Biology subjects

Laabei, M.

Publications and source records attributed to Laabei, M..

11 recordsLinked to original sources

bioRxiv2026

Expanding Macrocyclic Topology through Cysteine-to-N-Terminal Cyclisation Enables Covalent Peptide Inhibitor Discovery

Macrocyclic peptides are an attractive therapeutic modality capable of engaging challenging protein targets while retaining many favourable drug-like properties. Their high-affinity binding also provides an ideal framework for proximity-driven covalent inhibition through incorporation of latent electrophiles. Phage display enables the high-throughput screening of billion-member macrocyclic peptide libraries; however, existing libraries rely predominantly on cysteine-mediated cyclisation, restricting the range of macrocyclic topologies available for ligand discovery. Here, we report a mild and efficient cyclisation strategy based on a bromomethyl picolinaldehyde (BMP) linker that reacts with a cysteine side chain and the peptide N-terminus to generate a previously unexplored macrocyclic topology incorporating neighbouring pyridine and imidazolidinone rings. The chemistry is compatible with phage display and enabled screening of BMP-cyclised peptide libraries against plasma kallikrein, yielding a potent macrocyclic inhibitor. The BMP-cyclised peptide displayed substantially greater potency than analogous peptides cyclised through either a disulfide bond or the widely used linker 1,4-bis(bromomethyl)benzene (DBMB). Furthermore, comparison with an equivalent DBMB-cyclised library demonstrated that BMP-mediated cyclisation enabled access to binding motifs not identified by conventional cysteine-to-cysteine cyclisation. Finally, positional sulfur(VI) fluoride exchange (SuFEx) electrophile scanning converted the BMP-derived hit into a selective covalent macrocyclic activity-based probe capable of labelling plasma kallikrein in human plasma. Together, these findings establish BMP-mediated cyclisation as a versatile strategy for expanding the topological diversity of phage-displayed macrocycles and accelerating the discovery of both reversible and covalent macrocyclic peptide ligands.

biochemistry↗

bioRxiv2026

Prophage and metabolic determinants of Staphylococcus aureus survival to vancomycin identified via TraDIS screening

Reduced vancomycin susceptibility phenotypes in Staphylococcus aureus contribute to treatment failure, yet the genetic determinants of survival under inhibitory vancomycin exposure remain incompletely defined. We performed transposon directed insertion-site sequencing (TraDIS) on a methicillin resistant S. aureus (MRSA) ST398 mutant library following exposure to vancomycin at its minimum inhibitory concentration, identifying 52 genes whose disruption was associated with loss of population survival at inhibitory drug concentrations. Prophage associated loci were the largest functional group, spanning predicted structural and regulatory genes as well as multiple conserved hypothetical proteins. Targeted testing of defined transposon mutants in a USA300 background confirmed that disruption of selected loci impaired growth under vancomycin exposure. Our results highlight the contribution of diverse physiological processes, including metabolism, stress responses, and a prominent role for prophage-associated functions, rather than discrete resistance pathways. Together, these findings indicate that vancomycin tolerance is shaped by the general physiological state of the bacterial cell, including metabolic capacity and stress adaptation. ImportanceTreatment failure in Staphylococcus aureus infections often occurs in the absence of known antibiotic resistance determinants, suggesting that additional survival mechanisms influence therapeutic outcomes. In this study, we identify genetic determinants required for survival during inhibitory vancomycin exposure, revealing a broad role for metabolic functions, stress adaptation, and prophage-associated loci. The prominence of these diverse processes highlights that survival reflects global physiological adaptation rather than discrete resistance pathways. This insight underscores the need to consider cellular physiology and stress responses when developing strategies to prevent antibiotic tolerance and improve treatment efficacy.

microbiology↗

bioRxiv2026

Stress-induced DNA methylome plasticity and transcriptional re-programming in Staphylococcus aureus

