevent many diarrhea infections, such as traveler’s
diarrhea, infectious diarrhea, and antibiotic-related diarrhea (Elmer 2001; Lievin-Le
Moal 2007; Grandy et al. 2010; De Vrese et al. 2011; Ouwehand et al. 2014).
Crohn’s disease and ulcerative colitis conditions are known as inflammatory
bowel diseases. Chronic diarrhea is a common feature of both conditions. Micro-
organism Helicobacter pylori is known to cause ulcers in the stomach and duo-
denum. Studies have been performed that used probiotics to inhibit the growth of H.
pylori (Michetti et al. 1999; Pantoflickova et al. 2003; Gotteland et al. 2008). A
thorough review by Elmer (2001) on microorganisms Lactobacillus,S. boulardii,
and other probiotics revealed an in-depth understanding of the use of acidophiles
for varying forms of diarrhea, and presented them as helpful for mild diarrhea in
stable Crohn’s disease.
32 3 Therapeutic Implications of Extremophiles
The acid stability of some acidophiles has been studied through analysis of their
crystal structures. Acidophiles produce some substances that may be used in novel
drug treatments of those prone to infections from gastric cancers and stomach ulcers
(Lopez de Saro et al. 2013). To prevent fluctuations in the cellular membrane, these
organisms pump hydrogen ions into the membrane, which regulates the pH of the
cell (Irwin and Baird 2004). Nonetheless, exactly how acidophiles use these pumps
to maintain the pH and stability of their cells is still not understood (Irwin and Baird
2004). Bioenzymes have been found in the genomes of acidophiles, and researchers
are interested in cultivating them for use in biotechnology (Lu et al. 2010).
3.5 Mesophiles
Mesophiles are known for their ability to thrive at moderate temperatures and
pressures. These extremophiles are readily able to compose themselves in a way
that enables them to cope with the different surroundings and environments that
they have to endure. One major example of products that allow mesophiles to
survive is proteases. Proteases are enzymes that are used everyday in medical fields
(Fornbacke and Clarsund 2013) and can adapt to cold temperature changes easily
(Singh et al. 2009). They can also be used in combination with different ex-
tremophiles, such as psychrophiles, geophiles, and so on. Medical technicians are
now able to use the properties of mesophiles toward isolating, creating, and sup-
plying vaccines, which need to be kept at viable temperatures so that they do not
start to lyse. In other words, cold shock for mesophiles is an important part of the
cell cycle (Piette et al. 2012).
3.6 Psychrophiles
Psychrophiles adapt quickly to cold temperatures. These microorganisms have
immense potential for the medical field, as they can provide insights into how cells
cope with cold environments as well as actively protect and regulate the membrane
fluidity of the cell. The cell membrane is the most important factor when it comes to
psychrophiles, as it controls and regulates the homeostasis of the cell. If this
equilibrium is offset, it causes damage to the cell in the long run (Margesin and
Schinner 1994). It is important for psychrophiles to maintain certain temperatures
and conditions to manage changes in external cellular lipid saturation and the
disruption of intracellular organization (Margesin and Schinner 1994). Psychro-
philes are commonly used in protecting against diseases that require cold-mediated
immunity.
Polyunsaturated fatty acids produced from psychrophiles can be extensively
used in pharmaceutical agents. Proteases have been used in medicine for many
years in the treatment of blood disorders; they also have promising indications for
3.4 Acidophiles 33
treating digestion problems (pancrelipase) and muscle spasms, and as cosmeceu-
ticals. Proteases are an established and well-tolerated class of therapeutic agents
(Craik et al. 2011). Cold-adapted proteases have been used in a wide range of
applications, including molecular biology, cosmetics, and pharmaceuticals (Craik
et al. 2011; Gudmundsdottir and Palsdottir 2005; Marx et al. 2007). Cold-adapted
proteases have been reported to be particularly useful in low-water conditions and
high level of structural rigidity (Karan et al. 2012). Psychrophilic proteases have
been obtained from Atlantic cod (Gadus morhua) and Antarctic krill (Euphausia
superba). A wide variety of proteases have already been identified and genetically
expressed in microorganisms (Taguchi et al. 1998), and cold-adapted proteases
have been reviewed as an emerging class of potential therapeutics (Fornbacke and
Clarsund 2013). Certain psychrophiles have been studied for their potential to
prevent fungal infections (Garcia-Descalzo et al. 2013).
3.7 Geophiles
Geophilic or “soil-loving”microorganisms are a special type of microbial species;
they live in soil and cannot be reproduced in laboratory settings. Due to this fact,
geophiles can be useful in research that requires organisms in their natural settings.
