Bacto-Blood: A Bacterial
Blood Substitute
Austin Day, Josh Kittleson, and Lane Weaver
Abstract
Surgeries, severe injuries, chronic diseases, and a host of other conditions
generate a strong demand for blood transfusions worldwide. However, the current
system of relying on blood donations is fraught with difficulties, including a fluctuating
supply, potential contamination, and high expense. In developing countries, these
problems are particularly exacerbated, creating an environment where lives are
needlessly lost due to insufficient blood supplies. Although a range of blood substitutes
are currently being developed, none has emerged as a clear winner. To fill this vacuum,
and to develop a platform for additional therapeutic applications, we propose using E.
coli expressing human hemoglobin as a blood substitute. Although this approach faces a
variety of unique challenges, each can be overcome to ultimately yield a limitless supply
of blood substitute. Sufficient oxygen transporting capacity will be established by
controlled expression of both hemoglobin subunits, supported by a small array of
auxiliary proteins. Aversion of a fatal septic response to injected bacteria will be
accomplished by altering the structure of the offending molecule, while immune system
clearance will be ameliorated by encapsulation in a thick layer of polysaccharide.
Converting the cells into passive oxygen carriers, either through destruction of their DNA
or direct suppression of their ability to replicate, will prevent unintended infection.
Finally, the shelf life of transfusion-ready cells will be extended through exploration of
traditional preservation methods such as desiccation. Ultimately, the developed bacteria
will be tested in animal models to provide confidence that they will function as designed
in their final target, the human blood stream. Although the process of taking this concept
from mere idea to clinical reality could take many years, a prototype may be developed in
just a few months. Progress in this arena promises not only to aid millions of people, but
also to drive our understanding of bacterial systems, the human immune response, and
the role of erythrocytes forward in the years to come.
Part I: Motivation and Goal
Motivation
The global demand and importance for cheap, available, and disease free blood
substitutes is undisputed. Battlefield trauma, road traffic accidents, and blood loss during
surgery all currently require blood transfusions for successful treatment. However, there
are currently no red blood cell substitutes approved for clinical use in the US or the UK
and whole blood is almost always in short supply. 1
The global collection of blood per year is approximately 81 million units. However, only
27 million of these units are collected in low and medium income countries where 82%
of the world’s population lives. 2 The frequency of blood donation in such
underdeveloped countries has been reported to be less than 1% of the entire population. 3
To complicate matters even more, a significant portion of that population are disease
carriers.
As a result, cheap, effective red blood substitutes will provide the most benefit to
the populations of developing nations. The reality of the situation is that inexpensive
blood substitutes, even those that don’t meet the safety standards of blood substitutes in
the US and the UK, will allow patients to receive treatments ordinarily requiring blood
transfusion, and could dramatically improve the health in many developing countries. 4
Blood shortages are frequently reported even in the developed world and are due
primarily to the unpredictable nature of emergency situations, as well as the inconsistent
frequency of blood donors due to seasonal variation, holidays, and religious festivals. 5
To make matters worse, donated red blood cells only have a shelf life of about 35 days. 6
The health of the developed world can be improved by the introduction of a safe
blood substitute as well. The optimal transfusion is given to a patient before it is needed.
Presently, doctors might exercise restraint in giving patients a transfusion because there
are currently no quantitative guidelines for when a transfusion is needed and the doctors
must rely on somewhat of a “gut” instinct. Also, blood is expensive, has risks of disease
transmission or immune rejection7, and is usually in short supply. If doctors were able to
exercise less restraint due to cheaper and safer blood substitutes, that in itself could save
lives. 8
Goal
The ideal red blood cell substitute must satisfy the following criteria: It must
deliver oxygen efficiently. It must cause no undesirable side effects. It must require no
compatibility testing. It must remain stable during prolonged storage. It must persist in
circulation. It must be easily reconstituted, and it must be available at a reasonable cost. 9
Our project goal is meet all of the criteria for an ideal blood substitute using a
hemoglobin producing bacterium as the basis of a red blood cell substitute. By
expressing mutant human hemoglobins within E. Coli, we can achieve oxygen transport
with similar characteristics to real human red blood cells. The expression of the bacterial
K1 capsule and the O16 antigen will help to protect the bacterium from the immune
system to increase half life. This capsule combined with a modification to the lipid A
component of the lipopolysaccharide (LPS) layer will reduce sepsis, a fatal over-stimulus
of inflammatory responses. Such an artificial red blood cell would not require typing,
would be relatively inexpensive, and free from the possibility of disease transmission.
