Browsing by Author "Niu, Dongyan"

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    Biological insights into the parasitic nematode Heligmosomoides bakeri from bulk and single-cell transcriptomics
    (2023-12-05) Pollo, Stephen Michael Jerome; Wasmuth, James; Gilleard, John; Lynch, Tarah; Niu, Dongyan
    Heligmosomoides bakeri, a parasitic nematode (roundworm) of mice, is closely related to economically important parasites of livestock and the hookworm parasites of humans. As a murine parasite it is more amenable to being maintained and manipulated in a controlled laboratory environment than its relatives. The worm enters its host during its third larval stage and develops through another larval stage into adults that reside in the lumen of the small intestine to mate and lay their eggs. Unravelling these processes and other processes critical to H. bakeri survival could reveal new targets to use for drug discovery for controlling this group of parasites as well as refine previous predictions of immunomodulatory molecules, which have therapeutic potential. My thesis aims to describe the gene expression of H. bakeri during the parasitic phase of its lifecycle. I first used RNA sequencing to investigate how the (whole worm) expression of genes varies across the parasite’s infection. Using differential gene expression analyses I identified genes important for development and genes important for the males vs the females. I also uncovered evidence of an increased importance for anaerobic respiration in the adults compared to the larvae and hypothesize that aerobic conditions are important for the critical developmental processes of molting and cuticle synthesis. I then analyzed single-cell RNA-seq data from young adult male and female worms. Leveraging cell type markers from the model nematode C. elegans allowed me to putatively identify gamete, embryo, intestine, hypodermis, neuron, and muscle cells. The resulting cluster expression profiles can guide investigations into tissue-specific aspects of the adult worms. Putative intestinal transcription profiles suggest compartmentalization of function along the anterior-posterior axis of the worms, with an emphasis on protein synthesis in the anterior portion, as has been observed in other worms. Embryonic profiles are noticeably different from C. elegans embryogenesis, particularly with respect to paternal contributions to the early embryo. Overall, these datasets extend our understanding of how H. bakeri lives as a parasite and provide a public resource for further investigation into genes of interest. They also lay the groundwork for more comprehensive comparisons among the phylum Nematoda.
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    Development of a Gram-type Specific Method for Detection of Bovine Mastitis Pathogens by Combining Loop-mediated Isothermal Amplification (LAMP) and Split Trehalase Technologies
    (2022-07-20) Miao, Zhuohan; De Buck, Jeroen; Finney, Constance; Niu, Dongyan
    Bovine mastitis, which results mainly from intramammary infections (IMI) caused by Gram-positive or Gram-negative bacteria, is a huge burden on the global dairy industry. Oftentimes the presence of an infection can be deduced by measuring the somatic cell count (SCC) or released enzymes, or directly detected by the presence of mastitis pathogens. While many laboratory diagnostic methods (e.g., SCC determination, bacteria culturing, qPCR) exist, few have been modified and adopted for on-farm use due to the requirement of sterility, dedicated equipment and well-trained personnel for implementation, time restraints, and accuracy. As such, the development of new methods that can sensitively and specifically detect mastitis-causing pathogens on-farm would be very valuable. We aimed to detect and distinguish Gram-positive and Gram-negative mastitis bacteria to inform on the proper and prompter treatment/antibiotic to prescribe through two steps: (i) develop PCR and LAMP assay for a point-of-care (POC) amplification of Gram-positive and Gram-negative bacteria and (ii) use a modified amplicon binding split trehalase assay (ABSTA) to detect this amplification by a specific protein-DNA binding and the complementation of split trehalase. To do this, split trehalase fusion proteins HisTreA-C-SpoIIID and HisTreA-N-SpoIIID were purified, and the binding with modified oligonucleotides incorporated with SpoIII-specific recognition sequences was tested to verify and optimize the complementation conditions. Next, recognition sequences were introduced in Gram-type specific PCR primers, allowing for specific detection of PCR amplicons by ABSTA. We also integrated the recognition sequences in three Gram-type specific LAMP primer sets designed based on the pathogens’ 16S rRNA genes, allowing for specific detection of Staphylococcus, Streptococcus and Gram-negative bacteria LAMP products by ABSTA. The salt concentration, protein reagents versus DNA substrate ratio, direction and linker length of tandem recognition sites required for the complementation were optimized. Binding with purified PCR products demonstrated Gram-type detection specificity with the protein reagents, whereas the complementation appeared to be inhibited by agents in unpurified PCR products. The sensitivity of detection of bacterial genomic DNA of Escherichia coli, Staphylococcus devriesii and Streptococcus uberis ranged from 2 to 24. The sensitivity of detection of E. coli in spiked milk samples was 11 CFU/ml, but 4.9 × 104 CFU/ml and 2.0 × 105 CFU/ml for S. devriesei and S. uberis, respectively. The analytical specificity of the newly designed LAMP primer sets was evaluated with ten mastitis isolates at two genomic DNA copy number levels. In conclusion, the combination of bacterial genetic target amplification by LAMP and ABSTA demonstrated high sensitivity and specificity towards genomic DNA of Gram-positive and Gram-negative bacteria, enhancing the potential of developing a timesaving, user-friendly and cost-effective on-farm diagnostic method for educated treatment decisions of bovine mastitis causative pathogens.
