Thesis (Ph. D.)--University of Chicago, Dept. of Ecology and Evolution, December 2002. / Includes bibliographical references. Also available on the Internet.

This thesis documents the contribution of my bioinformatics research activities, including novel software development, to a range of research projects aimed at investigating the interactions between bacterial and viral pathogens and their hosts. The focus is largely on farm animal species and their pathogens, although some of the research has a wider scientific impact. RNA interference (RNAi) refers to a variety of related regulatory pathways present in animals, plants and insects. The major pathways are microRNAs (miRNAs), small-interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs). Marek’s disease virus is an important pathogen of poultry, causing T-cell lymphoma. We identified the presence and expression patterns of several MDV-encoded microRNAs, including the identification of 5 novel microRNAs. We also showed that not only do virus-encoded microRNAs dominate the mirNome within chicken cells, but also that specific host-microRNAs are down-regulated. We also identify novel virus-encoded microRNAs in other Herpesviridae and provide the first evidence of miRNA evolution by duplication in viruses. In related work, we present a novel microRNA generated by the canonical miRNA biogenesis pathway in Avian Leukosis Virus, another avian oncogenic virus, and publish data showing the expression pattern of known chicken microRNAs across a range of important avian cells. Two of the other RNAi pathways (siRNA and piRNA) form an important part of the antiviral response in arthropods. We have published work demonstrating an siRNA antiviral response to bluetongue virus and Schmallenberg virus in cells from the Culicoides midge, an important insect vector, as well as work demonstrating the importance of the piRNA pathway in the antiviral response to Semliki forest virus (SFV). Further work on flaviviruses in ticks demonstrates the active suppression of the siRNA response by Langat Virus, as well as a key difference between the siRNA responses in Mosquitos compared to ticks. Salmonella is one of the most important zoonoses, with an estimated 1.4 million cases of human salmonellosis per annum in the USA alone. Salmonella infections of farm animals are an important route into the human food chain. This thesis presents work on the comparative structure and function of 13 fimbrial operons within Salmonella enterica serovar Enteritidis as well as a genomic comparison of that serovar with Salmonella enterica serovar Gallinarum, a chicken-specific serovar. We characterised the global expression profile of Salmonella enterica serovar Typhimurium during colonization of the chicken intestine, and we have published the genomes of four strains of Salmonella eneterica serovars of well-defined virulence in food-producing animals. Our work in this area led to us publishing an important and comprehensive review of the automatic annotation of bacterial genomes. Finally, I present work on novel software development. ProGenExpress, a software tool that allows the easy and accurate integration and visualisation of quantitative data with the genome annotation of bacteria; Meta4 is a web application that allows data sharing of bacterial genome annotations from metagenomes; CORNA, a software tool that allows scientists to link together microRNA targets, gene expression and functional annotation; viRome, a software tool for the analysis of siRNA and piRNA responses in virus-infection studies; DetectiV, a software tool for the analysis of pathogen-detection microarray data; and poRe, a software tool that enables users to organise and analyse nanopore sequencing data.

Francisella tularensis is the causative agent of the disease tularemia and a potential bioterrorism agent. Few regulators have been identified in this organism and little is known about its genetic regulatory networks. In this dissertation project, culture-based and molecular methods were used to both determine the role of the RNA chaperone protein Hfq and identify potential novel small RNAs in F. tularensis subsp. novicida strain U112. The Hfq protein is recognized as an important regulatory factor in a variety of cellular processes, including stress resistance and pathogenesis, and has been shown in several bacteria to interact with small RNAs as a post-transcriptional regulator of mRNA stability and translation. Molecular methods were employed to determine that hfq is potentially transcribed in an operon with both the immediate up- and downstream genes. Phenotypic analysis of two transposon insertions within the hfq ORF revealed that the N-terminal region of the Hfq protein is more important for stress tolerance than the C-terminal end. Complete deletion of hfq resulted in a variety of growth defects under certain stress conditions such as heat-shock, low pH, and oxidative stress. Gene expression of hfq under several of these conditions changed significantly, further suggesting a role for the protein during stress tolerance. Because Hfq likely functions as a global regulator, the expression of several genes in the hfq mutant strain were compared to wild-type and some were significantly altered in particular growth backgrounds. The hfq mutant also exhibited a delayed entry into stationary phase and increased biofilm formation under certain conditions. Shotgun cloning and high-throughput sequencing were used to generate a list of potential sRNAs, an important class of regulators that had yet to be studied in F. novicida. Three candidates were selected and their expression verified using Northern blot analysis and self-ligating RACE. The sRNA transcript designated CISC-1 appears important for certain aspects of cell growth and is differently expressed under several stress conditions. ISC-2 is a transcript that has a minor effect on cell growth during exponential phase, but is upregulated during stationary phase. The third sRNA, ISC-16, is highly conserved among Francisella species and is potentially important for the biosynthesis of bacterial fatty acids. These sRNAs represent an important group of regulators that, along with the Hfq protein, could be important for controlling global gene expression in Francisella.