A red blood cell substitute made from E. Coli would require a bacterial chassis
which would not induce sepsis, even at high concentrations. Once this hurdle is
overcome, the possibilities of in vivo therapeutic bacteria applications become more
realistic. In a sense, a safe bacterial chassis would provide the much needed stepping
stone towards more advanced therapeutic bacteria applications. One such application
currently being researched is tumor killing bacteria, 10 but other projects such as artificial,
fast acting platelets, bacterial sensing, production, and delivery of protein therapeutics,
artery cleaning bacteria, toxin eliminating bacteria, and bacterial extensions of the human
immune system might follow. One might even envision potential near term applications
involving a sepsis free bacterial chassis with enzymes capable of breaking down or
sequestering toxins. However, the most immediate applications of a successful artificial
blood substitute include not only the obvious ones such as resuscitation and surgery, but
also ischemic disease, angioplasty, extracorporeal organ perfusion, cell culture media,
hematopoietic stimulation, cardioplegia, sickle-cell anemia, tumor therapy, and chronic
anemia. 11
Part II: Background
Cell-Free Hemoglobin Blood Substitutes
Because of the importance and need for blood transfusions in many situations,
there are many different substances currently under research as blood substitutes. Cell
free hemoglobin based oxygen carriers are solutions of structurally modified human
hemoglobin. The modifications aim to address the problems which occur when you take
the hemoglobin outside of the protective environment of the red blood cell. Due to being
suspended in solution, hemoglobin based blood substitutes are capable of reaching
capillaries that normal red blood cells have a hard time reaching, thus increasing the
ability to oxygenate tissues. 12 The hemoglobin is either extracted from animals or
produced in E. Coli cultures, and has potential to be readily available. 13 Hemoglobin
based substitutes degrade due to autoxidation, but it has also been suggested that
hemoglobin based blood substitutes can be stored for up to 36 months.14 The half life of
modified hemoglobin has been reported to be around 12-48 hours. 15
The first generation cell free hemoglobin based blood substitutes were riddled
with vasoconstriction problems, toxicity issues, oxidative damage issues, and binding
affinity mismatching. These problems are all currently being addressed through protein
structure engineering. 16 17 18
The primary mechanism thought to account for vasoconstriction is the free hemoglobin
binding and scavenging nitric oxide from the nearby environment of the blood vessels.
Nitric oxide is a well known stimulant of arterial relaxation. Many types of recombinant
hemoglobin mutants are being researched to correct this problem by reducing the
hemoglobin’s ability to bind nitric oxide, while leaving the ability to bind oxygen or
carbon dioxide intact. 19
Toxicity issues arise due to the alpha and beta subunits of hemoglobin
dissociating in dilute solutions, like how they are when suspended in free solutions. The
small size of the individual subunits of hemoglobin cause rapid uptake by the renal
system and can cause long term damage to the kidneys. Current research has concentrated
on cross linking the alpha and beta subunits of hemoglobin to decrease their dissociation,
and thus reduce their toxicity. 20
The oxidative damage caused by cell free hemoglobin blood substitutes is due to
autoxidation. 21 Hemoglobin, when outside its protective environment, undergoes
unhindered oxidation of its iron center. Spontaneous oxidation of the ferrous/oxy
derivative (HbFe2+) leads to non-functional ferric heme (HbFe3+) and a superoxide ion
(O2*), which subsequently dismutates to generate hydrogen peroxide, which can damage
the protein and/or the heme group itself. 22 There have been attempts to remedy this issue
either by conjugating antioxidant enzymes such as catalase and superoxide dismutase
onto the hemoglobin23, or to create mutants with inherently lower ability to autoxidize. 24
Because there are no free hemoglobin based blood substitutes currently on the
market, 25 the cost of them can’t be accurately determined. However, Robert Winslow, a
prominent figure among artificial blood substitutes field mentioned in his book that the
cost of hemoglobin based oxygen carriers is “probably high.” 26
Encapsulated Hemoglobin
Another potential blood substitute involves encapsulating free hemoglobin within
liposomes. Liposomes are synthetic lipid derivatives of poly(ethylene glycol) (PEG)
which are used to encapsulate high concentrations of mutant hemoglobin. The idea
behind encapsulation is that it would help to separate the hemoglobin from the
environment, similar to the function of a real erythrocyte membrane, and would thus
prevent dissociation of hemoglobin dimers and the subsequent renal toxicity. They are
considered the closest technology to creating artificial red blood cells. These liposomes
come in a range of sizes from less than 60nm to greater than 200nm. 27 Due to the small
size of these liposomes, they also can reach areas of the circulation that red blood cells
may have a hard time reaching, and similarly to cell free hemoglobin, they may also
enhance oxygen transport.