Staphylococcus aureus, a major human and livestock pathogen, is the second biggest cause of antimicrobial resistance-associated mortality. Although S. aureus transcriptional regulation has been extensively characterised, the potential role of DNA methylation in S. aureus transcriptional regulation and stress response remains largely undefined. We tackled this gap by combining genome-wide nanopore sequencing-derived DNA methylation data and transcriptomic data, acquired before and during exposure of methicillin-resistant Staphylococcus aureus strain USA300 to clinically relevant oxidative, antibiotic and nitrosative stresses. Stress-induced significant DNA methylation changes were rare, with <0.1% of cytosines/adenines undergoing [≥]20% change in methylation; these changes were enriched within genomic features (protein-coding genes, predicted promoter regions, ncRNAs). Transcription changes reflected metabolic, regulatory and stress-specific pathway adjustments. Many of the stress-induced DNA methylation changes occurred alongside transcription changes, although there was no obvious overarching relationship between the directions of changes in DNA methylation and transcription. Pre-stress treatment methylation entropy tended to be elevated at sites containing a base that underwent stress-induced change in methylation level, identifying focal sites of pre-existing methylome heterogeneity relative to both local and genome-wide backgrounds; the magnitude of the site-specific methylation entropy peak, moreover, correlated with the magnitude of subsequent methylation change. Genome-wide entropy levels were consistent with a finite number of methylation patterns, distinguished by key bases, that could correspond to clinically important S. aureus subpopulations exhibiting persistence, dormancy and immune evasion. These findings support the principle that DNA methylation is an important component of the regulatory machinery underpinning S. aureus adaptability and persistence. ImportanceStaphylococcus aureus survives antibiotic treatment and host immune attack partly through phenotypic diversity, yet the regulatory processes that support this adaptability remain incompletely understood. Here, we integrate genome-wide DNA methylation and transcriptomic profiling to examine how epidemic methicillin-resistant S. aureus USA300 responds to oxidative, antibiotic, and nitrosative stresses. Stress-induced methylation changes were rare but non-random, concentrated in coding and regulatory regions linked to stress defence, metabolism, persistence, virulence, biofilm formation, and host interaction. Sites that underwent stress-induced methylation changes tended to have unusually high pre-stress methylation pattern diversity relative to local and genome-wide pattern diversity. These findings support a model in which an S. aureus population contains a pre-stress repertoire of epigenetic states that stress selectively redistributes via bases that differentiate stable methylation patterns. By revealing a layer of heterogeneity that may support pathogen resilience, this work provides a framework for investigating whether bacterial methylation dynamics can be exploited clinically.

microbiology↗

bioRxiv2026

Beyond the Catalytic Serine: Selective Protease Engagement with Covalent Macrocyclic Activity-based Probes

Activity-based probes (ABPs) are widely used to profile serine protease activity - enzymes central to diverse physiological and pathological processes - but most rely on covalent modification of the conserved catalytic serine residue, often resulting in poor selectivity across related proteases. Here, we introduce covalent macrocyclic activity-based probes (cmABPs) that selectively target non-catalytic residues within serine protease active sites. By combining phage display with systematic electrophile scanning, we identify macrocyclic scaffolds that position sulfur(VI) fluoride (SuFEx) electrophiles to covalently engage alternative nucleophiles such as lysine and tyrosine. Applied to plasma kallikrein, this approach yielded a macrocyclic scaffold that was converted into covalent probes via fluorosulfate scanning. Remarkably, small changes in electrophile structure produced large, tuneable differences in covalent kinetics, with benzenesulfonyl fluoride derivative 23 achieving rapid and complete protein modification. Biochemical and mass spectrometry analyses confirmed selective modification of an active-site lysine by 23, along with robust performance in complex biological samples. Extension to urokinase plasminogen activator further demonstrates the generality of this strategy. More broadly, this work establishes electrophile scanning within macrocyclic scaffolds as a general approach for tuning covalent reactivity and provides a blueprint for designing selective probes that move beyond catalytic-residue targeting.

biochemistry↗

bioRxiv2023

Extensive re-modelling of the cell wall during the development of Staphylococcus aureus bacteraemia

