Biology subjects

Lovering, A. L.

Publications and source records attributed to Lovering, A. L..

16 recordsLinked to original sources

bioRxiv2026

A widespread bacterial toxin reveals a deeply divergent DNase family neutralized by diverse immunity proteins.

Bacteria compete for space and resources by delivering toxin proteins into neighboring cells. Many toxins remain uncharacterized because they lack similarity to proteins of known function. Here we identify Rehh as a widespread antibacterial toxin associated with multiple bacterial secretion systems and show that it is used in interbacterial competition. Purified recombinant Rehh functions as a manganese-dependent DNase, consistent with our in-vivo findings that Rehh damages target cell DNA, inducing the SOS response and the accumulation of double-strand breaks. The crystal structure of Rehh in complex with a cognate immunity protein reveals a positively charged histidine-rich metal-binding pocket embedded within a protein scaffold that bears no significant similarity to previously characterized nucleases. However, local structural analysis demonstrates that this pocket preserves the catalytic geometry characteristic of the HNH/His-Me-finger superfamily, revealing Rehh to be a deeply divergent HNH nuclease whose ancestry has been obscured through extensive structural remodeling. The cognate immunity protein neutralizes the toxin via an acidic surface that occludes the catalytic pocket, consistent with mimicry of the electrostatic properties of the DNA phosphate backbone. Comparative genomics further shows that Rehh homologs are associated with multiple unrelated immunity families, suggesting that distinct immunity domains repeatedly converge on a common strategy for inhibition. Together, these findings demonstrate how conflict-driven diversification can obscure enzyme ancestry while preserving catalytic function and reveal a possible case of convergent evolution among immunity proteins that independently exploit the same toxin biochemical vulnerability.

microbiology↗

bioRxiv2026

Direct targeting of the WAVE-regulatory complex and the actin cytoskeleton by the Anaplasma phagocytophilum effector, AnkA

The obligate intracellular tick-borne pathogen Anaplasma phagocytophilum is unusual in its tropism for neutrophils, within which it survives and replicates. Central to the ability of this bacterium to colonize this hostile niche are secreted effector proteins. One such effector protein, AnkA, interacts with multiple host ligands in both the cytoplasm and the nucleus, yet how these interactions promote infection is largely unknown. We discovered that AnkA directly binds both the WAVE Regulatory Complex (WRC), a signal-integration hub that regulates the formation of branched actin networks underpinning diverse cell functions, and to monomeric and filamentous actin. We determined that AnkA uses two forms of molecular mimicry, first by binding to the Abi1 component of the WRC via a WRC-Recruiting Abi-binding Peptide (WRAP), and second by binding to actin via a WH2-like Actin Binding Motif (ABM). Via these interactions, AnkA can exert multi-factorial influence on the actin cytoskeleton and signaling pathways converging on this host structure. AnkA and the WRC co-localise during bacterial internalization and during the late stages of infection, suggesting a central role in both host-cell colonization and bacterial dissemination. Our work reveals AnkA as the first example of a bacterial effector that directly targets the WRC. The co-occurrence of WRAP- and ABM-like motifs in other bacterial effector proteins suggests that this mechanism of actin cytoskeleton subversion may represent a conserved strategy used by other obligate intracellular bacteria.

microbiology↗

bioRxiv2026

A C-terminal Processing Protease Implicated in Flagellin Turnover and Developmental Progression in a Bacterial Predator

