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Apoptosis in Bacteria
Abstract:
Apoptosis is a phenomenon discovered by Professor John F. Kerr in 1972 in which the
cell, in response to specific stimuli, begins a suicide process in which the DNA fragments, the
chromatin condenses, the cytoskeleton fragments, and the cell breaks up into apoptotic bodies.
Though this phenomenon is most thoroughly understood in eukaryotic cells, researchers have
discovered a modified form of apoptosis in bacterial cells, in which the cell autolysis, or “self-
bursts”. The cell does this to perhaps provide nutrition for other cells as it functions as a
multicellular colony, or for nutrition of a germinating spore, or for DNA transformation
purposes. The markers of this process include DNA fragmentation, chromosome condensation,
and phosphotidylserine exposure. This process has genes and proteins that are distinct from
those that are present in eukaryotic apoptosis and are still under research at present. Research
into this field will allow us to fully understand the cell life cycle of a prokaryotic cell and
potentially have important clinical implications for the control of drug-resistant bacterial
infections and the development of new antibacterial drugs to combat this clinical issue. This
paper will discuss the presence of apoptosis in bacterial cells, as well as the genes involved and
the purpose of this action. Clinical applications of bacterial apoptosis will also be analyzed.
Introduction:
The life cycle of a prokaryotic cell differs significantly from the life cycle of a eukaryotic
cell. For example, reproduction in prokaryotic cell occurs strictly through a rudimentary division
process, known as binary fission, while reproduction in eukaryotic cells can be either through
mitosis or meiosis and is very complex in procedure. Bacterial cells tend to live fairly short lives
while they are in active form while eukaryotic cells can live for over a century, in the case of
neurons. However, there exist some parallels between the two life cycles. One of these
similarities is the method in which these cells die. Research is currently underway to clarify this
and still some controversy exists over this issue. However, it appears from current research that
bacterial cells undergo a form of apoptosis that is very similar to that process in eukaryotic
cells. Apoptosis is the process by which a cell, through a series of protein signals, commits
suicide. The process is similar in eukaryotes and prokaryotes in that it is induced the same way
by stress stimuli and exhibits similar hallmarks of the process which include DNA fragmentation,
chromatin condensation, and phosphotidylserine exposure, the exposure of this compound
which is usually contained on the interior of cells. Research is presently being conducted to fully
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clarify the specific genes and proteins that trigger and regulate apoptosis, as the process is not
fully understood. Further research into this subject can lead to new developments in clinical
therapy including methods of bacterial control and shows great promise for a more extensive
understanding of the entire bacterial cell life cycle.
Apoptosis in Eukaryotic Cells:
Apoptosis was first described in 1972 by biologist Dr. John F. Kerr. He realized that cells
were dying in a way that he called programmed cell death, in which the cell would commit
suicide once exposed to certain stimuli. Apoptosis has two distinct pathways that lead to the
execution phase of apoptosis in which DNA fragmentation, chromatin condensation, and
cytoskeleton dismantling takes place under the influence of specific proteins. The cell then goes
through a blebbing phase, where, as a result of the lack of structure usually provided by the
microfilaments, the cell bulges and stretches. Finally, the cell fragments into small apoptotic
bodies, plasma membrane enclosed cell debris, which are subsequently ingested by
neighboring cells via phagocytosis (Kerr 1972). In this way, the organismquickly and efficiently
rids itself of unwanted or damaged cells, without causing toxic waste to itself.
The Eukaryotic Apoptotic Pathways:
Apoptosis in eukaryotes has a few distinct signaling pathways that lead into the
execution phase. These pathways can be stimulated in an extrinsic or intrinsic manner. The
extrinsic pathway very simply is when an exterior stimuli binds to a ligand such as the Fas death
receptor binding to a Fas ligand, or a TNF receptor binding to a TNF ligand, on the surface of the
cell. Once this complex is formed, it will recruit additional cytoplasmic adaptor proteins to form
a Death signaling complex or DiSC. The DiSC will activate further proteins known as caspases
which will signal the cell to begin the execution phase of apoptosis (Elmore 2007).
