ORCID Profile
0000-0001-9266-5322
Current Organisation
University of Technology Sydney
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Infectious agents | Bacteriology | Microbiology | One health | Nanobiotechnology | Biological Physics | Microbiology | Bacteriology
Expanding Knowledge in the Biological Sciences | Infectious Diseases |
Publisher: Springer Science and Business Media LLC
Date: 18-10-2018
DOI: 10.1038/S41467-018-06887-7
Abstract: FtsZ is the main regulator of bacterial cell ision. It has been implicated in acting as a scaffolding protein for other ision proteins, a force generator during constriction, and more recently, as an active regulator of septal cell wall production. FtsZ assembles into a heterogeneous structure coined the Z-ring due to its resemblance to a ring confined by the midcell geometry. Here, to establish a framework for examining geometrical influences on proper Z-ring assembly and dynamics, we sculpted Escherichia coli cells into unnatural shapes using ision- and cell wall-specific inhibitors in a micro-fabrication scheme. This approach allowed us to examine FtsZ behavior in engineered Z-squares and Z-hearts. We use stimulated emission depletion (STED) nanoscopy to show that FtsZ clusters in sculpted cells maintain the same dimensions as their wild-type counterparts. Based on our results, we propose that the underlying membrane geometry is not a deciding factor for FtsZ cluster maintenance and dynamics in vivo.
Publisher: Public Library of Science (PLoS)
Date: 12-2022
Publisher: Springer Science and Business Media LLC
Date: 06-04-2023
DOI: 10.1007/S00294-023-01266-2
Abstract: Understanding where proteins are localized in a bacterial cell is essential for understanding their function and regulation. This is particularly important for proteins that are involved in cell ision, which localize at the ision septum and assemble into highly regulated complexes. Current knowledge of these complexes has been greatly facilitated by super-resolution imaging using fluorescent protein fusions. Herein, we demonstrate with FtsZ that single-molecule PALM images can be obtained in-vivo using a genetically fused nanotag (ALFA), and a corresponding nanobody fused to mEos3.2. The methodology presented is applicable to other bacterial proteins.
Publisher: American Society for Microbiology
Date: 02-2012
DOI: 10.1128/JB.06091-11
Abstract: To visualize the latter stages of cell ision in live Escherichia coli , we have carried out fluorescence recovery after photobleaching (FRAP) on 121 cells expressing cytoplasmic green fluorescent protein and periplasmic mCherry. Our data show conclusively that the cytoplasm is sealed prior to the periplasm during the ision event.
Publisher: Springer Science and Business Media LLC
Date: 08-07-2016
DOI: 10.1007/S00294-016-0630-2
Abstract: Bacterial cells are critically dependent on their ability to ide. The process of ision is carried out by a large and highly dynamic molecular machine, known as the isome. An understanding of the isomes' architecture is highly sought after, as it is essential for understanding molecular mechanisms and potentially designing antibiotic molecules that curb bacterial growth. Our current view, which is mainly based on high-resolution imaging of Escherichia coli, is that it is a patchy ring or toroid structure. However, recent super-resolution imaging has shown that the toroid structure contains at least three concentric rings, each containing a different set of proteins. Thus, the emerging picture is that the isome has different functional modules that are spatially separated in concentric rings.
Publisher: Royal Society of Chemistry (RSC)
Date: 2023
DOI: 10.1039/D2LC00794K
Abstract: Development of an inertial microfluidic device based on a zigzag configuration for rapid separation of bacteria from primary nasal s les.
Publisher: American Chemical Society (ACS)
Date: 31-07-2018
DOI: 10.1021/ACSSENSORS.8B00287
Abstract: Microbial biofilms possess intrinsic resistance against conventional antibiotics and cleaning procedures thus, a better understanding of their complex biological structures is crucial in both medical and industrial applications. Existing laboratory methodologies have focused on macroscopic and mostly indirect characterization of mechanical and microbiological properties of biofilms adhered on a given substrate. However, the kinetics underlying the biofilm formation is not well understood, while such information is critical to understanding how drugs and chemicals influence the biofilm formation. Herein, we report the use of localized surface plasmon resonance (LSPR) for real-time, label-free monitoring of E. coli biofilm assembly on a nanoplasmonic substrate consisting of gold mushroom-like structures. Our LSPR sensor is able to capture the signatures of biofilm formation in real-time by measuring the wavelength shift in the LSPR resonance peak with high temporal resolution. We employ this sensor feature to elucidate how biofilm formation is affected by different drugs, including conventional antibiotics (kanamycin and icillin) as well as rifapentine, a molecule preventing cell adhesion yet barely affecting bacterial viability and vitality. Due to its flexibility and simplicity, our LSPR based platform can be used on a wide variety of clinically relevant bacteria, thus representing a valuable tool in biofilm characterization and drug screening.
