title: The biochemistry and phylogeny of CysE: An antimicrobial target

authors: Oldham, Keely

subjects: CysE

abstract: Neisseria gonorrhoeae is an obligate human pathogen and the causative agent of the sexually transmitted infection (STI), gonorrhoea. There are over 87 million gonorrhoea cases per year globally, making it the second most prevalent bacterial STI. The rapid emergence of antimicrobial-resistant strains has led N. gonorrhoeae to being labelled as a priority pathogen for the development of new antimicrobials by the World Health Organisation. One strategy for identifying new antimicrobials is through targeting pathways required for virulence and mitigation of oxidative stress, such as sulphur assimilation pathways. This thesis investigates the potential of targeting the de novo cysteine biosynthetic enzyme, serine acetyltransferase, from N. gonorrhoeae (NgCysE) for new antimicrobials. 

In this thesis, we present the kinetic and structural characterisation of NgCysE.  NgCysE has serine acetyltransferase activity and is sensitive to feedback inhibition by L cysteine. Small-angle X-ray scattering demonstrates NgCysE is a single hexameric species in solution with subtle conformational changes upon binding of its inhibitor L-cysteine. Using X-ray crystallography, we present two NgCysE structures, with L-malate (2.01 Å) and substrate L-serine (2.8 Å), bound. NgCysE crystallises as a homohexamer with 3:2-fold symmetry with each monomer containing a N-terminal α-helical SATase and C-terminal left-handed β-helix domain. Additionally, we see extensive density for the C-terminal α-helical tails because of domain swapping, resulting in one of the most complete CysE crystal structures to date. 

The high resolution NgCysE structure was used for structure-based virtual inhibitor screening to identify potential inhibitors of NgCysE. Virtual screening produced 28 hit compounds that were tested in vitro, with the most potent inhibitor, compound 2, demonstrating inhibition in the low micromolar range. Analysis of compound 2 docking reveals interactions with both NgCysE substrate binding sites, serine and acetyl-CoA. This is the first reported inhibitor of NgCysE and provides a starting point for development of new antimicrobials for treating gonorrhoea infection. 
Lastly, we present the phylogenetic analysis of truncated and full-length CysE isoforms across the bacterial kingdom. We demonstrate that a truncated isoform has a discrete truncation of four α-helices from the N-terminal SATase domain and is the predominant isoform for Gram-positive bacteria. The functional implications of this truncation are explored using predictive modelling and demonstrate that CysE is likely to exist as a trimer rather than the hexamer seen for the full-length isoform. This highlights the diversity of CysE isoforms and paves the way for determining the evolutionary origin of these isoforms. 

Collectively, this thesis highlights the merit in the emerging field of CysE inhibition. Moreover, we provide the basis for the future development of not only NgCysE inhibitors, but also the development of CysE inhibitors for other bacterial pathogens.

text: The biochemistry and phylogeny of CysE: An antimicrobial target Neisseria gonorrhoeae is an obligate human pathogen and the causative agent of the sexually transmitted infection (STI), gonorrhoea. There are over 87 million gonorrhoea cases per year globally, making it the second most prevalent bacterial STI. The rapid emergence of antimicrobial-resistant strains has led N. gonorrhoeae to being labelled as a priority pathogen for the development of new antimicrobials by the World Health Organisation. One strategy for identifying new antimicrobials is through targeting pathways required for virulence and mitigation of oxidative stress, such as sulphur assimilation pathways. This thesis investigates the potential of targeting the de novo cysteine biosynthetic enzyme, serine acetyltransferase, from N. gonorrhoeae (NgCysE) for new antimicrobials. 

In this thesis, we present the kinetic and structural characterisation of NgCysE.  NgCysE has serine acetyltransferase activity and is sensitive to feedback inhibition by L cysteine. Small-angle X-ray scattering demonstrates NgCysE is a single hexameric species in solution with subtle conformational changes upon binding of its inhibitor L-cysteine. Using X-ray crystallography, we present two NgCysE structures, with L-malate (2.01 Å) and substrate L-serine (2.8 Å), bound. NgCysE crystallises as a homohexamer with 3:2-fold symmetry with each monomer containing a N-terminal α-helical SATase and C-terminal left-handed β-helix domain. Additionally, we see extensive density for the C-terminal α-helical tails because of domain swapping, resulting in one of the most complete CysE crystal structures to date. 

