bioRxiv (Cold Spring Harbor Laboratory), Mar 2, 2024
Discovering new bacterial signaling pathways offers unique antibiotic strategies. Here, through a... more Discovering new bacterial signaling pathways offers unique antibiotic strategies. Here, through an unbiased resistance screen of 3,884 gene knockout strains, we uncovered a previously unknown nonlytic bactericidal mechanism that sequentially couples three transporters and downstream transcription to lethally suppress respiration of the highly virulent P. aeruginosa strain PA14 -one of three species on the WHO's 'Priority 1: Critical' list. By targeting outer membrane YaiW, cationic lacritin peptide 'N-104' translocates into the periplasm where it ligates outer loops 4 and 2 of the inner membrane transporters FeoB and PotH, respectively, to suppress both ferrous iron and polyamine uptake. This broadly shuts down transcription of many biofilm-associated genes, including ferrous iron-dependent TauD and ExbB1. The mechanism is innate to the surface of the eye and is enhanced by synergistic coupling with thrombin peptide GKY20. This is the first example of an inhibitor of multiple bacterial transporters. 1F and S1A). Monitoring was continued after a buffer wash whereby disruptive peptides remove membrane lipid causing the SPR response to fall below the membrane-alone baseline by 600 s. SPR sensitivity is as little as 10 pg/ml 34 of dry mass. However, no significant disruption of the model membranes was detected (Figures and). Although the model 'Gram-positive' membrane appeared to show some slight disruption at two lower N-104 concentrations, it was not confirmed at the highest concentration (Figure ). The experiment was then repeated using quartz crystal microbalance with dissipation monitoring (QCM-D) in which a decrease of frequency is proportional to increase in wet mass 35 . This suggested a slight decrease in mass (Figures and) at levels not membrane disruptive. Thus, the bactericidal mechanism of the N-104 cleavage product is non-lytic. Some non-lytic antimicrobial peptides utilize multiple tryptophans or both arginines and prolines or a single helix disruptive proline to gain bacterial entry 36 . N-104 lacks arginine and contains only a single proline and tryptophan, both in a coiled-coil domain C-terminal to an amphipathic a-helix (Figures and). We synthesized ten N-104 analogs, each with a single serine substitution for lysines, leucines, phenylalanine and tryptophan. Most membrane disruptive by SPR were N-104:K111S and N-104:K116S, but neither substantially diminished growth of the highly virulent, multi-drug resistant PA14 strain 37 of P. aeruginosa as examined by colony forming unit (CFU) (Figures and) and C. elegans feeding assays (Figures and). Comparison of all ten analogs revealed no correlation between diminished CFU's and either SPR (Figure ) or QCM-D response (Figures and). Only N-104:L114S and N-104:L115S retained full bactericidal activity at a minimum inhibitory concentration (MIC) of 2 µM (3.6 µg/ml). Thus, all amino acids with basic side chains and most with nonpolar side chains contribute to the N-104 activity that is also not hemolytic (Figure ). Curious about resistance that often develops by 10 generations 38 -particularly when antibiotic exposure is below the minimal inhibitory concentration -we tested N-104 on PA14 through 30 generations. No stable resistance was apparent (Figure ). Despite newly apparent transcriptional effects downstream of deleted genes in bacterial libraries of single gene knockout strains 39 , unbiased screening of such libraries has proven to be powerfully insightful . Accordingly, we screened the Keio E. coli K12 collection of 3,884 single gene knockout strains 42 for resistance to N-104 (Figure ) using the reduction of resazurin in Alamar Blue as a measure of bacterial metabolic activity. Recognizing that the metabolic activity of each knockout strain may differ, a screening strategy was developed that compared the five hour Alamar Blue detectable metabolic activity of each before and after N-104 treatment (Figure ). A treated:untreated metabolic slope ratio of 0.75 or greater was considered to be indicative of N-104 resistance (Figure ) at a relatively high 100 µM (180 µg/ml) dose to minimize ambiguous outcomes. As respective negative and positive controls we took advantage of the 14-mer 'N-80/C-25' from an inactive region of lacritin (Figure ) and the beta-lactam antibiotic ampicillin, both at 100 µM (Figure ). Duplicate N-104 screens of the entire collection yielded 122 resistant candidates (Figure ). These were further screened twice (Figures and) thereby narrowing the list to 10 (Figure ) -each with a Z-score ≥ 1.85. Resistance was attributable to absence of: (i) the inner membrane ferrous iron transporter FeoB, (ii) the inner membrane polyamine transporter subunit PotH, (iii) the colanic acid polymerase WcaD involved in biofilm formation, (iv) N-acetyl transferase YjhQ, (v) 'putative glucose transporter regulator' YeeI (pseudogene), (vi) DUF986 protein YobD, or to one of four regulatory transcription factors: (vii) SgrR family member YbaE, (viii) ParB-like nuclease domain containing YbdM, (ix) GntR family member RluE
In this day and age, it's possible to find most anything on the internet. Besides delivering an e... more In this day and age, it's possible to find most anything on the internet. Besides delivering an endless stream of information from all over the world (the validity of which is dubious, at best, in many instances), it provides forums to discuss critical issues related to politics, news, natural disasters, science and, of course, celebrities. In a recent search for information on "autophagy" (or self-eating), the topic of this Special Issue of Experimental Eye Research, there was a discussion thread debating the question: "If I eat myself, will I grow twice as big, or disappear?" Not surprisingly there were many serious attempts to answer this question, usually involving the consumption of specific body parts, emphatic declarations that you would most certainly disappear, and even something about anti-matter. Luckily for us, this Special Issue actually may address this philosophical dilemma as we explore autophagy and its importance to homeostatic and disease mechanisms of the eye.
