DOI
https://doi.org/10.25772/DK9S-S466
Author ORCID Identifier
https://orcid.org/0000-0003-2427-5482
Defense Date
2022
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Pharmaceutical Sciences
First Advisor
Aaron E. May
Second Advisor
Malgorzata Dukat
Third Advisor
Michael Donnenberg
Fourth Advisor
Jiong Li,
Fifth Advisor
Mary Peace McRae
Abstract
There is an overall significance and need for new therapies targeting bacterial infections for numerous reasons, the most well-known among them is resistance. The CDC estimates that there were over 2.8 million infections and nearly 36,000 deaths caused by antibiotic-resistant bacteria in 2019, a 40% increase from 2013. This threat has resulted in many modern antibiotics becoming ineffective. Broad-spectrum antibiotics can also have serious detrimental effects, such as killing off important commensal bacteria that guard against secondary infections. Two potential methods to mitigate this problem are antivirulence therapeutics and narrow-spectrum/targeted antibiotics. These correspond to my two main areas of research: 1) the bacterial type III secretion system (T3SS), a Gram-negative pathogenesis mechanism, and 2) phage-related ribosomal protease (Prp), a novel antibiotic target in Gram-positive bacteria, respectively.
1) The Bacterial Type III Secretion System (T3SS). The T3SS functions as a molecular syringe in pathogenic Gram-negative bacteria to inject effector proteins directly into host cells to interfere with host mechanisms and machinery, resulting in more efficient infection and evasion of host immune responses. It is known that T3SS knockout bacteria have a highly attenuated ability to cause infection, but their growth is not affected. This means that selective pressure should be reduced against small-molecule T3SS inhibitors, thereby decreasing the rate of resistance formation to these agents. Additionally, since only pathogenic bacteria encoded the T3SS, commensal bacteria should be spared from the action of T3SS modulators. T3SS inhibition shows promise as a potential therapeutic target as small-molecule inhibition allowed hosts to clear infection better than placebo.
Notwithstanding the promise of the T3SS as an antivirulence target, many assays designed to study it are often expensive and inefficient. To mitigate this issue, our lab has developed a highly sensitive in vitro Glu-CyFur fluorescence reporter assay to monitor T3SS-related secretion that, unlike traditional secretion assays, does not require mammalian cell culture and can distinguish between direct and indirect inhibitors. I have contributed to a medium throughput screening effort using this assay to identify modulators of the T3SS in Citrobacter rodentium, a murine equivalent of enteropathogenic Escherichia coli, and gather insights on known T3SS inhibitors. I have also discovered another strength of our assay: it can observe dose-dependent activation of the T3SS and inhibition, another capability unseen in other standard T3SS secretion assays.
To complement this in vitro screening, an adapted version of the colonic epithelial permeability assay was developed to study infectious colitis. Traditional use of this assay requires euthanasia of animals at every timepoint during an infection to obtain blood and examine infected tissues histologically. This leads to a very high number of animals needed and an inability to track the course of infection within an individual. C. rodentium induces inflammation, hyperplasia, and increased colonic epithelial permeability in a T3SS-dependent manner. The modified version allows for tracking C. rodentium infection of mice over time within an individual to reduce animal numbers and create a cost-effective assay for in vivo T3SS research. It involves oral gavage of fluorescein isocyanate (FITC)-dextran followed by cheek bleed to observe FITC-dextran blood concentrations. This can then be correlated to colonic epithelial damage and, therefore, infection severity.
2) Phage-Related Ribosomal Protease (Prp). Phage-related ribosomal protease (Prp) is responsible for the post-translational cleavage of a supernumerary sequence on the N-terminus of the essential ribosomal protein L27. This cleavage is time-sensitive, as bacteria expressing either pre-cleaved or uncleavable L27 became unviable. Bioinformatic analysis has shown that only 6 of the more than 40 bacterial phyla encode or require Prp. These phyla contain many deadly antibiotic-resistant pathogens (e.g., Staphylococcus aureus, Streptococcus pneumoniae, and Clostridioides difficile). This, along with the lack of human homologs, makes Prp an attractive antimicrobial target.
The complete structure of S. aureus Prp (SaPrp) was not elucidated until 2015, when it was crystallized as a covalently bound complex with a chloromethylketone (CMK)-containing L27 peptide (PDB: 7KLD). This structure allowed for the first evaluation of SaPrp:L27 cleavage motif binding interactions and conformational changes of the L2 binding site clamp upon binding. A more recent structure with a catalytically inactive C34S SaPrp:L27 12mer peptide complex shows interactions of Prp with L27 both before and after the cleavage site. Analysis of these structures in conjunction has generated numerous valuable insights into Prp’s cleavage mechanism and binding selectivity.
I enzymatically characterized SaPrp via in vitro proteolysis fluorescence assays for selectivity and traditional Michaelis-Menten kinetics determination. Through this analysis, I have been able to identify the relatively high catalytic efficiency of SaPrp. This is potential evidence of SaPrp’s activity as a chaperone for L27 during ribosomal maturation. I also used the proteolysis assay to screen a library of traditional protease inhibitors. Surprisingly, none of these inhibitors significantly reduced SaPrp cleavage, indicating resistance to traditional inhibition, which may indicate further selectivity when designing narrow-spectrum antibacterial compounds targeting Prp.
Due to Prp’s apparent resistance to traditional protease inhibition, the exploitation of Prp’s proteolysis to reveal an antibiotic in a directed enzyme prodrug therapy (DEPT) may be an alternative. This would include coupling the L27 cleavage motif to an antibiotic such that the antibiotic’s activity would be quenched. Upon cleavage of the L27 cleavage motif by Prp, the antibiotic would be released in an active state inside the target bacterium. One advantage of this DEPT strategy is that the essential nature of Prp makes overcoming it highly difficult, as any pathogen would need to make complementary mutations to Prp and L27. Molecular docking studies have shown that due to structural constraints in the Prp active sight, a linker may be needed between the L27 motif and a fluorescent probe or antibiotic.
These targets both show the potential to change the way clinicians approach bacterial infection treatment. Targeting the T3SS may result in antivirulence agents that can be given prophylactically rather than antibiotics or in combination with antibiotics during an active infection to reduce the length or dose of antibiotic required for treatment. Prp, on the other hand, could be an excellent novel antibiotic target to expand the arsenal of modern medicine. Prp may also be exploited in a DEPT to create highly selective narrow-spectrum treatments that could fundamentally change how we treat bacterial infections. With these and many other advancements, the future of antibacterial medicine is promising and bright.
Rights
© Julia A. Hotinger
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
12-14-2022
Included in
Amino Acids, Peptides, and Proteins Commons, Animal Diseases Commons, Animals Commons, Bacteria Commons, Bacterial Infections and Mycoses Commons, Digestive System Diseases Commons, Enzymes and Coenzymes Commons, Infectious Disease Commons, Medicinal and Pharmaceutical Chemistry Commons, Medicinal Chemistry and Pharmaceutics Commons, Natural Products Chemistry and Pharmacognosy Commons, Pathogenic Microbiology Commons