ORCID Profile
0000-0002-4302-2440
Current Organisations
Emory University
,
University of Adelaide
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Publisher: Wiley
Date: 21-10-2016
DOI: 10.1111/MMI.13539
Abstract: Staphylococcus aureus SufT is composed solely of the domain of unknown function 59 (DUF59) and has a role in the maturation of iron‐sulphur (Fe‐S) proteins. We report that SufT is essential for S. aureus when growth is heavily reliant upon lipoamide‐utilizing enzymes, but dispensable when this reliance is decreased. LipA requires Fe‐S clusters for lipoic acid (LA) synthesis and a Δ sufT strain had phenotypes suggestive of decreased LA production and decreased activities of lipoamide‐requiring enzymes. Fermentative growth, a null clpC allele, or decreased flux through the TCA cycle diminished the demand for LA and rendered SufT non‐essential. Abundance of the Fe‐S cluster carrier Nfu was increased in a Δ clpC strain and a null clpC allele was unable to suppress the LA requirement of a Δ sufT Δ nfu strain. Over‐expression of nfu suppressed the LA requirement of the Δ sufT strain. We propose a model wherein SufT, and by extension the DUF59, is essential for the maturation of holo‐LipA in S. aureus cells experiencing a high demand for lipoamide‐dependent enzymes. The findings presented suggest that the demand for products of Fe‐S enzymes is a factor governing the usage of one Fe‐S cluster assembly factor over another in the maturation of apo‐proteins.
Publisher: Proceedings of the National Academy of Sciences
Date: 23-08-2018
Publisher: American Society for Microbiology
Date: 25-06-2019
Abstract: The outer membrane built by Gram-negative bacteria such as Escherichia coli forms a barrier that prevents antibiotics from entering the cell, limiting clinical options at a time of prevalent antibiotic resistance. Stress responses ensure that barrier integrity is continuously maintained. We have identified the Cpx signal transduction system as a stress response that monitors the trafficking of lipid-anchored lipoproteins to the outer membrane. These lipoproteins are needed by every machine that builds the outer membrane. Cpx monitors just one lipoprotein, NlpE, to detect the efficiency of lipoprotein trafficking in the cell. NlpE and Cpx were previously shown to play a role in resistance to copper. We show that copper blocks lipoprotein trafficking, reconciling old and new observations. Copper is an important element in innate immunity against pathogens, and our findings suggest that NlpE and Cpx help E. coli survive the assault of copper on a key outer membrane assembly pathway.
Publisher: Elsevier BV
Date: 07-2013
Publisher: Wiley
Date: 11-09-2012
Publisher: Microbiology Society
Date: 07-2012
Abstract: The IcsA autotransporter protein is a major virulence factor of the human intracellular pathogen Shigella flexneri. IcsA is distributed at the poles in the outer membrane (OM) of S. flexneri and interacts with components of the host actin-polymerization machinery to facilitate intracellular actin-based motility and subsequent cell-to-cell spreading of the bacterium. We sought to characterize the biochemical properties of IcsA in the bacterial OM. Chemical cross-linking data suggested that IcsA exists in a complex in the OM. Furthermore, reciprocal co-immunoprecipitation of differentially epitope-tagged IcsA proteins indicated that IcsA is able to self-associate. The identification of IcsA linker-insertion mutants that were negatively dominant provided genetic evidence of IcsA-IcsA interactions. From these results, we propose a model whereby IcsA self-association facilitates efficient actin-based motility.
Publisher: Oxford University Press (OUP)
Date: 16-03-2015
Abstract: Shigella species are the causative agents of human bacillary dysentery. These bacteria spread within the lining of the gut via a process termed actin-based motility whereby an actin 'tail' is formed at the bacterial pole. The bacterial outer membrane protein IcsA initiates this process, and crucially is precisely positioned on the bacterial polar surface. Lipopolysaccharide (LPS) O-antigen surface structure has been implicated as an augmenting factor of polarity maintenance due to the apparent dysregulation of IcsA polarity in O-antigen deficient strains. Due to Shigellae having long and short O-antigen chains on their surfaces, it has been proposed that O-antigen chain lengths are asymmetrically distributed to optimize IcsA exposure at the pole and mask exposure laterally. Additionally, it has been proposed that LPS O-antigen restricts IcsA diffusion from the pole by maintaining minimal membrane fluidity. This study utilizes minicells and quantitative microscopy providing data refuting the models of asymmetric masking and membrane diffusion, and supporting a model of symmetric masking of IcsA. We contend that IcsA surface distribution is equivalent between wild-type and O-antigen deficient strains, and that differences in cellular IcsA levels have confounded previous conclusions.
