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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2018

Electron microscopic analysis of the exo-skeleton of hadal zone amphipod Hirondellea gigas

The amphipod Hirondellea gigas inhabits the deepest regions of the oceans in extra high-pressure. However, the mechanisms by which they adapt to their high-pressure environments remain unknown. In this study, we investigated elements of the exoskeleton of H. gigas captured from the deepest points of the Mariana Trench. The H. gigas exoskeleton contained aluminum, as well as a major amount of calcium carbonate. Unlike other accumulated metals, aluminum was distributed on the surface of exoskeletons. To investigate how H. gigas obtains aluminum, we conducted a metabolome analysis and found that gluconic acid/gluconolactone was capable of extracting metals from the sediment under the habitat conditions of H. gigas. The extracted aluminum ions are transformed into the gel state of aluminum hydroxide in alkaline seawater, and this gel covers the body to protect the amphipod. The aluminum gel would be one of good materials to adapt to such high-pressure environment.

zoology↗

bioRxiv2018

On the prevalence of uninformative parameters in statistical models applying model selection in applied ecology

Research in applied ecology provides scientific evidence to guide conservation policy and management. Applied ecology is becoming increasingly quantitative and model selection via information criteria has become a common statistical modeling approach. Unfortunately, parameters that contain little to no useful information are commonly presented and interpreted as important in applied ecology. I review the concept of an uninformative parameter in model selection using information criteria and perform a literature review to measure the prevalence of uninformative parameters in model selection studies applying Akaikes Information Criterion (AIC) in 2014 in four of the top journals in applied ecology (Biological Conservation, Conservation Biology, Ecological Applications, Journal of Applied Ecology). Twenty-one percent of studies I reviewed applied AIC metrics. Many (31.5 %) of the studies applying AIC metrics in the four applied ecology journals I reviewed had or were very likely to have uninformative parameters in a model set. In addition, more than 40 % of studies reviewed had insufficient information to assess the presence or absence of uninformative parameters in a model set. Given the prevalence of studies likely to have uninformative parameters or with insufficient information to assess parameter status (71.5 %), I surmise that much of the policy recommendations based on applied ecology research may not be supported by the data analysis. I provide warning signals and a decision tree to help reduce the prevalence of uninformative parameters in studies applying model selection with information criteria. The four warning signals and decision tree should assist authors, reviewers, and editors to screen for uninformative parameters in studies applying model selection with information criteria. In the end, careful thinking at every step of the scientific process and greater reporting standards are required to detect uninformative parameters in studies adopting an information criteria approach.

ecology↗

bioRxiv2018

Antibiotic resistance is lower in Staphylococcus aureus isolated from antibiotic-free raw meat as compared to conventional raw meat

The frequent use of antibiotics contributes to antibiotic resistance in bacteria, resulting in an increase in infections that are difficult to treat. Livestock are commonly administered antibiotics in their feed, but there is current interest in raising animals that are only administered antibiotics during active infections. Staphylococcus aureus (SA) is a common pathogen of both humans and livestock raised for human consumption. SA has achieved high levels of antibiotic resistance, but the origins and locations of resistance selection are poorly understood. We determined the prevalence of SA and MRSA in conventional and antibiotic-free (AF) meat products, and also measured rates of antibiotic resistance in these isolates. We isolated SA from raw conventional turkey, chicken, beef, and pork samples and also from AF chicken and turkey samples. We found that SA contamination was common, with an overall prevalence of 22.64% (range of 2.78-30.77%) in conventional meats and 13.0% (range of 12.5-13.2%) in AF poultry meats. MRSA was isolated from 15.72% of conventional raw meats (range of 2.78-20.41%) but not from AF-free meats. The degree of antibiotic resistance in conventional poultry products was significantly higher vs AF poultry products for a number of different antibiotics, and while multi-drug resistant strains were relatively common in conventional meats none were detected in AF meats. The use of antibiotics in livestock contributes to high levels of antibiotic resistance in SA found in meat products. Our results support the use of AF conditions for livestock in order to prevent antibiotic resistance development in SA.

microbiology↗

bioRxiv2018

Collective radioresistance of T47D breast carcinoma cells is mediated by a Syncytin-1 homologous protein

It is generally accepted that radiotherapy must target clonogenic cells, i.e., those cells in a tumour that have self-renewing potential. Focussing on isolated clonogenic cells, however, may lead to an underestimate or even to an outright neglect of the importance of biological mechanisms that regulate tumour cell sensitivity to radiation. We develop a new statistical and experimental approach to quantify the effects of radiation on cell populations as a whole. In our experiments, we change the proximity relationships of the cells by culturing them in wells with different shapes, and we find that the radiosensitivity of T47D human breast carcinoma cells in tight clusters is different from that of isolated cells. Molecular analyses show that T47D cells express a Syncytin-1 homologous protein (SyHP). We observe that SyHP translocates to the external surface of the plasma membrane of cells killed by radiation treatment. The data support the fundamental role of SyHP in the formation of intercellular cytoplasmic bridges and in the enhanced radioresistance of surviving cells. We conclude that complex and unexpected biological mechanisms of tumour radioresistance take place at the cell population level. These mechanisms may significantly bias our estimates of the radiosensitivity of breast carcinomas in vivo and thereby affect treatment plans, and they call for further investigations.