Carboxy-terminal processing proteases (CTPs) are widely conserved bacterial proteases implicated in protein maturation, quality control, and stress responses, yet many family members functions remain unclear. Here, we characterise Bd0967, a previously unstudied CTP from the predatory bacterium Bdellovibrio bacteriovorus, and its role in flagellar function during predatory development. Crystal structures reveal a self-compartmentalised protease in which a PDZ domain forms a lid over a large internal cavity accessed through a proteolytic tunnel. Co-purifying peptides occupied two distinct substrate-binding sites, suggesting a coordinated recognition mechanism. Affinity pulldowns coupled with mass spectrometry identified several Bdellovibrio flagellins as candidate substrates, which were subsequently validated by biochemical assays and shown to be selectively degraded through recognition of a conserved C-terminal motif. A Bd0967-mCherry translational fusion localised to periplasmic foci during the intracellular predatory growth, consistent with flagellar resorption and flagellin turnover following prey invasion. Deletion of bd0967 caused developmental defects, including aberrant Bdellovibrio cell morphology and reduced predation efficiency. Together, these findings establish Bd0967 as a specialised CTP that couples flagellin degradation and developmental progression in a predatory bacterium.

biochemistry↗

bioRxiv2026

A Type VII-secreted toxin enables inter-mycobacterial competition

Summary ParagraphMost bacteria live in complex environments where resources are scarce and competition is fierce. These organisms have evolved mechanisms to compete with other bacteria, often through the specialised secretion of proteinaceous toxins. Mycobacteria have not previously been reported to engage in this form of competition. The thick and unusual cell wall of mycobacteria, comprised of peptidoglycan, arabinogalactan and mycolic acids, is generally thought to be highly protective to these bacteria. Many enzymes have evolved to maintain this structure, including the GH183 family, which cleaves arabinogalactan. Here, we establish for the first time that some mycobacteria have weaponised a subset of these endo-D-arabinanases to enable inter-bacterial competition. We show that mycobacteria secrete an endo-D-arabinanase effector via the type VII secretion system that specifically targets the arabinogalactan layer of the Mycobacteriales cell envelope. Using structural biology and biochemistry, we identify the molecular basis for this activity and reveal a new protein family that protects the bacterium from the activity of this toxin. Finally, our data uncover widespread T7-secreted toxins in the Mycobacteriales, pointing to extensive inter-mycobacterial competition.

microbiology↗

bioRxiv2025

TipA, a Bdellovibrio bacteriovorus BPI-like double-TULIP is opened by its adapter protein, TipB

Bdellovibrio bacteriovorus is a predatory bacterium that invades the periplasm of other Gram-negative bacteria and liberates prey biomolecules for replication. Bdellovibrio has a wealth of genes that encode unique proteins to enable this lifestyle. Using a series of x-ray structures, we show that the operonal pairing of bd2538 and bd2539, encode a double TULIP (tubular lipid binding protein) invasion protein A (TipA) and a small beta sandwich (TipB), respectively. TipA has a specialised N-terminal TULIP domain with a beta hairpin and helix that mediate homodimerisation through hairpin interdigitation. This dimerisation creates a large, continuous, enclosed lumen that we demonstrate to contain multiple lipid molecules. In addition, we show that TipB functions as a small adapter protein that binds to TipA using specialised loops that bury into the TipA hydrophobic core. This binding forms a clamp on the edge of the N-terminal beta sheet and induces a large 20 [A] conformational change, opening the TULIP fold to create an accessible interior. This study presents the first structural characterisation of lipid binding proteins in Bdellovibrio, and the first example of conformational change in TULIPs mediated by an adaptor protein. Synopsis O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=186 SRC="FIGDIR/small/675085v2_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@3ef907org.highwire.dtl.DTLVardef@4f555dorg.highwire.dtl.DTLVardef@6d560forg.highwire.dtl.DTLVardef@2cfe49_HPS_FORMAT_FIGEXP M_FIG C_FIG Bdellovibrio bacteriovorus is a predatory bacterium that invades the periplasm of other Gram-negative bacteria in order to consume them from within. To enable this lifestyle, it has an arsenal of predation-associated genes, including the operonal pair bd2538 and bd2539. We use structural and biophysical techniques to characterise two proteins produced by these genes, TipA and TipB. O_LITipA is a double tubular lipid binding protein (TULIP) that forms a homodimer that sequesters lipids in its lumen and binds to lipid bilayers C_LIO_LITipB is a small beta sandwich that interacts with TipA via two specialised loops C_LIO_LIInteraction of TipA with TipB opens the TULIP fold of TipA C_LI

biochemistry↗

bioRxiv2025

PNGaseA-mediated N-glycan stripping from peptides by infant-derived Bifidobacterium bifidum