This thesis reports investigations of the interactions between the intestinal epithelial barrier and the intracellular apicomplexan Eimeria spp., both in vivo and in vitro. Initially, conventional in vivo studies using genetically modified animals were used to investigate the contribution of innate lymphoid cells (ILCs) to immune protection of the intestinal barrier. Additionally, to understand complex epithelial host-pathogen interactions a novel in vitro model of small intestine organoids was developed. Data suggest that immunoprotection against Eimeria vermiformis infections is mediated by T cells. Furthermore, there is an indication that ILCs have a detrimental effect in Eimeria vermiformis-infected immunocompromised animals. However, the role for ILCs in the regulation of the immune response remains unclear. The life cycles of Eimeria vermiformis and Eimeria falciformis are highly complex, comprising multiple schizogonies followed by a gametogony. In vitro life cycle completion has not been achieved to date due to the limitations of monolayer cell line models. It is likely that for a successful parasite development the interaction of the different epithelial cell types present in intestinal organoids is required. The development of intestinal organoids by Sato and colleagues gave rise to a breakthrough in cellular studies, providing the tools to study complex interactions between host tissues and invading pathogens in vitro. I showed that small intestine-derived organoids grow exponentially after passage and that each organoid contains distinct specialised epithelial cell types, such as Paneth, Goblet or enteroendocrine cells, suggesting that the organoid model closely resembles the native intestinal epithelium and that Eimeria spp. benefit from the three-dimensional structure and physiological characteristics of the organoid model. Intestinal organoids were infected with E. vermiformis or E. falciformis sporozoites. These completed several rounds of asexual replication but did not proceed to the final gametogony. Despite the need for the development of sensitive techniques applicable to three-dimensional cell culture models, these results indicate that intestine-derived organoids are a promising model to study host-parasite interactions at the intestinal epithelial barrier at the cellular and molecular levels.

Pathogenic host-microbe interactions can result from continuous evolution of a host's ability to resist infection and a pathogen's ability to survive and replicate. Pseudomonas aeruginosa is a versatile and opportunistic pathogen, ubiquitous in soil, and capable of damaging plants, vertebrates, and invertebrates. Previous studies in nematodes suggest that the pathogenic effects of P. aeruginosa can result from multiple distinct pathways: a toxin-based effect that kills within a few hours and a generalized virulence that kills over the course of multiple days. Using experimental evolution in the highly polymorphic nematode Caenorhabditis remanei, I show that nematode resistance to the two modes of pathogenesis in P. aeruginosa evolves through genetically independent pathways. These results demonstrate that multiple virulence factors in a pathogen can result in multiple responses in the host, and the genetic lines established here create resources for further exploration of the genetic basis for resistance to P. aeruginosa.

