Functional Testing for Tranexamic Acid Duration of Action Using Modified Viscoelastometry

<b><i>Introduction:</i></b> Tranexamic acid (TXA) is the standard medication to prevent or treat hyperfibrinolysis. However, prolonged inhibition of lysis (so-called “fibrinolytic shutdown”) correlates with increased mortality. A new viscoelastometric test enables bedside quantification of the antifibrinolytic activity of TXA using tissue plasminogen activator (TPA). <b><i>Materials and Methods:</i></b> Twenty-five cardiac surgery patients were included in this prospective observational study. In vivo, the viscoelastometric TPA test was used to determine lysis time (LT) and maximum lysis (ML) over 96 h after TXA bolus. Additionally, plasma concentrations of TXA and plasminogen activator inhibitor 1 (PAI-1) were measured. Moreover, dose effect curves from the blood of healthy volunteers were performed in vitro. Data are presented as median (25–75th percentile). <b><i>Results:</i></b> In vivo TXA plasma concentration correlated with LT (<i>r</i> = 0.55; <i>p</i> &#x3c; 0.0001) and ML (<i>r</i> = 0.62; <i>p</i> &#x3c; 0.0001) at all time points. Lysis was inhibited up to 96 h (LT_TPA-test: baseline: 398 s [229–421 s] vs. at 96 h: 886 s [626–2,175 s]; <i>p</i> = 0.0013). After 24 h, some patients (<i>n</i> = 8) had normalized lysis, but others (<i>n</i> = 17) had strong lysis inhibition (ML &#x3c;30%; <i>p</i> &#x3c; 0.001). The high- and low-lysis groups differed regarding kidney function (cystatin C: 1.64 [1.42–2.02] vs. 1.28 [1.01–1.52] mg/L; <i>p</i> = 0.002) in a post hoc analysis. Of note, TXA plasma concentration after 24 h was significantly higher in patients with impaired renal function (9.70 [2.89–13.45] vs.1.41 [1.30–2.34] µg/mL; <i>p</i> &#x3c; 0.0001). In vitro, TXA concentrations of 10 µg/mL effectively inhibited fibrinolysis in all blood samples. <b><i>Conclusions:</i></b> Determination of antifibrinolytic activity using the TPA test is feasible, and individual fibrinolytic capacity, e.g., in critically ill patients, can potentially be measured. This is of interest since TXA-induced lysis inhibition varies depending on kidney function.

Dominant Vibrio cholerae phage exhibits lysis inhibition sensitive to disruption by a defensive phage satellite

Bacteria, bacteriophages that prey upon them, and mobile genetic elements (MGEs) compete in dynamic environments, evolving strategies to sense the milieu. The first discovered environmental sensing by phages, lysis inhibition, has only been characterized and studied in the limited context of T-even coliphages. Here, we discover lysis inhibition in the etiological agent of the diarrheal disease cholera, Vibrio cholerae, infected by ICP1, a phage ubiquitous in clinical samples. This work identifies the ICP1-encoded holin, teaA, and antiholin, arrA, that mediate lysis inhibition. Further, we show that an MGE, the defensive phage satellite PLE, collapses lysis inhibition. Through lysis inhibition disruption a conserved PLE protein, LidI, is sufficient to limit the phage produced from infection, bottlenecking ICP1. These studies link a novel incarnation of the classic lysis inhibition phenomenon with conserved defensive function of a phage satellite in a disease context, highlighting the importance of lysis timing during infection and parasitization.

Look Who’s Talking: T-Even Phage Lysis Inhibition, the Granddaddy of Virus-Virus Intercellular Communication Research

That communication can occur between virus-infected cells has been appreciated for nearly as long as has virus molecular biology. The original virus communication process specifically was that seen with T-even bacteriophages—phages T2, T4, and T6—resulting in what was labeled as a lysis inhibition. Another proposed virus communication phenomenon, also seen with T-even phages, can be described as a phage-adsorption-induced synchronized lysis-inhibition collapse. Both are mediated by virions that were released from earlier-lysing, phage-infected bacteria. Each may represent ecological responses, in terms of phage lysis timing, to high local densities of phage-infected bacteria, but for lysis inhibition also to locally reduced densities of phage-uninfected bacteria. With lysis inhibition, the outcome is a temporary avoidance of lysis, i.e., a lysis delay, resulting in increased numbers of virions (greater burst size). Synchronized lysis-inhibition collapse, by contrast, is an accelerated lysis which is imposed upon phage-infected bacteria by virions that have been lytically released from other phage-infected bacteria. Here I consider some history of lysis inhibition, its laboratory manifestation, its molecular basis, how it may benefit expressing phages, and its potential ecological role. I discuss as well other, more recently recognized examples of virus-virus intercellular communication.