The size of the liposome greatly determines its half life. The ideal liposome would
be as large as possible, while still retaining a PEG-mediated prolonged circulation half
life. By using charged lipids, researchers can enhance the encapsulation efficiency;
however, by doing this, they may be encouraging undesirable interactions with other
circulating proteins which can cause their rapid uptake by the reticulendothelial system
(RES). 28 There have been a few problems with toxicity issues as well, involving
pulmonary hypertension and other hypersensitivity reactions. 29 The manufacturing
technologies which create these encapsulations are currently high cost and have
difficulties in producing consistently sized particles. 30 This lack of large scale production
technology is the primary downfall of encapsulated hemoglobin.
The liposome encapsulation has been proven to be devoid of the vasoconstrictive
effects that are commonly seen with cell free hemoglobin substitutes. 31 Researchers have
obtained concentrations greater than 36g/dl within these liposome encapsulations. This is
significant compared to regular adult plasma, which contains approximately 15 g/dl.32
This artificial membrane also provides the potential to co-encapsulate allosteric modifiers
and antioxidants, to even more closely mimic a red blood cell in oxygen binding affinity,
as well as repression of reactive oxygen species. 33 The half life of the liposome particles
has been estimated to be about 42 hours34 and the shelf life estimated to be about 3-6
months. 35
Perfluorocarbon Emulsions
The most advanced, non-hemoglobin based blood substitutes are perfluorocarbon
emulsions. Perfluorocarbons are basically hydrocarbons with fluorine atoms in place of
hydrogens. They are chemically inert and appear as a clear liquid that readily dissolves
many gases, including oxygen and carbon dioxide. 36 Because these perfluorocarbons are
immiscible with water, they are emulsified using egg phospholipids. These
perfluorocarbon products are the most advanced in terms of FDA approval. OxygentTM,
made by Alliance Pharmaceuticals Corp, is already FDA approved and is being used with
a combination of volume expanders and hemoglobin based substitutes on a small scale. 37
The safety profiles of such perfluorocarbon emulsions aren’t published in the scientific
literature, but they form the basis to allow for clinical testing. 38 Perfluorocarbon
emulsions currently have no known metabolism in the human body and are cleared
through exhalation by the lungs or taken up by the reticuloendothelial system. 39 40 41
With droplet sizes of less than .2 µm 42, perfluorocarbon emulsions also show the same
enhanced oxygen transport over red blood cells as free hemoglobin and liposome
encapsulated blood substitutes do. 43
However, the gases aren’t bound to the perfluorocarbon chains like how they are
in hemoglobin. Gases are simply dissolved loosely in the liquid. An important benefit
arises because of this property. Perfluorocarbons have been shown to augment local
oxygen delivery much more than would be expected from the increase in oxygen content
in arterial blood. 44 The oxygen in the perfluorocarbons is taken up preferentially over
bound oxygen in hemoglobin based blood substitutes or even natural red blood cells. The
primary issue with perfluorocarbon emulsions is that the oxygen dissociation curve is
linear, as opposed to the sigmoidal curve seen for hemoglobin.
Figure 1: 45 Oxygen dissociation curves for cell-free hemoglobin and for the perfluorocarbon emulsion
OxygentTM. The linear curve of the perfluorocarbons is due to gases dissolving into the solution, as opposed
to being bound to a molecule. For any given partial pressure, hemoglobin will have higher oxygen content
than perfluorocarbon emulsions.
This linear oxygen dissociation curve for perfluorocarbons results in lower oxygen
capacities than hemoglobin based substitutes.
The half life of these perfluorocarbon emulsions are also shorter than the ideal
blood substitute, being about 9.4 hours. 46 However, it should also be noted that due to
their small sizes, it may take up to 2 years to fully clear the particles from the system.
Whole Blood
The most widely used substance for transfusion and resuscitation is still donated
human blood. The obvious benefit of whole blood transfusions is that you are replacing
lost blood with real blood, so there are not worries with oxygen binding affinities or
toxicity. If you plan an autologous transfusion for a planned surgery, the success rate is
very good. But more often, one must rely on donated blood, which introduces many
possible complications.
There are many things to consider when using donated blood. In developed
countries, there are many safeguards in place to prevent disease transmission. There is
sensitive screening prior to recruitment, disease testing, and post-donation product
quarantine. But even with these safeguards, disease transmission may still occur due to
human error or inadequate testing. Also, in developing countries, these safeguards aren’t
financially practical. Globally, over 13 million blood donations are not tested for human
immunodeficiency virus, hepatitis B, or hepatitis C, 47 and 39 countries reported issuing
some blood and blood components without testing for transfusion-transmissible
infections, owing to interruptions to supplies of test kits. 48
Disease testing procedures, along with pre-donation information literature,
deferral procedures, and donor tracing, notification when instances of disease
transmission are detected, storage, and shortages greatly increase the cost of whole blood.
