Contrasting nuclear dynamics of the caspase-activated DNase (CAD) in dividing and apoptotic cells

Although compelling evidence supports the central role of caspase-activated DNase (CAD) in oligonucleosomal DNA fragmentation in apoptotic nuclei, the regulation of CAD activity remains elusive in vivo. We used fluorescence photobleaching and biochemical techniques to investigate the molecular dynamics of CAD. The CAD-GFP fusion protein complexed with its inhibitor (ICAD) was as mobile as nuclear GFP in the nucleosol of dividing cells. Upon induction of caspase-3–dependent apoptosis, activated CAD underwent progressive immobilization, paralleled by its attenuated extractability from the nucleus. CAD immobilization was mediated by its NH2 terminus independently of its DNA-binding activity and correlated with its association to the interchromosomal space. Preventing the nuclear attachment of CAD provoked its extracellular release from apoptotic cells. We propose a novel paradigm for the regulation of CAD in the nucleus, involving unrestricted accessibility of chromosomal DNA at the initial phase of apoptosis, followed by its nuclear immobilization that may prevent the release of the active nuclease into the extracellular environment

Two Coregulated Efflux Transporters Modulate Intracellular Heme and Protoporphyrin IX Availability in Streptococcus agalactiae

Streptococcus agalactiae is a major neonatal pathogen whose infectious route involves septicemia. This pathogen does not synthesize heme, but scavenges it from blood to activate a respiration metabolism, which increases bacterial cell density and is required for full virulence. Factors that regulate heme pools in S. agalactiae are unknown. Here we report that one main strategy of heme and protoporphyrin IX (PPIX) homeostasis in S. agalactiae is based on a regulated system of efflux using two newly characterized operons, gbs1753 gbs1752 (called pefA pefB), and gbs1402 gbs1401 gbs1400 (called pefR pefC pefD), where pef stands for ‘porphyrin-regulated efflux’. In vitro and in vivo data show that PefR, a MarR-superfamily protein, is a repressor of both operons. Heme or PPIX both alleviate PefR-mediated repression. We show that bacteria inactivated for both Pef efflux systems display accrued sensitivity to these porphyrins, and give evidence that they accumulate intracellularly. The ΔpefR mutant, in which both pef operons are up-regulated, is defective for heme-dependent respiration, and attenuated for virulence. We conclude that this new efflux regulon controls intracellular heme and PPIX availability in S. agalactiae, and is needed for its capacity to undergo respiration metabolism, and to infect the host

Understanding heme stress sensing systems by pathogens, to design new antibiotics

Heme, a porphyrin containing an iron atom, is an essential cofactor of several bacterial functions. Heme is also toxic because of the reactivity of the iron generating reactive oxygen species. One of the main mechanisms of heme detoxification, in Gram-positive bacteria, relies on the expression of a heme efflux ABC transporter, HrtBA. The regulation of this transporter has been investigated in two opportunistic pathogens, Enterococcus faecalis and Staphylococcus aureus, two bacteria responsible for multiresistant nosocomial infections. In E. faecalis, a new TetR family regulator, FhtR, has been identified and characterized. The FhtR dependent transcriptional inhibition of hrtBA is lifted by its binding to heme. FhtR controls the intracellular heme pools as showed par the activity of the endogenous heme dependent catalase, KatA. FhtR is thus a master regulator of heme intracellular homeostasis in E. faecalis. In a mouse model of intestinal transit, HrtBA is expressed, demonstrating the relevance of this system in the gastrointestinal tract where E. faecalis is a commensal resident. In S. aureus, hrtBA transcription is controled by the two-component system, HssRS. The study of the mechanism of the membrane heme sensor HssS showed that the intracytoplasmic of the histidine kinase was responsible of the binding and heme signal transduction for HrtBA expression. Alltogether, these results demonstrate that while HrtBA is conserved among Gram positive bacteria, the regulating mechanisms leading to its expression are varied. This suggests that the host heme response is dependent of the bacteria lifestyle and underlies the importance of this cofactor in the host-pathogen relationship. Inhibiting heme effux by HrtBA or the heme sensing mechanisms could lead to new antibiotic strategies.L’hème, une structure porphyrique contenant un atome de fer, est un cofacteur essentiel à de nombreuses fonctions bactériennes. Cependant, le fer de l’hème génère des radicaux libres, ce qui explique sa toxicité. Un des mécanismes principaux de détoxification de l’hème est l’expression, par les bactéries à Gram positif, d’un transporteur d’efflux de la famille ABC, HrtBA. La régulation de ce transporteur a été étudiée chez deux bactéries pathogènes opportunistes Enterococcus faecalis et Staphylococcus aureus, responsables d’infections nosocomiales multirésistantes. Chez E. faecalis, un nouveau régulateur de la famille TetR, FhtR, a été identifié et caractérisé. L’inhibition de la transcription de hrtBA par FhtR est levée par sa liaison à l’hème. FhtR a aussi un impact important sur l’activité de la catalase à hème, KatA. FhtR a donc un rôle majeur dans l’homéostasie intracellulaire de l’hème. Dans un modèle de transit intestinal chez la souris, HrtBA est induit, suggérant que ce mécanisme est important pour E. faecalis, une bactérie commensale de l’intestin, dans cet environnement. Chez S. aureus, la transcription de hrtBA est régulée par un système à deux composants HssRS. L’étude du mécanisme du senseur membranaire HssS a révélé que la partie cytoplasmique de l’histidine kinase était impliquée dans la liaison et la transduction du signal hème pour l’expression de HrtBA. L’ensemble de ces résultats démontre que, bien que HrtBA soit conservée, plusieurs systèmes distincts régulent son expression, suggérant une adaptation complexe de la réponse bactérienne à l’hème de l’hôte et son importance dans la relation hôte-pathogène. Bloquer HrtBA ou la transduction du signal hème par ces deux senseurs d’hème pourrait constituer une cible pour la recherche antibiotique chez ces deux pathogènes