Publisher: Cold Spring Harbor Laboratory
Date: 28-11-2019
DOI: 10.1101/857813
Abstract: FtsZ, a highly conserved bacterial tubulin GTPase homolog, is a central component of the cell ision machinery in nearly all walled bacteria. FtsZ polymerizes at the future ision site and recruits greater than 30 proteins to assemble into a macromolecular complex termed the isome. Many of these isome proteins are involved in septal cell wall peptidoglycan (sPG) synthesis. Recent studies found that FtsZ polymers undergo GTP hydrolysis-coupled treadmilling dynamics along the circumference the ision site, driving the processive movement of sPG synthesis enzymes. How FtsZ’s treadmilling drives the directional transport of sPG enzymes and what its precise role is in bacterial cell ision are unknown. Combining theoretical modeling and experimental testing, we show that FtsZ’s treadmilling drives the directional movement of sPG-synthesis enzymes via a Brownian ratchet mechanism, where the shrinking end of FtsZ polymers introduces an asymmetry to rectify diffusions of single sPG enzymes into persistent end-tracking movement. Furthermore, we show that the processivity of this directional movement is dependent on the binding potential between FtsZ and the enzyme, and hinges on the balance between the enzyme’s diffusion and FtsZ’s treadmilling speed. This interplay could provide a mechanism to control the level of available enzymes for active sPG synthesis both in time and space, explaining the distinct roles of FtsZ treadmilling in modulating cell wall constriction rate observed in different bacterial species.
Publisher: Cold Spring Harbor Laboratory
Date: 18-12-2021
DOI: 10.1101/2021.12.17.473257
Abstract: Uropathogenic Escherichia coli (UPEC) cells can grow into highly filamentous forms during infection of bladder epithelial cells, but this process is poorly understood. Herein we found that some UPEC filaments released from infected bladder cells in vitro grew very rapidly and by more than 100 µm before initiating ision, whereas others did not survive, suggesting that filamentation is a stress response that promotes dispersal. The DamX bifunctional ision protein, which is essential for UPEC filamentation, was initially non-localized but then assembled at multiple ision sites in the filaments prior to ision. DamX rings maintained consistent thickness during constriction and remained at the septum until after membrane fusion was completed, like in rod cell ision. Our findings suggest a mechanism involving regulated dissipation of DamX, leading to ision arrest and filamentation, followed by its reassembly into ision rings to promote UPEC dispersal and survival during infection.
Publisher: Elsevier BV
Date: 07-2021
DOI: 10.1016/J.CUB.2021.04.041
Abstract: Dynamics of cell elongation and septation are key determinants of bacterial morphogenesis. These processes are intimately linked to peptidoglycan synthesis performed by macromolecular complexes called the elongasome and the isome. In rod-shaped bacteria, cell elongation and septation, which are dissociated in time and space, have been well described. By contrast, in ovoid-shaped bacteria, the dynamics and relationships between these processes remain poorly understood because they are concomitant and confined to a nanometer-scale annular region at midcell. Here, we set up a metabolic peptidoglycan labeling approach using click chemistry to image peptidoglycan synthesis by single-molecule localization microscopy in the ovoid bacterium Streptococcus pneumoniae. Our nanoscale-resolution data reveal spatiotemporal features of peptidoglycan assembly and fate along the cell cycle and provide geometrical parameters that we used to construct a morphogenesis model of the ovoid cell. These analyses show that septal and peripheral peptidoglycan syntheses first occur within a single annular region that later separates in two concentric regions and that elongation persists after septation is completed. In addition, our data reveal that freshly synthesized peptidoglycan is remodeled all along the cell cycle. Altogether, our work provides evidence that septal peptidoglycan is synthesized from the beginning of the cell cycle and is constantly remodeled through cleavage and insertion of material at its periphery. The ovoid-cell morphogenesis would thus rely on the relative dynamics between peptidoglycan synthesis and cleavage rather than on the existence of two distinct successive phases of peripheral and septal synthesis.