The cost of a single unit of whole blood is approximately $373. 49 With specialized
procedures, the cost can reach upwards of $500 a unit.
Immune rejection is also a notable risk with blood transfusions. Human error in serotype
choice is responsible for 2.2% of all fatal transfusion reactions. 50 There have been
successes in blood type conversion technology recently by using bacterial glycosidases to
cleave the A and B antigens from erythrocytes.51 This is an important advance because
hospitals still only allow the use of blood type O in emergency situations.
If the possibility of disease transmission, immune rejection, and a high cost
wasn’t enough, there is also the reality that red blood cells have a shelf life of about 35
days, and rely on regular donation to ensure an adequate supply. 52
Bacterial Blood Substitute
After considering the competition, a bacteria blood substitute introduces a
different set of problems for a blood substitute, the main issues being the half life and
control of sepsis. Even though these are limiting issues, they are unique limits in the
artificial blood field and provide another route by which an artificial blood substitute may
be developed. The remainder of the proposal will cover the details and planning involved
in building a bacterial blood substitute from a typical lab strain of bacteria.
Part III: Hemoglobin Expression
The first step in achieving a bacterial blood substitute is the expression of an
oxygen carrier within the bacterium. There are a few issues to consider when choosing an
oxygen carrier. Adult human hemoglobin seems like the most likely candidate because it
was made for this purpose, but free hemoglobin has a significantly lower p50 value
(partial pressure of oxygen at which 50% of the hemoglobin is saturated) than
erythrocytes, primarily due to the absence of an allosteric modifier, 2,3diphosphoglycerate (DPG). 53 It was reported that the p50 is directly proportional to the
amount of delivered oxygen. A p50 value which is too low will not deliver sufficient
oxygen.
There are two promising solutions to this problem. The first solution is to simply
express the eukaryotic enzyme, DPG mutase, in the bacterium so that it can create the
missing 2,3-DPG from the glycolysis pathway and hopefully correct the p50 value an
appreciable amount. This enzyme has already been expressed in E. coli, making this a
possibly quick solution. 54 Another way around this problem may be by the introduction
of mutations within the globin subunits themselves. There are various mutations in the
literature that increase p50, decrease auto-oxidation rates, and increase tetramer stability.
One mutation is termed a “Presbyterian mutation”, and involves the change of Asn-108
Lys in the β-subunit of hemoglobin. It was reported to create a mutant with decreased
oxygen binding affinity (increased p50) to a level comparable to that of native
hemoglobin bound to DPG in erythrocytes. 55 Chien Ho came across a set of mutations
capable of reducing the auto-oxidation of hemoglobin as well as increasing the p50. 56
Another issue is the level of soluble hemoglobin expression. The highest reported
amount of soluble hemoglobin expressed in E. coli found was .64g/dL.57 This is
significantly different than the ~15g/dL found in human blood. 58 The .64g/dL amount
was expressed in the present of three fold molar excess of heme, on a high copy number
plasmid, and used E. coli codon optimized synthetic hemoglobin genes. It should be
noted that in addition to the soluble hemoglobin, there was twice as much insoluble
hemoglobin produced. This may suggest room for improvement. We suggest a few tricks
that may be attempted to improve this estimate.
One method may be to increase the heme production from the endogenous
biosynthetic pathway. 59 This may seem redundant due to the excess of heme outside of
the cell, but it has been suggested that the hemoglobin subunits are stabilized by the
insertion of heme, and if the uptake of heme by the cell were limiting in any way, it may
cause less soluble protein to form. 60 61
It is mentioned that the alpha subunit of hemoglobin is unstable on its own in
solution and needs to either be incorporated quickly into a tetrameric hemoglobin, or it
will aggregate and precipitate. To aid in the proper folding of these subunits, coexpression of the alpha and beta subunits on the same cistron has been shown to improve
yield. 62
Another issue to consider is the presence of the extra N-terminal methionine
residue due to the protein being expressed in E. Coli. This extra residue affects the
binding affinity of the hemoglobin and changes its final conformation. 63 64 65 This may
be significant not only because it affects the oxygen binding affinity, but also because it
might be hindering the solubility of the protein, decreasing its yield. To correct this
problem, the genes for E. Coli methionine aminopeptidase (Met-AP) can be
overexpressed, which will cleave the extra methionine residue. 66
There also may be an issue with auto-oxidation of our heme centers. It is hard to
predict the rate of auto-oxidation of the hemoglobin mutants we want to use, but there
will most likely be some significant amount of auto-oxidation. It is most likely sufficient
to simply express the natural erythrocyte anti-oxidant enzymes catalase, superoxide
dismutase, and possibly a methemoglobin reductase enzyme, to prevent damage to the
heme centers of our hemoglobin.
