Understanding the role of hxc-type II secretion system of Pseudomonas aeruginosa in pathogenesis

Cross-kingdom interactions are ubiquitous in nature and play a key role in the survival of species in polymicrobial environments. The communication between the organisms in mixed infections are always complex with the antagonistic or synergistic nature of this interaction difficult to determine. Pseudomonas aeruginosa and Candida albicans are two opportunistic human pathogens commonly found together in vivo. These two organisms have been associated with host colonisation in several diseases including cystic fibrosis (CF), severe burn wounds and ventilator-associated pneumonia; where the nature of their interactions potentially influences virulence properties of individual populations and in turn the eventual outcome of the polymicrobial disease. Notwithstanding the frequent co-isolation of P. aeruginosa and C. albicans from mixed infections, the nature of their interactions and the molecules involved are still unclear. Towards this aim we developed a fungal culture system in the presence of supernatants from different strains of P. aeruginosa to identify potential changes in fungal behaviour by measuring optical density (OD) at 600 nanometres (nm). Our preliminary data showed that wild type (WT) P. aeruginosa Lausanne strain (PAO1-L) induce filamentation of C. albicans, a key virulence attribute of this fungus. Using appropriate P. aeruginosa mutants, data showed that this observation was not due to the production of rhamnolipids, exotoxin A, or dependent on the virulence regulator ToxR. Interestingly, promotion of filamentous growth was attenuated when using supernatants from a PAO1 strain, PAAMP1. PAAMP1 has a 58kb chromosomal deletion including genes encoding the Hxc-Type II secretion system (Hxc-T2SS), low molecular weight alkaline phosphatase (LapA), cyclic-di-GMP phosphodiesterase (BifA), and ToxR; indicating that the gene(s) responsible for promoting hyphal growth in C. albicans was encoded within this region. Using biochemical approaches, the hyphal-inducing activity in WT PAO1-L supernatant was shown to be heat-labile (95°C, 10 min) and was retained in ultra-filtrates of relative molecular weights (rMW) above 10 kDa. Furthermore, protein gel analysis showed the presence of a band with rMW between 25-30 kDa specific for WT. Although the hyphal induction effect of PAO1-L secretions was lost in the course of the study and thus the observation inconclusive, the preliminary data put together suggest that WT POA1-L possibly secretes a protein capable of hyphal growth induction in C. albicans in vitro. Alongside employing biochemical techniques to identify the molecular nature of the hyphal-inducing agent(s) in P. aeruginosa secretion, genetic approach was used to ascertain gene(s) involved in the hyphal-inducing activity. Based on the preliminary data showing hyphal induction to be independent of ToxR gene regulation and the active agent having a rMW between 25–30kDa, it was hypothesised that the hyphal-inducing agent in the WT supernatant was Hxc-T2SS dependent LapA, which has a rMW between 38-to-40 kDa. Towards this aim, an in-frame deletion mutant lacking the hxc-operon (Δhxc) was constructed in a PAO1-L background. Phenotypic characterisation showed that bacterial growth and virulence traits like biofilm formation and pyocyanin production were not detectably affected by the hxc deletion. Addition of exogenous phosphate to culture medium significantly increased bacterial growth as well as biofilm formation, but pyocyanin secretion was markedly reduced in both the mutant and the wild type PAO1-L. Transcriptional analyses of the hxc-operon using a bioluminescent reporter mini-CTX-lux inserted into PAO1-L (this thesis) confirmed an earlier study by Ball et al (2002) that the operon is tightly regulated by two promoter regions, PhxccV and PhxcT. However, contrary to previous findings that PhxcV transcriptional activity is five times less than PhxcT under phosphate limiting conditions, this work demonstrated that PhxcV is highly expressed, though earlier, and peaks at 5 hours of incubation while PhxcT peaks at 10 hours. Further, the data indicated that expression of both promoters is sustained longer when bacteria is in contact with epithelial cells or in complete media such as X-vivo15 or DMEM-F12 compared to LB rich medium or phosphate-limiting proteose peptone medium. This supports earlier reports that the hxc operon is expressed during acute infection. Using an in vitro infection model, it was demonstrated that PAO1-LΔhxc detectably thrives better than the WT after contact with differentiated and undifferentiated human bronchial epithelial cells at a multiplicity of infection of 50. Assessment of bacterial-induced cytokine production showed that bronchial epithelial cells respond to P. aeruginosa infection through the production of cytokines and chemokines including TNF-α, GM-CSF, G-CSF, IL-6 IL-17C and IL-8. This pattern was the same for WT and Δhxc. Overall, the findings from this study show tight regulation and high dependency on culture conditions for the transcription of genes encoding the Hxc-T2SS, which implies that this system plays an important role on the adaptation of P. aeruginosa to its environment. Considering that the P. aeruginosa devotes Hxc-T2SS to a single substrate whose expression is not controlled by quorum sensing, this study provides a platform for further research into the hxc operon and how it may contribute to P. aeruginosa fitness under different conditions