cancer biology↗

bioRxiv2018

Determination of biophysical parameters for enhancement of human osteoblast proliferation by mechanical vibration in the infrasonic frequency range

Experimental methods for studying an enhancement of osteoblast proliferation in vitro provide tools for the research of biochemical processes involved in bone turnover in vivo. Some of the current methods used for this purpose are based on the ability of the osteoblasts to enhance proliferation by mechanical stimulation. We describe an experimental approach of biomechanical stimulation of cultured human osteoblast-like cells by vibration. This method is based on the specially designed controlled vibration setup that consists of an electric actuator, with horizontally mounted well plate containing cell cultures. Previously this method found to be effective to enhance cell proliferation, but the exact mechanical parameters of effective vibration were elusive. The current low friction system for mechanical stimulation of osteoblast-like cells in vitro provides recording of narrow range mechanical parameters in the infrasonic spectrum.\n\nWe exposed human osteoblast-like cells in explant monolayer culture to mechanical vibration in the 10-70Hz range of frequencies and found that 50-70 Hz of vibration frequency is optimal for inducing osteoblast proliferation that was deduced from interrelation between unchanged cell number in culture samples with significant decrease in cell death rate (decreased LDH activity in culture media, p<.05) and with parallel decrease of their maturation level (p<.01).\n\nIn this report we determined the optimal mechanical parameters and excitation protocol for induction of osteoblast proliferation in vitro by using a tunable and versatile mechanical platform, which can be used in the research of cell mechanotransduction.

cell biology↗

bioRxiv2018

Heat Shock in C. elegans Induces Downstream of Gene Transcription and Accumulation of Double-Stranded RNA

We observed that heat shock of Caenorhabditis elegans leads to the formation of nuclear double-stranded RNA (dsRNA) foci, detectable with a dsRNA-specific monoclonal antibody. These foci significantly overlap with nuclear HSF-1 granules. To investigate the molecular mechanism(s) underlying dsRNA foci formation, we used RNA-seq to globally characterize total RNA and immunoprecipitated dsRNA from control and heat shocked worms. We find antisense transcripts are generally increased after heat shock, and a subset of both sense and antisense transcripts enriched in the dsRNA pool by heat shock overlap with dsRNA transcripts enriched by deletion of tdp-1, which encodes the C. elegans ortholog of TDP-43. Interestingly, transcripts involved in translation are over-represented in the dsRNAs induced by either heat shock or deletion of tdp-1. Also enriched in the dsRNA transcripts are sequences downstream of annotated genes (DoGs), which we globally quantified with a new algorithm. To validate these observations, we used fluorescence in situ hybridization (FISH) to confirm both antisense and downstream of gene transcription for eif-3.B, one of the affected loci we identified.

molecular biology↗

bioRxiv2018

Stressed mothers, tolerant daughters: a case study about the physiological responses and growth of sugarcane plants under water deficit

Drought stress can imprint marks in plants after a previous exposure, leading to a permissive state that facilitates a more effective response to subsequent stress events. Such stress imprints would benefit plants obtained from progenitors previously exposed to drought. Herein, our hypothesis was that daughter plants obtained from mother plants previously exposed to water deficit will perform better under water deficit as compared to those obtained from mothers that did not face stressful conditions. Sugarcane mother plants were grown under well-hydrated conditions or subjected to three cycles of water deficit by water withholding. Then, daughter plants produced through vegetative propagation were subjected to water deficit. Leaf gas exchange was reduced under water deficit and daughters from mothers that experienced water deficit presented a faster recovery of CO2 assimilation and higher instantaneous carboxylation efficiency after rehydration as compared to daughters from mothers that did not face water deficit. Plants obtained from mother plants that faced water deficit showed the highest leaf proline concentration under water deficit as well as higher leaf H2O 2 concentration and leaf ascorbate peroxidase activity regardless of water regime. Under well-watered conditions, daughters from mothers that faced stressful conditions presented higher root H2O2 concentration and root catalase activity than ones from mothers that did not experience water shortage. Such physiological changes were associated with improvements in leaf area and shoot and root dry matter accumulation in daughters from stressed mothers. Our results suggest that root H2O2 concentration is a chemical signal associated with stress memory and improved sugarcane growth. Such findings bring a new perspective to sugarcane production systems, in which stress memory can be explored for improving drought tolerance in rainfed areas.

plant biology↗

bioRxiv2018

Benefit-cost analysis of the policy of mandatory annual rabies vaccination of domestic dogs in rabies-free Japan