Publisher: Springer Science and Business Media LLC
Date: 28-07-2018
Publisher: Cold Spring Harbor Laboratory
Date: 31-05-2018
DOI: 10.1101/335356
Abstract: The FtsZ protein is a key regulator of bacterial cell ision. It has been implicated in acting as a scaffolding protein for other ision proteins, being a force generator during constriction, and more recently, as an active regulator of septal cell wall production. During an early stage of the ision cycle, FtsZ assembles into a heterogeneous structure coined the “Z-ring” due to its resemblance to a ring confined by the midcell geometry. While in vitro experiments on supported lipid bilayers have shown that purified FtsZ can self-organize into a swirling ring roughly the diameter of a bacterial cell, it is not known how, and if, membrane curvature affects FtsZ assembly and dynamics in vivo . To establish a framework for examining geometrical influences on proper Z-ring assembly and dynamics, we sculptured Escherichia coli cells into unnatural shapes, such as squares and hearts, using ision- and cell wall-specific inhibitors in a micro fabrication scheme. This approach allowed us to examine FtsZ behavior in engineered “Z-squares” and “Z-hearts”, and in giant cells up to 50 times their normal volume. Quantification of super-resolution STimulated Emission Depletion (STED) nanoscopy data showed that FtsZ densities in sculptured cells maintained the same dimensions as their wild-type counterparts. Additionally, time-resolved fluorescence measurements revealed that FtsZ dynamics were generally conserved in a wide range of cell shapes. Based on our results, we conclude that the underlying membrane environment is not a deciding factor for FtsZ filament maintenance and treadmilling in vivo .
Publisher: Springer Science and Business Media LLC
Date: 25-06-2022
DOI: 10.1038/S41467-022-31378-1
Abstract: During infection of bladder epithelial cells, uropathogenic Escherichia coli (UPEC) can stop iding and grow into highly filamentous forms. Here, we find that some filaments of E. coli UTI89 released from infected cells grow very rapidly and by more than 100 μm before initiating ision, whereas others do not survive, suggesting that infection-related filamentation (IRF) is a stress response that promotes bacterial dispersal. IRF is accompanied by unstable, dynamic repositioning of FtsZ ision rings. In contrast, DamX, which is associated with normal cell ision and is also essential for IRF, is distributed uniformly around the cell envelope during filamentation. When filaments initiate ision to regenerate rod cells, DamX condenses into stable rings prior to ision. The DamX rings maintain consistent thickness during constriction and remain at the septum until after membrane fusion. Deletion of damX affects vegetative cell ision in UTI89 (but not in the model E. coli K-12), and, during infection, blocks filamentation and reduces bacterial cell integrity. IRF therefore involves DamX distribution throughout the membrane and prevention of FtsZ ring stabilization, leading to cell ision arrest. DamX then reassembles into stable ision rings for filament ision, promoting dispersal and survival during infection.
Publisher: Cold Spring Harbor Laboratory
Date: 17-02-2023
DOI: 10.1101/2023.02.16.528894
Abstract: Urinary tract infections (UTIs) are a worsening global health problem. Multispecies bacterial UTIs are common but are poorly recognized and understood. Here we developed host cell co-infection models combined with high resolution imaging to compare single- and multi-species infections with common uropathogens, uropathogenic Escherichia coli (UPEC), Klebsiella pneumoniae and Enterococcus faecalis . We demonstrate cooperation and direct interactions specifically between UPEC and E. faecalis within host cells that corresponded to an increased frequency of host cell infection. 3D imaging showed UPEC rods orientated perpendicular to the major host cell axis within single-species intracellular bacterial communities (IBCs), whereas UPEC cells often enveloped E. faecalis in multispecies IBCs. Furthermore, during dispersal from host cells, only the Gram-negative bacteria underwent infection-related filamentation (IRF) in multispecies infections. These first insights into intra-species synergies during UTIs at a single cell level should provide research pathways towards better diagnosis and treatment.
Publisher: Wiley
Date: 21-08-2012
DOI: 10.1002/PRO.2131
Publisher: Cold Spring Harbor Laboratory
Date: 19-02-2023
DOI: 10.1101/2023.02.19.529144
Abstract: Understanding where proteins are localized in a bacterial cell is essential for understanding their function and regulation. This is particularly important for proteins that are involved in cell ision, which localize at the ision septum and assemble into highly regulated complexes. Current knowledge of these complexes has been greatly facilitated by super-resolution imaging using fluorescent protein fusions. Herein we demonstrate with FtsZ that single-molecule PALM images can be obtained in-vivo using a genetically fused nanotag (ALFA) and a corresponding nanobody fused to mEos3.2. The methodology presented is applicable to other bacterial proteins.
