Biology subjects

MacNeil, L. T.

Publications and source records attributed to MacNeil, L. T..

6 recordsLinked to original sources

bioRxiv2025

Butyrolactol A is a phospholipid flippase inhibitor that potentiates the bioactivity of caspofungin against resistant fungi

Fungal infections cause millions of deaths annually and are challenging to treat due to limited therapeutic options and rising resistance. Cryptococci are intrinsically resistant to the latest generation of antifungals, echinocandins, while Candida auris, a notorious global threat, is also increasingly resistant. We performed a natural product extract screen to rescue caspofungin fungicidal activity against Cryptococcus neoformans H99 and identified butyrolactol A, which restores echinocandin efficacy against resistant fungal pathogens, including multidrug-resistant C. auris. Mode of action studies revealed that butyrolactol A inhibits the phospholipid flippase Apt1-Cdc50, blocking phospholipid transport. Cryo-electron microscopy analysis of the Apt1*butyrolactol A complex revealed that the flippase is trapped in a dead-end state. Apt1 inhibition disrupts membrane asymmetry, vesicular trafficking, and cytoskeletal organization, thereby enhancing echinocandin uptake and potency. This study identifies lipid flippases as promising antifungal targets and demonstrates the potential of revisiting natural products to expand the antifungal arsenal and combat resistance.

biochemistry↗

bioRxiv2024

Temperate phages increase antibiotic effectiveness in a Caenorhabditis elegans infection model

The bactericidal nature of obligately lytic bacterial viruses (phages) is of increasing interest for the treatment of drug-resistant bacterial infections, either administered alone or in combination with antibiotics. In contrast, temperate phages are largely ignored in a therapeutic context due to their ability to lie dormant within the bacterial host. However, these phages often undergo a lytic cycle. Furthermore, even in their dormant state they can be a considerable burden to the bacterium - most famously by their ability to switch to lytic replication in response to environmental triggers, such as antibiotics, that stress the bacterial host. Recent reports of antibiotics synergizing with temperate phages in vitro, termed "temperate phage antibiotic synergy" (tPAS), present a potentially scalable opportunity to make use of these abundant entities for the treatment of bacterial infections. Here we employ Caenorhabditis elegans as a robust in vivo animal model for testing the efficacy of temperate phages as adjuvants to antibiotics. In vivo, temperate phage Hali alone results in 60% dormancy in the bacterial survivors. However, the antibiotic ciprofloxacin can abolish dormancy of temperate phage Hali - infecting a ciprofloxacin resistant Pseudomonas aeruginosa clinical strain. The phage Hali-ciprofloxacin pairing increased the lifespan of P. aeruginosa infected worms to that of the uninfected control, at doses where neither the phage nor the antibiotic had any effect alone. Complete rescue was also observed in worms infected with a phage-carrying strain treated with the otherwise ineffective antibiotic, supporting that the phage - even in its dormant form - can greatly enhance antibiotic effectiveness. This illustrates potential "accidental" phage therapy when antibiotics are prescribed. Our work establishes C. elegans as a suitable model for studying the in vivo efficacy of tPAS and is the first in vivo demonstration of this synergy, greatly expanding the therapeutic potential of temperate phages.

microbiology↗

bioRxiv2023

SHC-3: a previously unidentified C. elegans Shc family member functions in the insulin-like signaling pathway to enhance survival during L1 arrest

Shc proteins function in many different signaling pathways where they mediate phosphorylation-dependent protein-protein interactions. These proteins are characterized by the presence of two phosphotyrosine-binding domains, an N-terminal PTB and a C-terminal SH2. We describe a previously unrecognized C. elegans Shc gene, shc-3 and characterize its role of in stress response. Both shc-3 and shc-1 are required for long-term survival in L1 arrest, however, they do not act redundantly but rather play distinct roles in this process. SHC-3 function in survival during L1 arrest is DAF-16-dependent, demonstrating that like SHC-1, SHC-3 functions in the Insulin-like signaling pathway. In the absence of SHC-3, nuclear entry and exit are slowed suggesting that SHC-3 is required for rapid changes in DAF-16 signaling.

genetics↗

bioRxiv2021

Hyperpiliation reduces Pseudomonas aeruginosa pathogenicity by impeding type III secretion