We suspect that other factors which haven’t been extensively studied, such as the
expression rates, temperature, various other combinations of mutations, and the presence
of stabilizing chaperone proteins67 may also play important roles in the expression level
of soluble hemoglobin and may provide possible routes for future exploration. However,
the system we suggest for an initial trial would include a vector that co-expresses the
alpha and beta mutant human hemoglobin A, methionine aminopeptidase, catalase,
superoxide dismutase, methemoglobin reductase, AHSP (alpha Hb stabilizing protein),
and extra genes that encode for bottleneck molecules in the heme biosynthetic pathway.
Part IV: Bacterial Chassis
The first challenge for deliberate introduction of hemoglobin totting bacteria into
the bloodstream is development of a bacterial coat that minimizes immune response,
clearance rate, and risk of opportunistic infection. One of the primary concerns of
delivering a high load of bacteria intravenously is causing sepsis, which is an often fatal
overreaction of the immune system to bacteria and bacterial components68. In order to be
useful as a blood substitute, the bacteria need to remain in circulation for at least several
hours69. However, a normal immune system is capable of clearing the vast majority
(>90%) of bacteria in under an hour70. While immune response suppression may mitigate
both of these concerns, significantly weakening a patient’s immune defense for hours or
days risks opportunistic infection. Fortunately, the synthetic biology approach offers a
way to navigate these challenges: selection of an appropriate chassis, removal or
alteration of known surface antigens, and addition of a protective covering should do the
trick.
Selection of a Chassis
When considering what bacterial coat to use in a blood substitute, the first major
question to explore is whether gram positive or gram negative bacteria make more sense.
As illustrated in figure 2, there are several gross morphological differences between the
two types of bacteria. Where gram positive bacteria have an inner membrane surrounded
by a thick outer cell wall composed of petidoglycans, gram negative bacteria have an
inner membrane, a thin layer of peptidoglycan, and an outer membrane sporting an
asymmetric inner and outer leaflet. Both types of bacteria may further have an outer
coating, called a capsule. A rough measure of the relative ability of each type of bacteria
to survive in the blood can be garnered from the fact that the majority of bacteremia is
caused by gram positive bacteria71. However, they are also the leading cause of sepsis72.
Thus, a more detailed biochemical analysis of each cell type in the context of the immune
system is needed to determine which of the two provides a better option.
Figure 273 Differences between gram negative and gram positive cell envelope.
An in-depth examination of how gram positive bacteria survive so well in our
blood reveals a multi-faceted defense system. Group A Streptococcus inhibits
macrophage recruitment, blocks opsonization (biochemical labeling by the immune
system), prevents phagocytosis, resists phagocytotic killing mechanisms, and kills
macrophages74. Because the goal is to minimize the negative impact on the immune
system of injecting bacteria, only relatively unaggressive mechanisms associated with the
cell surface would be of use. Of the listed mechanisms, only a few are a consequence of
the cell surface and surface associate proteins: a hyaluronic acid capsule and a protein
called protein M that inhibits opsonization. Similarly, Staphylococcus aureus employs a
broad range of strategies to avoid the immune system75. Those relevant to the cell
surface are a protein (protein A) that blocks specific antibodies, a protein (ClfA) that
blocks opsonization by binding fibrinogen, and several modifications to the cell envelope
structure that make it resistant to both anti-microbial peptides and lysozyme. However,
in all of these cases, the peptidoglycan structure of the cell wall is left largely unaffected.
Since a critical component of the cell wall, lipotechoic acid, is recognized by a Toll-like
receptor, all gram positive bacteria are potent activators of sepsis76. Thus it seems
difficult to justify the use of gram positive bacteria.
Gram negative bacteria similarly rely on a capsule for protection, as well as a
variety of secreted proteins77. Also like gram positive bacteria, a core component of the
cell envelope, lipopolysaccharide (LPS, also known as endotoxin), induces a strong
immune system response, potentially leading to sepsis78. However, unlike in gram
positive bacteria, methods for reducing the toxicity of LPS have been described79.
Exploitation of this (and other) discoveries will be described in further detail below.
Given that gram-negative bacteria are a better choice for synthetic blood, the
question of what specific organism to use remains. Because of its loss of undesirable
virulence factors, ease of growth and manipulation, and well studied genetics, everyone’s
favorite lab strain of E. coli (E. coli K-12 MG1655) presents a very reason choice.
