Biology subjects

Al-Mayyah, Z.

Publications and source records attributed to Al-Mayyah, Z..

9 recordsLinked to original sources

bioRxiv2026

A structural trade-off balances mechanical resilience and supramolecular adaptability in type IV pili

Biological machines operate under mechanical load, requiring architectures that simultaneously resist force and remain functionally dynamic. Type IV pili (T4P) are bacterial filaments that experience large tensile forces during motor-driven retraction. Here, we combine cryo-electron microscopy, molecular dynamics simulations, optical tweezers, and functional analyses to define the structural basis of T4P mechanical adaptation. We determined a 2.8 [A] cryo-EM structure of the Pseudomonas aeruginosa T4P and integrated it with comparative all-atom simulations across six bacterial strains to identify a conserved force-bearing electrostatic network. Simulations predicted that this network tunes filament elasticity under load, a finding validated by single-filament force spectroscopy. Rewiring these interactions experimentally produced hyper-rigid pili that assembled normally but exhibited impaired twitching motility. Together, our findings uncover a structural trade-off between force-resistant architecture and reversible supramolecular adaptability.

biophysics↗

bioRxiv2026

Pyocyanin disrupts airway regeneration to favor chronic Pseudomonas aeruginosa infection

During chronic infections, host tissues undergo repeated cycles of injury and repair while being exposed to bacterial products. Although many virulence factors trigger immune responses, whether bacterial metabolites alter tissue architecture by disrupting regenerative programs remains largely unexplored. Here, using a primary-cell-derived human lung microtissue model, we show that the redox-active metabolite pyocyanin (PYO), produced by the opportunistic pathogen Pseudomonas aeruginosa, disrupts airway epithelial regeneration and reshapes tissue architecture, amplifying infection. PYO exposure impairs epithelial repair, leading to defects in mucociliary clearance and promoting bacterial growth. Single-cell transcriptomics and imaging reveal that PYO exposure during basal cell differentiation alters epithelial cell-type composition. Keratin-13-rich cells promote the emergence of squamous regions with defective ciliogenesis, resulting in functional defects. After characterizing how PYO remodels the epithelium, we tested how these changes impact bacterial fitness. Using transposon-insertion sequencing and live imaging of infections, we show that these regeneration defects improve the fitness of mutants that tend to form biofilms, a common feature of chronic infections. Together, our results reveal that PYO reshapes regenerative trajectories and tissue architecture in ways that subsequently alter infection outcomes, highlighting the role of secreted metabolites as potential modulators of tissue regeneration during chronic lung infections.

microbiology↗

bioRxiv2026

Pseudomonas aeruginosa balances cytotoxicity and motility to counter phagocytosis by macrophages

During chronic lung infections, Pseudomonas aeruginosa diversifies under selection from antibiotics, metabolic constraints, and host defenses. Macrophages are key sentinels of the innate immune system and play a central role in clearing airway pathogens. Yet, how they process heterogeneous bacterial populations remains poorly understood. Here, we investigate how P. aeruginosa evades phagocytosis under conditions that mimic chronic infection. We use an attenuated mutant lacking a functional type III secretion system (T3SS), which reduces macrophage killing, allowing us to isolate determinants of bacterial susceptibility to phagocytosis. Using transposon insertion sequencing (Tn-seq), we identify bacterial fitness factors under phagocytic selection. Our screen reveals that disruption of genes involved in swimming and twitching motility reduces uptake by macrophages. We find that motility defects interfere with the physical interactions between bacteria and macrophages. Live-cell imaging shows that motility-deficient bacteria exhibit reduced surface exploration and unstable attachment to macrophages, limiting their internalization. Clinical isolates with reduced swimming or twitching motility display similarly impaired uptake. Restoring T3SS activity in these motility mutants rescues cytotoxicity toward macrophages, with one notable exception: flagellum-less, hyper-piliated P. aeruginosa remains avirulent and resistant to phagocytosis due to their lack of engagement with macrophages. Together, these results support two distinct immune evasion strategies: during chronic infection, reduced motility promotes a "freeze"-like state that limits detection and engulfment, whereas during acute infection, P. aeruginosa adopts a "fight"-like strategy by activating its T3SS to eliminate macrophages. SummaryHow immune cells recognize and eliminate bacteria is typically explained by molecular signaling, yet the role of physical interactions remains unclear. We show that bacterial motility is a key determinant of phagocytosis by macrophages. Using functional genomics and live imaging, we find that swimming and twitching motility promote bacterial uptake by enabling effective surface exploration and stable physical engagement with macrophages. Loss of motility commonly observed in chronic P. aeruginosa infections reduces these interactions and allows bacteria to evade engulfment. In contrast, during acute infection, bacteria rely on T3SS-mediated killing of macrophages, independently of motility. These findings reveal that phagocytosis is governed not only by immune recognition but also by bacterial mechanical behavior, and identify a shift in host-pathogen interactions associated with chronic versus acute infection. More broadly, this work establishes mechanics as an important dimension of immune evasion.