The high resolution NgCysE structure was used for structure-based virtual inhibitor screening to identify potential inhibitors of NgCysE. Virtual screening produced 28 hit compounds that were tested in vitro, with the most potent inhibitor, compound 2, demonstrating inhibition in the low micromolar range. Analysis of compound 2 docking reveals interactions with both NgCysE substrate binding sites, serine and acetyl-CoA. This is the first reported inhibitor of NgCysE and provides a starting point for development of new antimicrobials for treating gonorrhoea infection. 
Lastly, we present the phylogenetic analysis of truncated and full-length CysE isoforms across the bacterial kingdom. We demonstrate that a truncated isoform has a discrete truncation of four α-helices from the N-terminal SATase domain and is the predominant isoform for Gram-positive bacteria. The functional implications of this truncation are explored using predictive modelling and demonstrate that CysE is likely to exist as a trimer rather than the hexamer seen for the full-length isoform. This highlights the diversity of CysE isoforms and paves the way for determining the evolutionary origin of these isoforms. 

Collectively, this thesis highlights the merit in the emerging field of CysE inhibition. Moreover, we provide the basis for the future development of not only NgCysE inhibitors, but also the development of CysE inhibitors for other bacterial pathogens.

year: 2024

title: Self-reported fever may be a poor proxy for the prevalence of malaria

authors: Alimi, Omoniyi Babatunde

abstract: About 90% of the 0.5 million global annual death toll from malaria occurs in Sub-Saharan Africa, especially amongst young children. While the most popular interventions for reducing the burden of malaria include insecticide-treated nets, improved dwellings, spraying and modification of mosquito habitat (e.g. drainage), use of some of these interventions has reached a plateau in some countries. Recently, a new anti-malaria intervention of reducing deforestation is claimed to be effective. The pathways through which deforestation can increase malaria include ecological effects that increase efficiency of the mosquito vectors and socio-economic changes such as migration that expose people with little tolerance to endemic forms of malaria. It is therefore argued in some studies that reducing deforestation can reduce the prevalence of malaria, presenting a win-win for health and the environment. The evidence on the effect of deforestation on malaria comes especially from linking remote sensing data on forest change to survey reports made by mothers of whether their child had a fever. For example, one influential study from Nigeria estimated the prevalence of malaria by asking mothers whether their child had been ill with a fever during the two weeks preceding the survey. Fever in children can have many causes other than malaria and so this proxy variable may lead to misleading relationships that divert attention from more effective anti-malaria interventions. In this study we use Demographic and Health Survey (DHS) data from Nigeria and Tanzania which has results from Rapid Diagnostic Tests (RDT) for parasite antigens in a blood drop, and tests using microscopic examination for Plasmodium in blood slides. We contrast the effects of local deforestation on these measured forms of malaria with the effects of deforestation on mother's report of children's fever (coming from the same DHS samples). In contrast to some recent studies, a positive impact of deforestation on malaria prevalence is not apparent when measured malaria (using microscopy or RDT) is the outcome variable. However, effects of deforestation on fever in children are apparent. The contrast between the impact on a measured health outcome and the impact on a proxy measure (the mother's report of fever in children) suggests that caution is needed when using proxy measures of health outcomes. In terms of policy implications, despite the potential attractiveness of adding child health reasons to the list of reasons for reducing the rate of deforestation, the available evidence for these two sub-Saharan African countries does not support this interpretation, in contrast to some recent studies.

text: Self-reported fever may be a poor proxy for the prevalence of malaria About 90% of the 0.5 million global annual death toll from malaria occurs in Sub-Saharan Africa, especially amongst young children. While the most popular interventions for reducing the burden of malaria include insecticide-treated nets, improved dwellings, spraying and modification of mosquito habitat (e.g. drainage), use of some of these interventions has reached a plateau in some countries. Recently, a new anti-malaria intervention of reducing deforestation is claimed to be effective. The pathways through which deforestation can increase malaria include ecological effects that increase efficiency of the mosquito vectors and socio-economic changes such as migration that expose people with little tolerance to endemic forms of malaria. It is therefore argued in some studies that reducing deforestation can reduce the prevalence of malaria, presenting a win-win for health and the environment. The evidence on the effect of deforestation on malaria comes especially from linking remote sensing data on forest change to survey reports made by mothers of whether their child had a fever. For example, one influential study from Nigeria estimated the prevalence of malaria by asking mothers whether their child had been ill with a fever during the two weeks preceding the survey. Fever in children can have many causes other than malaria and so this proxy variable may lead to misleading relationships that divert attention from more effective anti-malaria interventions. In this study we use Demographic and Health Survey (DHS) data from Nigeria and Tanzania which has results from Rapid Diagnostic Tests (RDT) for parasite antigens in a blood drop, and tests using microscopic examination for Plasmodium in blood slides. We contrast the effects of local deforestation on these measured forms of malaria with the effects of deforestation on mother's report of children's fever (coming from the same DHS samples). In contrast to some recent studies, a positive impact of deforestation on malaria prevalence is not apparent when measured malaria (using microscopy or RDT) is the outcome variable. However, effects of deforestation on fever in children are apparent. The contrast between the impact on a measured health outcome and the impact on a proxy measure (the mother's report of fever in children) suggests that caution is needed when using proxy measures of health outcomes. In terms of policy implications, despite the potential attractiveness of adding child health reasons to the list of reasons for reducing the rate of deforestation, the available evidence for these two sub-Saharan African countries does not support this interpretation, in contrast to some recent studies.