Geophiles are known for producing treatments for skin damage, stomach ulcers,
and gastric cancers. They have also been used to help find preventive techniques/
measures for decreasing the risk of infection (Dahl 1987). Products that are
involved with the mechanisms of geophiles are acid proteinases, keratinases to help
provide keratin, and antibiotic cells to prevent damage (Weitzman and Summerbell
1995). Arena et al. (2009) suggested that the biofilm formation of Geobacillus
thermodenitificans acts as an adjuvant agent in equilibrating immune response in
viral diseases.
Dermatophytes are a leading cause of fungal infections today (Achterman and
White 2011). Costs for treating these fungal infections can be high; geophiles may
make it possible to create an enduring treatment and cure for these types of fungal
infections. Understanding the virulence of the fungi involved is important for
coming up with the correct type of treatment, which is where the application of
geophiles comes into play (Achterman and White 2011). Geophiles now have great
promise for upcoming research in the field of therapeutics.
3.8 Barophiles
Barophiles survive at high-pressure levels, and can be found in environments such
as deep-sea vents, high mountain ranges, and places where there is less oxygen
(Kumar and Singh 2013). Barophiles are important in medicine because they can
thrive at high pressure and are not affected by frequent changes in pressure levels.
34 3 Therapeutic Implications of Extremophiles
Barophilic cells are most vulnerable to the aftereffects of pressure changes because
it is important for biological cells to have stable pressure. Pressure changes can
have detrimental effects that lead to cellular consequences (Tan et al. 2006). Bar-
ophilic microorganisms use mechanotransduction, a mechanism that senses the
changes in pressure and translates them to a signal that can be used by the cell
efficiently (Tan et al. 2006). Unwanted stress due to moderating pressures from
different environments can cause this to occur (Tan et al. 2006).
It has been known for some time that pressure is involved in treating diseases;
pressure affects all living organisms, and the right range of pressure is necessary for
cell stability (Kato and Bartlett 1997). Barophiles survive under high-pressure
ranges and can adapt to changes in pressure as well as changes in biochemical
processes (Kato and Bartlett 1997). Barophiles can play an important role in
therapeutics because of unusual characteristics such as abundance of pressure-
sensing mechanisms on their cellular membranes, osmotolerance, and pressure-
regulated operons (Marquis and Keller 1975). Tan et al. (2006) reviewed how
human and prokaryotic cells respond to mechanical forces in order to identify how
eye cells respond to pressure-induced glaucoma. Barophiles’ability to survive
under extreme pressure may provide insights to develop treatments for pressure-
induced injuries such as concussions and related athletic injuries.
3.8 Barophiles 35
Chapter 4
Challenges in Advancing Extremophiles
for Therapeutic Applications
Abstract Varying types of extremophiles have been identified, however, many
more are yet to be discovered from rare earth habitats. The challenges remain to
grow these bacteria in the laboratory without knowing the optimum growth media
and conditions. Advancements made in molecular biology of extremophiles are too
limited to investigate the routes extremophiles adopt for themselves at molecular
level under harsh environmental conditions. However, modern biology such as the
“–omics”making it easier for researchers to sequence entire genome of bacteria and
explore the systems biology approaches that enables extremophiles to cope with its
surroundings. Reference libraries for chemical properties of extremolytes would
make it easier to screen a variety of extremolytes against specific diseases.
Developing bioreactors for efficient production of extremolytes is among the major
challenges toward commerical benefits of extremophiles.
Keywords Isolation “-omics”Systems biology Genomics Proteomics
Metabolomics
Extremophiles have the potential to bring together multitudes of actors in the health
industry and in the greater field of health treatments. Recent advances in the study
of extremophiles show great promise for these organisms in therapeutic and medical
applications. However, several issues and challenges stand in the way of developing
useful medical applications of extremophiles.
First, the process of cultivating extremophiles in laboratory settings has been
extremely cumbersome. The slow growth of the organisms, low yield of the required
substances, and specialized equipment needed to cultivate them have limited the
production of these organisms. Second, although advances have been made in the
field of gene transfer, there are still no suitable gene transfer mechanisms for some
extremophile groups such as Archaea. Third, a common host for enabling gene
expression has not yet been found. Fourth, the review above indicates that the
interest of researchers has been highly oriented toward studying enzymes with
structural integrity that lend themselves to medical applications. Finally, in order to
©The Author(s) 2015
P. Babu et al., Extremophiles and Their Applications in Medical Processes,
Extremophilic Bacteria, DOI 10.1007/978-3-319-12808-5_4
37
use extremophiles in therapeutic applications, various species must be studied in the
extreme environmental conditions where they live. Such environments present harsh
working conditions (i.e., radiation, temperature, etc.) for scientists, however bene-
ficial the extremophiles may be (Mantelli 2003; Asker et al. 2011).