Publisher: Microbiology Society
Date: 12-2020
DOI: 10.1099/MIC.0.000996
Abstract: Fluorescent d -amino acids (FDAAs) are molecular probes that are widely used for labelling the peptidoglycan layer of bacteria. When added to growing cells they are incorporated into the stem peptide by a transpeptidase reaction, allowing the timing and localization of peptidoglycan synthesis to be determined by fluorescence microscopy. Herein we describe the chemical synthesis of an OregonGreen488-labelled FDAA (OGDA). We also demonstrate that OGDA can be efficiently incorporated into the PG of Gram-positive and some Gram-negative bacteria, and imaged by super-resolution stimulated emission depletion (STED) nanoscopy at a resolution well below 100 nm.
Publisher: Wiley
Date: 20-02-2014
DOI: 10.1111/MMI.12534
Publisher: American Chemical Society (ACS)
Date: 21-10-2019
DOI: 10.1021/ACSSENSORS.9B01580
Abstract: The extensive use of gold in sensing, diagnostics, and electronics has led to major concerns in solid waste management since gold and other heavy metals are nonbiodegradable and can easily accumulate in the environment. Moreover, gold ions are extremely reactive and potentially harmful for humans. Thus, there is an urgent need to develop reliable methodologies to detect and possibly neutralize ionic gold in aqueous solutions and industrial wastes. In this work, by using complementary measurement techniques such as quartz crystal microbalance (QCM), atomic force microscopy, crystal violet staining, and optical microscopy, we investigate a promising biologically induced gold biomineralization process accomplished by biofilms of bacterium
Publisher: Wiley
Date: 03-05-2023
DOI: 10.1111/MMI.13888
Abstract: The ision of Escherichia coli is mediated by a collection of some 34 different proteins that are recruited to the ision septum and are thought to assemble into a macromolecular complex known as 'the isome'. Herein, we have endeavored to better understand the structure of the isome by imaging two of its core components FtsZ and FtsN. Super resolution microscopy (SIM and gSTED) indicated that both proteins are localized in large assemblies, which are distributed around the ision septum (i.e., forming a discontinuous ring). Although the rings had similar radii prior to constriction, the in idual densities were often spatially separated circumferentially. As the cell envelope constricted, the discontinuous ring formed by FtsZ moved inside the discontinuous ring formed by FtsN. The radial and circumferential separation observed in our images indicates that the majority of FtsZ and FtsN molecules are organized in different macromolecular assemblies, rather than in a large super-complex. This conclusion was supported by fluorescence recovery after photobleaching measurements, which indicated that the dynamic behavior of the two macromolecular assemblies was also fundamentally different. Taken together, the data indicates that constriction of the cell envelope is brought about by (at least) two spatially separated complexes.
Publisher: American Society for Microbiology
Date: 15-01-2015
DOI: 10.1128/AEM.02733-14
Abstract: Bacterial ghosts are empty cell envelopes of Gram-negative bacteria that can be used as vehicles for antigen delivery. Ghosts are generated by releasing the bacterial cytoplasmic contents through a channel in the cell envelope that is created by the controlled production of the bacteriophage ϕX174 lysis protein E. While ghosts possess all the immunostimulatory surface properties of the original host strain, they do not pose any of the infectious threats associated with live vaccines. Recently, we have engineered the Escherichia coli autotransporter hemoglobin protease (Hbp) into a platform for the efficient surface display of heterologous proteins in Gram-negative bacteria, HbpD. Using the Mycobacterium tuberculosis vaccine target ESAT6 (early secreted antigenic target of 6 kDa), we have explored the application of HbpD to decorate E. coli and Salmonella ghosts with antigens. The use of different promoter systems enabled the concerted production of HbpD-ESAT6 and lysis protein E. Ghost formation was monitored by determining lysis efficiency based on CFU, the localization of a set of cellular markers, fluorescence microscopy, flow cytometry, and electron microscopy. Hbp-mediated surface display of ESAT6 was monitored using a combination of a protease accessibility assay, fluorescence microscopy, flow cytometry and (immuno-)electron microscopy. Here, we show that the concerted production of HbpD and lysis protein E in E. coli and Salmonella can be used to produce ghosts that efficiently display antigens on their surface. This system holds promise for the development of safe and cost-effective vaccines with optimal intrinsic adjuvant activity and exposure of heterologous antigens to the immune system.