Analysis of differential lipofection efficiency in primary and established myoblasts

Pampinella, FrancescaLechardeur, DelphineZanetti, ElenaMacLachlan, IanBenharouga, MohammedLukacs, Gergely LVitiello, LiberoengTI_A.142/Telethon/ItalyResearch Support, Non-U.S. Gov't2002/02/07 10:00Mol Ther. 2002 Feb;5(2):161-9.; In this study we have compared the process of lipid-mediated transfection in primary and established myoblasts, in an attempt to elucidate the mechanisms responsible for the scarce transfectability of the former. We determined the metabolic stability of cytoplasmically injected and lipofected DNA in primary and established myoblasts and carried out a comparative time course analysis of luciferase reporter-gene expression and DNA stability. The efficiency of the transcription-translation machinery of the two cell types was compared by intranuclear injection of naked plasmid DNA encoding luciferase. Subcellular colocalization of fluorescein-labeled lipopolyplexes with specific endosomal and lysosomal markers was performed by confocal microscopy to monitor the intracellular trafficking of plasmid DNA during transfection. The metabolic stability of plasmid DNA was similar in primary and established myoblasts after both lipofection and cytoplasmic injection. In both cell types, lipofection had no detectable effect on the rate of cell proliferation. Confocal analysis showed that nuclear translocation of transfected DNA coincided with localization in a compartment devoid of endosome- or lysosome-specific marker proteins. The residency time of plasmid DNA in this compartment differed for primary and established myoblasts. Our findings suggest that the lower transfectability of primary myoblasts is mostly due to a difference in the intracellular delivery pathway that correlates with more rapid delivery of internalized complex to the lysosomal compartment

Oligomerization state of the DNA fragmentation factor in normal and apoptotic cells

The caspase-activated DNase (CAD) is the primary nuclease responsible for oligonucleosomal DNA fragmentation during apoptosis. The DNA fragmentation factor (DFF) is composed of the 40-kDa CAD (DFF40) in complex with its cognate 45-kDa inhibitor (inhibitor of CAD: ICAD or DFF45). The association of ICAD with CAD not only inhibits the DNase activity but is also essential for the co-translational folding of CAD. Activation of CAD requires caspase-3-dependent proteolysis of ICAD. The tertiary structures of neither the inactive nor the activated DFF have been conclusively established. Whereas the inactive DFF is thought to consist of the CAD/ICAD heterodimer, activated CAD has been isolated as a large (>MDa) multimer, as well as a monomer. To establish the subunit stoichiometry of DFF and some of its structural determinants in normal and apoptotic cells, we utilized size-exclusion chromatography in combination with co-immunoprecipitation and mutagenesis techniques. Both endogenous and heterologously expressed DFF have an apparent molecular mass of 160-190 kDa and contain 2 CAD and 2 ICAD molecules (CAD/ICAD)2 in HeLa cells. Although the N-terminal (CIDE-N) domain of CAD is not required for ICAD binding, it is necessary but not sufficient for ICAD homodimerization in the DFF. In contrast, the CIDE-N domain of ICAD is required for CAD/ICAD association. Using bioluminescence resonance energy transfer (BRET), dimerization of ICAD in DFF was confirmed in live cells. In apoptotic cells, endogenous and exogenous CAD forms limited oligomers, representing the active nuclease. A model is proposed for the rearrangement of the DFF subunit stoichiometry in cells undergoing programmed cell death

