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Title: Utilisation of mucin sulphur by Pseudomonas aeruginosa : importance for cystic fibrosis
Author: Robinson, Camilla
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2013
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Pseudomonas aeruginosa is a common cause of chronic respiratory infection in cystic fibrosis (CF). Infection is established within the lung epithelial mucus layer, through adhesion to mucins. Terminal residues on mucin oligosaccharide chains are highly sulphated and sialylated, which increases their resistance to degradation by bacterial enzymes. However, a number of microbes display mucin sulphatase activity, including P. aeruginosa. Using ion chromatography, the levels of sulphation on different respiratory mucins and the availability of inorganic sulphate in CF sputum were quantified, and the ability of clinical P. aeruginosa isolates to desulphate mucin was tested by providing mucin as a sole sulphur source for growth. All tested P. aeruginosa strains isolated from the CF lung were able to use human respiratory mucin as source of sulphur for growth, whereas other non-clinical Pseudomonas species were not. However, measured levels of inorganic sulphate in CF sputum suggest that bacteria resident in the lung have sufficient inorganic sulphate for growth and are unlikely to require access to mucin-sulphur as a sulphur source during chronic infection. This was confirmed when expression of sulphate-repressed P. aeruginosa genes, atsK and msuE, were found by quantitative PCR to be repressed in CF sputum. These results indicate that sulphate-starvation is unlikely to occur in pathogens residing in CF sputum and, therefore, mucin desulphation may have an alternative purpose in the association between P. aeruginosa and CF airways.Transcriptomic studies showed enhanced expression of 5 main islands on the P. aeruginosa genome in the presence of mucin as a sulphur source, when compared to sulphate. These islands include general sulphur-starvation response gene clusters, encoding desulphurizing enzymes AtsA, SsuD and MsuD, plus the locus PA2083-PA2094. This locus has not been characterised but encodes a putative sulphonatase, an extracellular-function (ECF) type sigma factor, with associated TonB-dependent transducer, and Major Facilitator Superfamily transporters. Transcriptional studies of this locus in response to various sulphur sources revealed that the locus comprises two transcriptional units under sulphate-limited conditions, and putative σ70-type promoters were identified using 5’-RACE and sequence alignment. Transcriptional regulation of the locus is contributed to by the encoded ECF-type σ factor and anti-σ factor, as a mutant carrying only a disrupted copy of these genes displayed a lack of transcriptional downregulation of the locus in the presence of sulphate. The influence of mucin on transcription levels of the locus was also investigated by RT-qPCR, showing that for maximum transcriptional levels both sulphate-limitation and the presence of mucin are required. However, despite repression of P. aeruginosa sulphate-regulated genes in CF sputum, the level of expression of the locus PA2083-PA2094 in CF sputum was comparable to that of P. aeruginosa culture grown in sulphate-limited conditions. The influence of the lung environment may, therefore, have a greater impact on expression levels of the locus than seen in in vitro studies with mucin. To further investigate the role of the locus, mutants were constructed and screened for changes in their ability to utilise a range of sulphur sources, including mucin, for growth. However, none of the mutants showed significant change in their growth patterns in response to any of the other sulphur sources tested, suggesting that the locus may be involved in desulphurization of a compound not tested in this study or may be functionally replaced by other organosulphur utilisation pathways in its absence. With the aim of identifying genes involved in mucin desulphurization, a P. aeruginosa transposon library was generated, combining the high-throughput nature of a random library with the variable expression reporter capabilities afforded by a promoterless GFP insert. The GFP reporter transposon produced varying fluorescence levels over time during growth of individual mutants, based on the activity of the promoter upstream of the transposon insertion site. A preliminary method was devised using fluorescence-activated cell-sorting to isolate mutants displaying altered GFP expression levels in response to sulphate availability and to mucin. Overall, this work explores the prevalence and importance of mucin desulphurization by P. aeruginosa, with relation to cystic fibrosis lungs, and provides some insight into the transcriptional patterns of the P. aeruginosa locus PA2083 to PA2094.
Supervisor: Roberts, Ian; Kertesz, Michael Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Pseudomonas ; Mucin