Acinetobacter baumannii is an important microorganism involved in hospital-acquired infections with a remarkable ability to develop resistance to multiple antibiotics (multidrug-resistance, MDR) which makes it a highly troublesome pathogen in many hospitals around the world. Third-generation cephalosporins (such as ceftazidime) and carbapenems (such as imipenem and meropenem) represent important treatment options for infections caused by this microorganism. Nevertheless, the number of strains resistant to these antibiotics has been increasing during the last decade. The ability to capture, mobilise and regulate the expression of resistance-genes of this microorganism is a cornerstone factor in the development of the MDR, where the Mobilome, defined as “all the mobile genetic elements in a cell”, is responsible for its genetic plasticity. The aim of this work was to analyse the role of insertion sequences (ISs), transposon-like structures, resistance-plasmids and ISCR1-like elements in the resistance to carbapenems and ceftazidime in A. baumannii. Fifteen carbapanem-resistant strains of Acinetobacter baumannii isolated from Chile and two ceftazidime-resistant strains from the United Arab Emirates were studied. Different ceftazidime- and carbapenem-resistance genes were analysed and their genetic environments were characterised. The Mobilome in the carbapenem-resistant strains was composed of insertion sequences (ISs), specifically by ISAba1 associated with blaOXA-51-like, ISAba3 associated to blaOXA-58, which in turn was detected in two different plasmids, and ISAba15 interrupting ISAba3. In the case of the ceftazidime-resistant strain, the presence of an ISCR1 element was harbouring the blaPER-7, which was detected in a megaplasmid. The Mobilome, in the strains analysed, was composed of a wide variety of genetic elements, such as plasmids, insertion sequences, ISCR-like elements, which reflects the ability of A. baumannii to use different genetic platforms to capture and use resistance genes, making the Mobilome an important contributor in the resistance and the dissemination of resistance genes among nosocomial pathogens around the world.

M.Sc. / Barley (Hordeum vulgare) production is a vital constituent of the South African economy. Many pathogens reside on barley, which lead to low quality and yield. One of the most prominent barley pathogens, Fusarium graminearum, is the causal agent of small grain scab. F. graminearum resistance to barley is regulated by multiple genes referred to as quantitative trait loci (QTL), which makes it difficult to breed for resistance in new cultivars. Each of these genes contributes to a specific defence area and collectively counteracts Fusarium infection and spread in the barley plant. The aim of this project was to isolate and identify induced genes after infection of three leave stage barley with F. graminearum. These genes were isolated through the use of Suppression subtractive hybridisation (SSH), cloned and then sequenced. From this data set three transcript derived fragments (TDFs) sharing homology to known genes were selected and their expression profiles were studied through Northern blot analysis. Three TDFs shared homology with known genes namely a putative protease inhibitor-related protein, a senescence associated gene, and a manganese superoxide dismutase (MnSOD). These TDFs were previously also recognised for their function in host pathogen interactions. The expression analysis done using Northern blots showed up-regulation of the three fragments after inoculation. These results indicated that all the TDFs studied may play a role in the defence reaction of barley infected with F. graminearum, where both senescence and proteinase inhibitors could limit infection as well as spread and MnSODs might be a protective enzyme against oxidative stress. The results of this study indicated that all of the identified TDFs had database matches to proteins identified during stress responses. Furthermore, the Northern blot results indicated that all the TDFs studied could play a role in the defence reaction of F. graminearum infected barley. These TDFs will form the basis of further studies into the interaction between barley and F. graminearum. The results form this study will add to our knowledge of the interaction between barley and a necrotrophic pathogen. This will aid in understanding how cereal pathogens deal with pathogen attack and will aid in development of new more tolerant barley cultivars.

