Graduation Year

2020

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Biology (Integrative Biology)

Major Professor

Valerie J. Harwood, Ph.D.

Committee Member

Christina Richards, Ph.D.

Committee Member

Kathleen Scott, Ph.D.

Committee Member

Anita Wright, Ph.D.

Keywords

epigenetics, gene expression, methylase mutant, anthropogenic, sewage

Abstract

Vibrio vulnificus is an autochthonous inhabitant of warm, brackish coastal waters and is an opportunistic pathogen that causes the highest mortality of all seafood-related illnesses (1). These fatal infections are generally caused by the clinically-associated vcgC/16S rRNA type B genotypes within the biotype I group (2–5). However, the reasons for the heightened infectiousness of the clinically-associated strains over environmentally-associated ones remains elusive, as no unique clinically-associated virulence genes have been identified through genomic sequencing or other strategies. DNA methylation may contribute to regulation of virulence by affecting gene transcription, and was investigated in the highly virulent V. vulnificus strain CMCP6. The proportion of methylated nucleotides was altered under conditions that simulate free-living estuarine vs. infectious niches. Growth in seawater resulted in approximately 4x as many methylated bases as the human serum treatment, the majority of which were N4-methylcytosines (m4C) (6).

Significantly more methylated bases occurred in the seawater treatment across gene coding sequences, intergenic space, rRNA, and tRNA genes. Multiple DNA methylation sequence motifs were identified and one non palindromic motif was associated with a putative methylase and termed hsdM. No specific m4C motif was identified, but the largest change in methylations per kilobase occurred with an m4CHH methylation motif. Growth in seawater resulted in 5.5x more methylated m4CHH motifs than growth in human serum

Methylation’s role in regulating the transcription of six genes associated with virulence and survival (i.e. hupA, nptA, sodA-C, and wza) in the wildtype V. vulnificus CMCP6 and putative methylase mutants was assessed under the same conditions used in the methylation sequencing. When wild type and methylase mutant strains of V. vulnificus were grown in human serum the putative m4C methylase mutant (ΔdcmA) had significantly higher transcription of hupA, nptA and sodA-C. Abiotic factors similar to those found in the human host (i.e. pH 7.5, salinity 10 ‰, and temperature 37°C) tended to contribute to elevated transcription of most of the investigated genes.

Sewage was investigated as a potential driver of changes in V. vulnificus populations and gene transcription that would increase the risk of human disease. Sewage at a 1% concentration significantly increased the concentration of autochthonous V. vulnificus in estuarine water from Tampa Bay, FL (e.g. 8.12 x 104 + 1.03 x 104 GC/100 mL vs. 8.93 x 106 + 2.50 x 105 GC/100 mL). The presence of organic carbon and nitrogen in wastewater contributed the most to the observed growth. Wastewater significantly altered the population structure of environmentally- and clinically-associated genotypes (16S rRNA type A and B respectively) and had a stabilizing effect on the percentage of the 16S rRNA type A genotype. The oxidative stress response was altered when V. vulnificus CMCP6 was exposed to wastewater, resulting in significantly higher sodB transcription while repressing sodA. No significant effects on gene transcription were observed for sodC, hupA, nptA, or wza. This research has set the groundwork for investigating DNA methylation as an alternative mechanism, as opposed to possession of unique virulence genes, for regulating virulence in V. vulnificus while also demonstrating the important role of the environment on regulation of virulence-associated genes.

Download

Share

COinS