The intrinsic pathway of apoptosis in eukaryotic cells is targeted specifically to the
mitochondria. This occurs when many different stimuli are recognized by the cell that are not
mediated by a receptor. This could include a negative stimulus such as a lack of nutrients and
growth factors that the cell needs to thrive. If the cell has no access to the nutrients that it
needs, it will not be able to form the proteins needed to suppress the pro-apoptotic proteins
and the cell will be signaled to begin the cell death cycle. The cell could also receive a positive
stimulus such as the presence of radiation, toxins, hypoxia, hypothermia, a viral infection or
free radicals. Each of these stimuli will flood the mitochondria with a signal cascade and will
open the mitochondrial permeability transition pore (MPT). The loss of this membrane
potential releases two pre-apoptotic proteins from the mitochondria. The first group of
proteins are Cytochrome C, Smac/DIABLO and the serine protease HtrA2/Omi. These proteins
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activate the mitochondrial pathways that depend on caspases. Cytochrome C will bind and
activate Apaf 1 and procaspase 9 forming the apoptosome. The apoptosome complex will then
activate caspase 9. Smac/DIABLO and HtrA2/Omi further apoptosis by stopping the inhibitors of
the apoptosis proteins (Elmore 2007). Additional variations on these pathways are under
continued research as to the exact genes and proteins that are involved in this process as well
as the presence of variations in the signaling phase. In fact, the perforin/Granzyme B pathway
has been discovered to link the extrinsic and intrinsic pathways (Elmore 2007). Researchers
have attempted to establish homologs of these apoptotic pathways in prokaryotic cells so the
study of apoptosis in eukaryotic cells has many implications in the study of bacterial apoptosis.
If researchers have an idea of what to look for, then the study of bacterial apoptosis can be
more focused and researchers will not have to waste time establishing the concept of an
apoptotic pathway.
Apoptosis in Bacteria:
Current research is now trying to prove that this type of programmed cell death does
occur in bacterial cells as well. Though some academics maintain that technically apoptosis
does not occur in bacteria, many researchers have proven that bacteria do undergo a form of
programmed cell death. A study conducted two years ago suggested that many of the
morphological and biochemical markers that appear in eukaryotic apoptosis did occur in
bacterial cells when they were induced to begin the process of programmed cell death
(Hakensson et al. 2011). The experiment was conducted on Streptococcus pneumoniae. The
bacteria were induced to die by the introduction of a complex designated HAMLET, which
stands for "human alpha-lactaalbumin made lethal to tumor cells". The experiment showed
that bacteria reacted in a "dose-dependent manner" to the HAMLET which had a bactericidal
effect on the S. pneumoniae. The cells were subsequently examined for signs of apoptosis by
means of microscopy and gel electrophoresis. Microscopic evaluation revealed that the cells
exhibited the chromatin condensation that is characteristic of apoptosis in eukaryotic cells.
Streaming of the DNA by gel electrophoresis revealed a higher than normal count for DNA
fragments, meaning that the DNA fragmentation that occurs in eukaryotic apoptosis had also
occurred in the bacterial cells. This experiment was repeated with other bacteria of the
Streptococcus genus and with Haemophilus influenzae, a respiratory pathogen. DNA
fragmentation in this bacteria also was apparent. This proves that bacteria can be induced to
follow a death signaling pathway that is similar to apoptosis in eukaryotic cells. However,
further research has revealed that bacterial apoptosis differs in the genes and expressed
proteins that control the eukaryotic apoptotic cycle. (Hakensson et al 2011).
A study conducted in 2012 also proved that a form of apoptosis occurs in bacteria and
furthered research on the exact sequence. In eukaryotes, caspase proteins are the initiators
and regulators of the apoptotic pathways. These proteins function proteolytically, lysing
proteins, in many protein targets such as metabolic, structural, and DNA repair proteins. While
some distant sequence based cousins of caspase like proteins have been identified in various
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unicellular organisms, bacterial orthologs of these proteins are still under research to be
identified via their DNA sequences. The study attempted to determine if orthologs of caspases
existed in bacteria by looking for bacterial proteins that could bind synthetic caspase substrate
peptides after the bacteria had been subjected to antibacterial treatment which would induce
apoptosis in the cell. The study was conducted on E. coli. After treatment with norflaxacin,
ampicillin and other stimuli, a bacterial protein was expressed caspase like substrate specificity.
The researchers identified the proteins with affinity chromatography, and mass spectrometry of
the MMC-treated cells (Dwyer et al. 2012). This study proved that bacterial cells undergo a
programmed cell death process that is similar to apoptosis.