Publisher: American Society for Microbiology
Date: 2016
DOI: 10.1128/IAI.00994-15
Abstract: Shiga toxin (Stx)-producing Escherichia coli (STEC) infections can lead to life-threatening complications, including hemorrhagic colitis (HC) and hemolytic-uremic syndrome (HUS), which is the most common cause of acute renal failure in children in the United States. Stx1 and Stx2 are AB5 toxins consisting of an enzymatically active A subunit associated with a pentamer of receptor binding B subunits. Epidemiological evidence suggests that Stx2-producing E. coli strains are more frequently associated with HUS than Stx1-producing strains. Several studies suggest that the B subunit plays a role in mediating toxicity. However, the role of the A subunits in the increased potency of Stx2 has not been fully investigated. Here, using purified A1 subunits, we show that Stx2A1 has a higher affinity for yeast and mammalian ribosomes than Stx1A1. Biacore analysis indicated that Stx2A1 has faster association and dissociation with ribosomes than Stx1A1. Analysis of ribosome depurination kinetics demonstrated that Stx2A1 depurinates yeast and mammalian ribosomes and an RNA stem-loop mimic of the sarcin/ricin loop (SRL) at a higher catalytic rate and is a more efficient enzyme than Stx1A1. Stx2A1 depurinated ribosomes at a higher level in vivo and was more cytotoxic than Stx1A1 in Saccharomyces cerevisiae . Stx2A1 depurinated ribosomes and inhibited translation at a significantly higher level than Stx1A1 in human cells. These results provide the first direct evidence that the higher affinity for ribosomes in combination with higher catalytic activity toward the SRL allows Stx2A1 to depurinate ribosomes, inhibit translation, and exhibit cytotoxicity at a significantly higher level than Stx1A1.
Publisher: Proceedings of the National Academy of Sciences
Date: 30-01-2023
Abstract: The outer membrane (OM) is the defining feature of gram-negative bacteria and is an essential organelle. Accordingly, OM assembly pathways and their essential protein components are conserved throughout all gram-negative species. Lipoprotein trafficking lies at the heart of OM assembly since it supplies several different biogenesis machines with essential lipoproteins. The Escherichia coli Lol trafficking pathway relies on an inner membrane LolCDE transporter that transfers newly made lipoproteins to the chaperone LolA, which rapidly traffics lipoproteins across the periplasm to LolB for insertion into the OM. Strikingly, many gram-negative species (like Caulobacter vibrioides ) do not produce LolB, yet essential lipoproteins are still trafficked to the OM. How the final step of trafficking occurs in these organisms has remained a long-standing mystery. We demonstrate that LolA from C. vibrioides can complement the deletion of both LolA and LolB in E. coli , revealing that this protein possesses both chaperone and insertion activities. Moreover, we define the region of C. vibrioides LolA that is responsible for its bifunctionality. This knowledge enabled us to convert E. coli LolA into a similarly bifunctional protein, capable of chaperone and insertion activities. We propose that a bifunctional LolA eliminates the need for LolB. Our findings provide an explanation for why some gram-negative species have retained an essential LolA yet completely lack a dedicated LolB protein.