Management of Diabetes Patients across the Peri- Operative Pathway: A Systematic Review

Peri-operative environments are a hazardous setting for diabetes patients. A systematic review of literature regarding the management of diabetes patients across the peri-operative pathway has been undertaken to assess if the management of patients within this pathway is suitable and effective for patients. Methods A database search of Google Scholar, CINAHAL, Embase, OVID, Cochrane Library, Joanna Briggs institute and PUBMED was undertaken from 15 th of March 2019 to 30 th of March 2019. A total of 57 papers were found and reduced down to 11 final papers that answered the review question and met the inclusion and exclusion criteria. Inclusion criteria were: Full text, English language, human subjects, adult patients only and studies that focused on diabetes care in a section of the peri-operative pathway. Exclusion criteria: children or adults and children, studies that looked a one particular intervention or type of surgery. No date limit was set. PICO tool was used to frame the study question. Results Three main themes emerged from the literature. 1. Poor patient outcomes; 2. Longer length of stay (LOS); 3. Lack of adherence to guidance and or protocols and glycaemic control. Elective patients had advantageous outcomes compared to emergency surgical patients. Hyperglycaemia still remained a problem with an increase in other medical complications for diabetes patients. LOS in hospital was found to have increased due to medical complications. Adherence to protocols and guidance was found to be beneficial in monitoring and managing hyperglycaemia. However, this review found that best practice guidance and hospital protocol is not always adhered to. A liberal approach to glycaemic control is beneficial. Conclusion This systematic review investigated the management of diabetes patients across the peri-operative pathway. Three main themes emerged from the literature: poor patient outcomes; length of stay; and lack of adherence to guidance and or protocols and glycaemic control. We concluded the peri-operative environment is a hazardous setting for a diabetes patients. Elective patients had slightly more advantageous outcomes than emergency patients. Hyperglycaemia still remains a problem which leads to poor patient outcomes and longer LOS. Adherence to protocols and guidance was found to be beneficial in monitoring and managing hyperglycaemia

Effectiveness and Safety of Hyperbaric Oxygen Therapy (HBOT) in Treating Diabetic Foot Ulcers (DFUs)

Introduction: Diabetic Foot Ulcers (DFUs) are one of the more dangerous complications of diabetes, contributing to morbidity, mortality, and major financial strain, potentially affecting patients' quality of life. Therefore, an effective DFU treatment is needed to both heal and reduce severe consequences, such as amputation. Studies into effective multiple therapeutic interventions for DFUs indicate that Hyperbaric Oxygen Therapy (HBOT) may be a current alternative for treating patients presenting with DFUs

Clinically Relevant Insulin Degludec and its Interaction with Polysaccharides: A Biophysical Examination.