Irwin and Baird (2004) reported that protein function and structure are important
indicators of potential therapeutic applications. However, further research is needed
to explore the properties and uses of the enzymes derived from the proteins and
their corresponding extremophiles. Advances made in this area, although limited so
far, indicate that looking at the ways extremophiles work on a molecular level is the
first step toward finding cures for specific diseases. The research reviewed above
indicates that there has not been much research into products other than extremo-
lytes and extremozymes that might be useful for therapeutic reasons. The families
within these two types of products, such as ectoines and bioenzymes, show that
extremozymes have a multitude of possible uses in curing diseases as well as
providing insights and building connections between the medical world and the
extremophilic bacterial kingdom.
4.1 Isolation and Purification of Extremolytes
The limitations involved in the study of extremophiles can be observed in fields that
deal with particular types of extremophiles and the uses for which they are being
explored. One of the most challenging factors in the development of medical uses
for extremophiles is the difficulty of isolating and purifying extremolytes. Even
when appropriate extremolytes are isolated and their uses identified, their devel-
opment into specific drugs is a long and tedious process. Further, current regulatory
procedures hinder speedy development of these drugs. This is mainly due to the
challenges of limited functional analysis of specific molecules from extremophiles.
In the case of radio-resistant microbes, much research needs to be done before
any significant contribution can be made toward development of drugs. Singh and
Gabani (2011) reported that although radiation-resistant microbes contain com-
pounds that could be harnessed to produce radioprotective drugs, research efforts
remain insufficient. They suggested several reasons. First, unknown applications of
UVR reservoirs could have marginalized the market requirement for a strategic
product. Second, since the growth conditions and nutritional requirements of the
extremophiles are highly specific, isolation, and maintenance of radiation-resistant
microbes remains a challenge. Third, the instability in terms of their genomes,
possible mutation, and pathogenesis increases the risk for scientists cultivating them
in laboratory conditions. Finally, the limitations in terms of extraction and purifi-
cation of the enzymes place constraints on their production process.
38 4 Challenges in Advancing Extremophiles for Therapeutic …
4.2 Systems Biology of Extremophiles
The systems biology-based high-throughput experimental approach can analyze
global components from biological system (Ishii et al. 2007). These global com-
ponents may help predicting the systematic behavior of cellular development and
adaptation under extreme environmental conditions. This integrative approach of
systematic behavior allows researchers to study the complex metabolic and regu-
latory networks of extremophiles in the microbial system, which may lead to the
design of new value-added products of therapeutic significance. Now that 26,716
bacterial and 421 archaic genomes have been sequenced and abundant information
is available at NCBI (http://www.ncbi.nlm.nih.gov/genome/browse/), the next
challenge is to determine the function of each gene. Genome sequences of many
extremophiles revealed unique genetic elements of potential significance (Tyson
et al. 2004; Osorio et al. 2008; Lu et al. 2010; Cardenas et al. 2010; Lin and Xu
2013; Majhi et al. 2013; Jaubert et al. 2013; Wemheuer et al. 2013; Liljeqvist et al.
2013; Guo et al. 2014; Shin et al. 2014). Understanding of genetic roles in meta-
bolic and regulatory networks lies ahead to grasping the functionality of biological
system.
Functional “-omics”consists of high-throughput global experimental approaches
that make use of the information and reagents provided by structural genomics to
assess gene function (Hieter and Boguski 1997). This field has seen considerable
growth in recent years, encompassing areas such as transcriptomics (global gene
expression, i.e., mRNAs), proteomics (global proteins expression), and metabolo-
mics (global expression of primary and secondary metabolites). Comprehensive
transcriptome information for the extremophile Arabidopsis relative Thellungiella
salsuginea provides firsthand clues of functional genomics elements in plant stress
tolerance (Lee et al. 2013). A number of proteomics studies on a variety of ex-
tremophiles including Acidithiobacilus ferrooxidans (Chi et al. 2007; Osorio et al.