N-glycans are highly common sources of nutrition for human colonic-dwelling bacteria. These microbes have evolved a several methods to remove N-glycans from proteins; herein we describe the biochemical and structural characterisation of one such enzyme, a PNGaseA superfamily member produced by the infant-associated Bifidobacterium bifidum LMG13195. This PNGase was demonstrated to elicit activity against a wide variety of N-glycan structures yet exhibited a high preference for N-glycans attached to a peptide rather than to a denatured or native protein. This unusual specificity highlights how bacterial species tune their enzymology to different types of substrates. The structural characterisation of this PNGase reveals how its structure determines this specificity while being the first structure presented from the PNGaseA superfamily, revealing a unique ten-stand {beta}-sheet cradling a canonical PNGase catalytic module.

biochemistry↗

bioRxiv2023

The mycobacterial glycoside hydrolase LamH enables capsular arabinomannan release and stimulates growth

Mycobacterial glycolipids are important cell envelope structures that drive host-pathogen interactions. Arguably, the most important amongst these are lipoarabinomannan (LAM) and its precursor, lipomannan (LM), which are both trafficked out of the bacterium to the host via unknown mechanisms. An important class of exported LM/LAM is the capsular derivative of these molecules which is devoid of its lipid anchor. Here, we describe the identification of a glycoside hydrolase family 76 enzyme that we term LamH which specifically cleaves -1,6-mannoside linkages within LM and LAM, driving its export to the capsule releasing its phosphatidyl-myo-inositol mannoside lipid anchor. Unexpectedly, we found that the catalytic activity of this enzyme is important for efficient exit from stationary phase cultures where arabinomannan acts as a signal for growth phase transition. Finally, we demonstrate that LamH is important for Mycobacterium tuberculosis survival in macrophages. These data provide a new framework for understanding the biological role of LAM in mycobacteria.

microbiology↗

bioRxiv2022

Mining the human gut microbiome identifies mycobacterial D-arabinan degrading enzymes

Division and degradation of bacterial cell walls requires coordinated action of a myriad of enzymes. This particularly applies to the elaborate cell walls of acid-fast organisms such as Mycobacterium tuberculosis, which consist of a multi-layered cell wall that contains an unusual glycan called arabinogalactan. Enzymes that cleave the O_SCPLOWDC_SCPLOW-arabinan core of this structure have not previously been identified in any organism. We have interrogated the diverse carbohydrate degrading enzymes expressed by the human gut microbiota and uncovered four families of glycoside hydrolases with the capability to degrade the O_SCPLOWDC_SCPLOW-arabinan or O_SCPLOWDC_SCPLOW-galactan components of arabinogalactan. Using novel exo-O_SCPLOWDC_SCPLOW-galactofuranosidases from gut bacteria we generated enriched O_SCPLOWDC_SCPLOW-arabinan and used it to identify D. gadei as a D-arabinan degrader. This enabled the discovery of endo- and exo-acting enzymes that cleave D-arabinan. We have identified new members of the DUF2961 family (GH172), and a novel family of glycoside hydrolases (DUF4185) that display endo-O_SCPLOWDC_SCPLOW-arabinofuranase activity. The DUF4185 enzymes are conserved in mycobacteria and found in many microbes, suggesting that the ability to degrade mycobacterial glycans plays an important role in the biology of diverse organisms. All mycobacteria encode two conserved endo-O_SCPLOWDC_SCPLOW-arabinanases that display different preferences for the O_SCPLOWDC_SCPLOW-arabinan-containing cell wall components arabinogalactan and lipoarabinomannan, suggesting they are important for cell wall modification and/or degradation. The discovery of these enzymes will support future studies into the structure and function of the mycobacterial cell wall.