La PGPR Burkholderia phytofirmans PsJN (Bp) stimule la croissance de diverses plantes et les protège également contre certains stress environnementaux. L’objectif des travaux a été d’approfondir les connaissances sur l’interaction Bp-plante, en se focalisant sur l’aspect physiologique et métabolique des feuilles d’Arabidopsis thaliana. Nous avons également déterminé les mécanismes impliqués dans la réponse des feuilles suite à l’inoculation de cette bactérie lors d’un stress abiotique (froid) ou biotique (Pseudomonas syringae pv. tomato DC3000, Pst).Nos résultats montrent que l’induction de la promotion de croissance d’A. thaliana par Bp pourrait être liée à l’accumulation des teneurs en métabolites primaires (acides aminés, glucides solubles et vitamines) et la variation du niveau des hormones dans les feuilles. La physiologie et le métabolisme des feuilles sont modifiés localement et de façon distale par la colonisation épi- et endophytique de Bp. De plus, les modifications des taux de métabolites sont plus marquées après une interaction plante-bactéries relativement longue. Par ailleurs, l’inoculation de Bp peut réduire les dommages sur l’activité photosynthétique dus au froid par une limitation non-stomatique de la photosynthèse et l’accumulation de pigments photosynthétiques. Enfin, la présence de Bp entraîne localement un retard dans le développement initial de Pst. Cependant, l’inoculation par Bp ne protège pas l’appareil photosynthétique lors d’une attaque par Pst. Ces travaux soulignent donc que le temps de présence et la localisation d’une PGPR dans une plante influencent la physiologie, le métabolisme et la tolérance aux stress de cette même plante. / Endophytic PGPR Burkholderia phytofirmans PsJN (Bp) promotes growth of various plants and triggers protection against several environmental stresses. To get more insights into the interaction between plant and Bp, we focused on leaf physiological and metabolic aspects of Arabidopsis thaliana. We also determined the mechanisms involved in the defense of leaves after inoculation of the bacteria followed by an abiotic (cold) or a biotic (Pseudomonas syringae pv. tomato DC3000, Pst) stress. Our results show that the induction of growth promotion of A. thaliana by Bp could be related to the accumulation of primary metabolite levels (amino acids, soluble carbohydrates and vitamins) and to the variation of hormone levels in the leaves. Leaf physiology and metabolism are changed locally and distally by Bp epi- and endophytic colonization. In addition, changes in metabolite levels are more pronounced after a relatively long interaction between plant and bacteria.Moreover, Bp inoculation can also reduce cold injury on the photosynthetic activity by a non-stomatal limitation of photosynthesis and accumulation of photosynthetic pigments. Finally, the local presence of Bp causes a delay in the development of Pst, but only in the early stages of the infection. However, the inoculation with Bp does not protect the photosynthetic apparatus during Pst attack.Thus, our results emphasize that the time of presence of a PGPR and his location in the plant could influence the plant physiology and stress tolerance.

Phytopathogenic fungi parasitise plants for survival. Knowledge of the interactions between plants and these fungi is of importance in designing control strategies. In this thesis the interactions of Eucalyptus polygalacturonase-inhibiting proteins (PGIPs) and fungal polygalacturonases (PGs) were characterised. About thirty years of research on the interaction between PGIPs and PGs was reviewed in Chapter 1. The gene sequences of the mature PGIP peptides from selected Eucalyptus species were determined in Chapter 2. The PGIP genes have very high similarities among themselves, signifying conservation of the function of PGIPs in this genus. Molecular characterisation of the endoPG gene of C. cubensis was presented in Chapter 3. This endoPG gene occurs more than two times in the genome of C. cubensis. The gene sequences of the other endoPGs remain to be determined. EndoPG production by C. cubensis, a Eucalyptus canker pathogen, was presented in Chapter 4. It was found that polygalactw:onases probably have a minor role in determining the levels of pathogenicity in different C. cubensis isolates. However, the hypovirus CHVl-173 has a role in reducing polygalacturonase production in infected hypovirulent C. cubensis isolates. Chapter 5 dealt with the interaction between Eucalyptus PGIPs and endoPGs from different fungal pathogens. Coniothyrium zuluense and Botryosphaeria dothidea produced more PGs than C. cubensis and Phytophthora cinnamomi in liquid culture. Disease tolerant Eucalyptus TAG5 clones produced PGIPs that are more effective in inhibiting endoPGs from C. zuluense than the susceptible Eucalyptus ZG14 clones. In Chapter 6 the gene sequence of the endoPG of Fusarium circinatum was detennined. This :fungus causes the . pitch canker disease in pine trees. This endoPG gene occurs as a single copy and is related to those of other Fusarium spp. The results presented in this thesis add to the current scientific knowledge pertaining to the role of PGIPs and PGs in pathogenic interactions, especially, between Eucalyptus PGIPs and fungal endoPGs. Interaction of Eucalyptus PGIPs and fungal endoPGs was demonstrated; therefore it is possible to genetically engineer Eucalyptus for more disease tolerant Eucalyptus plants using PGIP genes, especially, against Coniothyrium zuluense. This means that more research is needed to identity genes that will give a more global protection against many fungal pathogens. / Thesis (PhD (Plant Biotechnology))--University of Pretoria, 2006. / Genetics / unrestricted

