Biology subjects

Kleanthous, C.

Publications and source records attributed to Kleanthous, C..

10 recordsLinked to original sources

bioRxiv2025

Ancestral neuronal receptors are bacterial accessory toxins

Symbiosis and horizontal gene transfer events were crucial in the emergence of eukaryotic and multicellular life. A striking example is the acquisition of Teneurins, putative surface-exposed toxins in bacteria that function as cell adhesion receptors in metazoan neuronal development. Here, we demonstrate the evolutionary relationships between metazoan and bacterial Teneurins. We show that bacterial Teneurins are accessory proteins, that harbour diverse encapsulated toxins. They predominate in species with complex social behaviours, suggesting roles in both cell-to-cell competition and cooperation. Metazoan Teneurins are therefore repurposed bacterial toxins central to cell-to-cell communication in nervous systems. Their acquisition was a foundational event in the evolution of metazoans.

biochemistry↗

bioRxiv2022

Mechanism of targeted killing of P. aeruginosa by pyocins SX1 and SX2

Pseudomonas aeruginosa is a common cause of serious hospital-acquired infections, the leading proven cause of mortality in people with cystic fibrosis and is associated with high levels of antimicrobial resistance. Pyocins are narrow spectrum protein antibiotics produced by P. aeruginosa that kill strains of the same species and have the potential to be developed as therapeutics targeting multi-drug resistant isolates. We have identified two novel pyocins designated SX1 and SX2. Pyocin SX1 is a metal-dependent DNase while pyocin SX2 kills cells through inhibition of protein synthesis. Mapping the uptake pathways of SX1 and SX2 shows these pyocins utilize a combination of the common polysaccharide antigen (CPA) and a previously uncharacterized TonB-dependent transporter (TBDT) PA0434 to traverse the outer membrane. In addition, TonB1 and FtsH are required by both pyocins to energise their transport into cells and catalyse their translocation across the inner membrane, respectively. Expression of PA0434 was found to be specifically regulated by copper availability and we have designated PA0434 as Copper Responsive Transporter A, or CrtA. To our knowledge these are the first S-type pyocins described that utilize a TBDT that is not involved in iron uptake.

microbiology↗

bioRxiv2022

Peptidoglycan maturation controls spatiotemporal organisation of outer membrane proteins in Escherichia coli

Linkages between the outer membrane of Gram-negative bacteria and the peptidoglycan layer are crucial to the maintenance of cellular integrity and enable survival in challenging environments1-5. The functionality of the outer membrane relies on outer membrane proteins (OMPs), which are inserted by the {beta}-barrel assembly machine, BAM6, 7. Previous work has shown that growing Escherichia coli cells segregate old OMPs towards the poles by an unknown mechanism8. Here, we demonstrate that peptidoglycan underpins the spatiotemporal organisation of OMPs. Mature, tetrapeptide-rich peptidoglycan binds to BAM components and suppresses OMP foldase activity. Nascent peptidoglycan, which is enriched in pentapeptides and concentrated at septa9, associates with BAM poorly and has little impact on its activity, leading to preferential insertion of OMPs at division sites. Synchronising OMP biogenesis to cell wall growth enables bacteria to replenish their OMPs by binary partitioning. Our study reveals that Gram-negative bacteria coordinate the assembly of two major cell envelope layers by rendering OMP biogenesis responsive to peptidoglycan maturation. This coordination offers new possibilities for the design of antibiotics that disrupt cell envelope integrity.

microbiology↗

bioRxiv2021

Bacterial competition systems share a domain required for inner membrane transport of the nuclease bacteriocin pyocin G