Human cellular prion protein hPrPC is sorted to the apical membrane of epithelial cells

Propagation of the scrapie isoform of the prion protein (PrP(Sc)) depends on the expression of endogenous cellular prion (PrP(C)). During oral infection, PrP(Sc) propagates, by conversion of the PrP(C) to PrP(Sc), from the gastrointestinal tract to the nervous system. Intestinal epithelium could serve as the primary site for PrP(C) conversion. To investigate PrP(C) sorting in epithelia cells, we have generated both a green fluorescent protein (EGFP) or hemagglutinin (HA) tagged human PrP(C) (hPrP(C)). Combined molecular, biochemical, and single living polarized cell imaging characterizations suggest that hPrP(C) is selectively targeted to the apical side of Madin-Darby canine kidney (MDCKII) and of intestinal epithelia (Caco2) cells

HssS activation by membrane heme defines a paradigm for 2-component system signaling in<i>Staphylococcus aureus</i>

Strict management of intracellular heme pools, which are both toxic and beneficial, can be crucial for bacterial survival during infection. The human pathogen Staphylococcus aureus uses a two-component heme sensing system (HssRS), which counteracts environmental heme toxicity by triggering expression of the efflux transporter HrtBA. The HssS heme sensor is a HisKA-type histidine kinase, characterized as a membrane-bound homodimer containing an extracellular sensor and a cytoplasmic conserved catalytic domain. To elucidate HssS heme sensing mechanism, a structural simulation of the HssS dimer based on Alphafold2 was docked with heme. In this model, heme is embedded in the membrane bilayer with its 2 protruding porphyrin propionates interacting with 2 conserved Arg94 and Arg163 that are located extracellularly. Mutagenesis of these arginines and of 2 highly conserved phenylalanines, Phe25 and Phe128, in the predicted hydrophobic heme binding pocket abolished the ability of HssS to induce HrtBA synthesis. This study gives evidence that exogenous heme interacts with HssS at the membrane/extracellular interface to initiate HssS activation to induce HrtBA-mediated heme extrusion from the membrane. This “gatekeeper” mechanism could limit intracellular diffusion of exogenous heme in S. aureus, and may serve as a paradigm for how efflux transporters control detoxification of exogenous hydrophobic stressors

HssS activation by membrane heme defines a paradigm for 2-component system signaling in<i>Staphylococcus aureus</i>

Strict management of intracellular heme pools, which are both toxic and beneficial, can be crucial for bacterial survival during infection. The human pathogen Staphylococcus aureus uses a two-component heme sensing system (HssRS), which counteracts environmental heme toxicity by triggering expression of the efflux transporter HrtBA. The HssS heme sensor is a HisKA-type histidine kinase, characterized as a membrane-bound homodimer containing an extracellular sensor and a cytoplasmic conserved catalytic domain. To elucidate HssS heme sensing mechanism, a structural simulation of the HssS dimer based on Alphafold2 was docked with heme. In this model, heme is embedded in the membrane bilayer with its 2 protruding porphyrin propionates interacting with 2 conserved Arg94 and Arg163 that are located extracellularly. Mutagenesis of these arginines and of 2 highly conserved phenylalanines, Phe25 and Phe128, in the predicted hydrophobic heme binding pocket abolished the ability of HssS to induce HrtBA synthesis. This study gives evidence that exogenous heme interacts with HssS at the membrane/extracellular interface to initiate HssS activation to induce HrtBA-mediated heme extrusion from the membrane. This “gatekeeper” mechanism could limit intracellular diffusion of exogenous heme in S. aureus, and may serve as a paradigm for how efflux transporters control detoxification of exogenous hydrophobic stressors