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    Effect of commercial slow-release urea product on in vitro rumen fermentation and ruminal microbial community using RUSITEC technique
    (2022-05-06) Guo, Yongmei; Xiao, Ling; Jin, Long; Yan, Sumei; Niu, Dongyan; Yang, Wenzhu
    Abstract Background The objectives of this study were to determine the effect of commercial slow-release urea (SRU) on in vitro fermentation characteristics, nutrient digestibility, gas production, microbial protein synthesis and bacterial community using a rumen simulation technique (RUSITEC). The experiment was a completely randomized design with four treatments and four replications of each treatment. Treatments were: control diet (no SRU addition), control diet plus 0.28% SRU (U28), or plus 0.56% SRU (U56), and control diet that was modified substituting a part of soybean meal equivalent to 0.35% SRU (MU35; dry matter [DM] basis). The experiment consisted of 8 d of adaptation and 7 d of data and sample collection. Rumen inoculum was obtained from three ruminally fistulated Angus cows fed the same diet to the substrate incubated. Results Digestibility of DM, organic matter (OM), crude protein (CP), fibre and starch was not affected, but daily production of gas (P < 0.07) and methane (P < 0.05) was quadratically increased with increasing SRU supplementation. The increase of SRU addition did not affect fermentation pH and total volatile fatty acid (VFA) production, whereas linearly (P < 0.01) decreased proportion of propionate, and linearly (P < 0.01) increased acetate to propionate ratio and ammonia nitrogen (N) concentration. The microbial N efficiency was also linearly (P < 0.03) improved with increasing supplementation of SRU. In comparison with control diet, the dietary substitution of SRU for part of soybean meal increased (P < 0.05) the digestibility of DM, OM and CP and decreased (P < 0.02) the total gas production. The total VFA production and acetate to propionate ratio did not differ between control and MU35, whereas the proportion of butyrate was lower (P < 0.05) and that of branched-chain VFA was greater (P < 0.05) with MU35 than control diet. Total and liquid-associated microbial N production as well as ammonia N concentration were greater (P < 0.03) with MU35 than control diet. Observed operational taxonomic units (OTUs), Shannon diversity index, and beta diversity of the microbial community did not differ among treatments. Taxonomic analysis revealed no effect of adding SRU on the relative abundance of bacteria at the phylum level, while at the genus level, the beneficial impact of SRU on relative abundance of Rikenellaceae and Prevotellaceae in feed particle-associated bacteria, and the abundance of Roseburia in liquid associate bacteria was greater (P < 0.05) with MU35. Conclusions Supplementation of a dairy cow diet with SRU showed potential of increase in ammonia N concentration and microbial protein production, and change fermentation pattern to more acetate production. Adding SRU in dairy cow diet also showed beneficial effect on improving digestibility of OM and fibre. The results suggest that SRU can partially substitute soybean meal in dairy cow diet to increase microbial protein production without impairing rumen fermentation.
