Mechanisms and roles of the LuxS system, methyl recycling, and DNA methylation on the physiology of Campylobacter jejuni

Campylobacter jejuni is one of the leading causes of human bacterial gastroenteritis, and campylobacteriosis in sheep. The genetic diversity of this organism, the potential for multiple sources to transmit C. jejuni to humans, and the possession of a variety of virulence factors and antimicrobial resistance mechanisms make C. jejuni a serious health problem worldwide. The autoinducer-2 (AI-2)/LuxS system has been the focus of several studies for its potential applications to attenuate C. jejuni virulence. A study from our group found that the LuxS enzyme plays a critical role in virulence and fitness of C. jejuni IA3902 and 11168 strains. Mutagenesis of the luxS gene negatively impacted C. jejuni colonization of the gastrointestinal tract of several host species. However, the physiologic basis for this colonization defect is unclear. In addition to AI-2 production, LuxS is also a key enzyme involved in the activated methyl cycle (AMC). The AMC is an important source for the formation of S-adenosylmethionine, a methyl donor crucial to biological processes like DNA methylation. DNA methylation has also been linked with a diverse number of important physiological and pathogenic mechanisms in many bacteria, but is poorly understood in C. jejuni. The collective work from this thesis attempts to address some of the knowledge gaps on the role of LuxS, methyl recycling, and DNA methylation in C. jejuni physiology. Collectively, results from our study showed that luxS mutation interrupted the AMC resulting in significant changes to intracellular concentrations of several key metabolites. However, the colonization-associated factors tested on our luxS mutants in this thesis do not show evidence of being the primary mechanisms responsible for the luxS mutant\u27s decreased colonization ability. We proceeded to analyze the role of LuxS on DNA methylation and found that the luxS mutation had no appreciable effect on the methylome profile of the mutant. We also compared the methylome profiles of three important C. jejuni strains and found significant strain variability in the methylomes, which suggest a potential role for DNA methylation in Campylobacter pathobiology. The methylome studies also revealed a novel putative methyltransferase which we later confirmed and definitively assigned to a specific methylation motif. While mutagenesis of the methyltransferase gene resulted in a loss of methylation of its cognate motif we were unable to show an effect on the growth or motility phenotypes tested in our study. In summary, the luxS mutation demonstrated physiological effects on the AMC, but the colonization mechanisms affected by the mutation are still unknown. However, DNA methylation studies revealed strain-specific methylation profiles, including a unique methyltransferase, which may serve a biological and/or pathogenic purpose specific to the strain

Shifts in the swine nasal microbiota following Bordetella bronchiseptica challenge in a longitudinal study

Bordetella bronchiseptica is a widespread, highly infectious bacterial pathogen that causes respiratory disease in swine and increases the severity of respiratory infections caused by other viral or bacterial pathogens. However, the impact of B. bronchiseptica infection on the swine respiratory microbiota has not been thoroughly investigated. Here, we aim to assess the influence of B. bronchiseptica infection on the community structure and abundance of members of the swine nasal microbiota. To do so, the nasal microbiota of a non-infected control group and a group infected with B. bronchiseptica (BB group) were characterized prior to B. bronchiseptica strain KM22 challenge (day 0) and on selected days in the weeks following B. bronchiseptica challenge (days 1, 3, 7, 10, 14, 21, 36, and 42). Bordetella bronchiseptica was cultured from nasal samples of the BB group to assess nasal colonization. The results showed that B. bronchiseptica colonization did not persistently affect the nasal bacterial diversity of either of the treatment groups (alpha diversity). However, the bacterial community structures (beta diversity) of the two treatment groups significantly diverged on day 7 when peak colonization levels of B. bronchiseptica were detected. This divergence continued through the last sampling time point. In addition, Pasteurella, Pasteurellaceae (unclassified), Mycoplasma, Actinobacillus, Streptococcus, Escherichia-Shigella, and Prevotellaceae (unclassified) showed increased abundances in the BB group relative to the control group at various time points. This study revealed that B. bronchiseptica colonization can disturb the upper respiratory tract microbiota, and further research is warranted to assess how these disturbances can impact susceptibility to secondary infections by other respiratory pathogens

Transcriptomic differences noted in Glaesserella parasuis between growth in broth and on agar.

