Functional analysis of signal transduction systems in clostridium perfringens

Clostridium perfringens is an anaerobic, Gram positive bacterium that is the etiological agent of a number of human and animal infections, such as gas gangrene, food poisoning and enterotoxemia. The pathogenesis of C. perfringens infections is mainly due to intoxication by a number of toxins and extracellular enzymes produced by the bacterium, such as α-toxin, β-toxin, ε-toxin, perfringolysin O, sialidase, collagenase, α- clostripain and hyaluronidase. Of these toxins, α-toxin and perfringolysin O have been shown to be important in the development of gas gangrene. The production of α-toxin, perfringolysin O, α-clostripain and collagenase has been shown to be under the transcriptional control of the VirSR two-component signal transduction system, which can regulate toxin gene transcription either directly or indirectly via the regulatory RNA molecule, VR-RNA. The C. perfringens strain 13 genome encodes 48 genes whose products are putatively involved in signal transduction. Research on the signal transduction pathways within C. perfringens has mainly focused on the VirSR two-component signal transduction system. The objective of this study was to investigate other signal transduction systems. Based on a bioinformatic analysis of the C. perfringens genome, three independent signal transduction pathways were chosen for further analysis based on their sequence similarity to other systems, their genetic organisation and their novelty. The systems chosen for further analysis were the RevR orphan response regulator, the MalNO two-component signal transduction pathway and the ReeS orphan sensor histidine kinase. Chapter Two reports the identification and functional characterisation of the first orphan response regulator shown to affect virulence in C. perfringens, the results of which were published in Infection and Immunity in June, 2011. RevR shares a similar domain architecture and amino acid sequence to members of the YycF, PhoB, PhoP and VicR family of response regulators from Gram-positive bacteria. A revR mutant was constructed by allelic exchange and was shown to have an altered cellular morphology, mutant cells produced long filaments compared to the consistently short rods produced by the wild-type. Complementation of the revR mutant with the wildtype revR gene restored the wild-type cellular morphology. To determine the effect of the revR mutation on gene expression, the transcriptome of the mutant was analysed using microarrays, which showed that more than 100 genes were differentially regulated in the mutant compared to wild-type, including some genes involved in cell wall metabolism and genes encoding potential virulence factors such as sialidase, hyaluronidase and α-clostripain, a cysteine protease. The altered expression of the sialidase, hyaluronidase and α-clostripain encoding genes was confirmed by QRT-PCR and was shown to correspond to a similar change in enzyme activity, which could be reversed following complementation. Based on the altered expression of several virulence-associated genes, the virulence of the revR mutant was tested using the mouse myonecrosis model. The results revealed a significant attenuation in virulence of the revR mutant compared to wild-type; attenuation that could be restored to wildtype levels following complementation. The identification of RevR represents the first orphan response regulator to be characterised in C. perfringens and only the second signal transduction system shown to control virulence. The identification of the MalNO two-component signal transduction system, which regulates the utilization of maltose in C. perfringens, is presented in Chapter Three. The MalNO system was originally named VirJI, based on a proposed role in virulence due to the upregulation of the plc gene (encoding α-toxin) during stationary phase, but for functional reasons we renamed this system as MalNO. To determine its role in virulence a malO mutant was constructed and used in the mouse myonecrosis model, which showed no difference in virulence between the mutant and wild type. Microarrays were used to determine gene expression differences in the mutant and showed a significant increase in the expression of genes involved in the uptake and metabolism of maltose. These results were confirmed using QRT-PCR and could be reversed following complementation with the wild-type malO gene. Growth of the mutant, complemented mutant and wild-type strains in medium with or without maltose showed that the malO mutant recovered more quickly from maltose deprivation compared to the wild-type and complemented strains. The characterisation of the MalNO two-component signal transduction system represents the first signal transduction system shown to be involved in carbohydrate catabolism in C. perfringens and provides a valuable insight into the complex regulatory networks that control nutrient acquisition in C. perfringens. The cpe1512 gene (later renamed reeS) was originally annotated as encoding a hybrid sensor histidine kinase response regulator protein, however, further analysis suggested that it functions as an orphan sensor kinase. A reeS mutant was constructed and microarrays were used to determine gene expression changes as a result of the reeS mutation. The microarrays, which were confirmed by QRT-PCR, revealed that the nanI and nanJ genes (encoding the major and minor sialidases, respectively) were significantly downregulated in the mutant, which could be reversed following complementation. The downregulation of the nanI and nanJ genes by ReeS appeared to occur independently of the VirSR system and corresponded to a significant decrease in sialidase activity in the mutant, which could be restored to wild-type levels following complementation. Virulence was unaffected by the reeS mutation however, its role in the pathogenesis of other C. perfringens infections remains to be determined. ReeS is the first orphan sensor histidine kinase to be characterised in C. perfringens and coupled with RevR, the work within this thesis has added more complexity to our current understanding of the regulation of sialidase production in C. perfringens. This thesis reports the identification and characterisation of three different signal transduction systems, which control various aspects of C. perfringens growth and pathogenesis and are all independent of the VirSR system. RevR was shown to be involved in the regulation of virulence and several potential virulence factors. Identification of ReeS led to the discovery of an additional regulator that controls sialidase production and MalNO, which is the first two-component signal transduction system to be shown to regulate carbohydrate catabolism in C. perfringens. When taken together this thesis adds valuable insight into the complex regulatory networks that exist within C. perfringens and expands our current knowledge of C. perfringens gene regulation.