microbiology↗

bioRxiv2025

Growth in confinement promotes Pseudomonas aeruginosa tolerance to antibiotics

Bacteria often proliferate within confined spaces imposed by host tissues, extracellular matrices, or their own biofilms. In such environments, cells press against surrounding materials and experience elevated mechanical stress, but whether these forces influence pathogen physiology and fitness remains unclear. Here, we show that Pseudomonas aeruginosa adapts to mechanical confinement by increasing resilience to antibiotics. Using synthetic hydrogels of tunable stiffness that restrict physical expansion without limiting nutrient access, we demonstrate that growth in elastic materials reduces P. aeruginosa sensitivity to multiple clinically relevant antibiotics in a stiffness-dependent manner. Although slower growth contributes to this decreased susceptibility, Tn-seq under antibiotic treatment identified key regulators of mechanically induced tolerance. We find that active efflux mediated by sodium- proton Sha antiporters, together with protective remodeling of the bacterial membrane, enhances the resilience of confined populations without impacting growth. These findings reveal that P. aeruginosa adapts to mechanical stress in ways that may promote treatment failure even in the absence of intrinsic antibiotic resistance.

microbiology↗

bioRxiv2022

Pseudomonas aeruginosa mechanosensing controls cell polarity during twitching by activating two antagonistic response regulators

The opportunistic pathogen Pseudomonas aeruginosa adapts to solid surfaces to enhance virulence and infect its host. Type IV pili (T4P), long and thin filaments that power surface-specific twitching motility, allow single cells to mechanosense surfaces. For example, cells sense T4P attachment to control the direction of twitching motility. In this process, they establish a local positive feedback that polarizes T4P distribution to the sensing pole. A complex chemotaxis-like system called Chp mediates this response. The signalling mechanism allowing for transduction of this spatially-resolved signal is however unresolved. Here we demonstrate that the two Chp response regulators PilG and PilH enable dynamic cell polarization by coupling their antagonistic functions on T4P extension. By precisely quantifying the localization of fluorescent protein fusions, we show that PilG polarizes in response to mechanosensing through phosphorylation by the histidine kinase ChpA. We find that PilH is not inherently required for reversals. However, PilH activation is necessary to break the local positive feedback established by PilG so that forward-twitching cells can reverse. To spatially resolve mechanical signals, Chp thus locally transduces signals with a main output response regulator, PilG. To respond to signal changes, Chp uses its second regulator PilH to break the local feedback. By identifying the molecular functions of two response regulators that dynamically control cell polarization, our work provides a rationale for the diversity of architectures often found in non-canonical chemotaxis systems.

microbiology↗

bioRxiv2022

Engineering Agrobacterium tumefaciens adhesion to target cells.

Agrobacterium tumefaciens is a plant pathogen commonly repurposed for genetic modification of crops. Despite its versatility, it remains inefficient at transferring DNA to many hosts, including to animal cells. Like many pathogens, physical contact between A. tumefaciens and host cells promotes infection efficacy. Thus, improving the strength and specificity of A. tumefaciens to target cells has the potential for enhancing DNA transfer for biotechnological and therapeutic purposes. Here we demonstrate a methodology for engineering genetically-encoded exogeneous adhesins at the surface of A. tumefaciens. We identified an autotransporter gene we named Aat, that is predicted to show canonical {beta}-barrel and passenger domains. We engineered the {beta}-barrel scaffold and linker (Aat{beta}) to display synthetic adhesins susceptible to rewire A. tumefaciens to alternative host targets. As a proof of concept, we leveraged the versatility of a VHH domain to rewire A. tumefaciens adhesion to yeast and mammalian hosts displaying a GFP target receptor. Finally, to demonstrate how synthetic A. tumefaciens adhesion can improve transfer to host cells, we showed improved protein translocation into HeLa cells using a sensitive split luciferase reporter system. Engineering A. tumefaciens adhesion has therefore a strong potential in generating complex heterogeneous cellular assemblies and in improving DNA transfer efficiency against non-natural hosts.

microbiology↗

bioRxiv2021

Mechanotaxis directs Pseudomonas aeruginosa twitching motility.