Introductory Paragraph / AbstractThe bloodstream represents a hostile environment that bacteria must overcome to cause bacteraemia. To understand how the major human pathogen Staphylococcus aureus manages this we have utilised a functional genomics approach to identify a number of new loci that affect the ability of the bacteria to survive exposure to serum, the critical first step in the development of bacteraemia. The expression of one of these genes, tcaA, was found to be induced upon exposure to serum, and we show that it is involved in the elaboration of a critical virulence factor, the wall teichoic acids (WTA), within the cell envelope. The activity of the TcaA protein alters the sensitivity of the bacteria to cell wall attacking agents, including antimicrobial peptides, human defence fatty acids, and several antibiotics. This protein also affects the autolytic activity and lysostaphin sensitivity of the bacteria, suggesting that in addition to changing WTA abundance in the cell envelope, it also plays a role in peptidoglycan crosslinking. With TcaA rendering the bacteria more susceptible to serum killing, while simultaneously increasing the abundance of WTA in the cell envelope, it was unclear what effect this protein may have during infection. To explore this, we examined human data and performed murine experimental infections. Collectively, our data suggests that whilst mutations in tcaA are selected for during bacteraemia, this protein positively contributes to the virulence of S. aureus through its involvement in altering the cell wall architecture of the bacteria, a process that appears to play a key role in the development of bacteraemia.

microbiology↗

bioRxiv2021

Significant Variability exists in the Toxicity of Global Methicillin-resistant Staphylococcus aureus Lineages.

Staphylococcus aureus is a major human pathogen where the emergence of antibiotic resistant lineages, such as methicillin-resistant S. aureus (MRSA), is a major health concern. While some MRSA lineages are restricted to the healthcare setting, the epidemiology of MRSA is changing globally, with the rise of specific lineages causing disease in healthy people in the community. In the past two decades, community-associated MRSA (CA-MRSA) has emerged as a clinically important and virulent pathogen associated with serious skin and soft-tissue infections (SSTI). These infections are primarily toxin driven, leading to the suggestion that hyper-virulent lineages/multi-locus sequence types (STs) exist. To examine this, we compared the toxic activity of 475 MRSA isolates representing five major MRSA STs (ST22, ST93, ST8, ST239 and ST36) by employing a monocyte-macrophage THP-1 cell line as a surrogate for measuring gross cytotoxicity. We demonstrate that while certain MRSA STs contain highly toxic isolates, there is such variability within lineages to suggest that this aspect of virulence should not be inferred from the genotype of any given isolate. Furthermore, by interrogating the accessory gene regulator (Agr) sequences in this collection we identified several Agr mutations that were associated with reduced toxicity. Interestingly, the majority of isolates that were attenuated in toxin production contained no mutations in the agr locus, indicating a role of other undefined genes in S. aureus toxin regulation.

microbiology↗

bioRxiv2021

The chemical synthesis of knob domain antibody fragments

Cysteine-rich knob domains found in the ultralong complementarity determining regions of a subset of bovine antibodies, are capable of functioning autonomously as 3-6 kDa peptides. While they can be expressed recombinantly in cellular systems, in this paper we show that knob domains are also readily amenable to chemical synthesis, with a co-crystal structure of a chemically synthesised knob domain in complex with antigen showing structural equivalence to the biological product. For drug discovery, following immunisation of cattle, knob domain peptides can be synthesised directly from antibody sequence data, combining the power and diversity of the bovine immune repertoire with the ability to rapidly incorporate non-biological modifications. We demonstrate that, through rational design with non-natural amino acids, paratope diversity can be massively expanded, in this case improving the efficacy of an allosteric peptide. As a potential route to further improve stability, we also performed head-to-tail cyclisation, exploiting the unusual proximity of the N- and C-termini to synthesise functional, fully cyclic antibody fragments. Lastly, we highlight the stability of knob domains in plasma and, through pharmacokinetic studies, use palmitoylation as a route to extend the plasma half-life of knob domains in vivo. This study presents an antibody-derived medicinal chemistry platform, with protocols for solid-phase synthesis of knob domains; together with characterisation of their molecular structures, in vitro pharmacology and pharmacokinetics.

biochemistry↗

bioRxiv2020

The allosteric modulation of Complement C5 by knob domain peptides

To overcome limited germline combinatorial diversity, bovines have evolved a subset of antibodies with ultra-long CDRH3 regions that harbour cysteine-rich knob domains. To produce affinity-maturated peptides, we previously isolated autonomous 3-6 kDa knob domains from bovine antibodies. Here, we show that binding of four knob domain peptides elicits a range of effects on the clinically validated drug target complement C5. Allosteric mechanisms predominated, with one peptide selectively inhibiting C5 cleavage by the alternative pathway C5 convertase, revealing a targetable mechanistic difference between the classical and alternative pathway C5 convertases. Taking a hybrid biophysical approach, we present C5-knob domain co-crystal structures and, by solution methods, observed allosteric effects propagating >50 [A] from the binding sites. This study expands the therapeutic scope of C5, presents new inhibitors and introduces knob domains as new, low molecular weight antibody fragments, with therapeutic potential.

immunology↗

bioRxiv2019

A small membrane protein critical to both the offensive and defensive capabilities of Staphylococcus aureus.