Protein polysaccharide complexes have been widely studied for multiple industrial applications and are popular due to their biocompatibility. Insulin degludec, an analogue of human insulin, exists as di-hexamer in pharmaceutical formulations and has the potential to form long multi-hexamers in physiological environment, which dissociate into monomers to bind with receptors on the cell membrane. This study involved complexation of two negatively charged bio-polymers xanthan and alginate with clinically-relevant insulin degludec (PIC). The polymeric complexations and interactions were investigated using biophysical methods. Intrinsic viscosity [η] and particle size distribution (PSD) of PIC increased significantly with an increase in temperature, contrary to the individual components indicating possible interactions. [η] trend was X > XA > PIC > A > IDeg. PSD trend was X>A>IDeg>XA>PIC. Zeta (ζ)- potential (with general trend of IDeg < A < XA < X ≈ PIC) revealed stable interaction at lower temperature which gradually changed with an increase in temperature. Likewise, sedimentation velocity indicated stable complexation at lower temperature. With an increase in time and temperature, changes in the number of peaks and area under curve were observed for PIC. Conclusively, stable complexation occurred among the three polymers at 4 °C and 18 °C and the complex dissociated at 37 °C. Therefore, the complex has the potential to be used as a drug delivery vehicle

Global Prevalence of Gestational Diabetes Mellitus: A Systematic Review and Meta-Analysis

1. Abstract 1.1. Background: Evidence suggests that diabetes in all forms are on the rise especially gesta-tional diabetes mellitus which increases the risk of maternal and neonatal morbidities; however global prevalence rates and geographical distribution of GDM remain uncertain. The aim of this study is to examine the global burden of gestational diabetes mellitus. 1.2. Methods: A systematic review and meta-analysis of studies reporting Randomised Clinical Trials (RCTs) in pregnant women who have GDM was conducted. Cochrane (Central), PubMed, Scopus, JBI, Medline, EMBASE and reference lists of retrieved studies were searched from inception to March 2019. Publications on prevalence of GDM irrespective of the baseline criteria used to diagnose GDM were included in the study. Studies were limited to English language, randomised control trials and women aged between 19-44 years inclusive. 1.3. Results: Eleven RCTs met the inclusion criteria for this review. The included studies collectively reported GDM rates of 13,450 pregnant women from 7 countries. The diagnostic criteria used in the studies were World Health Organisation (WHO) 1985 and 1999, International Association of Diabetes, Pregnancy Study Group (IADPSG), National Diabetes Data Group (NDDG), Carpenter-Coustan (C&C) and O'Sullivan's criteria. Seven RCTs screened for GDM in comparison with different diagnostic criteria in the same population while three studies used the same criteria for different groups. One study compared 100g, 3h OGTT to 75g, 2h OGTT for diagnosing GDM using Carpenter and Coustan criteria. All seven RCTs that compared different diagnostic criteria in the same population detected different prevalence rates of GDM. Three RCTs measured prevalence of GDM in the same population using WHO 1999 and IADPSG 2013 criteria. Using random effect model, data from three studies that compared IADPSG criteria to WHO 1999 showed an Odds Ratio (OR) of 0.52(0.15, 1.84), 95% Confidence Interval (CI) and high heterogeneity of 99%. In all three studies, prevalence of GDM measured by IADPSG criteria was higher than WHO 1999 criteria, although not significant (p= 0.31). Combining all the studies gave a global estimated prevalence of GDM to be 10.13% (95% CI, 7.33-12.94) with moderate heterogeneity of 27%. The highest prevalence of GDM wit

Exploration of temperature and shelf-life dependency of the therapeutically available Insulin Detemir

© 2020 Elsevier B.V. Purpose: Insulin, in typical use, undergoes multiple changes in temperature; from refrigerator, to room temperature, to body temperature. Although long-term storage temperature has been well-studied, the short term changes to insulin are yet to be determined. Insulin detemir (IDet) is a clinically available, slow-acting, synthetic analogue characterised by the conjugation of a C14 fatty acid. The function of this modification is to cause the insulin to form multi-hexameric species, thus retarding the pharmacokinetic rate of action. In this investigation, the temperature dependence properties of this synthetic analogue is probed, as well as expiration. Methods: Dynamic light scattering (DLS) and viscometry were employed to assess the effect of temperature upon IDet. Mass spectrometry was also used to probe the impact of shelf-life and the presence of certain excipients. Results: IDet was compared with eight other insulins, including human recombinant, three fast-acting analogues and two other slow-acting analogues. Of all nine insulins, IDet was the only analogue to show temperature dependent behaviour, between 20 °C and 37 °C, when probed with non-invasive backscatter dynamic light scattering. Upon further investigation, IDet observed significant changes in size related to temperature, direction of temperature (heated/cooled) and expiration with cross-correlation observed amongst all 4 parameters. Conclusions: These findings are critical to our understanding of the behaviour of this particular clinically relevant drug, as it will allow the development of future generations of peptide-based therapies with greater clinical efficacy

