Lawrence Livermore National Laboratory



Lawrence Dugan

Overview

Development of rapid pathogen-purification technologies from complex sample matrices (such as human fluids) without deactivating the organism being purified enables an increase in the number and types of follow-on assays that can be performed. Cell-wall binding domains are protein domains that bind to bacterial cell walls. They have demonstrated functionality as affinity reagents toward Gram-positive bacteria, but not Gram-negative bacteria (two classifications of bacteria based on cell wall properties) due to the presence of an outer membrane in the latter. A Bacillus amyloliquefaciens-infecting bacteriophage endolysin has displayed antimicrobial activity against Gram-negative bacteria. We generated this protein and demonstrated affinity to the Gram-negative bacteria Pseudomonas aeruginosa, but not toward Gram-negative Escherichia coli. Magnetic particle-conjugated cell-wall binding domains were used to recover B. amyloliquefaciens cells; however, recovery of P. aeruginosa and E. coli cells was negligible. Additional effort will be required to optimize this approach for purifying bacteria from bodily fluids.

Background and Research Objectives

The increasing incidence of antimicrobial-resistant bacteria, resulting in difficulty to accurately diagnose, treat, and prevent infections, is a serious threat to human health. The U.S. government issued a “Report to the President on Combating Antibiotic Resistance” in 2014 and a “National Action Plan for Combating Antibiotic-Resistant Bacteria” in 2015 to address this issue. Antimicrobial-resistant Enterobacteriaceae (such as Klebsiella pneumoniae and Escherichia coli) were specifically called out in these reports as URGENT threat-level pathogens, requiring aggressive action to reduce the incidence of the infection and the spread of these organisms.

Endolysin (a bacteriocidal substance within a leukocyte), autolysin (an enzyme), and related muramidase proteins hydrolyze and break down bacterial cell-wall components. They are involved in the bacteriophage release from host cells and bacterial growth and cell division. Lawrence Livermore National Laboratory researchers described one protein (BCZK2532) that displayed antimicrobial activity when applied to a bacterial culture (Bourguet et al. 2012). These proteins contain two functional domains: a C-terminal cell-wall binding domain (CBD) that binds to bacterial cell walls, and an N-terminal enzymatic activity domain (EAD) that degrades bacterial cell walls (Broendum et al. 2018). CBDs can be used as affinity reagents for Gram-positive (GP) bacteria (Ahmed et al. 2007). However, the presence of an impermeable lipopolysaccharide outer membrane on Gram-negative (GN) bacteria prevents the binding of CBDs to GN cell walls (Briers et al. 2014). Compounds that cause bacterial cell membranes to become permeable, such as chelating agents and surfactants, allow CBDs to access the GN cell wall (Orito et al. 2004); however, this results in non-viable bacteria, preventing downstream testing of live cells.

Purified endolysin from a bacteriophage specific to the GP bacterium Bacillus amyloliquefaciens has displayed antimicrobial activity against the GN bacterium Pseudomonas aeruginosa. This activity did not require chemical permeabilization, but it did require the CBD (Morita et al. 2001), indicating that the CBD is involved in targeting the endolysin to the bacteria. We intended to determine if it is feasible to use CBDs as affinity reagents (i.e., small molecules that specifically bind to larger target molecules) for the purification of viable GN bacteria from human bodily fluids for use in downstream assays. Bioinformatic tools were used to identify a panel of candidate endolysins and their CBD sequences similar to the B. amyloliquefaciens infecting bacteriophage endolysin (Morita D1) CBD. The CBDs were expressed using commercially-available cell-free in vitro translation kits and included an N-terminal 6X Histine tag (His-tag) to allow purification of the expressed protein.

The expressed proteins and CBDs were assayed to determine their antimicrobial activity, affinity to GN bacteria, and ability to purify GN bacteria. We focused on purifying Escherichia coli, one of the leading causes of community- and hospital-acquired urinary tract infections (Wilson and Gaido 2004), as well as Pseudomonas aeruginosa, also a cause of urinary tract infections. Full-length proteins and CBDs were spot tested for antimicrobial activity on continuous bacterial mats. His-tag CBDs were conjugated to magnetic beads and used to purify clinically-relevant concentrations of bacteria. Green fluorescent protein/CBD fusion proteins were incubated with bacterial cells and imaged microscopically to determine the affinity of the proteins to different bacteria.

Impact on Mission

Our research supports the NNSA goal of applying our science and technology capabilities to deal with broad national security challenges. This project also enhances the Laboratory’s core competency in bioscience and bioengineering, while supporting detection technology development in the chemical and biological countermeasures mission-focus area.

Conclusion

One of the endolysin CBDs we tested showed limited affinity to the GN bacterium Pseudomonas aeruginosa, but poor recovery of cells from the buffer. This protein did not show affinity to E. coli. Future studies should evaluate additional E. coli strains, additional endolysin cell binding domains, and optimization of the assay to improve results or confirm the ineffectiveness of this technology for isolating E. coli from bodily fluids.

References

Ahmed, A. B. F., et al. 2007. "Evaluation of Cell Wall Binding Domain of Staphylococcus Aureus Autolysin as Affinity Reagent for Bacteria and Its Application to Bacterial Detection." Journal of Bioscience and Bioengineering 104 (1): 55–61. doi: 10.1263/jbb.104.55.

Bourguet, F. A., et al. 2012. "Characterization of a Novel Lytic Protein Encoded by the Bacillus Cereus E33L Gene AmpD as a Bacillus Anthracis Antimicrobial Protein." Applied and Environmental Microbiology 78 (8): 3025–3027. doi: 10.1128/AEM.06906–11.

Briers, Y., et al. 2014. "Engineered Endolysin-Based ‘Artilysins’ to Combat Multidrug-Resistant Gram-Negative Pathogens." MBio 5 (4): e01379–01314. doi: 10.1128/mBio.01379-14.

Broendum, S. S., et al. 2018. "Catalytic Diversity and Cell Wall Binding Repeats in the Phage Encoded Endolysins." Molecular Microbiology, September. doi: 10.1111/mmi.14134.

Gibson, D. G., et al. 2009. "Enzymatic Assembly of DNA Molecules up to Several Hundred Kilobases." Nature Methods 6 (5): 343–345. doi: 10.1038/nmeth.1318.

Morita, M., et al. 2001. "Antibacterial Activity of Bacillus Amyloliquefaciens Phage Endolysin without Holin Conjugation." Journal of Bioscience and Bioengineering 91 (5): 469–473. doi: 10.1016/S1389–1723(01)80275-9.

——— . 2001. "Functional Analysis of Antibacterial Activity of Bacillus Amyloliquefaciens Phage Endolysin against Gram-Negative Bacteria." FEBS Letters 500 (1–2): 56–59. doi: 10.1016/S0014-5793(01)02587-X.

Orito, Y., et al. 2004. "Bacillus Amyloliquefaciens Phage Endolysin Can Enhance Permeability of Pseudomonas Aeruginosa Outer Membrane and Induce Cell Lysis." Applied Microbiology and Biotechnology 65 (1): 105–109. doi: 10.1007/s00253-003-1522-1.

Wilson, M. L., and L. Gaido. 2004. "Laboratory Diagnosis of Urinary Tract Infections in Adult Patients." Clinical Infectious Diseases 38 (8): 1150–1158. doi: 10.1086/383029.