Staphylococcus aureus is a major human pathogen, where the widespread emergence of antibiotic resistance is making infections more challenging to treat. Toxin induced tissue damage and resistance to the hosts immune system are well established as critical to its ability to cause disease. However, recent attempts to study S. aureus pathogenicity at a population level have revealed significant complexity and hierarchical levels of regulation. In an effort to better understand this we have identified and characterized a principle effector protein, MasA. The inactivation of this small highly-conserved membrane protein simultaneously disrupts toxin production and impairs S. aureus ability to resist several aspects of the innate immune system. These pleiotropic effects are mediated by both a change in the stability of the bacterial membrane and the dysregulation of iron homeostasis, which results in a significant impairment in the ability of S. aureus to cause infection in both a subcutaneous and a sepsis model of infection. That proteins with such major effects on pathogenicity remain unidentified in a bacterium as well studied as S. aureus demonstrates how incomplete our understanding of their ability to cause disease is, an issue that needs to be addressed if effective control and treatment strategies are to be developed.

microbiology↗

bioRxiv2017

Global phylogenomics of multidrug-resistant Staphylococcus aureus sequence type 772: the Bengal Bay clone

The global spread of antimicrobial resistance has been well documented in Gram-negative bacteria and healthcare-associated epidemic pathogens, often emerging from regions with heavy antimicrobial use. However, the degree to which similar processes occur with Gram-positive bacteria in the community setting is less well understood. Here we demonstrate the recent origin and global spread from the Indian subcontinent of a multidrug resistant Staphylococcus aureus lineage, sequence type 772 (Bengal Bay clone). Short-term outbreaks occurred following intercontinental transmission, typically associated with travel and family contacts, but ongoing endemic transmission was uncommon. Instrumental in the emergence of a single dominant clade in the early 1990s was the acquisition of a multidrug resistance integrated plasmid that did not appear to incur a significant fitness cost. The Bengal Bay clone therefore combines the multidrug resistance of traditional healthcare-associated clones with the epidemiological and virulence potential of community-associated clones.

genomics↗

bioRxiv2017

Epistasis mediated alleviation of the cost of antibiotic resistance for MRSA

Understanding how multi-drug resistant pathogens evolve is key to identifying means of curtailing their further emergence and dissemination. Fitness costs imposed on bacteria by resistance mechanisms are believed to hamper their dissemination in an antibiotic free environment, however, some have been reported to have little or no cost, which suggests there are few barriers preventing their global spread. One such apparently cost-free resistance mechanism acquired by the major human pathogen Staphylococcus aureus is to the clinically important antibiotic mupirocin, which is mediated by mutation of the highly-conserved and essential isoleucyl-tRNA synthethase (ileS) gene. In Genome Wide Association Studies (GWAS) on two genetically and geographically distinct MRSA lineages we have found this mutation to be associated with changes in bacterial virulence, driven through epistatic interactions with other loci. Given the potential dual effect of this mutation on both antibiotic resistance and virulence we adopted a proteomic approach and observed pleiotropic effects. This analysis revealed that the activity of the secretory apparatus of the PSM family of cytolytic toxins, the Pmt system, is affected in the mupirocin resistant mutant, which explains why it is less toxic. As an energetically costly activity, this reduction in toxicity masks the fitness costs associated with this resistance mutation, a cost that becomes apparent when toxin production is required. Given the widespread use of this antibiotic, and that this resistance often results from a single nucleotide substitution in the ileS gene, these hidden fitness costs provide an explanation for why this resistance mechanism is not more prevalent. This work also demonstrates how population-based genomic analysis of virulence and antibiotic resistance can contribute to uncovering hidden features of the biology of microbial pathogens.

microbiology↗