Contact lenses are widely prescribed for vision correction, and as such they are an attractive pl... more Contact lenses are widely prescribed for vision correction, and as such they are an attractive platform for drug delivery to the anterior segment of the eye. This manuscript explores a novel strategy to drive the reversible adsorption of peptide-based therapeutics using commercially available contact lenses. To accomplish this, thermo-sensitive elastin-like polypeptides (ELPs) alone or tagged with a candidate ocular therapeutic were characterized. For the first time, this manuscript demonstrates that Proclear Compatibles TM contact lenses are a suitable platform for ELP adsorption. Two rhodamine-labelled ELPs, V96 (thermo-sensitive) and S96 (thermo-insensitive), were employed to test temperature-dependent association to the contact lenses. During long-term release into solution, ELP coacervation significantly modulated the release profile whereby more than 80% of loaded V96 retained with a terminal half-life of ~4 months, which was only 1-4 days under solubilizing conditions. A selected ocular therapeutic candidate lacritin-V96 fusion (LV96), either free or lens-bound LV96, was successfully transferred to HCE-T cells. These data suggest that ELPs may be useful to control loading or release from certain formulations of contact lenses and present a potential for this platform to deliver a biologically active peptide to the ocular surface via contact lenses.
Genetic separation of the human lacritin gene ("LACRT") and triple A (Allgrove) syndrome on 12q13
Advances in experimental medicine and biology, 2002
Molecular mechanisms underlying the pathogenic decline of secretory output by the main lacrimal g... more Molecular mechanisms underlying the pathogenic decline of secretory output by the main lacrimal gland, and subsequently dry eye, are potentially multiple. Inflammatory expansion of B and T lymphocytes can lead to loss of lacrimal acini.1 Curiously, however, acinar volume loss often appears insufficient relative to the theoretical overcapacity of the main lacrimal gland. Estimates suggest a potential secretory output up to 10-fold greater than required to maintain a normal aqueous tear film layer.2,3 Other mechanisms, therefore, warrant attention, such as aberrant secretion of one or several common cytokines that may directly or indirectly alter lacrimal acinar cell function and/or lead to a decline in neural innervation.4 Novel autocrine/paracrine factor(s) released by lacrimal acinar cells into the tear film may be required for the health of the lacrimal secretory machinery, ductal system and corneal epithelium.5 The periacinar basement membrane is also required for normal secretor...
Alpha Helicity of Lacritin’s C-Terminal Syndecan-1 Binding Domain is Enhanced by Interaction with the Mutual Binding Region in Syndecan-1
Contact lenses are widely prescribed for vision correction, and as such they are an attractive pl... more Contact lenses are widely prescribed for vision correction, and as such they are an attractive platform for drug delivery to the anterior segment of the eye. This manuscript explores a novel strategy to drive the reversible adsorption of peptide-based therapeutics using commercially available contact lenses. To accomplish this, thermo-sensitive elastin-like polypeptides (ELPs) alone or tagged with a candidate ocular therapeutic were characterized. For the first time, this manuscript demonstrates that Proclear CompatiblesTM contact lenses are a suitable platform for ELP adsorption. Two rhodamine-labelled ELPs, V96 (thermo-sensitive) and S96 (thermo-insensitive), were employed to test temperature-dependent association to the contact lenses. During long-term release into solution, ELP coacervation significantly modulated the release profile whereby more than 80% of loaded V96 retained with a terminal half-life of ~4 months, which was only 1–4 days under solubilizing conditions. A selec...
In this day and age, it's possible to find most anything on the internet. Besides delivering an e... more In this day and age, it's possible to find most anything on the internet. Besides delivering an endless stream of information from all over the world (the validity of which is dubious, at best, in many instances), it provides forums to discuss critical issues related to politics, news, natural disasters, science and, of course, celebrities. In a recent search for information on "autophagy" (or self-eating), the topic of this Special Issue of Experimental Eye Research, there was a discussion thread debating the question: "If I eat myself, will I grow twice as big, or disappear?" Not surprisingly there were many serious attempts to answer this question, usually involving the consumption of specific body parts, emphatic declarations that you would most certainly disappear, and even something about anti-matter. Luckily for us, this Special Issue actually may address this philosophical dilemma as we explore autophagy and its importance to homeostatic and disease mechanisms of the eye.
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Papers by Gordon Laurie