2013; Almarcegui et al. 2014); Pyrococcus furiosus (Lee et al. 2009); Acidithio-
bacillus caldus (Mangold et al. 2011); Exiguobacterium sp. (Belfiore et al. 2013);
Sulfolobus solfataricus (Kort et al. 2013); Methylacidiphilum infernorum (Jamil
et al. 2014); Metallosphaera cuprina (Jiang et al. 2014) reveal multiprotein-med-
iated exertion in the survival mechanism of extremophiles under a variety of
environmental conditions. A global metabolomics study of extremophile is yet to
come. In the mean time, the development of scientific methods for identifying,
screening, and detecting microbial metabolites using advanced genomics, proteo-
mics, and metabolomics methods show increased promise for understanding the
structural and biochemical properties of extremophiles and extremozymes (Singh
2006; Karsten et al. 2009). Chemical reference libraries will help speedup progress
in screening newly identified extremozymes for their therapeutic potential.
Hendry (2006) predicted that advances in the use of extremophiles for medical
and therapeutic applications would require the use of novel methods to identify new
species of extremophiles and innovative ways to develop and employ extremo-
zymes. Mapping the genomic information of these useful organisms is still in its
4.2 Systems Biology of Extremophiles 39
infancy. However, since the financial cost of genome and proteome sequencing and
the time involved in the process have dramatically decreased in recent years, there
is a high probability that the broader categories of extremophiles will be identified
and their genomes and proteomes will be sequenced. Further, the altered products
of primary and secondary metabolites (i.e., the metabolome) in biochemical path-
ways could be tracked using traditional techniques (i.e., NMR, GC-MS, LC-MS,
etc.). This will further help in sequencing a full gamut of extremophiles, leading to
a new level of understanding of the nature and properties of extremophiles and their
use in medical applications.
4.3 Extremophiles Like Other Organisms
Given that the applied research on extremophiles has focused on a select few
organisms, such as E. coli and Helicobacter pylori in the case of the acidophiles,
exploration of the use of other species to translate the beneficial characteristics into
therapeutic applications remains a challenge (Baker-Austin and Dopson 2007;
Woappi et al. 2014). Further, due to recent developments in genome sequencing
techniques, much of the research has been on developing hypotheses related to the
Fig. 4.1 Extremophiles, representative chemical processes, and product development mechanisms
for medical applications
40 4 Challenges in Advancing Extremophiles for Therapeutic …
genomics data and little has been done to test these hypotheses. Baker-Austin and
Dopson (2007) highlighted the need for genetic tools to perform in-depth analysis
of the genetic and biochemical foundations of the observed phenomena and
defensive mechanisms. Another technological challenge in developing extremo-
philes is the specific peculiarities of these organisms, which are extremely difficult
to replicate in a natural environment. Further, a lack of genetic elements for vector
development in the genetic markers continues to pose serious challenges to the
development of therapeutic applications for compounds from extremophiles.
However, the challenge remains to move forward with serious applications of
extremophiles that will require innovative methods of prospecting these organisms
from their natural habitats, as well as developing genetic and biotechnological
approaches to understand the molecular and structural mechanisms and the bio-
chemical strategies that these organisms employ to survive in harsh and extreme
environmental conditions (Fig. 4.1).
4.3 Extremophiles Like Other Organisms 41
Chapter 5
Conclusion
Abstract Even with the amount of limitations that have come across in this
research and novel area of interest, a great amount of information has been found on
the coping mechanisms and thriving skills that extremophiles use in order to live in
their environments. With more interest coming to this area, it will allow to broaden
its horizons developing tools to help replicate the environments in which
extremophiles live. Collaborative efforts exploring extremophiles would ultimately
be of benefit to human society.
Keywords Extremolytes Extremozymes Human society Pharmaceutical
industries Sustainable therapeutics
Due to the complex properties of extremophiles and extremolytes, research in this
field will continue to expand. New extremozymes from extremophiles are being
identified and developed relatively slowly, since pharmaceutical industries are
driven by economic gains from their innovations. However, increased investment
for research into the characteristics of the various proteins and substances from
extremophiles can help in the process of developing sustainable therapeutic solu-
tions, as proposed in Fig. 4.1. Varying hypotheses regarding extremophiles’sur-
vival under harsh environmental conditions are still being explored. Therefore,
there is a need for collaborative efforts to study extremophiles to resolve medical
challenges in human society. Extremophiles have much untapped potential, and
future research must investigate types of extremophiles that have not been previ-
ously studied for their possible uses in therapeutic and medical processes.
©The Author(s) 2015
P. Babu et al., Extremophiles and Their Applications in Medical Processes,
Extremophilic Bacteria, DOI 10.1007/978-3-319-12808-5_5
43
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