Biology subjects

Hayouka, Z.

Publications and source records attributed to Hayouka, Z..

11 recordsLinked to original sources

bioRxiv2026

Rapid Electrochemical Biosensing of Listeria monocytogenes Using Rationally Designed Host-Pathogen Interface Peptides

Rapid, selective detection of bacterial pathogens remains a central challenge. Here we report a label-free electrochemical biosensing approach that leverages protein-protein interaction (PPI)-derived peptides as recognition elements for rapid detection of Listeria monocytogenes (LM). The sensor design is inspired by the interaction between the LM virulence factor Internalin A (InlA) and the human host receptor E-cadherin (E-Cad1). Peptides derived from the InlA-binding domain of E-Cad1 were engineered as molecular recognition elements, with the E-Cad1(15-24) peptide displaying micromolar affinity and selective binding towards LM. Immobilization of these peptides on gold electrodes enabled bacterial detection by electrochemical impedance spectroscopy within 10 minutes, without labels or external signal amplification. A low peptide surface density was associated with enhanced binding-site accessibility and may facilitate multivalent interactions between the bacterial surface and the immobilized peptides. The platform produced a detectable response at experimentally tested concentrations as low as 1 CFU mL {superscript 1} and exhibited excellent selectivity under the conditions examined. This work introduces a chemically programmable, PPI-inspired biosensing paradigm that uses a reductionist approach and could potentially be extended to other pathogen targets.

bioengineering↗

bioRxiv2026

Cross-Resistance Limits the Ability of Antimicrobial Peptide Combinations to Delay Resistance Evolution

AimsAntimicrobial peptide (AMP) combinations have been proposed to delay resistance evolution, but it remains unclear what properties of a peptide pair determine whether a combination reduces resistance evolution relative to its component AMPs used alone. One suggested factor is mode of action, yet this has rarely been tested experimentally. In the current study we have asked whether mode of action or physicochemical similarity between peptides better predicts which combinations delay resistance. MethodsWe evolved Staphylococcus aureus with six AMPs with reported membrane-targeting and intracellular-targeting activity, individually and in all 15 pairwise combinations. We quantified resistance evolution, cross-resistance and fitness costs across the full AMP panel, and performed whole-genome sequencing on 126 evolved lineages. ResultsResistance varied across AMPs and correlated with peptide chain length, not mode of action. Cross-resistance was associated with physicochemical similarity, and similar peptides selected for overlapping mutations. Most combinations reduced resistance relative to single-AMP treatment, but those whose components shared cross-resistance were less effective, channeling evolution into convergent trajectories that resolve both selective pressures at once. Notably, mode of action did not predict combination outcome. ConclusionsCross-resistance, not mode of action, is a key factor in determining AMP combination efficacy. Physicochemical distance between peptides may serve as a practical predictor for cross-resistance, enabling selection of AMP combinations that are more likely to constrain resistance evolution.

microbiology↗

bioRxiv2025

Psoriasin inhibits microbial growth in food by metal sequestering

Food spoilage is a significant economic and environmental concern, and it is estimated that [~]30% of fresh food is destroyed due to food spoilage between harvest to consumer. Current food preservatives are chemicals that are associated with various health risks and often have limited effectiveness under certain conditions like pH and temperature. Consequently, theres a growing need to develop effective, natural, and economical food preservatives. Herein, we studied the natural protein psoriasin as a potential food preservative. Psoriasin is naturally secreted in the oral cavity and has an effective and validated antimicrobial activity which makes it a potentially effective and safe protein-based food preservative. Indeed, our preliminary results show promising antimicrobial activity of the recombinant protein against food-related microbial organisms in vitro and in various food types. In addition, it is recombinantly expressed at high levels, which could set a cost-effective manufacturing process. These results set psoriasin as a safe and effective natural food preservative, addressing consumer demand for healthier food options and reducing food waste.

biochemistry↗

bioRxiv2024

Harnessing Random Peptide Mixtures to Overcome Multidrug-Resistant Fungal Infections

