Leon Espinosa

Giant Marseillevirus highlights the role of amoebae as a melting pot in emergence of chimeric microorganisms

Giant viruses such as Mimivirus isolated from amoeba found in aquatic habitats show biological sophistication comparable to that of simple cellular life forms and seem to evolve by similar mechanisms, including extensive gene duplication and horizontal gene transfer (HGT), possibly in part through a viral parasite, the virophage. We report here the isolation of “Marseille” virus, a previously uncharacterized giant virus of amoeba. The virions of Marseillevirus encompass a 368-kb genome, a minimum of 49 proteins, and some messenger RNAs. Phylogenetic analysis of core genes indicates that Marseillevirus is the prototype of a family of nucleocytoplasmic large DNA viruses (NCLDV) of eukaryotes. The genome repertoire of the virus is composed of typical NCLDV core genes and genes apparently obtained from eukaryotic hosts and their parasites or symbionts, both bacterial and viral. We propose that amoebae are “melting pots” of microbial evolution where diverse forms emerge, including giant viruses with complex gene repertoires of various origins.

Fe-S Cluster Biosynthesis Controls Uptake of Aminoglycosides in a ROS-Less Death Pathway

Unreactive Death A controversial proposal that all bactericidal antibiotics kill by reactive oxygen species (ROS) and not by their primary cell target has recently attracted high-profile refutations. The ROS-death pathway implicated overstimulation of the electron transport in respiratory chains; a malfunction that leads to ROS releasing Fe from Fe-S clusters and causing cell death via Fenton chemistry. Ezraty et al. (p. 1583) show that electron transport chains and Fe-S clusters are key to killing by aminoglycoside antibiotics but not for the reasons envisioned in the ROS theory. Fe-S clusters are essential for killing because they mature the respiratory chains that produce the necessary proton motive force for the energized uptake of aminoglycosides. Consequently, iron chelators protect against aminoglycosides, not because they scavenge the iron from Fenton chemistry, but because they block aminoglycoside uptake. The respiratory chain is required for antibiotic entry to the target cell rather than for its killing. All bactericidal antibiotics were recently proposed to kill by inducing reactive oxygen species (ROS) production, causing destabilization of iron-sulfur (Fe-S) clusters and generating Fenton chemistry. We find that the ROS response is dispensable upon treatment with bactericidal antibiotics. Furthermore, we demonstrate that Fe-S clusters are required for killing only by aminoglycosides. In contrast to cells, using the major Fe-S cluster biosynthesis machinery, ISC, cells using the alternative machinery, SUF, cannot efficiently mature respiratory complexes I and II, resulting in impendence of the proton motive force (PMF), which is required for bactericidal aminoglycoside uptake. Similarly, during iron limitation, cells become intrinsically resistant to aminoglycosides by switching from ISC to SUF and down-regulating both respiratory complexes. We conclude that Fe-S proteins promote aminoglycoside killing by enabling their uptake.

Repairing oxidized proteins in the bacterial envelope using respiratory chain electrons

The reactive species of oxygen and chlorine damage cellular components, potentially leading to cell death. In proteins, the sulfur-containing amino acid methionine is converted to methionine sulfoxide, which can cause a loss of biological activity. To rescue proteins with methionine sulfoxide residues, living cells express methionine sulfoxide reductases (Msrs) in most subcellular compartments, including the cytosol, mitochondria and chloroplasts. Here we report the identification of an enzymatic system, MsrPQ, repairing proteins containing methionine sulfoxide in the bacterial cell envelope, a compartment particularly exposed to the reactive species of oxygen and chlorine generated by the host defence mechanisms. MsrP, a molybdo-enzyme, and MsrQ, a haem-binding membrane protein, are widely conserved throughout Gram-negative bacteria, including major human pathogens. MsrPQ synthesis is induced by hypochlorous acid, a powerful antimicrobial released by neutrophils. Consistently, MsrPQ is essential for the maintenance of envelope integrity under bleach stress, rescuing a wide series of structurally unrelated periplasmic proteins from methionine oxidation, including the primary periplasmic chaperone SurA. For this activity, MsrPQ uses electrons from the respiratory chain, which represents a novel mechanism to import reducing equivalents into the bacterial cell envelope. A remarkable feature of MsrPQ is its capacity to reduce both rectus (R-) and sinister (S-) diastereoisomers of methionine sulfoxide, making this oxidoreductase complex functionally different from previously identified Msrs. The discovery that a large class of bacteria contain a single, non-stereospecific enzymatic complex fully protecting methionine residues from oxidation should prompt a search for similar systems in eukaryotic subcellular oxidizing compartments, including the endoplasmic reticulum.

