Molecular pathogenesis of infectious disease in domestic animals
AffiliationVeterinary and Agricultural Sciences Collected Works
Document TypePhD thesis
Access StatusThis item is embargoed and will be available on 2021-10-04. This item is currently available to University of Melbourne staff and students only, login required.
© 2019 Sara Mahdizadeh
Mycoplasma species are free living bacteria with minimal gene sets, and have been used as model organisms to study the basic requirements for life. M. gallisepticum and M. bovis are important pathogens of birds and cattle and cause considerable economic losses in production animals, in part because of the chronic nature of the diseases they cause in their hosts. The limitations of current methods for detection, treatment and control underline the need for more effective vaccines to control these pathogens. A better understanding of the functions of genes of mycoplasmas is required to identify targets to create live attenuated vaccines. Although the genome sequences of M. gallisepticum and M. bovis have been completed and could lead to the identification of such potential targets, the limited tools available for genetic engineering of mycoplasmas prevents us from ascertaining many of the functions of genes in these organisms. Genetic loci that control CRISPR-based immunity are predicted to be present in mycoplasmas chromosomes, usually in non-coding regions. The M. gallisepticum genome contains a set of cas genes coding for putative type II-A Cas proteins, as well as four CRISPR arrays, one of which is potentially functional. In M. gallisepticum, a putative PAM sequence, NNNAAAA, located downstream of the spacers has been proposed. However, an in silico search of protospacers during the studies described in this thesis suggested that the potential PAM sequence may be TANN and may be located upstream of the spacers. Endogenous CRISPR/Cas systems have been exploited for genomic engineering in a number of bacteria. This opens the possibility of performing targeted mutagenesis of M. gallisepticum genome, by directing its endogenous CRISPR/Cas system to specific sites. In the studies described here, three different CRISPR arrays were constructed with or without the putative PAM sequence identified by in silico analyses. As a proof of concept, the ksgA sequence was targeted because mutations in this gene confer resistance to the antibiotic kasugamycin, allowing development of an efficient screening method using this readily detectable phenotype. The sequence analysis of PCR products showed that there was a higher frequency of indels in ksgA amplicons derived from M. gallisepticum transformed with plasmids containing a synthetic CRISPR array targeting ksgA than in M. gallisepticum transformed with a control plasmid lacking the CRISPR arrays. This indicated that the CRISPR/Cas system in M. gallisepticum could be used to introduce indel mutations into a targeted location. Furthermore, a single spacer in the CRISPR array could also direct the induction of mutations in M. gallisepticum strain S6. This prompted the design of a second type of synthetic CRISPR array that targeted ksgA with one spacer and another target gene with the other spacer. The rationale for this approach was to use kasugamycin resistance as a marker for concurrently induced mutations in the second target. Because they lack enzymes for de novo synthesis of nucleic acid precursors, mycoplasmas rely on their intrinsic nuclease activity to scavenge nucleotides from the environment. The orthologue of the M. bovis major nuclease gene in M. gallisepticum, mnuA, was selected as the second target gene and a spacer was synthesised that targeted a sequence with the PAM sequence identified by other studies on the M. gallisepticum CRISPR-Cas system. Following transformation with an assembled CRISPR array targeting both the ksgA and mnuA genes, kasugamycin resistant colonies were tested for nuclease activity. These transformants had a complete or partial loss of nuclease activity compared to negative control transformants. This suggested that the assembled CRISPR array and the endogenous Cas system suppressed the MnuA nuclease activity. Interestingly, sequence analyses of the mnuA coding sequence of the mutants found that the Cas proteins had not affected the gene itself. However, RT-qPCR analyses of the mnuA mRNA in the CRISPR array transformants found that they had no detectable mnuA mRNA, even though this mRNA was detectable in M. gallisepticum transformed with the negative control plasmid, and that the region of the mRNA 3’ to the site targeted by the spacer was less abundant than the 5’ end of the mnuA mRNA in the CRISPR mutants, suggesting that the mRNA was being cleaved by the M. gallisepticum Cas protein (MgaCas9) at the site