Invasive fungal infections are associated with high mortality and are increasingly difficult to treat due to a limited antifungal arsenal and the rapid emergence of drug resistance. Novel therapeutic strategies that combine potent antifungal activity, low host toxicity, in vivo stability, and a reduced propensity for resistance development are urgently needed. Antimicrobial peptides (AMPs) stand out as a promising class of compounds to combat antimicrobial resistance. Leveraging the unique properties of AMPs, we previously developed a novel approach to synthesize random peptide mixtures (RPMs) with robust bactericidal activity against drug-resistant bacteria. Here, we evaluate the antifungal potential of RPMs and demonstrate species-dependent, broad-spectrum activity of FK20 (L-phenylalanine-L-lysine, 20-mer) against major human fungal pathogens, including Candida spp., Cryptococcus neoformans, and Aspergillus fumigatus, with particularly high potency against the multidrug-resistant pathogen Candida auris. Mechanistic analyses revealed rapid membrane and cell wall disruption accompanied by intracellular penetration, consistent with membrane-active antifungal activity. Importantly, experimental evolution assays demonstrated a markedly reduced capacity for resistance development in C. auris. FK20 inhibited biofilm formation and displayed substantial activity against mature, pre-formed biofilms, both alone and synergistically in combination with caspofungin. Finally, FK20 showed significant therapeutic efficacy in a murine model of systemic candidiasis. Collectively, these findings establish RPMs as a versatile antifungal platform with broad-spectrum activity, biofilm efficacy, and a low resistance footprint, highlighting their promise as a novel therapeutic strategy against drug-resistant fungal infections. ImportanceThe rising prevalence of invasive fungal infections, particularly among immunocompromised individuals, has become a critical public health concern. However, antifungal drug development has not kept pace with this growing need, and treatment options remain limited to a small number of drug classes. The emergence of multidrug-resistant fungal pathogens, such as Candida auris, further exacerbates this crisis by reducing the efficacy of existing therapeutics and increasing the risk of treatment failure. In this study, we evaluate the antifungal potential of FK20, a random peptide mixture (RPM) composed of L-phenylalanine and L-lysine. FK20 displays potent activity against C. auris and other clinically relevant human fungal pathogens, impairs biofilm formation, and exhibits synergy with caspofungin. Importantly, FK20 limits the emergence of resistance and demonstrates therapeutic efficacy in a murine model of systemic candidiasis. These findings establish RPMs as a promising new class of antifungals with broad-spectrum activity and clinical potential against drug-resistant fungal infections.

microbiology↗

bioRxiv2024

Peptide therapeutic leads for multi-target inhibition of inflammatory cytokines in Inflammatory Bowel Disease - computational design and in-vitro validation

Inflammatory Bowel Disease (IBD) are chronic and recurrent inflammatory disorders affecting the gastrointestinal tract, characterized by the involvement of numerous pro-inflammatory cytokines. These conditions profoundly impact both immune system dynamics and intestinal tissue integrity. Current therapeutic approaches predominantly rely on monoclonal antibodies, and frequently encounter limitations such as non-responsiveness, loss of efficacy over time, immunogenicity, adverse effects, and substantial cost. Consequently, there is a critical need for novel, targeted anti-inflammatory strategies. We present the computational structure guided design of peptidic inhibitors aimed at attenuating the activity of pivotal pro-inflammatory cytokines implicated in IBD pathogenesis, namely TNF, IL-1{beta}, and IL-6. These peptides were engineered to disrupt specific cytokine - receptor interactions, to block the release of pro-inflammatory cytokines. We structurally characterized key features in the studied interactions and used these to guide two computational design strategies, one based on the identification of dominant segments using our PeptiDerive approach, and one based on complementing fragments detected using our PatchMAN protocol. The designed peptides were synthesized and their efficacy was validated on Caco-2 intestinal epithelial cells and THP-1 macrophages, representative of the epithelial and immunological alterations typical of active IBD. The majority of the novel peptides effectively suppressed release of pro-inflammatory cytokines by both macrophages and intestinal epithelial cells, thereby reducing the risk of inflammation. This study underscores the efficacy of a rational design approach rooted in structural insights into inflammatory signaling complexes. Our findings demonstrate the potential of targeting key cytokines and receptor interaction with designed peptides as a promising therapeutic avenue for managing IBD and other inflammatory disorders.