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    Effects of brewers’ spent grain protein hydrolysates on gas production, ruminal fermentation characteristics, microbial protein synthesis and microbial community in an artificial rumen fed a high grain diet
    (2021-01-04) Ran, Tao; Jin, Long; Abeynayake, Ranithri; Saleem, Atef M; Zhang, Xiumin; Niu, Dongyan; Chen, Lingyun; Yang, Wenzhu
    Abstract Background Brewers’ spent grain (BSG) typically contains 20% – 29% crude protein (CP) with high concentrations of glutamine, proline and hydrophobic and non-polar amino acid, making it an ideal material for producing value-added products like bioactive peptides which have antioxidant properties. For this study, protein was extracted from BSG, hydrolyzed with 1% alcalase and flavourzyme, with the generated protein hydrolysates (AlcH and FlaH) showing antioxidant activities. This study evaluated the effects of AlcH and FlaH on gas production, ruminal fermentation characteristics, nutrient disappearance, microbial protein synthesis and microbial community using an artificial rumen system (RUSITEC) fed a high-grain diet. Results As compared to the control of grain only, supplementation of FlaH decreased (P < 0.01) disappearances of dry matter (DM), organic matter (OM), CP and starch, without affecting fibre disappearances; while AlcH had no effect on nutrient disappearance. Neither AlcH nor FlaH affected gas production or VFA profiles, however they increased (P < 0.01) NH3-N and decreased (P < 0.01) H2 production. Supplementation of FlaH decreased (P < 0.01) the percentage of CH4 in total gas and dissolved-CH4 (dCH4) in dissolved gas. Addition of monensin reduced (P < 0.01) disappearance of nutrients, improved fermentation efficiency and reduced CH4 and H2 emissions. Total microbial nitrogen production was decreased (P < 0.05) but the proportion of feed particle associated (FPA) bacteria was increased with FlaH and monensin supplementation. Numbers of OTUs and Shannon diversity indices of FPA microbial community were unaffected by AlcH and FlaH; whereas both indices were reduced (P < 0.05) by monensin. Taxonomic analysis revealed no effect of AlcH and FlaH on the relative abundance (RA) of bacteria at phylum level, whereas monensin reduced (P < 0.05) the RA of Firmicutes and Bacteroidetes and enhanced Proteobacteria. Supplementation of FlaH enhanced (P < 0.05) the RA of genus Prevotella, reduced Selenomonas, Shuttleworthia, Bifidobacterium and Dialister as compared to control; monensin reduced (P < 0.05) RA of genus Prevotella but enhaced Succinivibrio. Conclusions The supplementation of FlaH in high-grain diets may potentially protect CP and starch from ruminal degradation, without adversely affecting fibre degradation and VFA profiles. It also showed promising effects on reducing CH4 production by suppressing H2 production. Protein enzymatic hydrolysates from BSG using flavourzyme showed potential application to high value-added bio-products.
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    Genetics of Ribitol Catabolism in Rhizobium leguminosarum
    (2019-09-20) Buhlers, Deborah; Hynes, Michael F.; Gieg, Lisa M.; Wong, Sui-Lam; Oresnik, Ivan; Niu, Dongyan
    Rhizobium leguminosarum bv. viciae strain VF39SM contains six plasmids; a strain cured of both pRleVF39c and pRleVF39d is unable to grow on ribitol, whereas strains cured of only one of the plasmids can grow on this substrate. BLAST database searches and previous work show that the pRleVF39d plasmid ribitol catabolic genes are widely distributed among many R. leguminosarum strains. The less common pRleVF39c plasmid locus contains genes encoding a DeoR transcriptional regulator (rlcR), an ATPase component of an ABC transporter (rlcB), a periplasmic binding protein of an ABC transporter (rlcA), two ABC transporter permease components (rlcC, rlcF), a glycerophosphoryl diester phosphodiesterase (rlcG), a hydrolase (rlcH), a ribitol dehydrogenase (rlcD) and a ribulokinase (rlcK). The pRleVF39d locus contains genes encoding a reductase (rldE), a ribitol-2-dehydrogenase (rldD), an ABC transporter permease (rldC), the ATPase of an ABC transporter (rldB), the periplasmic binding protein of an ABC transporter (rldA), and an AraC-like regulator (rldR). Mutagenesis of the genes of these two loci showed that rlcK, rlcD, rldD, rldA, and rldR are required for the catabolism of ribitol. Three c and d plasmid double mutants, rlcK-rldA, rlcK-rldD and rlcK-rldR, were also unable to grow in the presence of ribitol as sole carbon and energy source. The d plasmid ribitol catabolic genes comprise as single operon, while the c plasmid ribitol catabolic genes are in multiple operons, with rlcR and rlcB comprising a divergently transcribed operon, and the remaining c plasmid ribitol catabolic genes making up additional transcripts. The c plasmid ribitol catabolic genes are induced by ribitol and seed exudates of peas, beans and lentils. Nodulation competition assays using ribitol catabolic gene single and double mutants, demonstrated that ribitol catabolism is required for nodulation competitiveness on peas (cv. Little Marvel) and lentils (cv. Marble), but not for vetch or faba beans (cv. Windsor). Ribitol catabolic gene induction during interaction of R. leguminosarum VF39SM with lentils and vetch seedlings also showed that rldR, but not rlcA was induced during early stages of interaction with the roots of lentil seedlings, and that rlcA is induced within the nodules of vetch.