year: 2021

title: Lavinia and Henry Dunbar Johnson

authors: Hart, Philip

abstract: Rawinia Manukau, of Ngati Tamatera, married Henry Dunbar Johnson in 1868, when aged 21. Johnson had been a storekeeper at Coromandel from 1863 onwards and after 1866 had the first store at the site of the future Paeroa. In both places, but particularly in the latter, there was always the fear of conflict with the local Maori population, despite his being protected by a rangatira. After spending time at the new Thames goldfield, from 1871 onwards he was a partner in another Paeroa store, being able to erect a house because a rangatira related to his wife wished her to settle there. Lavinia (as she was known to Pakeha) obtained interests in several blocks of land in Ohinemuri, and her husband also acquired some land for a farm. He prospected Karangahake mountain from 1866 onwards, despite Maori opposition, and in 1875 and the following year actively mined there, unprofitably; Lavinia was not involved with this field, but did acquire interests in two claims when the Te Aroha one opened.
A petty squabble with a Thames neighbour resulted in Lavinia telling the latter to go back to England – she was feisty in defending her heritage. Through his marriage and close contact with Maori, Johnson understood and admired the Maori language, leading to his being appointed a licensed interpreter in 1872. After being a leading pioneer of Paeroa, in 1879 he went to Wellington to work in the Native Office, leaving his wife and family behind for a while; after then she was employed by Pakeha as a nurse and midwife. 
In 1885 Johnson was appointed to oversee the development of Rotorua, where he attempted to have Maori children educated (as his own were) and had to cope with the aftermath of the 1886 Tarawera eruption. Retrenched in 1888, he farmed for a while on his wife’s land at Te Aroha West, becoming involved in local issues and local politics. During the 1890 he obtained more official positions, and from 1896 to 1906 was a land court judge.
The Johnson family was well integrated into the dominant culture, all his daughters marrying Pakeha apart from one who married a ‘half-caste’ who had been brought up as a Pakeha. Johnson was not a Pakeha Maori in the original sense, but was accepted by Maori when living in Paeroa and as a judge tried to be kind to poverty-stricken Maori, although in time he viewed Maori as becoming lazy compared with those he had lived amongst during his first two decades in New Zealand.

text: Lavinia and Henry Dunbar Johnson Rawinia Manukau, of Ngati Tamatera, married Henry Dunbar Johnson in 1868, when aged 21. Johnson had been a storekeeper at Coromandel from 1863 onwards and after 1866 had the first store at the site of the future Paeroa. In both places, but particularly in the latter, there was always the fear of conflict with the local Maori population, despite his being protected by a rangatira. After spending time at the new Thames goldfield, from 1871 onwards he was a partner in another Paeroa store, being able to erect a house because a rangatira related to his wife wished her to settle there. Lavinia (as she was known to Pakeha) obtained interests in several blocks of land in Ohinemuri, and her husband also acquired some land for a farm. He prospected Karangahake mountain from 1866 onwards, despite Maori opposition, and in 1875 and the following year actively mined there, unprofitably; Lavinia was not involved with this field, but did acquire interests in two claims when the Te Aroha one opened.
A petty squabble with a Thames neighbour resulted in Lavinia telling the latter to go back to England – she was feisty in defending her heritage. Through his marriage and close contact with Maori, Johnson understood and admired the Maori language, leading to his being appointed a licensed interpreter in 1872. After being a leading pioneer of Paeroa, in 1879 he went to Wellington to work in the Native Office, leaving his wife and family behind for a while; after then she was employed by Pakeha as a nurse and midwife. 
In 1885 Johnson was appointed to oversee the development of Rotorua, where he attempted to have Maori children educated (as his own were) and had to cope with the aftermath of the 1886 Tarawera eruption. Retrenched in 1888, he farmed for a while on his wife’s land at Te Aroha West, becoming involved in local issues and local politics. During the 1890 he obtained more official positions, and from 1896 to 1906 was a land court judge.
The Johnson family was well integrated into the dominant culture, all his daughters marrying Pakeha apart from one who married a ‘half-caste’ who had been brought up as a Pakeha. Johnson was not a Pakeha Maori in the original sense, but was accepted by Maori when living in Paeroa and as a judge tried to be kind to poverty-stricken Maori, although in time he viewed Maori as becoming lazy compared with those he had lived amongst during his first two decades in New Zealand.

year: 2016