bioinformatics↗

bioRxiv2024

The evolution of antimicrobial peptide resistance in Pseudomonas aeruginosa is severely constrained by random peptide mixtures

The prevalence of antibiotic-resistant pathogens has become a major threat to public health, requiring swift initiatives for discovering new strategies to control bacterial infections. Hence, antibiotic stewardship and rapid diagnostics, but also the development, and prudent use, of novel effective antimicrobial agents are paramount. Ideally, these agents should be less likely to select for resistance in pathogens than currently available conventional antimicrobials. The usage of antimicrobial Peptides (AMPs), key components of the innate immune response, and combination therapies, have been proposed as strategies to diminish the emergence of resistance. Herein, we investigated whether newly developed random antimicrobial peptide mixtures (RPMs) can significantly reduce the risk of resistance evolution in vitro to that of single sequence AMPs, using the ESKAPE pathogen Pseudomonas aeruginosa (P. aeruginosa) as a model Gram-negative bacterium. Infections of this pathogen are difficult to treat due the inherent resistance to many drug classes, enhanced by the capacity to form biofilms. P. aeruginosa was experimentally evolved in the presence of AMPs or RPMs, subsequentially assessing the extent of resistance evolution and cross-resistance/collateral sensitivity between treatments. Furthermore, the fitness costs of resistance on bacterial growth were studied, and whole-genome sequencing used to investigate which mutations could be candidates for causing resistant phenotypes. Lastly, changes in the pharmacodynamics of the evolved bacterial strains were examined. Our findings suggest that using RPMs bears a much lower risk of resistance evolution compared to AMPs and mostly prevents cross-resistance development to other treatments, while maintaining (or even improving) drug sensitivity. This strengthens the case for using random cocktails of AMPs in favour of single AMPs, against which resistance evolved in vitro, further providing an alternative to classic antibiotics worth pursuing.

microbiology↗

bioRxiv2023

Nature-Inspired Peptide of MtDef4 C-terminus Tail Enables Protein Delivery in Mammalian Cells

Cell-penetrating peptides hold great promise as versatile tools for the intracellular delivery of therapeutic agents. Various peptides have originated from natural proteins with antimicrobial activity. In this study, we investigated the mammalian cell-penetrating properties of a 16-residue peptide derived from the C-terminus tail of the Medicago truncatula defensin protein, with the sequence GRCRHGFRRRCFCTTHC. We evaluated the ability of this peptide to penetrate multiple types of cells. Our results demonstrate that the peptide efficiently penetrates mammalian cells within minutes and at a sub-micromolar concentration. Moreover, upon N-terminal fusion to the fluorescent protein GFP, the peptide efficiently delivers the GFP into the cells. Despite its remarkable cellular penetration, the peptide has only a minor effect on cellular viability, making it a promising candidate for the development of a cell-penetrating peptide, with potential therapeutic applications.

cell biology↗

bioRxiv2023

Reactivating the complement system using peptidic bacterial labeling tag

The immune system plays a critical role in protecting the host against pathogens, including bacteria, viruses, and parasites. However, pathogens have evolved mechanisms to evade the immune system, for example by altering their surface proteins or by producing enzymes that can interfere with the immune response. These evasion strategies enable pathogens to escape detection and destruction by the immune system, which allows them to establish serious infections. Thus, there is a critical need for new strategies for developing antimicrobial agents. Here, we describe a novel strategy for targeting pathogens, by labeling them with a general peptide functioning as a bacterial binder, conjugated to a protein tag recognizable by the complement system, thereby activating the immune system against the target pathogen. To that end, we screened several pathogenic bacteria to find complement-resistant bacterial strain. A selected peptide binder was crosslinked with the C3b complement protein using glutaraldehyde. We show by an ELISA assay that the resulting complex binds the C5 complement protein with high affinity. We posited that by binding C5, this complex will be capable of initiating the alternative complement downstream proteolytic cascade, thereby inducing the formation of the membrane attack complex. Using this methodology, we were able to eradicate 90% of complement-resistant E. coli bacterial cells. By showing enhancement of complement sensitivity in complement-resistant pathogens, this work demonstrates the basis for new therapeutic approach capable of targeting pathogenic bacteria and activating the immune system against them.