Removal of Remaining Unwanted Structures
Although lab strains of E. coli generally lack virulence factors, they are still
capable of creating structures that can contribute to virulence and be recognized by the
immune system. Specifically, they can create fimbriae (pili) and flagella80,81. Because
fimbriae are involved in adhesion and localization and flagella are involved in motility,
and ideally a blood substitute would neither stick to anything nor move of its own accord,
it seems to make sense to knock out the genes responsible in the E coli chassis being
developed. The fimbriae of E. coli have been extremely well studied, and as such, the
genes responsible for both their structure and formation have been identified82. Deletion
of all of the fim genes will be accomplished by well-established genomic deletion
methods83,84,85. The flagella of E. coli have also been extensively studied, and the
structural and regulatory genes involved identified86. Rather than attempting to remove
all of the genes involved, deletion of the master activators of the flagellum, flhD and
flhC87, by standard methods will ensure that no flagella are expressed.
Reducing Sepsis by Altering Lipid A Biosynthesis
Septic shock, as noted above, is a frequently deadly overreaction of the immune
system to components of a pathogen. The routes of activation of septic shock have been
extensively studied88,89, and in the case of gram negative bacteria, the lipid A moiety of
lipopolysaccharide (see Figure 3) is the primary bioactive component responsible for
causing sepsis90. However, several alterations to the canonical lipid A structure have
been identified that reduce the toxicity of lipid A. In experiments using multiple
indicators of septic shock, Zhang and coworkers demonstrated that synthetic lipids with
modifications such as shorter lipid chains, modified phosphorylation patterns, and added
palmitoyl groups had toxicities reduced by as much as 1000-fold91. It has also been noted
that underacylated lipid A molecules only have about 1% the toxicity of the canonical
structure in both in vivo and in vitro models92. Enzymes responsible for these types of
modifications have been identified in natural pathogens and commensal bacteria. In
Salmonella typhimurium, PagL and PagP are responsible for removing one acyl chain
and adding a different palmitoyl group, as illustrated in figure 4. These changes result in
a roughly 100 fold decrease in toxicity93. In E. coli, a mutant was identified with a nonfunctional msbB gene, which was incapable of adding one of the acyl groups to lipid A.
The resulting lipid A precursor, illustrated in figure 5, was originally reported to have a
1,000-10,000 fold reduction in toxicity94. However, further studies more properly
characterized both the MsbB mutant lipid A and another under-acylated form of lipid A
created by Porphyromonas gingivalis as antagonists of the human response to canonical
lipid A, even going so far to suggest their use as a therapeutic95. Finally, recent work has
challenged the minimal structure of lipid A needed for bacterial viability. Meredith and
coworkers isolated a viable mutant defective in an early part of the lipid A biosynthesis
process that resulted in synthesis halting at lipid IVa (see figure 6), which also appears to
be an antagonist of the septic response96.
Presented with this variety of mechanisms for reducing lipid A toxicity, the most
straightforward approach is to simply delete msbB from our chassis. A known
complication of the mutation, however, is that it reduces the ability of the bacterium to
effectively express a K-capsule97 (discussed in greater detail below). Since some
evidence suggests that this is due to problematic assembly of the outer membrane98, two
approaches to solving the problem seem viable. First, simple overexpression of the
components of the capsule may overcome the assembly difficulty. As that may equally
well exacerbate the problem, a second approach is to mutate the gene partly responsible
for lipid A transport and assembly, msbA. Such mutations have previously been shown
to rescue defects in lipid A assembly because of upstream synthesis mutations,
presumably because of a shift in transporter specificity toward the precursor rather than
the final lipid A product99.
Figure 3100 Basic Structure of Lipopolysaccharide.
Figure 4101 Modification of lipid A by Salmonella typhimurium
Figure 5102. Lipid A molecule of MsbB E. coli mutant.
Figure 6103. Lipid IVa, Precursor to Lipid A lacking 2 acylations and core connections.
Reducing Clearance through Encapsulation
The initial, rapid clearance of bacteria by the immune system is mediated by the
complement system. This component of the immune response relies on multiple
pathways to opsonize (chemically label) pathogens, as illustrated in figure 7 104. While
the classical complement pathway relies on the presence of pathogen-specific antibodies,
both the lectin and alternative pathways are part of the innate immune response and
recognize pathogens and particulates non-specifically. Opsonization can lead either to
direct killing of cells via a membrane attack complex, or more often, killing by
phagocytosis105.
Figure 6J106 Three Pathways of Complementation Activation. a The classical pathway triggers through
binding of specific antibodies. b The lectin pathway triggers through binding to LPS associated
carbohydrates and other surface molecules c The alternative pathway is triggered by non-specific cleavage
and adhesion of the C3 protein.