Bacteria exploit a variety of attack strategies to gain dominance within ecological niches. Prominent amongst these are contact-dependent inhibition (CDI), type VI secretion (T6SS) and bacteriocins. The cytotoxic endpoint of these systems is often the delivery of a nuclease to the cytosol. How such nucleases translocate across the cytoplasmic membrane of Gram-negative bacteria is unknown. Here, we identify a small, conserved, 15-kDa domain, which we refer to as the inner membrane translocation (IMT) domain that is common to T6SS and bacteriocins and linked to nuclease effector domains. Through fluorescence microscopy assays using intact and spheroplasted cells, we demonstrate that the IMT domain of the Pseudomonas aeruginosa specific bacteriocin pyocin G (PyoG) is required for import of the toxin nuclease domain to the cytoplasm. We also show that translocation of PyoG into the cytosol is dependent on inner membrane proteins FtsH, a AAA+ATPase/protease, and TonB1, the latter more typically associated with transport of bacteriocins across the outer membrane. Our study reveals that the IMT domain directs the cytotoxic nuclease of PyoG to cross the cytoplasmic membrane and, more broadly, has been adapted for the transport of other toxic nucleases delivered into Gram-negative bacteria by both contact dependent- and contact-independent means. ImportanceNuclease bacteriocins are potential antimicrobials for the treatment of antibiotic resistant bacterial infections. While the mechanism of outer membrane translocation is beginning to be understood, the mechanism of inner membrane transport is not known. This study uses PyoG as a model nuclease bacteriocin and defines a conserved domain which is essential for inner membrane translocation and which is widespread in other bacterial competition systems. Additionally, the presented data links two membrane proteins, FtsH and TonB1, with inner membrane translocation of PyoG. These findings point to the general importance of this domain to the cellular uptake mechanisms of nucleases delivered by otherwise diverse and distinct bacterial competition systems. The work is also of importance for the design of new protein antibiotics.

microbiology↗

bioRxiv2021

Colicin-mediated transport of DNA through the iron transporter FepA

Colicins are protein antibiotics used by bacteria to eliminate competing Escherichia coli. Colicins frequently exploit outer membrane (OM) nutrient transporters to penetrate through the strictly impermeable bacterial cellular envelope. Here, applying live-cell fluorescence imaging we were able to follow colicin B (ColB) into E. coli and localize it within the periplasm. We further demonstrate that single-stranded DNA coupled to ColB is also transported into the periplasm, emphasizing that the import routes of colicins can be exploited to carry large cargo molecules into bacteria. Moreover, we characterize the molecular mechanism of ColB association with its OM receptor FepA, applying a combination of photo-activated crosslinking, mass spectrometry, and structural modeling. We demonstrate that complex formation is coincident with a large-scale conformational change in the colicin. Finally In vivo crosslinking experiments and supplementary simulations of the translocation process indicate that part of the colicin engages active transport by disguising itself to part of the cellular receptor.

microbiology↗

bioRxiv2021

Passive receptor dissociation driven by porin threading establishes active colicin transport through Escherichia coli OmpF

Bacteria deploy weapons to kill their neighbours during competition for resources and aid survival within microbiomes. Colicins were the first antibacterial system identified yet how these bacteriocins cross the outer membrane of Escherichia coli is unknown. Here, by solving the structures of translocation intermediates and imaging toxin import, we uncover the mechanism by which the Tol-dependent nuclease colicin E9 (ColE9) crosses the outer membrane. We show that threading of ColE9s disordered domain through two pores of the trimeric porin OmpF causes the colicin to disengage from its primary receptor, BtuB, and reorganise the translocon either side of the membrane. These rearrangements prime the toxin for import through the lumen of a single OmpF subunit, which is driven by the proton motive force-linked TolQ-TolR-TolA-TolB assembly. Our study explains why OmpF is a better translocator than OmpC and reconciles the mechanisms by which Ton- and Tol- dependent bacteriocins cross the bacterial outer membrane.

biochemistry↗

bioRxiv2021

Spatiotemporal organization of BamA governs the pattern of outer membrane protein distribution in growing Escherichia coli cells

The outer membrane (OM) of Gram-negative bacteria is a robust protective barrier that excludes major classes of antibiotics. The assembly, integrity and functioning of the OM is dependent on {beta}-barrel outer membrane proteins (OMPs), the insertion of which is catalyzed by BamA, the core component of the {beta}-barrel assembly machine (BAM) complex. Little is known about BamA in the context of its native OM environment. Here, using high-affinity fluorescently-labelled antibodies in combination with diffraction-limited and super-resolution fluorescence microscopy, we uncover the spatial and temporal organization of BamA in live Escherichia coli K-12 cells. BamA is clustered into ~150 nm diameter islands that contain an average of 10-11 BamA molecules, in addition to other OMPs, and are distributed throughout the OM and which migrate to the poles during growth. In stationary phase cells, BamA is largely confined to the poles. Emergence from stationary phase coincides with new BamA-containing islands appearing on the longitudinal axis of cells, suggesting they are not seeded by pre-existing BamAs but initiate spontaneously. Consistent with this interpretation, BamA-catalyzed OMP biogenesis is biased towards non-polar regions. Cells ensure the capacity for OMP biogenesis is uniformly distributed during exponential growth, even if the growth rate changes, by maintaining an invariant density of BamA-containing OMP islands (~9 islands/m2) that only diminishes as cells enter stationary phase, the latter change governing what OMPs predominate as cells become quiescent. We conclude that OMP distribution in E. coli is driven by the spatiotemporal organisation of BamA which varies with the different phases of growth. SignificanceThe integrity and functioning of the outer membrane (OM) of Gram-negative bacteria depends on the {beta}-barrel assembly machinery (BAM). Although the structure and the mechanism of the complex have been widely explored, little information exists about the organization of the BAM complex and how it dictates protein distribution in the OM. Here, we utilized highly specific monoclonal antibodies to study the spatiotemporal organization of BamA, the key component of this complex. We reveal that BAM organization is dynamic and tightly linked to the cells growth phase. We further discover that the rate of BAM facilitated OMP biogenesis is significantly reduced near the poles. In turn, these features govern the biogenesis patterns and the distribution of OMPs on the cell surface.