Publisher: Springer Science and Business Media LLC
Date: 09-09-2016
DOI: 10.1038/SREP33138
Abstract: The source of constriction required for ision of a bacterial cell remains enigmatic. FtsZ is widely believed to be a key player, because in vitro experiments indicate that it can deform liposomes when membrane tethered. However in vivo evidence for such a role has remained elusive as it has been challenging to distinguish the contribution of FtsZ from that of peptidoglycan-ingrowth. To differentiate between these two possibilities we studied the early stages of ision in Escherichia coli , when FtsZ is present at the ision site but peptidoglycan synthesizing enzymes such as FtsI and FtsN are not. Our approach was to use correlative cryo-fluorescence and cryo-electron microscopy (cryo-CLEM) to monitor the localization of fluorescently labeled FtsZ, FtsI or FtsN correlated with the septal ultra-structural geometry in the same cell. We noted that the presence of FtsZ at the ision septum is not sufficient to deform membranes. This observation suggests that, although FtsZ can provide a constrictive force, the force is not substantial at the onset of ision. Conversely, the presence of FtsN always correlated with membrane invagination, indicating that allosteric activation of peptidoglycan ingrowth is the trigger for constriction of the cell envelope during cell ision in E. coli .
Publisher: Wiley
Date: 23-05-2016
DOI: 10.1111/MMI.13400
Publisher: Springer Science and Business Media LLC
Date: 27-01-2021
DOI: 10.1038/S41467-020-20873-Y
Abstract: The FtsZ protein is a central component of the bacterial cell ision machinery. It polymerizes at mid-cell and recruits more than 30 proteins to assemble into a macromolecular complex to direct cell wall constriction. FtsZ polymers exhibit treadmilling dynamics, driving the processive movement of enzymes that synthesize septal peptidoglycan (sPG). Here, we combine theoretical modelling with single-molecule imaging of live bacterial cells to show that FtsZ’s treadmilling drives the directional movement of sPG enzymes via a Brownian ratchet mechanism. The processivity of the directional movement depends on the binding potential between FtsZ and the sPG enzyme, and on a balance between the enzyme’s diffusion and FtsZ’s treadmilling speed. We propose that this interplay may provide a mechanism to control the spatiotemporal distribution of active sPG enzymes, explaining the distinct roles of FtsZ treadmilling in modulating cell wall constriction rate observed in different bacteria.
Publisher: Oxford University Press (OUP)
Date: 22-12-2022
Abstract: araC pBAD is a genetic fragment that regulates the expression of the araBAD operon in bacteria, which is required for the metabolism of L-arabinose. It is widely used in bioengineering applications because it can drive regulatable and titratable expression of genes and genetic pathways in microbial cell factories. A notable limitation of araC pBAD is that it generates a low signal when induced with high concentrations of L-arabinose (the maximum ON state). Herein we have lified the maximum ON state of araC pBAD by coupling it to a synthetically evolved translation initiation region (TIREVOL). The coupling maintains regulatable and titratable expression from araC pBAD and yet increases the maximal ON state by & -fold. The general principle demonstrated in the study can be applied to lify the signal from similar genetic modules. Graphical Abstract
Publisher: Wiley
Date: 28-01-2020
DOI: 10.1002/MBO3.999
Publisher: American Society for Microbiology
Date: 2019
DOI: 10.1128/JB.00396-18
Abstract: YidC proteins are membrane-localized chaperone insertases that are universally conserved in all bacteria and are traditionally studied in the context of membrane protein insertion and assembly. Both YidC paralogs of the cariogenic pathogen Streptococcus mutans are required for proper envelope biogenesis and full virulence, indicating that these proteins may also contribute to optimal biofilm formation in streptococci. Here, we show that the deletion of either yidC results in changes to the structure and physical properties of the EPS matrix produced by S. mutans , ultimately impairing optimal biofilm development, diminishing its mechanical stability, and facilitating its removal. Importantly, the universal conservation of bacterial yidC orthologs, combined with our findings, provide a rationale for YidC as a possible drug target for antibiofilm therapies.
Location: No location found
Location: Japan
Start Date: 2017
End Date: 2019
Funder: Japan Society for the Promotion of Science
View Funded ActivityStart Date: 2020
End Date: 2023
Funder: University of Technology Sydney
View Funded ActivityStart Date: 2014
End Date: 2015
Funder: Stiftelsen Olle Engkvist Byggmästare
View Funded ActivityStart Date: 07-2023
End Date: 06-2027
Amount: $828,774.00
Funder: Australian Research Council
View Funded ActivityStart Date: 06-2022
End Date: 05-2025
Amount: $448,619.00
Funder: Australian Research Council
View Funded Activity