The mechanism of force transmission at bacterial focal adhesion complexes

Various rod-shaped bacteria mysteriously glide on surfaces in the absence of appendages such as flagella or pili. In the deltaproteobacterium Myxococcus xanthus, a putative gliding motility machinery (the Agl–Glt complex) localizes to so-called focal adhesion sites (FASs) that form stationary contact points with the underlying surface. Here we show that the Agl–Glt machinery contains an inner-membrane motor complex that moves intracellularly along a right-handed helical path; when the machinery becomes stationary at FASs, the motor complex powers a left-handed rotation of the cell around its long axis. At FASs, force transmission requires cyclic interactions between the molecular motor and the adhesion proteins of the outer membrane via a periplasmic interaction platform, which presumably involves contractile activity of motor components and possible interactions with peptidoglycan. Our results provide a molecular model of bacterial gliding motility.

Value of in vivo T2 measurement for myocardial fibrosis assessment in diabetic mice at 11.75 T.

Objective:The aim of the study was to assess the value of in vivo T2 measurements to noninvasively quantify myocardial fibrosis in diabetic mice at 11.75 T. Diabetic cardiomyopathy is characterized by extracellular matrix alteration and microcirculation impairment. These conditions might provide electrical heterogeneity, which is a substrate for arrhythmogenesis. T1 mapping has been proposed to quantify diffuse myocardial fibrosis in cardiac diseases but has several limitations. T2 measurement may represent an alternative for fibrosis quantification at high magnetic field. Materials and Methods:A magnetic resonance imaging protocol including in vivo T2 measurements at 11.75 T was performed in 9 male C57BL/6J mice after 8 weeks of streptozotocin-induced diabetes and in 9 control mice. Programmed ventricular stimulation was performed in both groups. T2 measurements were compared with histologic quantification of fibrosis using picrosirius red staining. Results:Myocardial T2 was significantly lower in diabetic mice (13.8 ± 2.8 ms) than in controls (18.9 ± 2.3 ms, P < 0.001). There was a good correlation between T2 and fibrosis area obtained by histopathology (R2 = 0.947, P < 0.001). During programmed ventricular stimulation, 3 nonsustained ventricular tachycardias were induced in diabetic mice versus none in the control group. Conclusions:The in vivo T2 relaxation time strongly correlated with myocardial fibrosis area assessed with histologic staining in diabetic mice.

Functional organization of a multimodular bacterial chemosensory apparatus.

Chemosensory systems (CSS) are complex regulatory pathways capable of perceiving external signals and translating them into different cellular behaviors such as motility and development. In the δ-proteobacterium Myxococcus xanthus, chemosensing allows groups of cells to orient themselves and aggregate into specialized multicellular biofilms termed fruiting bodies. M. xanthus contains eight predicted CSS and 21 chemoreceptors. In this work, we systematically deleted genes encoding components of each CSS and chemoreceptors and determined their effects on M. xanthus social behaviors. Then, to understand how the 21 chemoreceptors are distributed among the eight CSS, we examined their phylogenetic distribution, genomic organization and subcellular localization. We found that, in vivo, receptors belonging to the same phylogenetic group colocalize and interact with CSS components of the respective phylogenetic group. Finally, we identified a large chemosensory module formed by three interconnected CSS and multiple chemoreceptors and showed that complex behaviors such as cell group motility and biofilm formation require regulatory apparatus composed of multiple interconnected Che-like systems.

Evolution and Design Governing Signal Precision and Amplification in a Bacterial Chemosensory Pathway.

Understanding the principles underlying the plasticity of signal transduction networks is fundamental to decipher the functioning of living cells. In Myxococcus xanthus, a particular chemosensory system (Frz) coordinates the activity of two separate motility systems (the A- and S-motility systems), promoting multicellular development. This unusual structure asks how signal is transduced in a branched signal transduction pathway. Using combined evolution-guided and single cell approaches, we successfully uncoupled the regulations and showed that the A-motility regulation system branched-off an existing signaling system that initially only controlled S-motility. Pathway branching emerged in part following a gene duplication event and changes in the circuit structure increasing the signaling efficiency. In the evolved pathway, the Frz histidine kinase generates a steep biphasic response to increasing external stimulations, which is essential for signal partitioning to the motility systems. We further show that this behavior results from the action of two accessory response regulator proteins that act independently to filter and amplify signals from the upstream kinase. Thus, signal amplification loops may underlie the emergence of new connectivity in signal transduction pathways.

Defective monocyte dynamics in Q fever granuloma deficiency.