microbiology↗

bioRxiv2020

Pyocin efficacy in a murine model of Pseudomonas aeruginosa sepsis

SynopsisO_ST_ABSBackgroundC_ST_ABSBloodstream infections with antibiotic resistant Pseudomonas aeruginosa are common and increasingly difficult to treat. Pyocins are naturally occurring protein antibiotics produced by P. aeruginosa that have potential for human use. ObjectivesTo determine if pyocin treatment is effective in a murine model of sepsis with P. aeruginosa. MethodsRecombinant pyocins S5 and AP41 were purified tested for efficacy in a Galleria mellonella infection model and a murine model of P. aeruginosa sepsis. ResultsBoth pyocins produced no adverse effects when injected alone into mice and showed good in vitro antipseudomonal activity. In an invertebrate model of sepsis using Galleria mellonella, both pyocins significantly prolonged survival. Following injection into mice, both showed extensive distribution into different organs. When administered 5 hours after infection, both pyocins reduced mortality, with pyocin S5 being more effective than AP41. ConclusionsPyocins S5 and AP41 show in vivo biological activity and can improve survival in a murine model of P. aeruginosa infection. They hold promise as novel antimicrobial agents for treatment of multi-drug resistant infections with this microbe.

microbiology↗

bioRxiv2019

Pyocin S5 import into Pseudomonas aeruginosa reveals a generic mode of bacteriocin transport

Pyocin S5 (PyoS5) is a potent protein bacteriocin that eradicates the human pathogen P. aeruginosa in animal infection models, but its import mechanism is poorly understood. Here, using crystallography, biophysical and biochemical analysis and live-cell imaging, we define the entry process of PyoS5 and reveal links to the transport mechanisms of other bacteriocins. In addition to its C-terminal pore-forming domain, elongated PyoS5 comprises two novel tandemly repeated kinked three helix bundle domains that structure-based alignments identify as key import domains in other pyocins. The central domain binds the lipid-bound common polysaccharide antigen, allowing the pyocin to accumulate on the cell surface. The N-terminal domain binds the ferric pyochelin transporter FptA while its associated disordered region binds the inner membrane protein TonB1, which together drive import of the bacteriocin across the outer membrane. Finally, we identify the minimal requirements for sensitizing Escherichia coli towards PyoS5, as well as other pyocins, and suggest that a generic pathway likely underpins the import of all TonB-dependent bacteriocins across the outer membrane of Gram-negative bacteria.

biochemistry↗

bioRxiv2019

Pal stabilises the bacterial outer membrane during constriction by a mobilisation-and-capture mechanism

Coordination of outer membrane constriction with septation is critical to faithful division in Gram-negative bacteria and vital to the barrier function of the membrane. Recent studies suggest this coordination is through the active accumulation of the peptidoglycan-binding outer membrane lipoprotein Pal at division sites by the Tol system, but the mechanism is unknown. Here, we show that Pal accumulation at Escherichia coli division sites is a consequence of three key functions of the Tol system. First, Tol mobilises Pal molecules in dividing cells, which otherwise diffuse very slowly due to their binding of the cell wall. Second, Tol actively captures mobilised Pal molecules and deposits them at the division septum. Third, the active capture mechanism is analogous to that used by the inner membrane protein TonB to dislodge the plug domains of outer membrane TonB-dependent nutrient transporters. We conclude that outer membrane constriction is coordinated with cell division by active mobilisation-and-capture of Pal at division septa by the Tol system.

microbiology↗