Genes involved in bacterial apoptosis:
A study performed in 2012 discovered that RecA, which acted as a multifunctional
regulator, was the most probable identity of an unidentified protein that was present in large
quantities in E. coli cells that have undergone apoptosis. RecA has the ability to bind caspase
substrates. A link had to be established between this protein and the proteolysis that causes
apoptosis in bacteria. The researchers discovered that in E. coli, proteins are lysed by Lon and
FtsH polypeptides and ClpP and HsIV complexes. The researchers set out to determine which of
these enzymes would result in the trademarks of apoptosis, chromatin condensation, DNA
fragmentation, and phosphotidylserine exposure. They determined that when ClpP was present
in the cell, it was the most consistent with apoptotic markers. The researchers then
experimented to prove that RecA was responsible for DNA fragmentation, an apoptotic marker
by observing that when a cell did not have RecA activity because of gene suppression, it did not
react apoptotically to antibiotic treatment. There was a decrease of approximately 80% of
these cells, proving that RecA is pivotal in apoptosis of bacteria. Also, those cells that had DNA
fragmentation were very efficient at producing hydroxide ions in response to norflaxacin
treatment, further proving that the cell was committing apoptosis in response to the
antibiotics. To summarize their study, RecA was identified as a key participant in the apoptotic
response. Also, the ClpXP protease complex which is responsive to stress was identified as an
apoptotic enzyme. A further protein, the SOS stress response regulon, was identified to
produce phenotypic apoptotic markers. These three proteins interact to bring about
physiological changes when the bacteria experiences stress. The ClpXP protein acts as a
regulator of RecA and had been shown to reshape cellular proteomes after DNA damage. The
study revealed that these three proteins will act together to change the cell’s action when the
cell is too stressed by regulating the function of target proteins that are involved in the
apoptotic pathway. (Dwyer et al. 2012)
In another experiment, researchers conducted studies on penicillin resistant bacteria.
These bacteria had mutations of the gene in a psa locus that was not fully understood.
However, these bacteria, E. faecalis notably had a mutation in the gene vncS (Novak 1998).
These bacteria were resistant to many antibiotics that specifically targeted the cell wall, certain
enzymes, and the ribosomes. This shows that the gene vncS plays a role in apoptosis if a
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defective vncS gene will resist apoptosis. It may also play a role in apoptotic signaling that
affects the cell wall, those proteins, and the ribosomes. In studies done on S. pneumonia,
mutations of the autolysin LytA had tolerances to penicillin (Wells 1998). Again, this may prove
that LytA plays a role in the inducement of apoptosis. Each bacterial species has a specific
genetic pathway to programmed cell death so trials must be conducted with each species of
bacteria to fully identify the genes and expressed proteins that regulate autolysis. This will
require extensive research and experimental trials.
Researchers have further clarified the pathway that a bacterial cell takes to
programmed cell death. Evidence suggests that there are two main pathways to programmed
cell death in bacteria. The first is when bactericidal stress in induced. DNA damage occurs. RecA
is activated by ClpXP and RecA mRNA from an inactive form to an active form. Once activated,
RecA leads the cell to the execution phase of apoptosis and the DNA fragments, the chromatin
condenses, the membrane loses strength, and the phosphotidyserine is exposed on the outer
surface of the cell membrane. The other pathway is when there are multiple stresses are
involved which include DNA damage, excessive heat, oxidative stress from free radicals,
nutrient starvation, or a viral infection. In this case, ClpXP activates the Extracellular Death
Factor or EDF. The EDF then stimulates the MazF protein. There is some controversy what
happens at this point. Research suggests that the pathway proceeds directly to apoptosis with
all the requisite characteristics of cell death. There is also a school of thought that MazF
stimulates RecA mRNA which activates RecA which then activates apoptosis. To clarify, the
controversy is if MazF or RecA is the final executor of apoptosis (Carmona-Guiterrez, Kroemer,
and Madeo 2012)
Apoptotic Stimulators:
Bacterial cells have been shown by experimentation to commit to apoptosis in response
to bactericidal antibiotics, such as ampicillin and norflaxacilin, or any of the eukaryotic
apoptotic stimuli, which include chemicals, hypoxia, and starvation, or UV radiation treatment
(Dwyer et al. 2012).