Publisher: American Society for Microbiology
Date: 11-2010
DOI: 10.1128/IAI.00801-10
Abstract: Subtilase cytotoxin (SubAB) was first isolated from a Shiga toxigenic Escherichia coli (STEC) strain that was responsible for an outbreak of hemolytic-uremic syndrome and is the prototype of a new family of AB 5 cytotoxins. SubAB is a subtilase-like serine protease, and upon uptake by host cells, it is trafficked to the endoplasmic reticulum (ER), where it cleaves the essential ER chaperone BiP (GRP78) with high specificity. Previous work has shown that BiP cleavage by SubAB initiates ER stress-signaling pathways in host cells that eventuate in cell death associated with DNA fragmentation, a hallmark of apoptosis. The present study has investigated the role of the Bcl-2 protein family, which has been shown to regulate ER stress-induced apoptosis in other model systems. Examination of the cytotoxicity of SubAB for wild-type and bax −/− / bak −/− mouse embryonic fibroblasts and comparison of apoptotic markers in these cells revealed that SubAB cytotoxicity can be predominantly attributed to the activation of apoptotic pathways activated by Bax/Bak. The results of the present study further our understanding of the molecular mechanism whereby SubAB kills eukaryotic cells and contributes to STEC pathogenesis, in addition to consolidating the roles of Bcl-2 family members in the regulation of ER stress-induced apoptosis.
Publisher: Wiley
Date: 25-03-2023
DOI: 10.1111/MMI.15054
Abstract: Bacterial two‐component signal transduction systems provide sensory inputs for appropriately adapting gene expression. These systems rely on a histidine kinase that phosphorylates a response regulator which alters gene expression. Several two‐component systems include additional sensory components that can activate the histidine kinase. In Escherichia coli , the lipoprotein NlpE was identified as a sensor for the Cpx cell envelope stress response. It has remained unclear how NlpE signals to Cpx in the periplasm. In this study, we used a combination of genetics, biochemistry, and AlphaFold2 complex modeling to uncover the molecular details of how NlpE triggers the Cpx response through an interaction with the CpxA histidine kinase. Remarkably, only a short loop of NlpE is required to activate the Cpx response. A single substitution in this loop inactivates NlpE signaling to Cpx and abolishes an in vivo biochemical NlpE:CpxA interaction. An independent AlphaFold multimer prediction supported a role for the loop and predicted an interaction interface at CpxA. Mutations in this CpxA region specifically blind the histidine kinase to NlpE activation but preserve the ability to respond to other cell envelope stressors. Hence, our work additionally reveals a previously unrecognized complexity in signal integration by the CpxA periplasmic sensor domain.
Publisher: MDPI AG
Date: 02-2020
DOI: 10.3390/JCM9020394
Abstract: Patients with chronic kidney disease (CKD) have altered physiologic processes, which result in different treatment outcomes compared with the general population. We aimed to systematically evaluate the efficacy of clinical interventions in reducing mortality of patients with CKD. We searched PubMed, MEDLINE, Embase, and Cochrane Database of Systematic Reviews for meta-analyses of randomized controlled trials (RCT) or observational studies (OS) studying the effect of treatment on all-cause mortality of patients with CKD. The credibility assessment was based on the random-effects summary estimate, heterogeneity, 95% prediction intervals, small study effects, excess significance, and credibility ceilings. Ninety-two articles yielded 130 unique meta-analyses. Convincing evidence from OSs supported mortality reduction with three treatments: angiotensin-converting-enzyme inhibitors or angiotensin II receptor blockers for patients not undergoing dialysis, warfarin for patients with atrial fibrillation not undergoing dialysis, and (at short-term) percutaneous coronary intervention compared to coronary artery bypass grafting for dialysis patients. Two treatment comparisons were supported by highly credible evidence from RCTs in terms of all-cause mortality. These were high-flux hemodialysis (HD) versus low-flux HD as a maintenance HD method and statin versus less statin or placebo for patients not undergoing dialysis. Most significant associations identified in OSs failed to be replicated in RCTs. Associations of high credibility from RCTs were in line with current guidelines. Given the heterogeneity of CKD, it seems hard to assume mortality reductions based on findings from OSs.