Analysis of insulin glulisine at the molecular level by X-ray crystallography and biophysical techniques

© 2021, The Author(s). This study concerns glulisine, a rapid-acting insulin analogue that plays a fundamental role in diabetes management. We have applied a combination of methods namely X-ray crystallography, and biophysical characterisation to provide a detailed insight into the structure and function of glulisine. X-ray data provided structural information to a resolution of 1.26Å. Crystals belonged to the H3 space group with hexagonal (centred trigonal) cell dimensions a = b = 82.44 and c = 33.65Å with two molecules in the asymmetric unit. A unique position of D21Glu, not present in other fast-acting analogues, pointing inwards rather than to the outside surface was observed. This reduces interactions with neighbouring molecules thereby increasing preference of the dimer form. Sedimentation velocity/equilibrium studies revealed a trinary system of dimers and hexamers/dihexamers in dynamic equilibrium. This new information may lead to better understanding of the pharmacokinetic and pharmacodynamic behaviour of glulisine which might aid in improving formulation regarding its fast-acting role and reducing side effects of this drug

Understanding the role of hxc-type II secretion system of Pseudomonas aeruginosa in pathogenesis

Cross-kingdom interactions are ubiquitous in nature and play a key role in the survival of species in polymicrobial environments. The communication between the organisms in mixed infections are always complex with the antagonistic or synergistic nature of this interaction difficult to determine. Pseudomonas aeruginosa and Candida albicans are two opportunistic human pathogens commonly found together in vivo. These two organisms have been associated with host colonisation in several diseases including cystic fibrosis (CF), severe burn wounds and ventilator-associated pneumonia; where the nature of their interactions potentially influences virulence properties of individual populations and in turn the eventual outcome of the polymicrobial disease. Notwithstanding the frequent co-isolation of P. aeruginosa and C. albicans from mixed infections, the nature of their interactions and the molecules involved are still unclear. Towards this aim we developed a fungal culture system in the presence of supernatants from different strains of P. aeruginosa to identify potential changes in fungal behaviour by measuring optical density (OD) at 600 nanometres (nm). Our preliminary data showed that wild type (WT) P. aeruginosa Lausanne strain (PAO1-L) induce filamentation of C. albicans, a key virulence attribute of this fungus. Using appropriate P. aeruginosa mutants, data showed that this observation was not due to the production of rhamnolipids, exotoxin A, or dependent on the virulence regulator ToxR. Interestingly, promotion of filamentous growth was attenuated when using supernatants from a PAO1 strain, PAAMP1. PAAMP1 has a 58kb chromosomal deletion including genes encoding the Hxc-Type II secretion system (Hxc-T2SS), low molecular weight alkaline phosphatase (LapA), cyclic-di-GMP phosphodiesterase (BifA), and ToxR; indicating that the gene(s) responsible for promoting hyphal growth in C. albicans was encoded within this region. Using biochemical approaches, the hyphal-inducing activity in WT PAO1-L supernatant was shown to be heat-labile (95°C, 10 min) and was retained in ultra-filtrates of relative molecular weights (rMW) above 10 kDa. Furthermore, protein gel analysis showed the presence of a band with rMW between 25-30 kDa specific for WT. Although the hyphal induction effect of PAO1-L secretions was lost in the course of the study and thus the observation inconclusive, the preliminary data put together suggest that WT POA1-L possibly secretes a protein capable of hyphal growth induction in C. albicans in vitro. Alongside employing biochemical techniques to identify the molecular nature of the hyphal-inducing agent(s) in P. aeruginosa secretion, genetic approach was used to ascertain gene(s) involved in the hyphal-inducing activity. Based on the preliminary data showing hyphal induction to be independent of ToxR gene regulation and the active agent