Antimicrobials and antimicrobial resistance

In order to curb the concerning rise in antimicrobial resistance, novel antimicrobials, particularly against multi-drug resistant Gram-negative bacteria, must be developed. The Gram-negative outer membrane (OM) and its biosynthetic pathways presents a number of promising drug targets. The OM is complex and asymmetrical, with an outer leaflet packed lipopolysaccharide (LPS) which provides an impermeable barrier against various antimicrobials. Asymmetry is thought to be maintained by the Mla system, a multiprotein complex which removes outer-leaflet OM phospholipids, traffics and reinserts them into the inner membrane (IM). Firstly, this thesis examines the IM MlaFEDB complex, and periplasmic MlaC. Quartz crystal microbalance and thin layer chromatography were used to examine a MlaFEDB-tethered bilayer model, to determine the directionality of transfer of phospholipid at the IM. Strikingly, MlaC was shown to acquire, rather than deposit, phospholipid from MlaFEDB at the membrane, suggesting that the Mla system is involved in anterograde, rather than retrograde phospholipid transport. This may constitute a novel pathway in which phospholipids are trafficked to the OM, for which no mechanism is currently understood. Secondly, the development of an LPS-containing membrane model using the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and its amenability for experiments using solid state magic angle spinning NMR, was explored. DMPC was found to be a potentially useful substitute for more fluid lipids in studying LPS structure and membrane dynamics. Moreover, this work reports the first successful 13C-labelling of smooth-type Escherichia coli LPS. These advances open avenues for developing a more biologically relevant, LPS-containing membrane model for studying the OM. This work could next be directly applied to replicating the MlaFEDB experiments on a model OM, with the OM component, MlaA, to better determine the directionality of phospholipid transport, and thus the overall biological function of the Mla system

Exploring the structure, regulation, and function of the surface tethered Pseudomonas aeruginosa virulence factor, AaaA, and its role in maintaining chronic wound infections

Pseudomonas aeruginosa is a leading cause of bacterial wound infections and is associated with disproportionately high mortality in burn patients, and morbidity in immunosuppressed and diabetic people, due to its ability to establish chronic wound infections. In this study, an in vitro synthetic wound model was used to examine the role of arginine-specific aminopeptidase of P. aeruginosa A (AaaA), a highly conserved, surface-tethered autotransporter which is known to be important for virulence during murine chronic wound infections. AaaA has potential as an antimicrobial drug or vaccine target due to its accessibility and immunogenicity, yet its mechanism of action is unclear. It is known that AaaA cleaves N-terminal arginine from peptides, which serve as a nutrient source in the oxygen and nutrient-limited environments of chronic wounds. Additionally, arginine can act as a signalling molecule in biofilm regulation, and it not yet clear what role AaaA may play in this regard. This study aimed firstly, to elucidate the structure and conservation of AaaA using both bioinformatic tools and by purifying AaaA for crystallography, and secondly to probe the gene expression and function of AaaA in a synthetic chronic wound (SCW) model. In this study, AaaA was found to be highly conserved, with zero missense mutations in its active site residues in over 3000 P. aeruginosa genomes. A few, likely non-deleterious amino acid substitutions were identified to be common in almost all genomes except for PAO1, highlighting the deviation of this lab strain from other P. aeruginosa isolates. Some progress was made towards purifying a truncated version of AaaA, which did not contain the membrane-localising β-barrel, with some evidence that it exists as both a monomer and an SDS-resistant dimer, though further verification is needed. Using transcriptional reporters and enzymatic AaaA activity assays, this study showed that AaaA is preferentially upregulated in the SCW, compared to in planktonic culture. AaaA was also shown to confer a modest but significant survival advantage in the SCW, as seen previously in vivo. This highlights the usefulness of this more disease-relevant model in studying important virulence factors which have marginal phenotypes in planktonic conditions. Analysis using RT-qPCR showed that rpoS, but not arginine metabolism genes, was upregulated ~3-fold in an aaaA mutant, suggesting that loss of aaaA leads to an increased starvation response. Transcriptomics by RNA-Seq identified further quorum-sensing and RpoS-repressed genes involved in phenazine and alkyl-quinolone production, as well as possible chronic-infection-specific virulence factors, which were downregulated in the aaaA mutant, possibly as a result of increased RpoS expression. This is the first time a link between AaaA and RpoS has been demonstrated, both of which have likely distinct roles in nutrient foraging, potentially on either axis of acute or chronic infection phenotypes. Further study, particularly using proteomics, is required to better understand this relationship and how it relates to the growth differences seen in the aaaA mutant in the SCW. Using a colorimetric quantification assay, no difference in extracellular arginine between wild-type and aaaA mutant-infected SCWs was detected. However, arginine levels in uninfected SCWs were ~3-fold higher, suggesting that arginine is being utilised in the SCW. A higher resolution approach, such mass spectrometry is required to detect differences related to AaaA. Finally, the Spytag-Spycatcher tagging system was used to localise AaaA in P. aeruginosa at the single cell level, with the potential that this technology could be used to localise AaaA in a multi-species biofilm in the future. In summary, this study has created a number of tools for studying AaaA in chronic wound infections, including validating the use of the SCW. It has also uncovered new links between AaaA, RpoS and quorum sensing, to create an updated model of AaaA regulation in a chronic wound environment