targeted by the guide RNA transcribed from the synthetic CRISPR array. The similarities between the amino acid sequence of MgaCas9 and that of the Staphylococcus aureus SauCas9, which is capable of targeting both DNA and RNA, suggest the possibility that MgaCas9 may be able to cleave both DNA and RNA. Further experiments will be required to further characterise the MgaCas9 protein and explore its dependence on one or more PAM sequences. Metabolomics has recently been shown to be a useful tool for determining gene function in M. gallisepticum and M. bovis. The simple genomes of mycoplasmas make them ideal candidates for metabolomic studies. Because of the genome reduction that has occurred in mycoplasmas during their evolution, many biosynthetic pathways are missing resulting in their dependence on transporter proteins to provide nutrients from extracellular sources to supply their metabolic pathways. Therefore, many of the nutrient transport systems in mycoplasmas are likely to be essential for virulence, and thus are potentially targets for development of attenuated vaccines. Here, the polar metabolomes of two transposon mutants were characterised and compared to those of the relevant M. gallisepticum and M. bovis wild type strains. The first mutant selected for this study was a ΔmalF strain of M. gallisepticum with a transposon inserted into the malF gene. The MalF protein is predicted to be a component of an ABC transport system and has been reported to be essential for persistence and pathogenicity of M. gallisepticum strain Ap3AS in vivo. Bioinformatic studies and database searches confirmed that MalF was a component of an ABC transporter protein. Comparison of the steady state metabolite profile of the ΔmalF mutant to that of the wild type strain using gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) suggested that MalF was involved in glycerol transport. Labelling experiments utilising 13C-glycerol were used to demonstrate a change in glycerol metabolism in the absence of MalF. To assess the regulatory role of MalF, RT-qPCR studies were carried out on selected genes predicted to be involved in glycerol metabolism, demonstrating upregulation of expression of gtsA and the PTS system in the malF mutant, suggesting the predicted transporter encoded by gtsA and the PTS system were compensating for the loss of MalF, although the 13C- glycerol labelling experiments suggest that GtsA is more likely to be importing glycerol-3-phosphate than glycerol. The ΔmalF mutant produced reduced levels of hydrogen peroxide (H2O2) in vitro than the wild type, presumably due to the changes in glycerol metabolism, which may explain the reduced virulence of this strain in vivo that has been described previously. Electron microscopy demonstrated that the cells of the malF mutant were significantly larger and more elongated, and suggested the loss of the electron dense attachment organelle in the ΔmalF mutant, which may also explain its reduced capacity to colonise and attach to tracheal mucosa in studies described previously. Another mutant selected for metabolomic studies was a strain of M. bovis containing a transposon inserted into a putative component of a phosphotransferase system (PTS) transporter (MBOVPG45_0735). Bioinformatic analyses demonstrated more than 90% similarity to a putative pentitol phosphotransferase enzyme IIA component of M. agalactiae. The steady state metabolite profile of the ΔMBOVPG45_0735 mutant and that of the wild type were compared using both GC/MS and LC/MS, and 13C-glucose labelling experiments were also performed. The significantly lower levels of galactitol and sorbitol in the ΔMBOVPG45_0735 mutant suggested that MBOVPG45_0735 might be involved in transport or metabolism of these sugar alcohols. However, because of technical limitations, very few phosphorylated sugars were detected in the steady state analysis, and 13C labelling was not detected in phosphorylated sugars on either of the platforms, restricting assessment of the PTS function(s). In conclusion, the studies described in this thesis demonstrated the potential for use of the endogenous CRISPR/Cas system of M. gallisepticum to induce targeted mutations in putative virulence genes. This is a significant advance in the tools available to genetically manipulate mycoplasmas, which will progress understanding of the molecular pathogenesis of this organism, as well as assist in studies to synthesise a minimal bacterial genome based on this species. Furthermore, these studies show that metabolomics has potential to assist in understanding gene function in mycoplasmas, a key step forward in designing effective vaccines.
Keywordsmycoplasma gallisepticum; mycoplasma bovis; metabolomics; CRISPR/Cas
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