biochemistry↗

bioRxiv2021

NamZ1 and NamZ2 from the oral pathogen Tannerella forsythia are peptidoglycan processing exo-β-N-acetylmuramidases with distinct substrate specificity

The Gram-negative periodontal pathogen Tannerella forsythia is inherently auxotrophic for N-acetylmuramic acid (MurNAc), which is an essential carbohydrate constituent of the peptidoglycan (PGN) of the bacterial cell wall. Thus, to build up its cell wall, T. forsythia strictly depends on the salvage of exogenous MurNAc or sources of MurNAc, such as polymeric or fragmentary PGN, derived from cohabiting bacteria within the oral microbiome. In our effort to elucidate how T. forsythia satisfies its demand for MurNAc, we recognized that the organism possesses three putative orthologs of the exo-{beta}-N-acetylmuramidase BsNamZ from Bacillus subtilis, which cleaves non-reducing end, terminal MurNAc entities from the artificial substrate pNP-MurNAc and the naturally-occurring disaccharide substrate MurNAc-{beta}-1,4-N-acetylglucosamine (GlcNAc). TfNamZ1 and TfNamZ2 were successfully purified as soluble, pure recombinant His6-fusions and characterized as exo-lytic {beta}-N-acetylmuramidases with distinct substrate specificities. The activity of TfNamZ1 was considerably lower compared to TfNamZ2 and BsNamZ, in the cleavage of pNP-MurNAc and MurNAc-GlcNAc. When peptide-free PGN glycans were used as substrates, we revealed striking differences in the specificity and mode of action of these enzymes, as analyzed by mass spectrometry. TfNamZ1, but not TfNamZ2 or BsNamZ, released GlcNAc-MurNAc disaccharides from these glycans. In addition, glucosamine (GlcN)-MurNAc disaccharides were generated when partially N-deacetylated PGN glycans from B. subtilis 168 were applied. This characterizes TfNamZ1 as a unique disaccharide-forming exo-lytic {beta}-N-acetylmuramidase (exo-disaccharidase), and, TfNamZ2 and BsNamZ as sole MurNAc monosaccharide-lytic exo-{beta}-N-acetylmuramidases. IMPORTANCETwo exo-{beta}-N-acetylmuramidases from T. forsythia belonging to glycosidase family GH171 (www.cazy.org) were shown to differ in their activities, thus revealing a functional diversity within this family: NamZ1 releases disaccharides (GlcNAc-MurNAc/GlcN-MurNAc) from the non-reducing ends of PGN glycans, whereas NamZ2 releases terminal MurNAc monosaccharides. This work provides a better understanding of how T. forsythia may acquire the essential growth factor MurNAc by the salvage of PGN from cohabiting bacteria in the oral microbiome, which may pave avenues for the development of anti-periodontal drugs. On a broad scale, our study indicates that the utilization of PGN as a nutrient source, involving exo-lytic N-acetylmuramidases with different modes of action, appears to be a general feature of bacteria, particularly among the phylum Bacteroidetes.

microbiology↗

bioRxiv2021

Asymmetric peptidoglycan editing generates the curvature of predatory bacteria, optimizing invasion and replication within a spherical prey niche

The vibrioid predatory bacterium Bdellovibrio bacteriovorus secretes prey wall-modifying enzymes to invade and replicate within the periplasm of Gram-negative prey bacteria. Studying self-modification of predator wall peptidoglycan during predation, we discover that Bd1075 generates self-wall curvature by exerting LD-carboxypeptidase activity in the vibrioid B. bacteriovorus strain HD100 as it grows inside spherical prey. Bd1075 localizes to the outer curved face of B. bacteriovorus, in contrast to most known shape-determinants. Asymmetric protein localization is determined by the novel function of a nuclear transport factor 2-like (NTF2) domain at the protein C-terminus. The solved structure of Bd1075 is monomeric, with key differences to other LD-carboxypeptidases. Rod-shaped {Delta}bd1075 mutants invade prey more slowly than curved wild-type predators, and stretch and deform the invaded prey cell from within. Vibrioid morphology increases the evolutionary fitness of wild predatory bacteria, facilitating efficient prey invasion and intracellular growth of curved predators inside a spherical prey niche.

microbiology↗

bioRxiv2021

Structures of Bdellovibrio bacteriovorus phosphoglucose isomerase reveal novel rigidity in the active site of a selected subset of enzymes upon substrate binding.