The opportunistic pathogen Pseudomonas aeruginosa explores surfaces using twitching motility powered by retractile extracellular filaments called type IV pili. Single cells twitch by successive pili extension, attachment and retraction. However, whether and how single cells control twitching migration remains unclear. We discovered that P. aeruginosa actively directs twitching in the direction of mechanical input from type IV pili, in a process we call mechanotaxis. The Chp chemotaxis-like system controls the balance of forward and reverse twitching migration of single cells in response to the mechanical signal. On surfaces, Chp senses type IV pili attachment at one pole thereby sensing a spatially-resolved signal. As a result, the Chp response regulators PilG and PilH control the polarization of the extension motor PilB. PilG stimulates polarization favoring forward migration, while PilH inhibits polarization inducing reversal. Subcellular segregation of PilG and PilH efficiently orchestrates their antagonistic functions, ultimately enabling rapid reversals upon perturbations. This distinct localization of response regulators establishes a signaling landscape known as local-excitation, global-inhibition in higher order organisms, identifying a conserved strategy to transduce spatially-resolved signals. Our discovery finally resolves the function of the Chp system and expands our view of the signals regulating motility.

microbiology↗

bioRxiv2020

The membrane microenvironment regulates the sequential attachment of bacteria to host cells.

Pathogen attachment to host tissue is critical in the progress of many infections. Bacteria use adhesion in vivo to promote colonization and regulate the deployment of contact-dependent virulence traits. To specifically target host cells, they decorate themselves with adhesins, proteins that bind to mammalian cell surface receptors. One common assumption is that adhesin-receptor interactions entirely govern bacterial attachment. However, how adhesins engage with their receptors in an in vivo-like context remains unclear, in particular under the influence of a heterogeneous mechanical microenvironment. We here investigate the biophysical processes governing bacterial adhesion to host cells using a tunable adhesin-receptor system. By dynamically visualizing attachment, we found that bacterial adhesion to host cell surface, unlike adhesion to inert surfaces, involves two consecutive steps. Bacteria initially attach to their host without engaging adhesins. This step lasts about one minute during which bacteria can easily detach. We found that at this stage, the glycocalyx, a layer of glycosylated proteins and lipids, shields the host cell by keeping adhesin away from their receptor ligand. In a second step, adhesins engage with their target receptors to strengthen attachment for minutes to hours. The active properties of the membrane, endowed by the actin cytoskeleton, strengthen specific adhesion. Altogether, our results demonstrate that adhesin-ligand binding is not the sole regulator of bacterial adhesion. In fact, the host cells mechanical microenvironment relatively strongly mediated host-bacteria physical interactions, thereby playing an essential role in the onset of infection.

microbiology↗

bioRxiv2020

Biofilms mechanically damage epithelia by buckling.

During chronic infections and in microbiota, bacteria predominantly colonize their hosts as multicellular structures called biofilms. Despite their ubiquity in vivo, we still lack a basic understanding of how they interact with biological tissues, and ultimately how they influence host physiology. A common assumption is that biofilms interact with their hosts biochemically. However, the contributions of mechanics, while being central to the process of biofilm formation, have been vastly overlooked as a factor influencing host physiology. Specifically, how biofilms form on soft, tissue-like materials remains unknown. Here we show that biofilms can deform soft substrates by transmission of internally-generated mechanical stresses. We found that biofilms from both Vibrio cholerae and Pseudomonas aeruginosa can induce large deformations of soft synthetic hydrogels. Using a combination of mechanical measurements and mutants in matrix components, we found that biofilms deform their substrates by simultaneous buckling and adhesion. Specifically, mechanical constraints opposing growth causes biofilm buckling, while matrix components maintaining surface adhesion transmit buckling forces to the substrate. Finally, we demonstrate that biofilms can generate sufficient mechanical stress to deform and disrupt soft epithelial cell monolayers, suggesting that these forces can damage a host independently of typical virulence factors. Altogether, our results illustrate that forces generated by bacterial communities play an important role not only in biofilm morphogenesis but also in host physiology, suggesting a mechanical mode of infection.

microbiology↗