Antimicrobials and antimicrobial resistance

In order to curb the concerning rise in antimicrobial resistance, novel antimicrobials, particularly against multi-drug resistant Gram-negative bacteria, must be developed. The Gram-negative outer membrane (OM) and its biosynthetic pathways presents a number of promising drug targets. The OM is complex and asymmetrical, with an outer leaflet packed lipopolysaccharide (LPS) which provides an impermeable barrier against various antimicrobials. Asymmetry is thought to be maintained by the Mla system, a multiprotein complex which removes outer-leaflet OM phospholipids, traffics and reinserts them into the inner membrane (IM). Firstly, this thesis examines the IM MlaFEDB complex, and periplasmic MlaC. Quartz crystal microbalance and thin layer chromatography were used to examine a MlaFEDB-tethered bilayer model, to determine the directionality of transfer of phospholipid at the IM. Strikingly, MlaC was shown to acquire, rather than deposit, phospholipid from MlaFEDB at the membrane, suggesting that the Mla system is involved in anterograde, rather than retrograde phospholipid transport. This may constitute a novel pathway in which phospholipids are trafficked to the OM, for which no mechanism is currently understood. Secondly, the development of an LPS-containing membrane model using the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and its amenability for experiments using solid state magic angle spinning NMR, was explored. DMPC was found to be a potentially useful substitute for more fluid lipids in studying LPS structure and membrane dynamics. Moreover, this work reports the first successful 13C-labelling of smooth-type Escherichia coli LPS. These advances open avenues for developing a more biologically relevant, LPS-containing membrane model for studying the OM. This work could next be directly applied to replicating the MlaFEDB experiments on a model OM, with the OM component, MlaA, to better determine the directionality of phospholipid transport, and thus the overall biological function of the Mla system

Exploring the structure, regulation, and function of the surface tethered Pseudomonas aeruginosa virulence factor, AaaA, and its role in maintaining chronic wound infections