Introduction: Emerging pathogens (EP) in cystic fibrosis (CF) include organisms that have infected individuals with CF for many years e.g. Burkholderia multivorans and Mycobacterium abscessus and more recently identified potential pathogens in CF e.g. Pneumocystis jirovecii and Pandoraea spp. The clinical implications of infection with these organisms are emerging but much remains unknown. Current evidence suggests that infection with some EP is associated with a worse prognosis. This thesis aimed to investigate the epidemiology, prevalence and clinical impact of EP in adults with CF.Methods: (1) The prevalence of P. jirovecii was determined in adults attending Manchester Adult Cystic Fibrosis Centre (MACFC) who were clinically stable versus those experiencing an acute pulmonary exacerbation (PEx). (2) The prevalence of M. abscessus at MACFC was determined, isolates of M. abscessus were strain typed, and cross infection risk was assessed. The clinical impact of Gram-negative EP was assessed by: (3) assessing their prevalence and determining if any patients attending MACFC harboured identical strains and had opportunities for cross infection to occur, and by (4) following these patients longitudinally and comparing outcome with age, gender and FEV1 matched Pseudomonas aeruginosa infected controls. Results: (1) P. jirovecii was detected via sputum PCR in 10 (4.4%) of 226 samples tested from 111 patients. P. jirovecii was more likely to be detected in samples taken from an acute pulmonary exacerbation compared with samples taken from stable patient visits (7 (9.2%) of 76 exacerbations samples versus 3 (2%) of 150 stable visit samples, p = 0.033). (2) Prevalence of M. abscessus was stable at ≤3.6% from 2010 to 2015. 21 patients (91.3%) with a positive culture for M. abscessus since 2010 were infected with M. abscessus subsp abscessus. 2 clusters of 7 and 6 patients harboured strains with identical variable number tandem repeat profiles and some of these patients had opportunities for cross infection to occur. 28.6% of patients developed M. abscessus pulmonary disease, 38.1% were persistently culture positive with no related pulmonary disease, and 33.3% spontaneously cleared M. abscessus from their sputum. (3) Prevalence of Gram-negative EP ranged from 1.9% (Ralstonia spp.) to 6.2% (B. multivorans). Small numbers of patients shared strains of B. multivorans; Stenotrophomonas S. maltophilia and Achromobacter; Ralstonia and Pandoraea species. Epidemiological connections consistent with possible cross infection were found in patients infected with Pandoraea and Ralstonia species. (4) Patients with B. multivorans; S. maltophilia; Ralstonia spp. and Pandoraea spp. had higher antibiotic requirements than P. aeruginosa infected matched controls. B. multivorans; Achromobacter spp.; Ralstonia spp. and Pandoraea spp patients had median FEV1 (% predicted) values ≥10% (absolute) lower than the overall median FEV1.Conclusion: Prevalence of all EP investigated at MACFC was low. P. jirovecii was approximately 5 times more likely to be detected in patients with acute PEx compared with stable patients suggesting it may be a cause of PEx. Results suggest that some patients attending MACFC may have acquired infection with M. abscessus subsp abscessus, Pandoraea spp. or Ralstonia spp. through cross infection. Patient numbers are too small to establish this with certainty and a common environmental source is possible. Gram-negative EP other than Achromobacter spp. were associated with higher acute antibiotic requirements than P. aeruginosa matched controls suggesting these EP are associated with an increased risk of PEx. The fact that many Gram-negative EP were associated with lower median lung function may indicate that these EP cause accelerated lung function decline or that patients with more advanced disease are at most risk of acquiring EP.