Not surprisingly, natural pathogens have evolved a variety of mechanisms for
evading opsonization107. The presence of an outer capsule composed of high molecular
weight polysaccharides, known as the K capsule (see figure 8), can help evade all three
mechanisms of opsonization, and as such, represents a powerful way to prolong the
lifetime of bacteria in the bloodstream. More specifically, a variant of the K-capsule
known as K-1 has been shown to be particularly effective at resisting
complementation108. Because the K-1 capsule contains sialyic acid, a component
expressed on the surface of some human cells, it does not induce a strong specific
response from the immune system109,110. It also prevents non-specific complementation
mechanisms by both hiding underlying cell surface structures and binding to factor H,
which interferes with the signaling cascade necessary for effective complementation111.
The biosynthesis of K capsules in general, and the K-1 capsule in particular, have
been extensively studied112. Figure 9 shows the island needed for K-1 capsule synthesis,
which includes 14 genes113. In order to protect the artificial blood cells, our chassis will
be altered by insertion of this island of genes into the genome using published
techniques114. Should direct manipulation of the entire island prove technically difficult,
serial addition of smaller fragments of the island can be used instead. The efficiency of
the bacteria at expressing K-capsule can be selected for by using serum to kill those that
haven’t developed resistance115.
A
B
Figure 8116 A Cartoon representation of the capsule layer of gram negative bacteria. B Electron micrograph
of the actual K1 capsule of E coli.
Figure 9117 Genes required for K1 capsule synthesis.
Part V: Safety Mechanisms
Safety, of course, must play a paramount role in the development of a surrogate E.
coli blood cell. To effectively address concerns arising from the introduction of a foreign
organism into the blood stream, two strategies are proposed.
Strategy I: Genomic Destruction
The first strategy entails complete destruction of the E. coli genome, prior to
intravenous administration of the substitute blood solution. This provides three benefits:
(1) Without any genetic material to pass on to progeny, the cell cannot reproduce, thus
eliminating the possibility of unchecked growth; (2) the cell is also unable to acquire
mutations which could be passed horizontally (via plasmids) to other cells, abrogating a
primary evolutionary mechanism of pathogenesis; and, finally, (3) without the genetic
machinery necessary to sense and interact with the environment, the cell is much more
likely to play the role of a passive hemoglobin transporter, rather than perturbing the
blood environment and possibly inducing an immune response.
A simple design is proposed to accomplish this goal, as outlined in Figure 10.
Various restriction endonucleases (in this example BamHI, PstI, and EcoRI) are placed
under the control of a tightly repressed promoter (e.g. pBAD/pBAD+fim). Thus, the
default expression from the promoter will be “off,” and the cell will function normally.
However, when induced with arabinose, repression of the promoter will be relieved and
the restriction enzymes will be expressed. To assay genomic destruction, we will place
several reporter genes (with LVA degradation tags for quick response) throughout the
genome. Alternatively (or additionally), we may perform RT-PCR and/or southern blot
analysis on several candidate genes. Using these assays, we will build a small library of
ribosome binding sites placed upstream of our restriction enzymes to obtain expression
levels and subsequent genomic destruction in congruence with timescales relevant to our
experiment.
Figure 10. Strategy for (measuring) genomic destruction
Depending on the results of these experiments, a more radical approach
incorporating Deoxyribonucleases (DNase), which indiscriminately cleave
phosphodiester linkages in DNA, may need to be employed.
However, the structural integrity of a cell (in particular that of its cell wall) in the
absence of DNA is somewhat uncertain. A report linking methylglyoxal (a metabolite
that induces genomic DNA destruction) production to cell viability118 demonstrated that
turbidity of a culture appears to stay constant while its viability decreases (a proxy for
genomic destruction), suggesting that cells may stay intact even after their DNA is
degraded (see Figure 11). However, it should be noted that this was not the main result
of the report and the correlation is far from definitive.
Figure 1: Kinetics of growth inhibition and killing by xylose and camp. Open circles: experimental,
Squares: control.
Even in the presence of DNA, the cell wall is highly dynamic. Cell wall recycling
is a well-established phenomenon that is often exploited in antibiotic mechanisms of
action. These antibiotics, particularly the -lactams, interfere with cell wall recycling,
consequently triggering lysis as a result of osmotic stress. While these antibiotics are
only effective in growing cells, it is difficult to predict how the complete loss of genetic
control might perturb the complex recycling system.
To measure cell-stability, we will induce DNA destruction in a hemoglobinproducing cell via the aforementioned methods and measure structural stability at
multiple time points by centrifuging the cells, running the supernatant on an SDS-PAGE
gel and staining for heme.
Strategy 2: Growth Suppression
If there is found to be hemoglobin leakage from cells, an alternative strategy, in
which the cells are kept viable, but in a quiescent, or non-growing state, may be pursued.
