Student Theses and Dissertations

Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Hang Laboratory


The intestinal microbiota consists of diverse bacterial species and their effectors that play key roles in regulating human health. Interestingly, cell wall, or peptidoglycan, fragments from commensal and pathogenic bacteria can activate host immunity. The mechanism(s) by which immunologically active peptidoglycan fragments are generated, however, are not well-understood. In this regard, peptidoglycan hydrolases are ubiquitous in bacteria and possess diverse activities to remodel the cell wall during cell growth and division. These peptidoglycan hydrolases can also generate cell wall fragments in this process that are shed or recycled and available for triggering host immunity. In this thesis, we describe methodologies for the biochemical characterization of the NlpC/p60 family of peptidoglycan hydrolases to harness their catalytic activity in multiple therapeutic applications involving commensal bacteria, and to target these proteins in pathogenic bacteria. In Chapter 1, we introduce the NlpC/p60 protein family and review the functions of NlpC/p60 hydrolases in bacterial cell division, their diverse biochemical activities, structurefunction relationships, and novel functions beyond bacterial cell division. In Chapter 2, we describe methods for the biochemical characterization of NlpC/p60 hydrolase activity. These methods encompass bacterial expression and purification of recombinant NlpC/p60 proteins, large scale isolation of peptidoglycan, and in vitro activity assays of NlpC/p60 hydrolase activity. Our optimized methodologies were employed to determine the peptidoglycan substrate specificity of the NlpC/p60 hydrolase SagA from the commensal bacterium Enterococcus faecium. These methods also enabled the evaluation of features identified in the SagA-NlpC/p60 domain structure that may be important for binding peptidoglycan substrates. Moreover, SagA-generated peptidoglycan products can activate a pattern recognition receptor in mammalian cells, which directly links SagA NlpC/p60 hydrolase activity to the activation of host immune pathways, as observed with SagA+ bacteria in mouse models of enteric infection. Our results emphasize the utility of the biochemical analysis of NlpC/p60 hydrolase activity. In Chapter 3, we uncover the localization of SagA in Enterococcus and describe structurefunction studies of the SagA-NlpC/p60 domain. Imaging studies of SagA+ Enterococcus species and fluorescently-tagged SagA constructs implicated SagA in peptidoglycan remodeling in actively dividing Enterococcus cells. Comparative analysis of full-length SagA and SagANlpC/ p60 indicated that the coiled-coil SagA N-terminus may play a scaffolding or targeting function during peptidoglycan remodeling as opposed to an autoinhibitory role. A peptidoglycanbound model of the SagA-NlpC/p60 structure along with alanine screening revealed key ligandprotein interactions that may govern Enterococcus peptidoglycan turnover by SagA-NlpC/p60. From this analysis, we define the catalytic dyad in the NlpC/p60 domain of SagA from multiple commensal enterococci. Together, these results highlight the possible role of SagA in E. faecium viability and provide mechanistic insight into how SagA NlpC/p60 hydrolase activity processes peptidoglycan and generates immunostimulatory cell wall fragments. In Chapter 4, we explore SagA-mediated activation of host immunity and improved responsiveness to anticancer therapy in mouse tumor models. Enterococcus species and strains were recovered from cancer patients demonstrating responsiveness to cancer immunotherapy, but the molecular mechanism(s) behind this association were not understood. Here, we show that Enterococcus species and strains possessing SagA orthologs with highly conserved NlpC/p60 domains specifically enhanced efficacy of cancer immunotherapy by checkpoint blockade. Our optimized biochemical methods were applied to show that the active enterococci share conserved SagA expression, cell wall composition, and in vitro NlpC/p60 hydrolase activity of the respective SagA orthologs. In this analysis, I characterized the peptidoglycan hydrolase activity of a SalA from the non-protective Enterococcus faecalis. Heterologous expression of SagA in E. faecalis showed that SagA is sufficient for improving the antitumor activity of several antibodies. Moreover, using engineered strains of the probiotic Lactococcus lactis that heterologously expressed SagA constructs, we validated that SagA-mediated antitumor activity is dependent on secretion of catalytically active SagA. These results provide further evidence of the therapeutic applicability of SagA NlpC/p60 hydrolase activity. In Chapter 5, we explore targeting NlpC/p60 hydrolase activity in the context of multi-drug resistant Enterococcus therapy. Strains of E. faecium Com12 displaying resistance to a particular phage encoded sagA mutations localized at the NlpC/p60 domain. Phage resistance upon sagA mutation coincided with an antibiotic fitness tradeoff that was exploited to discover the synergistic effect of phage-antibiotic combination therapy on these strains. We show that while the phage resistant strains share similar SagA secretion and cell wall profiles compared to wild type, the respective mutations abrogate NlpC/p60 hydrolase activity in vitro. These results imply that the phage resistant E. faecium Com12 strains produce catalytically impaired SagA mutants, which manifests as enhanced antibiotic susceptibility and raises the possibility of targeting NlpC/p60 hydrolase activity to treat multi-drug resistant strains of E. faecium. In Chapter 6, we identify and biochemically characterize two NlpC/p60 proteins, 501F and 51B9 CwlT, as candidate multi-drug resistant E. faecium targets. Using our established biochemical methods, we confirm that the proteins are functional peptidoglycan hydrolases in vitro, thus laying the groundwork for future functional studies of these enzymes in pathogenic E. faecium. Our results collectively indicate that NlpC/p60 hydrolase activity may serve as a marker for the viability of pathogenic bacteria. In Chapter 7, we summarize our work on NlpC/p60 hydrolases from commensal and pathogenic bacteria. We also discuss our ongoing efforts to develop high throughput activity assays for NlpC/p60 hydrolases and describe the next frontier of these enzymes as multi-drug resistant Enterococcus targets. Together, the work described in this thesis underscore the importance of NlpC/p60 peptidoglycan hydrolase activity in mediating host-microbe interactions.


A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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