Pseudomonas aeruginosa is a leading cause of bacterial wound infections and is associated with disproportionately high mortality in burn patients, and morbidity in immunosuppressed and diabetic people, due to its ability to establish chronic wound infections. In this study, an in vitro synthetic wound model was used to examine the role of arginine-specific aminopeptidase of P. aeruginosa A (AaaA), a highly conserved, surface-tethered autotransporter which is known to be important for virulence during murine chronic wound infections. AaaA has potential as an antimicrobial drug or vaccine target due to its accessibility and immunogenicity, yet its mechanism of action is unclear. It is known that AaaA cleaves N-terminal arginine from peptides, which serve as a nutrient source in the oxygen and nutrient-limited environments of chronic wounds. Additionally, arginine can act as a signalling molecule in biofilm regulation, and it not yet clear what role AaaA may play in this regard. This study aimed firstly, to elucidate the structure and conservation of AaaA using both bioinformatic tools and by purifying AaaA for crystallography, and secondly to probe the gene expression and function of AaaA in a synthetic chronic wound (SCW) model. In this study, AaaA was found to be highly conserved, with zero missense mutations in its active site residues in over 3000 P. aeruginosa genomes. A few, likely non-deleterious amino acid substitutions were identified to be common in almost all genomes except for PAO1, highlighting the deviation of this lab strain from other P. aeruginosa isolates. Some progress was made towards purifying a truncated version of AaaA, which did not contain the membrane-localising β-barrel, with some evidence that it exists as both a monomer and an SDS-resistant dimer, though further verification is needed. Using transcriptional reporters and enzymatic AaaA activity assays, this study showed that AaaA is preferentially upregulated in the SCW, compared to in planktonic culture. AaaA was also shown to confer a modest but significant survival advantage in the SCW, as seen previously in vivo. This highlights the usefulness of this more disease-relevant model in studying important virulence factors which have marginal phenotypes in planktonic conditions. Analysis using RT-qPCR showed that rpoS, but not arginine metabolism genes, was upregulated ~3-fold in an aaaA mutant, suggesting that loss of aaaA leads to an increased starvation response. Transcriptomics by RNA-Seq identified further quorum-sensing and RpoS-repressed genes involved in phenazine and alkyl-quinolone production, as well as possible chronic-infection-specific virulence factors, which were downregulated in the aaaA mutant, possibly as a result of increased RpoS expression. This is the first time a link between AaaA and RpoS has been demonstrated, both of which have likely distinct roles in nutrient foraging, potentially on either axis of acute or chronic infection phenotypes. Further study, particularly using proteomics, is required to better understand this relationship and how it relates to the growth differences seen in the aaaA mutant in the SCW. Using a colorimetric quantification assay, no difference in extracellular arginine between wild-type and aaaA mutant-infected SCWs was detected. However, arginine levels in uninfected SCWs were ~3-fold higher, suggesting that arginine is being utilised in the SCW. A higher resolution approach, such mass spectrometry is required to detect differences related to AaaA. Finally, the Spytag-Spycatcher tagging system was used to localise AaaA in P. aeruginosa at the single cell level, with the potential that this technology could be used to localise AaaA in a multi-species biofilm in the future. In summary, this study has created a number of tools for studying AaaA in chronic wound infections, including validating the use of the SCW. It has also uncovered new links between AaaA, RpoS and quorum sensing, to create an updated model of AaaA regulation in a chronic wound environment

Isolation of Methicillin-resistant Staphylococcus aureus and Multidrug-resistant Elizabethkingia meningoseptica From Neonates Within Minutes of Birth.

Pediatr Infect Dis J 2017 Jan; 36(1):123-124

Surface-tethered planar membranes containing the β-barrel assembly machinery : a platform for investigating bacterial outer membrane protein folding

The outer membrane of Gram-negative bacteria presents a robust physicochemical barrier protecting the cell from both the natural environment and acting as the first line of defense against antimicrobial materials. The proteins situated within the outer membrane are responsible for a range of biological functions including controlling influx and efflux. These outer membrane proteins (OMPs) are ultimately inserted and folded within the membrane by the β-barrel assembly machine (Bam) complex. The precise mechanism by which the Bam complex folds and inserts OMPs remains unclear. Here, we have developed a platform for investigating Bam-mediated OMP insertion. By derivatizing a gold surface with a copper-chelating self-assembled monolayer, we were able to assemble a planar system containing the complete Bam complex reconstituted within a phospholipid bilayer. Structural characterization of this interfacial protein-tethered bilayer by polarized neutron reflectometry revealed distinct regions consistent with known high-resolution models of the Bam complex. Additionally, by monitoring changes of mass associated with OMP insertion by quartz crystal microbalance with dissipation monitoring, we were able to demonstrate the functionality of this system by inserting two diverse OMPs within the membrane, pertactin, and OmpT. This platform has promising application in investigating the mechanism of Bam-mediated OMP insertion, in addition to OMP function and activity within a phospholipid bilayer environment

Evidence for phospholipid export from the bacterial inner membrane by the Mla ABC transport system

The Mla pathway is believed to be involved in maintaining the asymmetrical Gram-negative outer membrane via retrograde phospholipid transport. The pathway is composed of three components: the outer membrane MlaA–OmpC/F complex, a soluble periplasmic protein, MlaC, and the inner membrane ATPase, MlaFEDB complex. Here, we solve the crystal structure of MlaC in its phospholipid-free closed apo conformation, revealing a pivoting β-sheet mechanism that functions to open and close the phospholipid-binding pocket. Using the apo form of MlaC, we provide evidence that the inner-membrane MlaFEDB machinery exports phospholipids to MlaC in the periplasm. Furthermore, we confirm that the phospholipid export process occurs through the MlaD component of the MlaFEDB complex and that this process is independent of ATP. Our data provide evidence of an apparatus for lipid export away from the inner membrane and suggest that the Mla pathway may have a role in anterograde phospholipid transport