Use this URL to cite or link to this record in EThOS:
Title: Bacterial nitric oxide metabolism in the pathogenesis of meningococcal sepsis
Author: Haque, Md Risat Ul
ISNI:       0000 0004 5917 9332
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2015
Availability of Full Text:
Access from EThOS:
Access from Institution:
Neisseria meningitidis is the causative agent of fatal meningococcal sepsis in humans, characterised by high bacterial loads in blood, and collapse of the microcirculatory system. The organism is adapted to colonise the human nasopharynx, an environment which is oxygen poor but rich in nitric oxide (NO), a gas vital for the regulation of essential physiological processes such as vasorelaxation, antimicrobial and innate immune responses by the host. Furthermore, during sepsis caused by meningococcaemia, high concentrations of nitrite can be measured in the blood, derived from activated circulating monocytes and endothelial cells. Meningococci express a partial denitrification pathway comprising of a nitrite reductase (AniA) and a nitric oxide reductase (NorB) to survive and thrive in an oxygen deficient niche such as the nasopharynx. The aniA and norB genes are negatively regulated by an NO sensitive repressor, NsrR. Studies from our group have shown that NorB is critical for counteracting the antimicrobial and innate immune response of the host. As NO based regulation requires a tightly regulated equilibrium, this could have far reaching consequences on the NO mediated signalling processes, and is likely to be relevant to survival of the organism within NO-enriched nasopharyngeal mucosae and blood. Previously, it was shown that bacterial NO detoxification reduces the concentration of host-cell S-nitrosothiol (SNO), a vital post-translational modification akin to phosphorylation, in murine macrophages in vitro. To investigate if similar meningococcal NO metabolism mediated SNO depletion persists in vivo, we established a murine model of early acute meningococcal sepsis. We showed that bacterial burden correlates positively with plasma SNO and hepatic NO2- but negatively with hepatic NOx. However, bacterial NO metabolism did not differentially modulate SNO and other NO metabolite profile of murine blood and liver tissue. Since there is no information to date on the effect of multiple meningococcal denitrification genes (aniA and norB) on the cellular pathology of meningococcal sepsis, we constructed and characterised a combination of NO metabolising gene mutants (ΔaniA/ΔnorB, ΔnsrR/ΔnorB, ΔaniA/ΔnorB/ΔnsrR) using the isocloning method. Differentiated human primary bronchial airway epithelial cells cultured at an air-liquid interface (HPEC-ALI) are polarised cells with tight junctions, possessing similar characteristics to the nasopharyngeal epithelial cells with which meningococci have to interact during colonisation and pathogenesis. HPEC-ALIs were infected with the newly created mutants (ΔaniA/ΔnorB and ΔaniA/ΔnorB/ΔnsrR) to examine the role of bacterial NO metabolism on the barrier function and immune response, functions known to be modulated by high concentrations of NO present in the airway epithelium. We demonstrated bacterial burden inversely correlates with the barrier function (TER) but positively with the cytokine profile (IL-8, TNFα). However, meningococcal denitrification does not have any differential role in the regulation of barrier function and cytokine profile of the HPEC-ALIs in the experimental system we used. The role of meningococcal denitrification in biofilm formation in vitro was also investigated. Preliminary data showed when biofilm formation was induced by nutrient starvation, ΔaniA/ΔnorB showed a significantly reduced biofilm forming ability compared to the Wt strain measured by the crystal violet staining. To investigate the role of aniA in differential regulation of biofilm formation, reverse complemented strains (ΔaniA/aniAIPTG+ and ΔaniA/aniA+) were created. Characterisation data showed functional activation was restored in ΔaniA when aniA was complemented along with the upstream regulatory elements such as the endogenous promoter (ΔaniA/aniA+) but not when aniA coding region was complemented under the control of an IPTG inducible lac promoter (ΔaniA/aniAIPTG+).
Supervisor: Read, Rob Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available