Purpose of Apoptosis in bacteria:
The original discovery of apoptosis was made when research was conducted on the
development of cells in C. elegans. There, it was discovered that apoptosis was a crucial stage in
ontogenesis. As a parallel to this in a bacterial format, the autolysis of certain cells is a crucial
stage in the developmental process in a few select species of bacteria. For example, in Bacillus
subtilis, a sporulating species of bacteria, the sporangium is actively lysed before the release of
the spore. Three autolysin hormones have been discovered that contribute to the lysis of the
sporangium. CwlB is the most important autolysin that is produced at the end of the log phase
of the bacterial growth curve. However, these autolysins are not enough for the lysis of the
sporangium. It requires an additional, undefined factor that would function as an autolysin
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activator. CwlB would, as a matter of course, exterminate all cells, both the mother and the
spore cells, at the end of the log phase. After the spore is formed, an additional factor is
released that activates the autolysins to target only the mother cell. (Lewis 2000). The autolysis
in this situation is the best understood of bacterial apoptosis as this phenomenon is currently
under extensive research. It seems intuitive that the reason that a sporangium conducts
apoptosis is to rid the developing spore of the constrictive surrounding barrier. However,
Professor Lewis theorizes that the reason that the mother cell self-destructs during the
germination of the spore is to provide nutrients for the other bacteria in the environment so
that they too can begin the process of sporulation. Sporulation is the process by which certain
bacteria, commonly bacilli, will form an inner spore that contains the nucleoid and ribosomes
surrounded by protective coverings of peptidoglycan and proteins. Sporulation usually occurs in
the situation of extreme stress to the bacteria, such as periods of an extreme lack of nutrition,
an excess or lack of heat, radiation, chemical disinfection, or desiccation. In this theory, the
mother cell will provide for other nearby cells to undergo sporulation by breaking herself down
into nutrients to provide for this process. However, certain bacteria act in a completely
opposite manner. In experimentation with the bacteria Fibrobacter succinogenes, normally
lysed a significant portion of cells when it was involved in a period of logarithmic growth.
However, when it was deprived of nutrients, it automatically secreted proteins to inhibit
autolysis (Wells, Russell 1996). This bacteria, when it was deprived of nutrients, did not break
down altruistically to provide nutrients for neighboring cells as B. subtilis did (Lewis 2000).
Another example of apoptosis in bacteria is the development of formation and
sporulation that was observed in Myxococcus bacteria. Fatty acids called autocides “induce
autolysis in dense cultures of Myxococcus Xanthus and are required for normal fruiting body
development and sporulation (Lewis 2000). This process is not well understood.
One more example quoted by Professor Lewis is that when cells lyse by bacterial
apoptosis, their released DNA is absorbed by other DNA. This process is called transformation
and it is one of the ways that bacteria conduct genetic recombination (Lewis 2000). The gene
identified for S. pneumoniae’s autolysin is lytA which is actually very close to the gene recA
which mediates “homologous recombination with the incoming DNA” (Mortier Barriere 1998)
Why would a single celled bacteria posses the potential for suicide? Many bacteria
develop into complex relationships with many other cells of the same species or similar species,
forming diverse and composite complexes, such as biofilms, that closely resemble multicellular
organisms. Further research also suggests that programmed cell death in prokaryotes evolved
as a means of destroying defective cells that would be harmful to the rest of the colony. For
example, those under the effects of a strong antibiotic or those infected by a viral
bacteriophage. This would ensure that those bacterial cells with damaged DNA would not
survive to perpetuate a damaged and defective species. Or, “In the case of serious damage by
toxic factors, cells will donate their nutrients to their neighbors instead of draining resources
from their kin in a futile attempt to repair themselves.”(Lewis 2000).
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Programmed cell death is most commonly seen when a cell is exposed to antibiotics.
The antibiotic induces the bacterial cell to begin autolysis of the cell wall. This is a process of
self-digestion of the cell wall by peptidoglycan hydrolases that are called autolysins. When the
cell originally builds the cell wall, it must simultaneously synthesize and hydrolyze
peptidoglycan strands. Autolysins are involved in this activity. Therefore, this proves that some
form of programmed cell death is involved in the development of the bacterial cell (Lewis 2000)
Clinical applications:
There exist some cells that are defective in the sense that they do not posess the
potential to perform programmed cell death. In the introduction of antibiotics which attempt to
trigger apoptosis in the bacterial cell, the bacteria will remain functional. This may play a role in
the development of antibacterial resistant bacterial strains that are such a dangerous
component in infection, especially in hospitals where there is an abundance of bacteria
undergoing antibiotic treatment. It appears as though the treatment of these hidden bacterial
mutants is what causes the MRSA and Staph infections that are so feared in hospitals. (Lewis
2000). Another factor in bacterial resistance to antibiotics may arise from a fact stated above.
