Genomics and Molecular Approaches to Delineate Pathogenesis of Aeromonas Hydrophila, Aeromonas Veronii, and Edwardsiella Piscicida Infections in Fish

The U.S. aquaculture industry has become well established in the last three decades, and channel catfish aquaculture is the most significant component of this industry. Virulent Aeromonas hydrophila has been a serious disease problem since 2009 in the U.S. catfish aquaculture, and Aeromonas veronii and Edwardsiella piscicida are emerging pathogens of catfish. Therefore, this study aims to address fundamental questions on virulence mechanisms of these three fish pathogens, which I expect to support the development of control measures for preventing these diseases. In this study, E. piscicida and virulent Aeromonas hydrophila (vAh) genomes were sequenced, and comparative analyses were conducted using the genome sequences. Average nucleotide identity (ANI) calculations showed that E. piscicida strains share high sequence identity, yet they are from diverse host species and geographic regions. vAh isolates share very high sequence identity, while the other A. hydrophila genomes are more distantly related to this clonal group. We applied several comparative genomics approaches to evaluate E. piscicida genomes and E. ictaluri genomes, providing valuable information about unique and shared features of these two important pathogens in the Edwardsiella genus. Comprehensive secretion system analysis of 55 A. hydrophila genomes and deletion of tssD and tssI core elements of T6SS from vAh isolate ML09-119 has provided new knowledge. We sequenced the genome of virulent Aeromonas veronii strain ML09-123 from catfish indicated that it was highly similar to an A. veronii strain from China. Evaluation of all 41 A. veronii genomes available in the National Center for Biotechnology Information (NCBI) provides a base platform to investigate in detail the molecular mechanism of A. veronii biology and virulence. Lastly, we constructed deletion mutants vAhΔsia, vAhΔent, vAhΔcol, vAhΔhfq1, vAhΔhfq2, and vAhΔhfq1Δhfq2 to determine roles of A. hydrophila secreted proteins and regulatory proteins on virulence in catfish. Results showed that sialidase (vAhΔsia) and enterotoxin (vAhΔent) mutants were significantly attenuated

Comparative Genomic Studies of Catfish and Zebrafish Strains of Edwardsiella ictaluri

Edwardsiella ictaluri is a gram negative bacterium that is the causative agent of enteric septicemia of catfish. In 2011, this bacterium was identified as the causative agent of massive death in zebrafish populations in U.S. In this project, we found that isolates of E. ictaluri from zebrafish comprise a unique strain that differs from the type strain of E. ictaluri phenotypically as well as genetically. Also, strains of E. ictaluri from zebrafish are non-infectious in channel catfish Ictalurus punctatus by immersion. Here we sequenced the zebrafish strains of E. ictaluri LADL11-100 and LADL11-194 and compared the potential virulence genes in these strains with their homologous genes from the catfish strain LADL93-146. One of the major differences between the catfish strain and the zebrafish strain was found in the LPS related genes, more specifically, the O-antigen biosysnthesis cluster. The catfish strain and the zebrafish strain each contained unique genes in their O-antigen biosynthesis cluster. Three of the genes were shared by both strains but with relatively low identities. In contrast, the entire O-antigen biosynthesis cluster of the zebrafish strain of E. ictaluri was nearly identical to that of E. piscicida C07-087 with higher than 90% similarity. The differences in the O antigen were further confirmed by observing the different banding patterns of the purified LPS samples from the catfish and the zebrafish strain of E. ictaluri. Comparative genomic DNA analysis revealed that the major part of the type III secretion system is present and consistent among the zebrafish strains and the catfish strain. One variable portion is the effector EseI and its potential chaperone EscD are missing from the zebrafish strains. Also, there are more than twenty single nucleotide polymorphisms (SNPs) in one of the type III secretion system regulators esrA, and two genes eseC and eseD, which encode translocon proteins. These variations in the type III secretion system may contribute to the differences in virulence between the zebrafish and catfish strains. The type IV secretion system harbored the most variations between the catfish strain and the zebrafish strain compared to the other secretion systems. The sequences of the putative proteins in this type IV secretion system of both the zebrafish and the catfish strain were aligned and very low similarities were observed for most of the proteins in this system. In fact, two of the putative proteins in the zebrafish strain of E. ictaluri, had different conserved domains than their related proteins in the catfish strain, indicating possible different functions of these proteins. Other potential virulence related systems, the type VI secretion system and the urease system, are conserved between the catfish and zebrafish strains of E. ictaluri with only few SNPs. In addition, to protect against outbreaks of edwardsiellosis in zebrafish populations, the wild type zebrafish strain of E. ictaluri was mutated with the goal of generating attenuated strains that could serve as live attenuated vaccines. Both of our mutants, the ureG and esrC mutant, were proven to be fully attenuated by immersion in zebrafish. Further study is needed to test their efficacy as live attenuated vaccines

Investigation into the mobile genetic elements of Clostridium difficile

Clostridium difficile is a pathogenic bacterium that can colonise both humans and various animals. Toxin production leads to clinical symptoms ranging from mild to severe diarrhoea and can result in potentially fatal pseudomembranous colitis. These symptoms are caused by the disruption of the cytoskeleton and tight junctions of gut epithelial cells by the toxins. Genomic sequencing of C. difficile has indicated the chromosome carries a number of mobile genetic elements including conjugative transposons, which can encode antibiotic resistance genes. Analysing the sequence of a number of C. difficile strains indicated that each genome carries at least one and often multiple conjugative transposons. For many of the genes on these elements, functions were predicted using various bioinformatic tools. The study of conjugative transposons in C. difficile has been limited by the lack of resistance genes encoded by the elements. Therefore, an antibiotic resistance gene was inserted into six of the elements in strains 630 and R20291 and filter-matings performed. Conjugative transfer was shown for all elements from strain 630 but not for Tn6103 from R20291. The study of transconjugants of these matings showed the pathogenicity locus, encoding the two major toxins of C. difficile, to transfer at a low frequency into a non-toxigenic recipient strain. Whole genome sequencing of transconjugants determined that the transfer is not limited to the pathogenicity locus but includes varying sizes of chromosomal DNA flanking the pathogenicity locus. RNA-seq was used for the comparison of mutants for transcriptional regulators of conjugative transposons CTn2 and CTn4, however no significant differential expression was detected. Furthermore, strain 630Δerm, a commonly used laboratory strain for the generation of knockout mutants, was compared to the wildtype strain 630. A predicted oxidative stress operon was upregulated in 630Δerm which raises the question of the biological impact of these results on the knockout model