having a rMW between 25–30kDa, it was hypothesised that the hyphal-inducing agent in the WT supernatant was Hxc-T2SS dependent LapA, which has a rMW between 38-to-40 kDa. Towards this aim, an in-frame deletion mutant lacking the hxc-operon (Δhxc) was constructed in a PAO1-L background. Phenotypic characterisation showed that bacterial growth and virulence traits like biofilm formation and pyocyanin production were not detectably affected by the hxc deletion. Addition of exogenous phosphate to culture medium significantly increased bacterial growth as well as biofilm formation, but pyocyanin secretion was markedly reduced in both the mutant and the wild type PAO1-L. Transcriptional analyses of the hxc-operon using a bioluminescent reporter mini-CTX-lux inserted into PAO1-L (this thesis) confirmed an earlier study by Ball et al (2002) that the operon is tightly regulated by two promoter regions, PhxccV and PhxcT. However, contrary to previous findings that PhxcV transcriptional activity is five times less than PhxcT under phosphate limiting conditions, this work demonstrated that PhxcV is highly expressed, though earlier, and peaks at 5 hours of incubation while PhxcT peaks at 10 hours. Further, the data indicated that expression of both promoters is sustained longer when bacteria is in contact with epithelial cells or in complete media such as X-vivo15 or DMEM-F12 compared to LB rich medium or phosphate-limiting proteose peptone medium. This supports earlier reports that the hxc operon is expressed during acute infection. Using an in vitro infection model, it was demonstrated that PAO1-LΔhxc detectably thrives better than the WT after contact with differentiated and undifferentiated human bronchial epithelial cells at a multiplicity of infection of 50. Assessment of bacterial-induced cytokine production showed that bronchial epithelial cells respond to P. aeruginosa infection through the production of cytokines and chemokines including TNF-α, GM-CSF, G-CSF, IL-6 IL-17C and IL-8. This pattern was the same for WT and Δhxc. Overall, the findings from this study show tight regulation and high dependency on culture conditions for the transcription of genes encoding the Hxc-T2SS, which implies that this system plays an important role on the adaptation of P. aeruginosa to its environment. Considering that the P. aeruginosa devotes Hxc-T2SS to a single substrate whose expression is not controlled by quorum sensing, this study provides a platform for further research into the hxc operon and how it may contribute to P. aeruginosa fitness under different conditions

Clinically Relevant Insulin Degludec and Its Interaction with Polysaccharides: A Biophysical Examination

Protein polysaccharide complexes have been widely studied for multiple industrial applications and are popular due to their biocompatibility. Insulin degludec, an analogue of human insulin, exists as di-hexamer in pharmaceutical formulations and has the potential to form long multi-hexamers in physiological environment, which dissociate into monomers to bind with receptors on the cell membrane. This study involved complexation of two negatively charged bio-polymers xanthan and alginate with clinically-relevant insulin degludec (PIC). The polymeric complexations and interactions were investigated using biophysical methods. Intrinsic viscosity [&eta;] and particle size distribution (PSD) of PIC increased significantly with an increase in temperature, contrary to the individual components indicating possible interactions. [&eta;] trend was X &gt; XA &gt; PIC &gt; A &gt; IDeg. PSD trend was X &gt; A &gt; IDeg &gt; XA &gt; PIC. Zeta (&zeta;)- potential (with general trend of IDeg &lt; A &lt; XA &lt; X &asymp; PIC) revealed stable interaction at lower temperature which gradually changed with an increase in temperature. Likewise, sedimentation velocity indicated stable complexation at lower temperature. With an increase in time and temperature, changes in the number of peaks and area under curve were observed for PIC. Conclusively, stable complexation occurred among the three polymers at 4 &deg;C and 18 &deg;C and the complex dissociated at 37 &deg;C. Therefore, the complex has the potential to be used as a drug delivery vehicle