From 2007.igem.org

Latest revision as of 02:10, 27 October 2007

The Chassis

There are several core issues associated with introducing E. coli into the bloodstream of human beings or other animals. First is the issue of sepsis. E. coli possesses a species called lipid X, or endotoxin, in its outer membrane which causes the release of TNFalpha in humans. This is an essential process of the innate immune system, but high doses of lipid X can be lethal. Bactoblood must have some ability to avoid this series of events. Similarly, there are a variety of additional features in E. coli that can elicit strong adaptive immune responses including the pili and flagella. From the bacterium's perspective, the interaction with the bloodstream is no more desirable. The complement system, another core component of the innate immune response, can kill bacteria directly. Additionally, phagocytic cells including macrophages and neutrophils, can engulf and kill E. coli. Fortunately for our purposes, there are a variety of modifications we can make to circumvent these problems.

The E. coli Outer Surface

To understand these modifications, we must first understand what features are present in E. coli strain MC1061, our starting point for Bactoblood. Like most strains of E. coli used in the lab, MC1061 comes from the MG1655 lineage and is a "rough" strain. Unlike other "smooth" strains, MC1061 lacks surface-displayed capsular polysaccharides known as K capsules and O antigens. It retains the general 2-membrane architecture present in gram-negative bacteria. In between these membranes is the periplasmic space which is composed of a gel-like carbohydrate-rich polymer called peptidoglycan. The inner membrane is composed of a lipid bilayer and a variety of proteins. The outer membrane similarly is a lipid bilayer, and the lipid component of it is called lipopolysaccharide, or LPS. The structure of LPS at it's core is a 6 fatty acid lipid called lipid X. When O antigen polymer chains are present, they are covalently attached to the outer leaf of LPS. K capsules are similarly embedded in the outer leaf of the outer membrane, but they are not directly attached to LPS. Other components of the outer membrane include a structural protein, LPP, and a variety of other proteins. This outer surface is the critical region of the bacterium for understanding how it interacts physically with the outside world. When the bloodstream "looks" at E. coli, what it "sees" is the outer membrane because everything else is stuck inside. Modifications such as O antigens and K capsules therefore have dramatic effects on the bacterium's interactions with the outside world.

Capsular Polysaccharides

The carbohydrates embedded in the outer membrane are extremely diverse within the E. coli species. Both K capsules and O antiens are linear carbohydrates polymer, but at least 150 chemically-distinct O antigens exist in one E. coli strain or another. Similarly, at least 100 chemically-distinct K capsules have been described. Almost all pathogenic strains of E. coli have some sort of capsular polysaccharide and are referred to as "smooth" strains. The rough vs. smooth distinction refers to a visibly discernible quality of their colonies. The particular choice of carbohydrate present in a bacterium is essential to its ability to survive in its living environment. For pathogenic and commensal bacteria, specific O or K carbohydrates are appropriate for distinct areas of the body (blood stream, urinary tract, intestines) and also for distinct animal types (birds, pigs, humans, cows, etc.). Over 90% of human cases of E. coli bacteremia (the clinical word for having bacteria in the bloodstream) are caused by strains that have a specific type of K capsule called K1. K1 is a long linear polymer of sialic acid that extends about half the diameter of the bacterium beyond its surface. Because polysialic acid is a frequent coating on mammalian cells, the human immune system does not recognize K1 as foreign. Bacteria with a K1 capsule are therefore resistant to both innate and adaptive immune responses. Proper display of a K1 capsule requires the concomitant expression of any of several O antigens. For our studies, we have chosen O16. Genetically, the K1 capsule requires 14 genes encoded within a 20kb cassette. The O16 antigen requires 11 genes encoded within a 12kb cassette. Together, these surface modifications allow the bacterium to avoid detection by the immune system and should extend the serum half-life of Bactoblood to several hours rather than the less-than-5 minutes observed with rough strains. Both of these gene clusters have been installed into the genome of MC1061 in the course of preparing our chassis strain, MC828U.

Lipid X and Its Variants

Additional Cell-Surface Epitopes

Growth Control by Iron Restriction

Characterization of the Chassis, MC828U

The genotype of our chassis organism is:

MC828U delta(araA-leu)7697 araD139 delta(codB-lac)=deltalac74 galK16   
galE15 mcrA0 relA1 rpsL150 spoT1 mcrB9999 hsdR2 O16(deltawbbL)   
K1(deltaneuS) deltamsbB deltafim deltatonB deltaflhCD   
upp::(Ptet-wbbL-neuS)  

To illustrate the function of our chassis we demonstrate its ability to survive in serum we also show the loss of a non essential, highly a immunogenic component, the flagella.

The serum assay shows the chassis' ability to survive in horse serum due to its K1:O16 capsule addition. Horse serum contains proteins of the complement immune system. Cells will be attacked by these proteins and lyse. However, the K1:O16 capsule allows our cells to survive this attack of the immune system. The cells were incubated in horse serum for 2-3 hours, plated on LB plates, and the number of surviving colonies were counted.