This method has the advantage of utilizing the cells’ natural genetic machinery to keep it
structurally intact, but of course suffers from accompanying risks of introducing a viable
organism into the body. However, recent studies119 in which cells are administered
intravenously to target and destroy tumors, have lent precedent to this approach.
Regulator of Cell Division (Rcd) is a small RNA that, when over-expressed in an
hns– E. coli strain, causes the cell cycle to arrest, resulting in a stable quiescent state120.
Overexpression of Rcd additionally results in an increase in production of plasmid-borne
proteins (in some instances constituting as much as 40% of total cellular protein) for up
to 10 hours. Morphologically, these cells are non-filamentous, and grow on average to
about four times normal E. coli cell length.
Thus, overexpression of Rcd results in a large, non-growing, high proteinproducing cell that is stable for many hours, making it an excellent candidate for a
surrogate RBC.
To create an hns- strain, the -red recombination method can be used. This
background strain would be employed for subsequent capsular and LPS modification
work. In addition, Rcd will be placed under control of a strongly repressed promoter,
such as pCI. When co-transformed with a temperature-sensitive CI repressor protein, this
strain can be grown at permissive ambient temperatures (<30C), but will not grow when
transferred to the bloodstream (>30C)121.
In a therapeutic scenario, these quiescent bacteria would need to be eliminated
after the initial period of emergency has passed. While application of externally
administered antibiotics would be effective clearance agents, it would be desirable to
have a means of control that is specific to the engineered bacteria. CcdB (controller of
cell death protein B) is a cytotoxic killer protein that is used in many current cloning
strategies to decrease background122. This gene can be controlled by a switch-like
system, such as the fim Inversion system, in which there is no leaky expression in the uninduced state123.
Part VI: Storage
The allure of an E. coli based blood substitute is the easy with which it can be
made, transported, and stored, and the low cost of this process. One can envision a
scenario where master culture is periodically used to inoculate a preparative culture that
will be induced to synthesize hemoglobin and subsequently induced to degrade its DNA.
Two alternatives routes of storage prior to administration are then proposed.
In the first, the cells may be desiccated or lyophilized. Cell viability has been
shown to reach levels up to ~80% after being desiccated for timescales of days to
hundreds of years, and industry commonly freeze-dries starter cultures for dairy
products124. Additionally, capsular polysaccharides may also aid in desiccation
tolerance125. Judging from this viability data, it is likely that intracellular proteins are
able to be reversibly dried without major loss of function.
The second option would be to centrifuge the preparative culture, resuspend in a
volume expander, and store as-is prior to administration. Because the main constituent of
the volume expander is albumin, which is stable at room temperature for years126, this
solution may be refrigerated or possibly even stored on the shelf.
Part VII: In Vivo Testing
Following the construction of the requisite E. coli blood cells and several in vitro
preliminary tests (oxygen-delivery, immune response), the ability of the cells to act as a
blood surrogate will be tested in an in vivo blood stream environment.
Protocols from previous studies will be adapted for this purpose. Hemoglobin
containing vesicles (HbV) are the most analogous blood substitute to E. coli cells, thus its
protocols can be employed for these studies127 128.
The E. coli cells will be reconstituted in a human albumin (HSA, 25%) and saline
solution to regulate the colloid osmotic pressure similar to that in blood vessels
(~40mmHg) Data from Hemoglobin expression studies will be used to match hemoglobin
concentrations close to levels observed in the blood (10 g/dL)126.
These solutions will then be administered to specified animals (mice, hamsters,
and/or rabbits) suffering from hemorrhagic shock127. Cardiac, circulatory, and
pulmonary variables will then be measured, including Mean Arterial Pressure (MAP),
heart rate, cardiac index, tissue oxygenation, and blood gas concentrations (pO2, pCO2).
Part VIII: Timeline
Pursuing all of the proposed lines of investigation to the point where a therapeutic
product would be ready for use in humans would require a vast amount of time and
money. We thus consider instead a timeline for development of a minimal system that
demonstrates some of the key characteristics needed for a successful blood substitute,
including expression of hemoglobin, protection from the immune system, reduced
immune response to lipid A, and possibly stalled growth due to DNA destruction. This
minimal system can then be tested in an animal model to suggest what challenges remain
insufficiently addressed. Note that the encapsulation work has already been successfully
performed by Chris Anderson.
Concluding Remarks
The proposed project to develop E. coli into a bacterial blood substitute presents a variety
of interesting problems. In exploring how to overcome those difficulties, significant inroads will
be made into both bacterial systems and mammalian systems, and importantly, their interaction.
Should the project be successful, millions of people stand to benefit worldwide, and the future of
therapeutic bacteria will be bright indeed.
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