Glycolysis and gluconeogenesis are central pathways of metabolism across all domains of life. A prominent enzyme in these pathways is phosphoglucose isomerase (PGI) which mediates the interconversion of glucose-6-phosphate and fructose-6-phosphate (F6P). The predatory bacterium Bdellovibrio bacteriovorus leads a complex lifecycle, switching between intraperiplasmic replicative and extracellular "hunter" attack-phase stages. Passage through this complex lifecycle involves different metabolic states. Here we present the unliganded and substrate bound structures of the Bdellovibrio bacteriovorus PGI, solved to 1.74[A] and 1.67 [A], respectively. These structures reveal that an induced-fit conformational change within the active site is not a pre-requisite for the binding of substrates in some PGIs. Crucially, we suggest a phenylalanine residue, conserved across most PGI enzymes but substituted for a glycine in Bdellovibrio and other select organisms, is central to the induced-fit mode of substrate recognition for PGIs. This enzyme also represents the smallest conventional PGI characterised to date and likely represents the minimal requirements for a functional PGI.

biochemistry↗

bioRxiv2020

Global query on bacterial sirtuin CobB interactants reveals crosstalk with PRPP synthase Prs

In bacteria, post-translational acetylation of lysine residues affects the activities of numerous proteins, thus constituting an important regulatory mechanism that influences bacterial growth and adaptation to various environments. Conversely, the modification level of some lysine residues is controlled by deacetylases that remove the acetyl group. Most bacterial deacetylases are homologous to eukaryotic sirtuins, which utilize NAD+ as a cofactor. The Escherichia coli genome encodes a single sirtuin - CobB. However, both the regulation of CobB activity and the role that reversible acetylation plays in the control of protein functions and cellular pathways are not fully understood. In this study, we demonstrate that CobB forms a stable complex with PRPP synthase Prs. PRPP synthase is responsible for providing precursor metabolite for the synthesis of certain amino acids as well as nucleotides, including the cofactor NAD+. This association stimulates the deacetylation rate by CobB and protects it from inhibition by its reaction byproduct nicotinamide. We present evidence indicating that acetylation of Prs is physiologically significant and impacts E. coli metabolism and global protein acetylation level, although it does not affect CobB binding in vitro. In turn, Prs can be deacetylated by CobB, while formation of CobB-Prs complex formation safeguards PRPP synthase activity under non-optimal conditions. We propose that formation of the complex regulates deacetylation of other protein substrates by CobB.

molecular biology↗

bioRxiv2020

Structure-function characterization of the conserved regulatory mechanism of the Escherichia coli M48 metalloprotease BepA

The asymmetric Gram-negative outer membrane (OM) is the first line of defence for bacteria against environmental insults and attack by antimicrobials. The key component of the OM is lipopolysaccharide, which is transported to the surface by the essential lipopolysaccharide transport (Lpt) system. Correct folding of the Lpt system component LptD is regulated by a periplasmic metalloprotease, BepA. Here we present the crystal structure of BepA from Escherichia coli, solved to a resolution of 2.18 [A], in which the M48 protease active site is occluded by an active site plug. Informed by our structure, we demonstrate that free movement of the active site plug is essential for BepA function, suggesting that the protein is auto-regulated by the active site plug, which is conserved throughout the M48 metalloprotease family. Targeted mutagenesis of conserved residues reveals that the negative pocket and the TPR cavity are required for function and degradation of the BAM complex component BamA under conditions of stress. Lastly, we show that loss of BepA causes disruption of OM lipid asymmetry, leading to surface exposed phospholipid. ImportanceM48 metalloproteases are widely distributed in all domains of life. E. coli possesses four members of this family located in multiple cellular compartments. The functions of these proteases are not well understood. Recent investigations revealed that one family member, BepA, has an important role in the maturation of a central component of the LPS biogenesis machinery. Here we present the structure of BepA and the results of a structure guided mutagenesis strategy, which reveal the key residues required for activity.