Previously stated was the questions of how programed cell death would be a logical idea for a
single celled organism. The rationale was that bacterial cells form complexes with many other
cells and function as multicellular organisms. However, if the entire colony is exposed to a toxic
agent, how will a mass suicide benefit the colony? Therefore, the phenomenon of drug
resistant bacteria has arisen when individual bacterial cells begin to develop tolerance to the
antibiotic. If enough bacterial cells develop this tolerance, entire classes of antibiotics will be
rendered obsolete as bacteria will no longer be affected.
In fact, there is significant evidence of this occurrence. In a study done, 15 years ago, the
emergence of antibiotic resistant bacteria had just emerged. An experiment was conducted on
a library of pneumococcal mutants. The study revealed that in a library of these bacteria, 17
individuals failed to die with the introduction of penicillin, which meant that they had
developed a tolerance to antibiotics. Further experimentation on these mutants revealed that
the vncS gene had mutated, which produced a “loss of function of the VncS histidine kinase of a
two component sensor-regulation systemin S. pneumonia produced tolerance to vancomycin
and other classes of antibiotic” (Novak 1999). This class of antibiotic works by inhibiting cell wall
synthesis. It accomplishes this by “relying on activation of bacterially encoded death effectors”
(Novak 1999). This shows a link between bacterial gene mutation and resistance to antibiotics.
This study was particularly alarming because these bacteria displayed resistance to vancomycin
which is the “last resort” antibiotic. An article from last year confirms clinician’s concerns over
vancomycin resistant bacteria. A solution has not been discovered as of yet. However, the
article reported an experiment that the researchers had conducted to the purpose of furthering
research on the resistance to antibiotics. They introduced vancomycin to methicillin resistant
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bacteria and closely monitored the metabolic activity. The results were that these bacteria
displayed an “upregulated capsular gene expression”. This overproduction of the capsule
proteins may play a role in protecting the bacterial cell from the effects of the antibiotic. The
researchers offered a clinical application of these results. They suggest that “selected genes of
the capsule locus could be used as diagnostic targets for monitoring patients undergoing
treatment with vancomycin therapy, as an increase in their expression may indicate progressive
development of low-level resistance” (Awad et al 2013). This means that when these genes are
detected in a bacterial infection, the bacteria may possess antibiotic resistance. With this
technology, clinicians can treat accordingly.
Conclusion:
In conclusion, bacterial cells seemto posess the potential to carry out a programmed
cell death cycle similar to apoptosis in eukaryotic cells. The genes that are involved in these
autolytic mechanisms are still under research but a clear apoptotic pathway has been identified
for bacteria. Possible purposes of this apoptotic cycle could include the altruistic suicide of a
diseased cell to protect the colony, a vegetative cell breaking itself up to provide nutrition for
its newly emerged spore, or to aid in the process of bacterial transformation to the goal of
genetic recombination. Most research currently conducted deals with the newly emerged issue
of antibiotic resistant bacteria. Research into this topic shows potential to deepen our
understanding of the bacterial cell life cycle as well as contribute to important clinical
applications in bactericidal compounds and drug resistant bacteria.
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Awad, S., Alharbi, A. E., Alshami, I., “Exposure of Vancomycin sensitive Staphylococcus aureus
to subinhibitory levels of vancomycin leads to upregulated capsular gene expression”
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Ancestral Apoptotic Network in Bacteria”, Molecular Cell 46.5 8 June 2012 ,552-554
Web. 8 April 2014.
Dwyer, Daniel J., Camacho, Diogo M., Kohanski, Michael A. , Callura, Jarred M., Collins, James J.,
“Antibiotic Induced Bacterial Cell Death Exhibits Physiological and Biochemical
Hallmarks of Apoptosis”, Molecular Cell 46.5 8 June 2012 561-572, Web. 12 April, 2014.
Elmore Susan, “Apoptosis: A Review of Programmed Cell Death” Toxicologic Pathology 35.4
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Hakansson, Anders P., Roche-Hakanson, Hazeline, Mossberg, Ann-Kristin, Svanborg, Catharina,
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Complex” PLoS ONE 6.3 10 March 2011, Web. 13 April 2014.
Kerr, John F., Wyllie, A.H., Currie, A.R., “Apoptosis: A Basic Biological Phenomenon with Wide
Ranging Implications in Tissue Kinetics”, The National Center for Biotechnology
Information, The British Journal of Cancer 26. Pg. 239 April 1972, Web. 8 April 2014.
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RecA is required for Full recombination Proficiency during Transformation in
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