Date of Degree

9-2021

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor

Nicolas Biais

Committee Members

Luis Quadri

Edward Kennelly

Ryan Murelli

Sean Brady

Subject Categories

Biochemistry | Molecular Biology | Pathogenic Microbiology

Keywords

Neisseria gonorrhoeae, Bacillus, Antibiotics, DedA

Abstract

Bacterial human pathogens cause severe infectious diseases which are the second most common cause of death next to cancer and cardiovascular diseases in the world, especially in developing countries. Gonorrhea particularly, is the second most common sexually transmitted infection (STI) which is caused by the microorganism Neisseria gonorrhoeae (GC). Centers for Disease Control and Prevention (CDC) estimates that more than 1.6 million new gonorrhea cases emerged in USA in 2018 (“Detailed STD Facts - Gonorrhea” n.d.). Also, the WHO (World Health Organization) shows that gonorrhea is the most antibiotic resistant STI (“PAHO/WHO | Gonorrhea” n.d.), highlighting the shortage of efficient antibiotics act against N. gonorrhoeae nowadays.

Gonorrhea is a growing public health concern worldwide due to the rapid development of antibiotic resistance. At present, the only recommended way to efficiently cure or control the condition of gonorrhea is the combination usage (dual therapy) of ceftriaxone and azithromycin (Unemo and Nicholas 2012). Nonetheless, there are some recently reported “superbugs” which are resistant in a high level to antibiotics known to act against gonorrhoeae, such as GC strains F89 (Unemo et al. 2012) and H041 (Tomberg et al. 2013). This means gonorrhea may become untreatable if no novel antibiotics or substitute therapy is developed in the immediate future and a superbug gains resistance to all those antibiotic and spreads in the population. Thus, the most urgent effort that we should make is to find or design alternative drugs to solve the gonorrhea crisis that people are facing or get it under control even temporarily. However, the discovery of new antibiotics is usually challenging and slow, especially after the “golden era” stimulated by Selman Waksman in the 1940s, not only for large-scale screening of natural products, but also for synthetic compounds (K. Lewis 2020).

As a serendipity, Dr. Nicolas Biais and I noticed a contaminant on a bacterial survival plate, which shows specifically strong inhibition activity to N. gonorrhoeae compared to other strains. This “penicillin-discovery-like” observation triggered my central hypothesis of the thesis: this contaminant produces a narrow spectrum antimicrobial compound which effectively acts against Neisseria gonorrhoeae due to a molecular mechanism to be elucidated.

My thesis has been organized in the following way: In Chapter 1, I will introduce the context and familiarizing the reader with N. gonorrhoeae, antimicrobial products, and species’ interaction, as well as presenting the aims and broad experimental design of this research. I isolated the strain and named it as “Bacillus S” based on phylogenetic study, allowing me to characterize it, identify the active compound, and investigate its interaction with N. gonorrhoeae.

Chapter 2 begins by characterizing Bacillus S. I used genomic sequencing and phylogenetic study to identify that it is a species of the Bacillus genus, close to the subtilis and amyloliquefaciens species. The disc diffusion assay of its bacterial supernatant indicates that the compound that inhibits gonorrhoeae (anti-GC) can be excreted to the extracellular environment. The following part is determining the active compound, by using a combination of molecular biology assays, bioinformatic analysis, mass spectrometry, and nuclear magnetic resonance (NMR). I finally identified that anti-GC compound is oxydifficidin produced by Bacillus S, this new antimicrobial activity has not been published before.

Lastly, I will turn to the questions of target of oxydifficidin in N. gonorrhoeae and the potential mechanism of action. In Chapter 3, I used transposon mutagenesis and insertion site mapping to successfully find that the target of this compound is an ancient trans-membrane protein – DedA. This protein family is poorly investigated since it has been discovered. Surprisingly, previous research shows DedA promotes antibiotic resistance in bacterial cells, which shows discrepancy to what I describe in this study as DedA is promoting sensitivity to oxydifficidin in N. gonorrhoeae. Through the membrane integrity assay, membrane potential assay, and molecular modeling of oxydifficidin – DedA interaction, I propose that oxydifficidin may bind to the trans-membrane DedA protein on N. gonorrhoeae, subsequently modifying cell structure and metabolism which then motivates cell death. In addition, pili dynamic assays show that the type IV pili dynamics of N. gonorrhoeae is relative to the DedA protein, the annex of this thesis provides a deeper understanding of type IV pili as a side project.

This work starts with isolating an antibiotic producing strain, aims to identify and characterize the active compound, and understand the interaction between this small molecule and N. gonorrhoeae cell. For the reason that new treatments of gonorrhea are urgently needed, I suggest that this molecule’s type and inhibition method can be considered as a novel or alternative direction for future drug development of gonorrhea, and even other pathogens.

This work is embargoed and will be available for download on Wednesday, March 30, 2022

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