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    Genomic and Virulence Profiling of Erysipelothrix rhusiopathiae Isolated from Widespread Muskox Mortalities in Arctic Archipelago
    (2023-04-21) Seru, Lakshmi Vineesha; Niu, Dongyan; Kutz, Susan; Mohamed, Faizal Abdul Careem; Forde, Taya L
    Muskoxen are an important food and economic resource for the indigenous people in the Canadian Arctic; however, in recent years this species has experienced substantial disease-related population declines. A single strain (‘Arctic clone’) of Erysipelothrix rhusiopathiae was associated with mass mortalities of muskoxen on Banks and Victoria Islands in 2010-2013, and various wild species on Prince Patrick Island in 2017. In 2021, an outbreak of E. rhusiopathiae was reported for the first time in muskoxen on Ellesmere Island. The predominance of the Arctic clone in the Arctic raises the question of virulence of this lineage. Objectives of this study were to characterize E. rhusiopathiae isolates from Ellesmere Island and identify amino acid sequence variations among 17 virulence genes, pathogenicity islands and prophages among 28 Arctic clone and 31 other closely related E. rhusiopathiae genomes. In addition, unique genetic contents of the Arctic clone that may encode virulence traits were determined via pan-genome wide association studies. Comparison of virulence gene sequences among 59 E. rhusiopathiae genomes offered insights into amino acid variations unique to the Arctic clone. I found that 16 of 17 virulence genes investigated were present and 4 of 17 were highly conserved among all the genomes. Putative virulence gene sequences of adhesin, rhusiopathiae surface protein-A, choline binding protein-B and leucine rich repeat protein had amino acid sequence variants unique to the Arctic clone. These genes encoded proteins that help E. rhusiopathiae to attach to the host endothelial cells and form biofilms. Characterizing pathogenicity islands revealed a novel finding of 12/28 Arctic clone isolates harbouring toxin-B. Toxin-B is an exotoxin produced by Clostridiodes difficile and causes pathogenesis via cytolysis. None of the prophages harboured virulence genes. The core genome alignment of 59 E. rhusiopathiae whole genomes provided evidence that the Arctic clone might be associated with the mortality on Ellesmere Island. The newly isolated E. rhusiopathiae belong to the Arctic clone, which was found to contain unique amino acid sequences for known virulence genes. The Arctic clone may have gained new virulence traits via mobile genetic elements mediated.
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    Host responses in laying hens following infectious bronchitis vaccination: Comparison of two vaccination strategies
    (2021-06-03) Buharideen, Sabrina Marsha; Abdul Careem, Mohamed Faizal; Czub, Markus; Niu, Dongyan; Gedamu, Lashitew
    The infectious bronchitis virus (IBV) causes infectious bronchitis (IB) and causes nephritis and reproductive tract abnormalities depending on the infecting IBV strain. Vaccination for the control of IB has been practiced for decades. Although it has been shown that administration of inactivated vaccine following priming with live attenuated vaccines in pullets induces protection of laying hens against IB, the immunological basis of this protective response has not been investigated adequately. The first objective was to inactivate and formulate the IBV Massachusetts (Mass) variant that was isolated from a flock with shell-less egg syndrome (SES) as an in-house adjuvanted vaccine and test whether it induces an antibody-mediated immune response. Although we observed that the in-house adjuvanted inactivated IBV Mass variant vaccine induces a positive antibody-mediated immune response, it does not induce an antibody-mediated immune response similar to that of a commercial inactivated IBV Mass vaccine. The second objective of the study was to compare two vaccination strategies adopted by the Canadian poultry industry in terms of their ability to induce an adequate immune response in the IBV-impacted tissues in laying hens. Vaccination strategy 1 (multiple live attenuated vaccines) and vaccination strategy 2 (combination of live attenuated vaccines given multiple times and one inactivated vaccine) were given to pullets between 3 and 16 weeks of age. Serum anti-IBV antibodies, recruitment of T cell subsets, and interferon (IFN)-γ mRNA expression were measured at 10 weeks post-last vaccination, in selected tissues. We observed that vaccination strategy 2 induced higher serum anti-IBV antibody response and IFN-γ mRNA expression in the lungs, kidneys and reproductive tract. Both vaccination strategies 1 and 2 recruited CD4+ T cells in the lungs and isthmus and CD8+ T cells in all the examined tissues except the uterus. Serum collected from chickens vaccinated with vaccination strategy 2 was able to neutralize the IBV Mass variant that caused SES in Western Canadian layers, indicating the potential ability of vaccination strategy 2 to protect laying hens against this IBV variant. Overall, our findings indicate that administration of live attenuated vaccines followed by an inactivated vaccine, induces better host responses in laying hens.