Glaesserella parasuis is the cause of Glӓsser's disease in pigs and is a significant contributor to post-weaning mortality in the swine industry. Prevention of G. parasuis disease relies primarily on bacterin vaccines, which have shown good homologous protection and variable heterologous protection. Bacterin production involves large scale growth of the bacteria and proteins produced during the proliferation phase of production become important antigens that stimulate the immune response. In order to evaluate genes activated during G. parasuis growth on different media substrates, the transcriptome of broth and agar grown G. parasuis strain 29755 were sequenced and compared. The transcription of most purported virulence genes were comparable between broth and agar grown G. parasuis; however, four virulence-associated genes, including ompA and vapD, had elevated expression under agar growth, while six virulence-associate genes had elevated expression during broth growth, including several protease genes. Additionally, there were metabolic shifts toward increased protein and lipid production and increased cellular division in broth grown G. parasuis. The results contribute to the understanding of how growth substrate alters gene transcription and protein expression, which may impact vaccine efficacy if immunogens important to the protective immune response are not produced under specific in vitro conditions. While the results of this work are unable to fully elucidate which growth medium presents a transcriptome more representative of in vivo samples or best suited for bacterin production, it forms a foundation that can be used for future comparisons and provides a better understanding of the metabolic differences in broth and agar grown bacteria

Resilience of swine nasal microbiota to influenza A virus challenge in a longitudinal study

Abstract Influenza A virus (IAV) is an important contributing pathogen of porcine respiratory disease complex (PRDC) infections. Evidence in humans has shown that IAV can disturb the nasal microbiota and increase host susceptibility to bacterial secondary infections. Few, small-scale studies have examined the impact of IAV infection on the swine nasal microbiota. To better understand the effects of IAV infection on the nasal microbiota and its potential indirect impacts on the respiratory health of the host, a larger, longitudinal study was undertaken to characterize the diversity and community composition of the nasal microbiota of pigs challenged with an H3N2 IAV. The microbiome of challenged pigs was compared with non-challenged animals over a 6-week period using 16S rRNA gene sequencing and analysis workflows to characterize the microbiota. Minimal changes to microbial diversity and community structure were seen between the IAV infected and control animals the first 10 days post-IAV infection. However, on days 14 and 21, the microbial populations were significantly different between the two groups. Compared to the control, there were several genera showing significant increases in abundance in the IAV group during acute infection, such as Actinobacillus and Streptococcus. The results here highlight areas for future investigation, including the implications of these changes post-infection on host susceptibility to secondary bacterial respiratory infections

Silicon-doped β-Ga2O3 films grown at 1 µm/h by suboxide molecular-beam epitaxy

We report the use of suboxide molecular-beam epitaxy (S-MBE) to grow β-Ga2O3 at a growth rate of ∼1 µm/h with control of the silicon doping concentration from 5 × 1016 to 1019 cm−3. In S-MBE, pre-oxidized gallium in the form of a molecular beam that is 99.98% Ga2O, i.e., gallium suboxide, is supplied. Directly supplying Ga2O to the growth surface bypasses the rate-limiting first step of the two-step reaction mechanism involved in the growth of β-Ga2O3 by conventional MBE. As a result, a growth rate of ∼1 µm/h is readily achieved at a relatively low growth temperature (Tsub ≈ 525 °C), resulting in films with high structural perfection and smooth surfaces (rms roughness of <2 nm on ∼1 µm thick films). Silicon-containing oxide sources (SiO and SiO2) producing an SiO suboxide molecular beam are used to dope the β-Ga2O3 layers. Temperature-dependent Hall effect measurements on a 1 µm thick film with a mobile carrier concentration of 2.7 × 1017 cm−3 reveal a room-temperature mobility of 124 cm2 V−1 s−1 that increases to 627 cm2 V−1 s−1 at 76 K; the silicon dopants are found to exhibit an activation energy of 27 meV. We also demonstrate working metal–semiconductor field-effect transistors made from these silicon-doped β-Ga2O3 films grown by S-MBE at growth rates of ∼1 µm/h