In a swarming assay we demonstrate the effect of flagella deletion. As flagella are essential for E. coli movement, wild type MC1061 (left plate) was able to swim out much farther than MC828U (right plate).

References

J. Christopher Anderson et al., “Environmentally Controlled Invasion of Cancer Cells by Engineered Bacteria”, J. Mol. Biol., 355, 619-627, 2006.Takada et al., “Structure-function relationships of lipid A. In Bacterial endotoxic lipopolysaccharides”, Molecular Biochemistry and Cellular Biology, Vol. 1., pp. 107-130, 1992.

Pohlman, T.H. et al., “Deacylated lipopolysaccharide inhibits neutrophil adherence to endothelium induced by lipopolysaccharide in vitro”, J. Exp. Med. 165:1393-1402, 1987.

C Galanos et al., “Synthetic and natural Escherichia coli free lipid A express identical endotoxic activities”, European Journal of Biochemistry, Vol. 148, 1-5, 1985.

Edwin S. Van Amersfoort, “Receptors, Mediators, and Mechanisms Involved in Bacterial Sepsis and Septic Shock”, Clinical Microbiology Reviews, pp. 379-414, July 2003.

Jonathan Cohen, “The Immunopathogenesis of Sepsis”, Nature 420, 885-891, December, 2002.

Judith Hellman et al, “Outer Membrane Protein A, Peptidoglycan-Associated Lipoprotein, and Murein Lipoprotein Are Released by Escherichia coli Bacteria into Serum”, Infection and Immunity, pp. 2566-2572, May 2000.

Ernst T. Rietschel et al, “Bacterial endotoxin: molecular relationships of structure to activity and function”, FASEB, Vol. 8, February, 1994.

Poltorak, A. et al., “Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4gene”, Science 282, 2085-2088, 1998.

Poltorak, A. et al., “Physical contact between LPS and TLR4 revealed by genetic complementation”, Proc. Natl Acad. Sci. 97, 2163-2167, 1999.

Edwin S. Van Amersfoort, “Receptors, Mediators, and Mechanisms Involved in Bacterial Sepsis and Septic Shock”, Clinical Microbiology Reviews, pp. 379-414, July 2003.

Golenbock, D.T. et al., “Surface expression of human CD14 in Chinese hamster ovary fibroblasts imparts macrophage-like responsiveness to bacterial endotoxin.” J. Biol. Chem. 268:2205-22059, 1993Lee, J. D. et al., “Transfection of CD14 into 70Z/3 cells dramatically enhances the sensitivity to complexes to lipopolysaccharide (LPS) and LPS binding protein.” J. Exp. Med. 175:1697-1705, 1992.

Richard P. Darveau et al., “Ability of Bacteria Associated with Chronic Inflammatory Disease To Stimulate E-Selectin Expression and Promote Neutrophil Adhesion”, Infection and Immunity, pp. 1331-1317, April 1995.

Kiyoshi Kawasaki et al., “3-O-Deacylation of Lipid A by PagL, a PhoP/PhoQ-regulated Deacylase of Salmonella typhimurium, Modulates Signaling through Toll-like Receptor 4”, The Journal of Biological Chemistry, Vol. 279, No. 19, May, 2004

Anthony P. Moran et al., “Structural Characterization of the Lipid A Component of Helicobacter pylori Rough- and Smooth- Form Lipopolysaccharides”, Journal of Bacteriology, pp. 6453-6463, October 1997.

Tomohiko Ogawa et al., “Immunobiological activities of chemically defined lipid A from Helicobacter pylori LPS in comparison with Prophyromonas gingivalis lipid A and Escherichia coli-type synthetic lipid A (compound 506)”, Vaccine, Vol. 15, No. 15, pp. 1598-1605, 1997.

Timothy H. Pohlman et al., “Deacylated lipopolysaccharide inhibits neutrophil adherence to endothelium induced by lipopolysaccharide in vitro”, J. Exp. Med., Vol. 163, May 1987.

Mingfang Lu et al., “Lipopolysaccharide deacylation by an endogenous lipase controls innate antibody responses to Gram-negative bacteria”, Nature Immunology, Vol. 6, No. 10, October 2005.

John E. Somerville et al., “A Novel Escherichia coli Lipid A Mutant That Produces an Antiinflammatory Lipopolysaccharide”, J. Clin. Invest., Vol. 97, Number 2, pp. 359-365, January 1996.

Edwin S. Van Amersfoort, “Receptors, Mediators, and Mechanisms Involved in Bacterial Sepsis and Septic Shock”, Clinical Microbiology Reviews, pp. 379-414, July 2003.

John E. Somerville et al., “Escherichia coli msbB Gene as a Virulence Factor and a Therapeutic Target”, Infection and Immunity, pp. 6583-6590, December, 1999.

<<< Return to UC Berkeley iGEM 2007

<<Previous Section: Oxygen Transport | Next Section: Controller>>