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    Investigations of Methanobrevibacter spp. Culture to Enable Archaea Virus Propagation, and Comparison of the Efficacy of Individual Bacteriophages and their Cocktails against Escherichia coli O157
    (2022-01-06) Fletcher, Jane; van der Meer, Frank; Niu, Dongyan; McAllister, Tim
    Viruses that target prokaryotes are a growing area of interest for control of pathogens and other detrimental microorganisms. Archaeal viruses were identified as an option for the mitigation of methane produced by Methanobrevibacter ruminantium, which lives in ruminants such as cattle. In this study we tested three protocols to culture Methanobrevibacter spp. While a PCR was able to confirm the presence of M. ruminantium in these cultures, we could not achieve replicable growth. Unfortunately, an anti-methanogen virus could not be isolated from rumen fluid, and the culture of the archaea was found to be challenging. Another microbial target, Escherichia coli, was also explored in this study. Phages are used to control pathogenic E. coli, however, while phages can be used as support or as an alternative to antibiotic therapy, pre-existing or induced anti-phage mutations in the targeted bacteria can reduce the efficacy of this approach. Phage cocktails have been used as a strategy to reduce the likelihood that phage resistance would develop. However, in some cases, individual phages were found to be more effective at lysing bacteria and at reducing the frequency of resistance development. Therefore, we tested individual (T1, T4, T5, and rV5) and cocktail phage treatments against six strains of E. coli O157, including EDL933, R508N, CO281-31N, E32511, H4420N, and E318N. EDL933 was selected for further experiments as this is a well characterized human pathogenic strains. Our results showed that T5 and cocktail T1+T4+T5+rV5 treatments were effectively lysing bacteria, however, we have strong indications that phage resistance developed in both individual and cocktail-exposed EDL933. Cocktail T1+T4+rV5 was more effective at lysing EDL933 compared to T1, T4, or rV5 treatments. Initially the concentration of T1 in T1+T4+rV5, and T5 in cocktail T1+T4+T5+rV5 decreased before increasing to the levels of replication observed in individual treatments. T1 in T1+T4+rV5 decreased over time until it was not detectable. Sequencing of exposed strains and the phages is needed to reveal the genetic basis of resistance. More research into strategic combinations of phages, based on receptor usage or lytic activity, is required to increase the susceptibility of pathogenic E. coli to phages or antibiotics.
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    Isolation, Characterization, And Application Of Novel Bacteriophages For Preventing Experimental Infection With Mycobacterium avium subspecies paratuberculosis (MAP) Of Dairy Calves
    (2023-10-24) Harman-McKenna, Victoria Katharine; De Buck, Jeroen; Niu, Dongyan; van deer Meer, Frank
    Johne's disease (JD), a chronic enteritis affecting ruminants, poses a substantial economic burden of $28 million CAD annually to the Canadian dairy industry. The disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP) and primarily spreads to youngstock through the ingestion of contaminated feed and water. Due to JD's prolonged subclinical phase and the absence of a cure, conventional test-based culling has proven ineffective in curbing its spread. Mycobacteriophages, as potential anti-mycobacterial agents, offer promise in controlling mycobacterial infections. Isolating new MAP-lysing bacteriophages is a crucial step toward implementing phage-based JD control. In pursuit of novel mycobacteriophages for JD control, we optimized an isolation protocol involving fecal spiking and varied isolation solution compositions. This enhanced method led to a significant boost in phage yield, increasing it from less than 1% to an 59% recovery of the spiked phages from fecal samples. We screened 475 environmental samples, enriched with MAP and fast-growing M. smegmatis, sourced from farms with documented MAP presence. These samples encompassed diverse environments, including soil, manure pits, lactation barns, feces, milk, and drain water, resulting in the isolation of 14 phages. Restriction enzyme profiling facilitated the identification of 12 distinct and novel phages. Further investigations into host range revealed that 8 of these phages could lyse genetically diverse MAP strains. We also explored aspects such as cross-resistance, lysogeny, and the influence of pH. Subsequently, we assessed the efficacy of these mycobacteriophages in preventing MAP infection in dairy calves through oral administration in an experimental calf infection trial. This research demonstrated the potential of mycobacteriophages to prevent MAP infection effectively. Our findings highlight the discovery and characterization and application of eight novel mycobacteriophages. This research opens new avenues for the utilization of these newly isolated mycobacteriophages in strategies aimed at curbing the spread of Johne's disease on dairy farms.
