Determining the mechanisms of antimicrobial peptide LL-37 protection against Klebsiella pneumoniae in a murine model of necrotizing enterocolitis (NEC).
Necrotizing enterocolitis (NEC) is an intestinal inflammatory disorder that frequently occurs in premature babies born less than 33 weeks [10] and weighing less than 1500g [3]. This disease is characterized by inflammation and tissue necrosis. This leads to bacterial translocation, subsequent tissue infection and further tissue damage within the small intestine. Affecting approximately 9,000 of 480,000 [4, 7] premature births in the United States annually, necrotizing enterocolitis accounts for approximately 33% [6] of premature deaths.
While the cause of necrotizing enterocolitis remains unknown, three risk factors have been identified for the disease including: premature birth, formula feeding and bacterial dysbiosis. Premature infants are highly susceptible to infection and bacterial dysbiosis due to the immaturity of their immune systems [2]. Previous studies on neonates have shown that the primary intestinal and fecal organisms found in patients diagnosed with NEC were Klebsiella pneumoniae, Escherichia coli and Staphylococcus epidermidis [1]. Of these bacteria, K. pneumoniae and E. coli species are both well-known for causing nosocomial infections and developing antibiotic resistance.
With multidrug-resistant bacteria on the rise, new avenues for treating bacterial infections are now being explored. This includes antimicrobial peptides (AMPs), which have the potential to be an alternative to antibiotics in the future. Antimicrobial peptides are a diverse class of naturally occurring molecules that are produced as a first line of defense by all organisms from prokaryotes to humans and serve a fundamental role in innate immunity [8,11]. Common examples of antimicrobial peptides in the small intestine include α- and β-defensins and cathelicidin LL-37, which comprise the two major classes of AMPs in mammals.
Through host defense mechanisms, LL-37 can modulate inflammatory responses, promote cellular proliferation, and signal chemo-attracting cells to wound/infection sites [5,9]. These mechanisms help protect against pathogenic bacterial infection and are crucial to maintaining the homeostatic balance found between the healthy and unhealthy microbiota. Our preliminary data shows that LL-37 treatment can prevent development of NEC-like injury in newborn mice. However, it remains unclear if LL-37 is preventing NEC-like injury in the small intestines by its use of antimicrobial properties or another mechanism. We hypothesize that LL-37 is working though both antimicrobial and non-antimicrobial effects. To study this hypothesis, we will use the following Aims:
1) Determine LL-37’s antimicrobial activity against Klebsiella pneumoniae in vitro. We will test this aim by use of minimum inhibitory concentration (MIC) testing, and live-dead staining techniques in order to quantify the concentration of bacteria killed by each amount of cathelocidin LL-37. Because of Klebsiella pneumoniae’s ability to cause inflammatory induction, it was chosen as the target bacteria of our focus.
2) To determine the effects of LL-37 on the distribution of ZO-1 and subsequent barrier function in the small intestine in vivo. We will test this through staining small intestinal samples for ZO-1 staining and with FITC-dextran barrier function testing.
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References
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1. Bell, M., Ternberg, J., R, F., Keating, R., Marshall, R., Barton, L., & Brotherton, T. (1978). Neonatal necrotizing enterocolitis. Theraputic decisions based upon clinical staging. Annals of Surgery, 187(1), 1-7. Retrieved June 28, 2019
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2. Bokulich, N., Mills, D., & Underwood, M. (2013). Surface microbes in the neonatal intensive care unit: changes with routine cleaning over time. Journal of Clinical Microbiology, 51(8), 2617-24. doi:10.1128/JCM.00989-13
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3. Gephart, S. M., McGrath, J. M., Effken, J. A., & Halphern, M. D. (2013, April 1). Necrotizing Enterocolitis Risk: State of the Science. Advanced Neonatal Care, 12(2), 77-89. doi:10.1097/ANC.0b013e31824cee94
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4. Hamilton, B., Martin, J., & Ventura, S. (2010). Births: Preliminary Data for 2010. CDC, Division of Vital Statisitics. National Vital Statistic Reports. Retrieved July 1, 2019, from http://www.cdc.gov/nchs/data/nvsr/nvsr60/novsr60_02.pdf
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5. Kao, C., Lin, X., Yi, G., Zhang, Y., Rowe-Magnus, D. A., & Bush, K. (2016). Cathelicidin Antimicrobial Peptides with Reduced Activation of Toll-Like Receptor Signaling Have Potent Bactericidal Activity against Colistin-Resistant Bacteria. (G. A. Jacoby, Ed.) American Society for Microbiology, 7(5). doi:10.1128/mBio.01418-16
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6. McElroy, S. J., Underwood, M. A., & Sherman, M. P. (2013). Paneth Cells and Necrotizing Enterocolitis: A novel hypothesis for disease pathogenesis. Neonatology, 103(1), 10-20. doi:10.1159/000342340
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7. NICHD. (2016, 12 1). NIH. Retrieved from How many infants are affected by or at risk of Necrotizing enterocolitis (NEC)?: https://www.nichd.nih.gov/health/topics/nec/conditioninfo/risk#f2
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8. Wang, S., Xiangfang, Z., Yang, Q., & Qiao, S. (2016). Antimicrobial peptides as potential alternatives to antibiotics in food animal industry. (A. Piozzi, Ed.) International Journal of Molecular Sciences, 17(5), 603. doi:10.3390/ijms17050603
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9. Xhindoli, D., Pacor, S., Benincasa, M., Scocchi, M., Gennaro, R., & Tossi, A. (2016). The human cathelicidin LL-37--A pore-forming antibacterial peptide and host-cell modulator. Biochimica et Biophysica Acta, 546-566. doi:10.1016/j.bbamem.2015.11.003
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10. Yee, W. H., Soraisham, A. S., Shah, V. S., Aziz, K., Yoon, W., Lee, S. K., & Network, t. C. (2012, January 23). Incidence and Timing of Presentation of Necrotising Enterocolitis in Preterm Infants. Pediatrics, 129(2), 2011-2022. doi: 10.1542/peds.2011-2022
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11. Zhang, L.-j., & Gallo, R. L. (2018). Antimicrobial peptides. Current Biology, 26(1), 14-19. doi:10.1016/j.cub.2015.11.017
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