Thursday, November 21, 2024
11/21/2024
Project Summary Streptococcus pneumoniae (Spn, the pneumococcus) are Gram-positive bacteria and the leading cause of community-acquired pneumonia worldwide. The World Health Organization estimates >1.6 million deaths are the result of Spn infection each year, with children and the elderly being the most susceptible populations. Major problems of pneumococcal disease include the acquisition of antimicrobial resistance and the global spread of resistant clones. In addition, these problems are magnified by the major disadvantages of the current capsular polysaccharide-based vaccines, such as serotype specificity and the resulting incomplete coverage. An emerging way to address the growing antimicrobial resistance problem is the use of bacteriophage endolysins. These enzymes are capable of degrading the bacterial peptidoglycan, killing and dispersing biofilm bacteria and its matrix. In preliminary studies, we have developed a chimeric derivative of the well characterized Cpl-1 endolysin that displays >100-fold increase in antimicrobial activity, termed ClyX-1. We also show the ability of this new endolysin to lyse planktonic Spn, and importantly we also observed that ClyX-1 was able to kill biofilm Spn, as well as disperse the biofilm matrix. In addition, we have shown that upon Spn nasopharyngeal colonization, activation of programmed necrosis, i.e. necroptosis, leads to development of antigen-specific antibodies. Of note, treatment of colonized mice with ClyX-1 further promoted necroptosis activation, suggesting endolysin treatment may enhance development of protective immunity against Spn. Herein, we aim to address three overall hypotheses: a) that endolysins are efficient pneumococcal anti-biofilm agents, b) that endolysins can be an effective way to prevent Spn colonization in a serotype-independent manner, and c) that intranasal treatment with endolysins promotes protective immunity, to prevent re-colonization and severe disease. We will use a combination of in vitro and in vivo studies with static and dynamic biofilms, mice, biochemistry (characterize and benchmark pneumococcal endolysins), transgenic mice (to define the role of programmed cell death in protective immunity), molecular and immunological techniques and next generation technologies (proteomics, single cell transcriptomics) to establish better understanding of the effects of endolysin treatments against pneumococcal disease in vivo and test their effectiveness in development of long-term serotype-independent protective immunity.
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