Type IVa pili (T4aP) are important virulence factors for many bacterial pathogens. Previous studies suggested that the retraction ATPase, PilT, modulates pathogenicity due to its critical role in pilus dynamics and twitching motility. Here we use a Caenorhabditis elegans slow killing model to show that hyperpiliation, not loss of pilus retraction, reduces virulence of Pseudomonas aeruginosa strains PAK and PA14 by interfering with function of the contact-dependent type III secretion system (T3SS). Hyperactivating point mutations in the P. aeruginosa PilSR two-component system that controls transcription of the major pilin gene, pilA, increased levels of surface pili to the same extent as deleting pilT, without impairing twitching motility. These functionally hyperpiliated PilSR mutants had significant defects in pathogenicity that were rescued by deleting pilA or by increasing the length of T3SS needles via deletion of the needle-length regulator, PscP. Hyperpiliated pilT deletion or pilO point mutants showed similar PilA-dependent impairments in virulence, validating the phenotype. Together, our data support a model where a surfeit of pili prevents effective engagement of contact-dependent virulence factors. These findings suggest that the role of T4aP retraction in virulence should be revised. SIGNIFICANCEPseudomonas aeruginosa is a major contributor to hospital-acquired infections and particularly problematic due to its intrinsic resistance to many front-line antibiotics. Strategies to combat this and other important pathogens include development of anti-virulence therapeutics. We show that the pathogenicity of P. aeruginosa is impaired when the amount of type IVa pili (T4aP) expressed on the cell surface increases, independent of the bacterias ability to twitch. We propose that having excess T4aP on the cell surface can physically interfere with productive engagement of the contact-dependent type III secretion toxin delivery system. A better understanding of how T4aP modulate interaction of bacteria with target cells will improve the design of therapeutics targeting components involved in regulation of T4aP expression and function, to reduce the clinical burden of P. aeruginosa and other T4aP-expressing bacteria.

microbiology↗

bioRxiv2020

C. elegans ELT-3 regulates cuticle collagen expression in response to environmental stimuli

The nematode Caenorhabditis elegans is protected from the environment by the cuticle, an extracellular collagen-based matrix that encloses the animal. Over 170 cuticular collagens are predicted in the C. elegans genome, but the role of each individual collagen is unclear. Stage-specific specialization of the cuticle explains the need for some collagens, however, the large number of collagens suggests that specialization of the cuticle may also occur in response to other environmental triggers. Missense mutations in many collagen genes can disrupt cuticle morphology, producing a helically twisted body causing the animal to move in a stereotypical pattern described as rolling. We find that environmental factors, including diet, early developmental arrest, and population density can differentially influence the penetrance of rolling in these mutants. These effects are in part due to changes in collagen gene expression that are mediated by the GATA family transcription factor ELT-3. We propose a model by which ELT-3 regulates collagen gene expression in response to environmental stimuli to promote the assembly of a cuticle specialized to a given environment.

developmental biology↗

bioRxiv2017

Pseudomonas aeruginosa type IV minor pilins and PilY1 regulate virulence by modulating FimS-AlgR activity

Type IV pili are expressed by a wide range of prokaryotes, including the opportunistic pathogen Pseudomonas aeruginosa. These flexible fibres mediate twitching motility, biofilm maturation, surface adhesion, and virulence. The pilus is composed mainly of major pilin subunits while the low abundance minor pilins FimU-PilVWXE and the putative adhesin PilY1 prime pilus assembly and are proposed to form the pilus tip. The minor pilins and PilY1 are encoded in an operon that is positively regulated by the FimS-AlgR two-component system. Independent of pilus assembly, PilY1 is proposed to be a mechanosensory component that - in conjunction with minor pilins - triggers up-regulation of acute virulence phenotypes upon surface attachment. Here, we investigated the link between the minor pilins and virulence. pilW, pilX, and pilY1 mutants had reduced virulence towards Caenorhabditis elegans relative to wild type or a major pilin mutant, implying a role in pathogenicity that is independent of pilus assembly. We hypothesized that loss of specific minor pilins relieves feedback inhibition on FimS-AlgR, increasing transcription of the minor pilin operon and other members of the AlgR regulon. Reporter assays confirmed that FimS-AlgR were required for the increased expression from the minor pilin operon promoter upon loss of select minor pilins. Overexpression of AlgR or its hyperactivation via point mutation reduced virulence, and the virulence defects of pilW, pilX, and pilY1 mutants were dependent on FimS-AlgR expression and activation. We propose that PilY1 and the minor pilins inhibit their own expression, and that loss of these proteins leads to FimS-mediated activation of AlgR and reduced expression of acute-phase virulence factors. This mechanism could contribute to adaptation of P. aeruginosa in chronic lung infections, as mutations in the minor pilin operon result in the loss of piliation and increased expression of AlgR-dependent virulence factors - such as alginate - that are characteristic of such infections.\n\nAuthor summaryPseudomonas aeruginosa causes dangerous infections, including chronic lung infections in cystic fibrosis patients. It uses many strategies to infect its hosts, including deployment of grappling hook-like fibres called type IV pili. Among the components involved in assembly and function of the pilus are five proteins called minor pilins that - along with a larger protein called PilY1 - may help the pilus attach to surfaces. In a roundworm infection model, loss of PilY1 and specific minor pilins delayed killing, while loss of other pilus proteins did not. We traced this effect to increased activation of the FimS-AlgR regulatory system that inhibits expression of virulence factors used to initiate infections, while positively regulating chronic infection traits such as alginate production, a phenotype called mucoidy. A disruption in the appropriate timing of FimS-AlgR-dependent virulence factor expression when select minor pilins or PilY1 are missing may explain why those pilus-deficient mutants have reduced virulence compared with others whose products are not under FimS-AlgR control. Increased FimS-AlgR activity upon loss of PilY1 and specific minor pilins could help to explain the frequent co-occurrence of the non-piliated and mucoid phenotypes that are hallmarks of chronic P. aeruginosa lung infections.

microbiology↗