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    Microbiological and Molecular Identification and Characterization of Avian Pathogenic Escherichia coli from Poultry Farms, Alberta
    (2024-01) Wang, Yanqi; Niu, Dongyan; Niu, Dongyan; Abdul-Careem, Mohamed Faizal; Fairbrother, John; Zhang, Kunyan
    Avian pathogenic Escherichia coli (APEC) is the etiological agent of avian colibacillosis, leading to significant economic losses in Canada and elsewhere. Avian fecal E. coli (AFEC) is considered to serve as a potential reservoir for APEC given shared genetic features. This study aimed to profile phenotypic and genotypic features of causative APEC in Alberta. Here, 100 APEC isolates from diseased and dead chickens (broiler and layer) and turkeys from 45 farms collected between 2021 and 2023 were subjected to serogroup typing, APEC-associated virulence gene (VG) profiling, antimicrobial susceptibility testing, multilocus sequence typing, and phylogenetic analysis. In addition, 121 AFEC isolates from five layer flocks were collected to investigate APEC-associated VG carriage in healthy birds. Furthermore, genetic similarity between 22 AFEC strains identified as potential APEC (poAPEC) and APEC strains was assessed through phylogenetic analysis. We identified 27 serogroups, 38 known and two novel sequence types (STs), and varied VG distribution across APEC isolates from chickens and turkeys. Interestingly, variations in VG among AFEC strains were found across distinct egg laying stages or housing systems. Antimicrobial resistance (AMR) genes, especially extended-spectrum beta-lactamase genes were detected in APEC and poAPEC strains across all poultry species. Moreover, phylogenetic analysis revealed close clonal relationships among APEC and poAPEC strains. Notably, we identified a poAPEC isolate that shared ST155 and VGs with an APEC strain. This study is the first to report the characteristics of APEC and poAPEC isolates that cause or potentially cause colibacillosis morbidity and mortalities in chickens and turkeys in Alberta. A variety of APEC phenotypes and genotypes can lead to colibacillosis in poultry farms and may originate from the commensal E. coli found in the gastrointestinal tract of birds.
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    The Virus Resistance Mechanism of Abortive Infection K in Lactococcus lactis
    (2023-07) Du, Amy; Hynes, Michael Francis; Fraser, Marie Elizabeth; Harrison, Joe Jonathan; Niu, Dongyan; Fortier, Louis-Charles
    In nature, there is a constant battle between viruses and their hosts known as the evolutionary arms race, where they both continuously evolve to attempt to gain an advantage over the other. This evolutionary arms race has resulted in many mechanistic inventions, such as abortive infection (Abi) mechanisms in bacteria. In Abi mechanisms, viruses inject their genetic material into cells, but bacteria block phage replication by preventing one of the steps of phage maturation, typically resulting in cell death. This study looks at the Abi mechanism called AbiK, which is controlled by a protein of the same name that was discovered on a plasmid in Lactococcus lactis. A large portion of the study focuses on what happens in vivo during the AbiK mechanism. The normal wild-type infection, the abortive infection mediated by the AbiK protein, and the escape of the AbiK mechanism by a mutant phage are all studied for comparative analyses. Observations have shown that phage DNA replication is inhibited, and that transcription of the phage genes is delayed during the AbiK mechanism. The mutant phage can bypass these inhibitions, and successfully replicate its DNA and complete the phage lytic cycle. RNA-Sequencing experiments were conducted to narrow down the mechanism behind AbiK, and preliminary results indicate that AbiK depletes the nucleotide resources available in the cell, eventually resulting in cell death and inhibition of the phage replication cycle. A second part of this study focuses on the biochemistry of the AbiK protein. The AbiK protein has a polymerase activity that polymerizes an untemplated long single-stranded DNA that is covalently attached to the AbiK protein. This study identified which amino acid is used as a primer, which is tyrosine 44 on the AbiK protein. The release of protein structure prediction databases allowed for the analysis of a generated model of the AbiK protein, allowing for the elucidation of other biochemical functions the AbiK protein has. In addition to the polymerization activity, AbiK is hypothesized to bind to nucleotides or other proteins. The culmination of these studies allowed for insight on the AbiK mechanism, generating a hypothesis for future studies.