Dominant Vibrio cholerae phage exhibits lysis inhibition sensitive to disruption by a defensive phage satellite

Bacteriophages and their bacterial hosts are locked in a dynamic evolutionary arms race. Phage satellites, selfish genomic islands which exploit both host bacterium and target phage, further complicate the evolutionary fray. One such tripartite system involves the etiological agent of the diarrheal disease cholera – Vibrio cholerae, the predominant phage isolated from cholera patients – ICP1, and a phage satellite – PLE. When ICP1 infects V. cholerae harboring the integrated PLE genome, PLE accelerates host lysis, spreading the PLE while completely blocking phage production protecting V. cholerae at the population level. Here we identify a single PLE gene, lidI, sufficient to mediate accelerated lysis during ICP1 infection and demonstrate that LidI functions through disrupting lysis inhibition – an understudied outcome of phage infection when phages vastly outnumber their hosts. This work identifies ICP1-encoded holin and antiholin genes teaA and arrA respectively, that mediate this first example of lysis inhibition outside the T-even coliphages. Through lysis inhibition disruption, LidI is sufficient to limit the number of progeny phage produced from an infection. Consequently, this disruption bottlenecks ICP1 evolution as probed by recombination and CRISPR-Cas targeting assays. These studies link novel characterization of the classic phenomenon of lysis inhibition with a conserved protein in a dominant phage satellite, highlighting the importance of lysis timing during infection and parasitization, as well as providing insight into the populations, relationships, and evolution of bacteria, phages, and phage satellites in nature.ImportanceWith increasing awareness of microbiota impacting human health comes intensified examination of, not only bacteria and the bacteriophages that prey upon them, but also the mobile genetic elements (MGEs) that mediate interactions between them. Research is unveiling evolutionary strategies dependent on sensing the milieu: quorum sensing impacts phage infection, phage teamwork overcomes bacterial defenses, and abortive infections sacrifice single cells protecting populations. Yet, the first discovered environmental sensing by phages, known as lysis inhibition (LIN), has only been studied in the limited context of T-even coliphages. Here we characterize LIN in the etiological agent of the diarrheal disease cholera, Vibrio cholerae, infected by a phage ubiquitous in clinical samples. Further, we show that a specific MGE, the phage satellite PLE, collapses LIN with a conserved protein during its anti-phage program. The insights gleaned from this work add to our expanding understanding of microbial fitness in natural contexts beyond the canonical bacterial genome and into the realm of antagonistic evolution driven by phages and satellites.

The LastrLocus Unveiled: T4 RIII Is a Cytoplasmic Antiholin

ABSTRACTThe latent period of phage T4, normally ∼25 min, can be extended indefinitely if the infected cell is superinfected after 5 min. This phenomenon, designated lysis inhibition (LIN), was first described in the 1940s and is genetically defined by mutations in diverse T4rgenes. RI, the main effector of LIN, has been shown to be secreted to the periplasm, where, upon activation by superinfection with a T-even virion, it binds to the C-terminal periplasmic domain of the T4 holin T and blocks its lethal permeabilization of the cytoplasmic membrane. Anotherrlocus,rIII, has been the subject of conflicting reports. In this study, we show that RIII, an 82-amino-acid protein, is also required for LIN in bothEscherichia coliB strains andE. coliK-12 strains. In T4ΔrIIIinfections, LIN was briefly established but was unstable. The overexpression of a clonedrIIIgene alone impeded T-mediated lysis temporarily. However, coexpression ofrIIIandrIresulted in a stable LIN state. Bacterial two-hybrid assays and pulldown assays showed that RIII interacts with the cytoplasmic N terminus of T, which is a critical domain for holin function. We conclude that RIII is a T4 antiholin that blocks membrane hole formation by interacting directly with the holin. Accordingly, we propose an augmented model for T4 LIN that involves the stabilization of a complex of three proteins in two compartments of the cell: RI interacting with the C terminus of T in the periplasm and RIII interacting with the N terminus of T in the cytoplasm.IMPORTANCELysis inhibition is a unique feature of phage T4 in response to environmental conditions, effected by the antiholin RI, which binds to the periplasmic domain of the T holin and blocks its hole-forming function. Here we report that the T4 generIIIencodes a cytoplasmic antiholin that, together with the main antiholin, RI, inhibits holin T by forming a complex of three proteins spanning two cell compartments.

Proteomic profiles and kinetics of development of bacteriophage T4 and its rI and rIII mutants in slowly growing Escherichia coli

Bacteriophage T4 survival in its natural environment requires adjustment of phage development to the slow bacterial growth rate or the initiation of mechanisms of pseudolysogeny or lysis inhibition (LIN). While phage-encoded RI and probably RIII proteins seem to be crucial players in pseudolysogeny and LIN phenomena, the identity of proteins involved in the regulation of T4 development in slowly growing bacteria has remained unknown. In this work, using a chemostat system, we studied the development of wild-type T4 (T4wt) and its rI (T4rI) and rIII (T4rIII) mutants in slowly growing bacteria, where T4 did not initiate LIN or pseudolysogeny. We determined eclipse periods, phage propagation times, latent periods and burst sizes of T4wt, T4rI and T4rIII. We also compared intracellular proteomes of slowly growing Escherichia coli infected with either T4wt or the mutants. Using two-dimensional PAGE analyses we found 18 differentially expressed proteins from lysates of infected cells. Proteins whose amounts were different in cells harbouring T4wt and the mutants are involved in processes of replication, phage–host interactions or they constitute virion components. Our data indicate that functional RI and RIII proteins – apart from their already known roles in LIN and pseudolysogeny – are also necessary for the regulation of phage T4 development in slowly growing bacteria. This regulation may be more complicated than previously anticipated, with many factors influencing T4 development in its natural habitat.