BACKGROUND The outcome of Q fever, an infectious disease caused by Coxiella burnetii, is associated with granuloma formation. Granulomas are present in patients with resolutive Q fever but are lacking in patients with chronic Q fever. METHODS Study of granuloma formation requires invasive approaches. Here, we took advantage of a recently described method that enables in vitro generation of human granulomas specific for C. burnetii. RESULTS Circulating mononuclear cells progressively accumulated around beads coated with C. burnetii extracts, and complete granulomas were generated in 8 days. Granuloma cells consisted of macrophages, lymphocytes, and, to a lesser extent, epithelioid cells and multinucleated giant cells. Early events that govern granuloma formation were studied using live-imaging microscopy. Monocytes migrated toward C. burnetii-coated beads independently of the presence of T lymphocytes and then recruited T lymphocytes. About 90% of patients with chronic Q fever failed to form granulomas. This deficiency was associated with defective migration of monocytes toward coated beads. CONCLUSIONS Monocytes were involved in the early stages of granuloma formation and recruited T lymphocytes to complete granuloma formation. This article describes a direct relationship between defective granuloma formation and defective migration of monocytes.

Opposite and coordinated rotation of amphitrichous flagella governs oriented swimming and reversals in a magnetotactic spirillum

UNLABELLED Current knowledge regarding the mechanism that governs flagellar motor rotation in response to environmental stimuli stems mainly from the study of monotrichous and peritrichous bacteria. Little is known about how two polar flagella, one at each cell pole of the so-called amphitrichous bacterium, are coordinated to steer the swimming. Here we fluorescently labeled the flagella of Magnetospirillum magneticum AMB-1 cells and took advantage of the magnetically controllable swimming of this bacterium to investigate flagellar rotation in moving cells. We identified three motility behaviors (runs, tumbles, and reversals) and two characteristic fluorescence patterns likely corresponding to flagella rotating in opposite directions. Each AMB-1 locomotion mode was systematically associated with particular flagellar patterns at the poles which led us to conclude that, while cell runs are allowed by the asymmetrical rotation of flagellar motors, their symmetrical rotation triggers cell tumbling. Our observations point toward a precise coordination of the two flagellar motors which can be temporarily unsynchronized during tumbling. IMPORTANCE Motility is essential for bacteria to search for optimal niches and survive. Many bacteria use one or several flagella to explore their environment. The mechanism by which bipolarly flagellated cells coordinate flagellar rotation is poorly understood. We took advantage of the genetic amenability and magnetically controlled swimming of the spirillum-shaped magnetotactic bacterium Magnetospirillum magneticum AMB-1 to correlate cell motion with flagellar rotation. We found that asymmetric rotation of the flagella (counterclockwise at the lagging pole and clockwise at the leading pole) enables cell runs whereas symmetric rotation triggers cell tumbling. Taking into consideration similar observations in spirochetes, bacteria possessing bipolar ribbons of periplasmic flagella, we propose a conserved motility paradigm for spirillum-shaped bipolarly flagellated bacteria.

HIV-1 Infection and First Line ART Induced Differential Responses in Mitochondria from Blood Lymphocytes and Monocytes: The ANRS EP45 “Aging” Study

Background The ANRS EP45 “Aging” study investigates the cellular mechanisms involved in the accelerated aging of HIV-1 infected and treated patients. The data reported focus on mitochondria, organelles known to be involved in cell senescence. Methods 49 HIV-1 infected patients untreated with antiretroviral therapy, together with 49 seronegative age- and sex-matched control subjects and 81 HIV-1 infected and treated patients, were recruited by 3 AIDS centres (Marseille, Montpellier, Nice; France; http://clinicaltrials.gov/, NCT01038999). In more than 88% of treated patients, the viral load was <40 copies/ml and the CD4+ cell count was >500/mm3. ROS (reactive oxygen species) production and ΔΨm (inner membrane potential) were measured by flow cytometry in blood lymphocytes and monocytes (functional parameters). Three mitochondrial network quantitative morphological parameters were computed using confocal microscopy and image analysis. Three PBMC mitochondrial proteins (porin and subunits 2 and 4 of cytochrome C oxidase encoded by mtDNA or nuclear DNA, respectively) were analysed by western blotting. Results Quantitative changes in PBMC mitochondrial proteins were not induced by either HIV-1 infection or ART. Discriminant analysis integrating functional (ROS production and ΔΨm) or morphological (network volume density, fragmentation and branching) parameters revealed HIV-1 infection and ART differential effects according to cell type. First line ART tended to rescue lymphocyte mitochondrial parameters altered by viral infection, but induced slight changes in monocytes. No statistical difference was found between the effects of three ART regimens on mitochondrial parameters. Correlations between functional parameters and viral load confirmed the damaging effects of HIV-1 in lymphocyte mitochondria. Conclusions In patients considered to be clinically stable, mitochondria exhibited functional and morphological modifications in PBMCs resulting from either direct or indirect effects of HIV-1 infection (lymphocytes), or from first line ART (monocytes). Together with other tissue impairments, these changes may contribute to global aging. Trial Registration ClinicalTrials.gov NCT01038999 NCT01038999