microbiology↗

bioRxiv2022

Quorum sensing peptidic inhibitor rescue host immune system eradication: a novel QS infectivity mechanism

Subverting the host immune system is a major task for any given pathogen to assure its survival and proliferation. For the opportunistic human pathogen Bacillus cereus (Bc), immune evasion enables the establishment of potent infections. In various species of the Bc group, the pleiotropic regulator PlcR and its cognate cell-cell signaling peptide PapR7 regulates virulence genes expression in response to fluctuations in population density, i.e., a quorum-sensing (QS) system. However, how QS exerts its effects during infections, and whether PlcR confers the immune evading ability remain unclear. Herein, we report how interception of the QS communication in Bc obliterates the ability to control the host immune system. Here we designed a peptide-based QS inhibitor that suppresses PlcR-dependent virulence factor expression and attenuates Bc infectivity in mouse models. We demonstrate that the QS peptidic inhibitor blocks host immune system-mediated eradication by reducing the expression of PlcR-regulated major toxins. Our findings provide the first evidence that Bc infectivity is regulated by QS circuit mediated destruction of the host immunity, thus reveal a new strategy to limit Bc virulence and enhance host defense. This peptidic quorum-quenching agent constitutes readily accessible chemical tool for studying how other pathogen QS systems modulate host immunity and forms a basis for development of anti-infective therapeutics.

microbiology↗

bioRxiv2022

The C-terminal tail of CSNAP attenuates the CSN complex

Protein degradation is one of the essential mechanisms that enables reshaping of the proteome landscape in response to various stimuli. The largest E3 ubiquitin ligase family that targets proteins to degradation by catalyzing ubiquitnation is the cullin-RING ligases (CRL). Many of the proteins that are regulated by CRLs are central to tumorigenesis and tumour progression, and dysregulation of the CRL family is frequently associated with cancer. The CRL family comprises [~]300 complexes all of which are regulated by the COP9 signalosome complex (CSN). Therefore, the CSN is considered an attractive target for therapeutic intervention. Research efforts for targeted CSN inhibition have been directed towards inhibition of the complex enzymatic subunit, CSN5. Here, we have taken a fresh approach focusing on CSNAP, the smallest CSN subunit. Our results show that the C-terminal region of CSNAP is tightly packed within the CSN complex, in a groove formed by CSN3 and CSN8. We show that a 16 amino acid C-terminal peptide, derived from this CSN interacting region, can displace the endogenous CSNAP subunit from the complex. This, in turn, leads to a CSNAP null phenotype that attenuates CSN activity and consequently CRLs function. Overall, our findings emphasize the potential of a CSNAP-based peptide for CSN inhibition as a new therapeutic avenue.

biochemistry↗

bioRxiv2022

Combination treatment can hinder the evolution of resistance to antimicrobial peptides

Antibiotic resistant microbial pathogens are becoming a major threat to human health. Therefore, there is an urgent need to develop new alternatives to conventional antibiotics. One such promising alternative is antimicrobial peptides (AMPs), which are produced by virtually all organisms and typically inhibit bacteria via membrane disruption. However, previous studies demonstrated that bacteria can rapidly develop AMP resistance. Here, we study whether combination therapy, known to be able to inhibit the evolution of resistance to conventional antibiotics, can also hinder the evolution of AMP resistance. To do so, we evolved the opportunistic pathogen S. aureus in the presence of individual AMP, AMP pairs, and a combinatorial antimicrobial peptide library. Treatment with some AMP pair indeed hindered the evolution of resistance compared with individual AMPs. In particular, resistance to pairs was delayed when resistance to the individual AMPs came at a cost of impaired bacterial growth, and did not confer cross-resistance to other tested AMPs. The lowest level of resistance evolved during treatment with the combinatorial antimicrobial peptide library termed random antimicrobial peptide mixture, which contains more than a million different peptides. A better understanding of how AMP combinations affect the evolution of resistance is a crucial step in order to design resistant proof AMPs cocktails that will offer a sustainable treatment option for antibiotic resistant pathogens.

evolutionary biology↗