Publisher: American Society for Microbiology
Date: 02-05-2018
Abstract: The outer membrane (OM) bilayer of Gram-negative bacteria is biologically unique in its asymmetrical organization of lipids, with an inner leaflet composed of glycerophospholipids (PLs) and a surface-exposed outer leaflet composed of lipopolysaccharide (LPS). This lipid organization is integral to the OM’s barrier properties. Perturbations of the outer leaflet by antimicrobial peptides or defects in LPS biosynthesis or transport to the OM cause a compensatory flipping of PLs to the outer leaflet. As a result, lipid asymmetry is disrupted and OM integrity is compromised. Recently, we identified an Escherichia coli mutant that exhibits aberrant accumulation of surface PLs accompanied by a cellular increase in LPS production. Remarkably, the observed hyperproduction of LPS is PldA dependent. Here we provide evidence that the fatty acids generated by PldA at the OM are transported into the cytoplasm and simultaneously activated by thioesterification to coenzyme A (CoA) by FadD. The acyl-CoAs produced ultimately inhibit LpxC degradation by FtsH. The increased levels of LpxC, the enzyme that catalyzes the first committed step in LPS biosynthesis, increases the amount of LPS produced. Our data suggest that PldA acts as a sensor for lipid asymmetry in the OM. PldA protects the OM barrier by both degrading mislocalized PLs and generating lipid second messengers that enable long-distance signaling that prompts the cell to restore homeostasis at a distant organelle. IMPORTANCE The outer membrane of Gram-negative bacteria is an effective permeability barrier that protects the cell from toxic agents, including antibiotics. Barrier defects are often manifested by phospholipids present in the outer leaflet of this membrane that take up space normally occupied by lipopolysaccharide. We have discovered a signaling mechanism that operates across the entire cell envelope used by the cell to detect these outer membrane defects. A phospholipase, PldA, that functions to degrade these mislocalized phospholipids has a second, equally important function as a sensor. The fatty acids produced by hydrolysis of the phospholipids act as second messengers to signal the cell that more lipopolysaccharide is needed. These fatty acids diffuse across the periplasm and are transported into the cytoplasm by a process that attaches coenzyme A. The acyl-CoA molecule produces signals to inhibit the degradation of the critical enzyme LpxC by the ATP-dependent protease FtsH, increasing lipopolysaccharide production.
Publisher: Proceedings of the National Academy of Sciences
Date: 29-02-2016
Abstract: The cell envelope of Gram-negative bacteria contains two membranes and a cell wall located in the aqueous compartment between them. The outer membrane (OM) functions as a barrier that contributes to antibiotic resistance. We describe a dominant mutation in a gene for an OM lipoprotein that leads to cell death under starvation conditions in medium with limited cation concentrations. We show that death occurs not by rapid cell lysis but by a previously uncharacterized mechanism involving flow of material from the inner membrane to the OM that results in rupture of the inner membrane and the slow leakage of cytoplasmic contents. Our study highlights the vital need for balanced synthesis across the Gram-negative envelope and may empower the development of new therapeutics.
Publisher: American Society for Microbiology
Date: 07-2008
DOI: 10.1128/JB.00093-08
Abstract: The IcsA (VirG) protein of Shigella flexneri is a polarly localized, outer membrane protein that is essential for virulence. Within host cells, IcsA activates the host actin regulatory protein, neural Wiskott-Aldrich syndrome protein (N-WASP), which in turn recruits the Arp2/3 complex, which nucleates host actin to form F-actin comet tails and initiate bacterial motility. Linker insertion mutagenesis was undertaken to randomly introduce 5-amino-acid in-frame insertions within IcsA. Forty-seven linker insertion mutants were isolated and expressed in S. flexneri Δ icsA strains. Mutants were characterized for IcsA protein production, cell surface expression and localization, intercellular spreading, F-actin comet tail formation, and N-WASP recruitment. Using this approach, we have identified a putative autochaperone region required for IcsA biogenesis, and our data suggest an additional region, not previously identified, is required for N-WASP recruitment.
Publisher: American Society for Microbiology
Date: 15-02-2022
DOI: 10.1128/JB.00574-21
Abstract: Purcell and colleagues offer new insights into a major mechanism of polymyxin resistance in Gram-negative bacteria. Inactivating a single lipid recycling enzyme causes accumulation of waste lipid by-products that inhibit a key factor responsible for polymyxin resistance.
Publisher: Elsevier BV
Date: 11-2017
Publisher: Hindawi Limited
Date: 09-2008
No related grants have been discovered for Kerrie Leanne May.