Table of Contents
Introduction
DNA minor-groove alkylating drugs are commonly used to target malignant cells and their efficacy in disrupting cell division1 and promoting mutagenesis is well studied. Far fewer studies have been conducted to demonstrate the effects of these drugs on enteric microflora and how chemotherapeutic use leads to disruption of bacterial metabolisms and diversity. Like antibiotics, DNA alkylating drugs inhibit certain bacteria that are incapable of repairing alkylation damage or which do not encode polymerases capable of bypassing DNA lesions. In the human gut, consideration of these effects is especially important because synergistic relationships and stable population numbers are required for prevention of pathogenesis.
Like eukaryotic cells, bacteria also rely on DNA-repair mechanisms to provide protection against cell death. Limited knowledge on alkylation-response pathways and their associated proteins in bacteria other than Escherichia coli and pathogenic Salmonella enterica highlights an opportunity to investigate these survival mechanisms in greater detail.
My proposed research introduces a method to: (1) study the cytotoxic effects of DNA minor-groove alkylators on select species of common enteric and enteropathogenic bacteria and (2) to quantify their individual, coupled, and triplet susceptibilities to these chemical agents. Furthermore, I will also: utilize the acquired susceptibility data to select the most resilient enteropathogenic bacterial species for further analysis. This subsequent investigation aims to identify alkylation-response pathways analogous to those already found in E. coli and Salmonella enterica ser. Enteritidis.
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Background
The DNA minor-groove is an important receptor for enzymes and proteins involved in gene expression. Specific types of small molecules such as active metabolites and minor-groove alkylating drugs can disrupt minor-groove DNA-protein interactions,1 and lead to deregulation of nucleic acid repair. Among minor-groove alkylating drugs, adozelesin1 and bizelesin1 are currently used as experimental chemotherapeutic agents. In vivo, the former serves as a monofunctional alkylator capable of inducing single-stranded lesions and the latter as a bifunctional alkylator capable of inducing single-stranded and double-stranded DNA cross-linkages.1
In E. coli, genes expressed via the adaptive response (ada, alkA, alkB, aidA) have been studied and encode proteins responsible for counteracting the effects of DNA alkylation lesions. However, no studies have demonstrated E. coli adaptive response functionality when cells are exposed to dosages of chemotherapeutic agents chosen for their lethality in malignant tumor cells. Because the two chemical agents adozelesin and bizelesin are experimental antitumor drugs capable of preventing colon tumor cell division,1 their likelihood of infiltrating microbial biofilms is significant.
Aims
Aim 1: Establishing minimal inhibitory concentrations for isolate, paired, triplet cultures
To establish cytotoxic drug concentrations, I will grow E. coli MG1655, Lactobacillus acidophilus, Bifidobacterium bifidum, Bacteroides fragilis, and Enterococcus faecalis on solid media in the presence of five different concentrations (based on tumoricidal activity) of only adozelesin and only bizelesin. Measurement of colony-forming units (CFUs) on each of the plates will determine a minimal inhibitory concentration (MIC) for each drug in each bacterium. I hypothesize that genetic variance will account for different levels of resistance to minor-groove alkylation, but also that all five species will be inhibited by at least one of the concentrations for each drug. To model a microbiome, I will culture the bacteria in pairs and triples of all possible combinations.
The drug concentrations will be the same and measurement of CFUs will require sub-culturing of isolated colonies from the pair and triplet plates onto selective and differential media containing nutrients specific to the isolation of each bacterium. In this manner, I seek to determine the degree to which a synergistic response to minor-groove alkylation has the ability to promote survival. I hypothesize that variations in levels of resistance demonstrated by each species will result in pair and triplet cultures with one bacterium growing, all growing, or a combination growing due to the uptake and depletion of the drug by one species in higher concentrations before the others begin growing.
Aim 2: Establishing minimal inhibitory concentrations for select enteropathogenic species
To establish cytotoxic drug concentrations for select enteropathogenic bacteria, I will repeat the procedure from paragraph 1 of Aim 1, and culture clinical isolates of Campylobacter jejuni, Salmonella enterica ser. Enteritidis, Shigella sonnei, E. coli O157:H7, and E. coli O104:H4 on solid blood media. I hypothesize that various resistances will be demonstrated, but also that S. enterica ser. Enteritidis and others will be resistant to a majority or all concentrations of both adozelesin and bizelesin. I support this hypothesis with knowledge of constitutive Tag and Ogt enzyme expression in S. enterica ser.
Enteritidis (which confer resistance to DNA alkylation cytotoxicity),5 and the prospect of analogous adaptive response mechanisms existing in the other enteric pathogens. Following determination of minimal inhibitory concentrations, I will culture the resistant pathogens with sets of bacteria from the triplet plates which resulted in successful growth at the same drug concentrations. In this manner I can model the addition of a pathogen to a stressed microbiome and answer two questions: (1) Can a resistant pathogen significantly outcompete a resistant non-pathogen given the selected drug conditions? and (2) Can the presence of a resistant pathogen result in growth of a non-pathogen with previously limited or no growth?
Aim 3: Confirming existence of analogous adaptive response mechanisms in pathogens
With cytotoxicity data collected, I will select the most resistant pathogen, excluding S. enterica ser. Enteritidis, and perform molecular analysis to identify if an adaptive response mechanism analogous to those in E. coli and S. enterica ser Enteritidis is present. I will first extract the pathogen’s DNA and react it with Type II restrictive nucleases6 to recover the genome sequences of interest. These sequences will then be amplified via PCR using synthesized DNA primer strands that demonstrate complementarity to portions of the ada, ogt, and tag gene sequences. Finally, the recovered PCR products will be gel-eluted to isolate the desired genome sequences and sequenced to verify their analogy. If PCR fails and no such analogous systems are found, the lack thereof will serve as a basis on which a subsequent hypothesis to identify novel systems may be formed.
Intellectual Merit
My familiarity with culturing of clinically significant bacteria as well as the ability to perform all included molecular techniques will facilitate this study. All guidance to interpret my results and progress the experiment will be acquired through molecular biology courses and direct mentoring from my research advisor and her post-doctoral fellow.
Broader Impacts
The ability of this study to determine enteric bacterial resistance to minor-groove alkylation in both isolate and microbiome-like conditions provides a reliable model to ascertain how experimental antitumor drugs with minor-groove alkylating properties may impact enteric pathogenesis in patients requiring chemotherapy. As adaptive response mechanisms in any pathogenic bacteria are of medical significance, this study can serve as a model for subsequent analyses of other pathogens commonly found in conjunction with malignancy.
Citations
- Pei-rang Chao et al. (2003) Mol. Cancer Thera., 2(7)
- Ivana Bjedov et al. (2007) Genetics, 176(3)
- Z. Moravek et al. (2002) Nucleic Acid Res., 30(5)
- Damian Mielecki et al. (2014) FEMS Microbio. Lett. 355(1)
- Gerard Alvarez et al. (2010) J. of Bac. 192(7)
- Alfred Pingoud et al. (2001) Nucleic Acid Res. 29(18)