microbiology↗

bioRxiv2019

Structure-function analysis of the Escherichia coli β-barrel assembly enhancing protease BepA suggests a role for a self-inhibitory state

The asymmetric Gram-negative outer membrane (OM) is the first line of defence for the bacteria against environmental insults and attack by antimicrobials. The key component of the OM barrier is the surface exposed lipopolysaccharide, which is transported to the surface by the essential lipopolysaccharide transport (Lpt) system. Correct folding of the Lpt system OM component, LptD, is essential and is regulated by a periplasmic metalloprotease, BepA. Here we present the crystal structure of BepA, solved to a resolution of 1.9 [A]. Our structure comprises the zinc-bound m48 protease domain and a tetratricopeptide repeat (TPR) domain, consisting of four 2-helix TPR motifs and four non-TPR helices, leading to a nautilus-like shape in which the TPR repeats cup the protease domain. Using targeted mutagenesis approaches, we demonstrate that the protein is auto-regulated by the active-site plug. Further to this we reveal that mutation of a negative pocket, formed at the interface between the m48 and TPR domains, impairs BepA activity suggesting the pocket as a possible substrate binding site. We also identify a potential protein interaction site within the TPR cavity as being important for BepA function. Lastly, we provide evidence to show that increased antibiotic susceptibility in the absence of correctly functioning BepA occurs through disruption of OM lipid asymmetry, leading to reduced barrier function and increased cell permeability.

microbiology↗

bioRxiv2018

Evidence for phospholipid export from the gram-negative inner membrane: time to rethink the Mla pathway?

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 3 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 novel pivoting {beta}-sheet mechanism which functions to open and close the phospholipid-binding pocket. Using the apo form of MlaC we provide evidence that the Mla pathway functions in an anterograde rather than a retrograde direction by showing the inner membrane MlaFEDB machinery exports phospholipids and transfers them to MlaC in the periplasm. We confirm that the lipid export process occurs through the MlaD component of the MlaFEDB complex. This lipid export process is shown to be independent of ATP. Our data provides, for the first time, evidence of an apparatus for lipid export to the outer membrane.

biophysics↗

bioRxiv2018

Mycobacteria recycle their peptidoglycan via a novel pathway which influences antimicrobial resistance and limits proliferation in macrophages.

Growth and division by most bacteria requires remodeling and cleavage of their cell wall. A byproduct of this process is the generation of free peptidoglycan (PG) fragments known as muropeptides. These muropeptides are recycled in many model organisms, where the bacteria can harness their unique nature as a signal for cell wall damage. These molecules also serve as important signals for hosts where binding to specific receptors reports on the presence of intracellular bacteria. Despite this critical role for muropeptides, it has long been thought that pathogenic mycobacteria such as Mycobacterium tuberculosis do not recycle their PG. Herein we show for the first time that M. tuberculosis and Mycobacterium bovis BCG are able to recycle components of their PG. We demonstrate that the core-mycobacterial gene lpqI, encodes an authentic NagZ {beta}-N-acetylglucosaminidase and that it is essential for PG-derived amino sugar recycling via an unusual pathway. By characterizing an M. bovis BCG strain lacking lpqI we are also able to show that stem-peptide recycling proceeds independent of amino sugar recovery and loss of lpqI leads to antimicrobial resistance in vitro. Together these data provide a critical first step in understanding how mycobacteria recycle their peptidoglycan.

microbiology↗