Japan is one of the few rabies-free countries/territories which implement the policy of mandatory vaccination of domestic dogs. In order to assess the economic efficiency of such policy in reducing the economic burden of a future canine rabies outbreak in Japan, a benefit-cost analysis (BCA) was performed using probabilistic decision tree modelling. Input data derived from simulation results of published mathematical model, field investigation conducted by the authors at prefectural governments, literature review, international or Japanese database and empirical data of rabies outbreaks in other countries/territories. The current study revealed that the annual costs of implementing the current vaccination policy would be US$160,472,075 (90% prediction interval [PI]: $149,268,935 - 171,669,974). The economic burden of a potential canine rabies outbreak in Japan were estimated to be US$1,682,707 (90% PI: $1,180,289 - 2,249,283) under the current vaccination policy, while it would be US$5,019,093 (90% PI: $3,986,882 - 6,133,687) under hypothetical abolition of vaccination policy, which is 3-fold higher. Under a damage-avoided approach, the annual benefits of implementing the current vaccination policy in expected value were estimated to be US$85.75 (90% PI: $55.73 - 116.89). The benefit-cost ratio (BCR) was estimated to be 5.35 x 10-7 (90% PI: 3.46 x 10-7 - 7.37 x 10-7), indicating that the implementation of the current policy is very economically inefficient for the purpose of reducing the economic burden of a potential canine rabies outbreak. In worse-case scenario analysis, the BCR would become above 1 (indicating economic efficiency) if the risk of rabies introduction increased to 0.04 corresponding to a level of risk where rabies would enter Japan in 26 years while the economic burden of a rabies outbreak under the abolition of vaccination policy increased to $7.53 billion. Best-case analysis further revealed that the economic efficiency of the current policy could be improved by decreasing the vaccination price charged to dog owners, relaxing the frequency of vaccination to every two to three years and implementing the policy on a smaller scale, e.g. only in targeted prefectures instead of the whole Japan.

epidemiology↗

bioRxiv2018

Whole blood and hypertonic saline resuscitation improve the outcomes of controlled hemorrhagic shock combined with penetrating brain injury in rat

One of the most frequent injury patterns leading to early death of polytraumatized patients is hemorrhagic shock combined with traumatic brain injury. Currently, there is no agreement on the best treatment protocol of such injury. This study was aimed to compare the outcomes of resuscitation with various volumes of hypertonic saline (HTS), whole blood (WB) and fresh frozen plasma (FFP) in a rat model of penetrating brain injury combined with controlled hemorrhagic shock in order to look for optimal treatment. Anesthetized rats were intubated and ventilated. 30% of circulating blood volume was withdrawn following open brain injury, and intravenous resuscitation with various volumes of HTS, WB and FFP (3, 9, and 18 ml/kg) was administered. Blood samples were collected during the experiment for measurements of lactate and base excess levels, and before sacrifice for neutrophil counts. Twenty four and 48 hours after the injury, rats neurological status was examined. Rats were, then, sacrificed and pathological studies of brain and lung sections were performed. Regardless of fluid type, resuscitation improved the mean arterial pressure and lactate levels in accordance to fluids volume. However, base excess was improved only when rats were treated with WB and FFP. Improvement in behavioral performance of the rats was observed when treated with 3 and 9 ml/kg HTS or with 18 ml/kg WB or FFP. Blood and lung neutrophils were reduced in rats treated with 9 or 18 ml/kg WB. Mortality rates were reduced in rats administered with 9 and 18 ml/kg of HTS or WB. Taken together, resuscitation with WB at 9-18ml/kg or HTS at 9ml/kg is optimal for treatment of combined injury.

physiology↗

bioRxiv2018

The outer membrane proteins OmpA, FhuA, OmpF, EstA, BtuB and OmpX have unique lipopolysaccharide fingerprints.

The outer membrane of Gram-negative bacteria has a highly complex asymmetrical architecture, containing a mixture of phospholipids in the inner leaflet and in the outer leaflet they contain almost exclusively lipopolysaccharide (LPS) molecules. In E. coli, the outer membrane contains a wide range proteins with a beta barrel architecture, that vary in size from the smallest having eight strands to larger barrels composed of twenty-two strands. Here we report coarse-grain molecular dynamics simulations of six proteins from the E. coli outer membrane OmpA, OmpX, BtuB, FhuA, OmpF and EstA in a range of membrane environments, which are representative of the in vivo for different strains of E. coli. We show that each protein has a unique pattern of interaction with the surrounding membrane, which is influenced by the composition of the protein, the level of LPS in the outer leaflet and the differing mobilities of the lipids in the two leaflets of the membrane. Overall we present analyses from over 200 microseconds of simulation for each protein.\n\nAuthor summaryWe present data from over 200 microseconds of coarse-grain simulations that show the complexities of protein-lipid interactions within the outer membranes of Gram-negative bacteria. We show that the slow movement of lipolysaccharide molecules necessitate simulations of over 30 microsecond duration to achieve converged properties such as protein tilt angle. Each of the six proteins studied here shows a unique pattern of interactions with the outer membrane and thus constitute a fingerprint or signature.

biophysics↗

bioRxiv2018

Computational modeling reveals dynamics of brain metastasis in non-small cell lung cancer and provides a tool for personalized therapy

Brain metastases (BMs) are associated with poor prognosis in non-small cell lung cancer (NSCLC), but are only visible when large enough. Therapeutic decisions such as whole brain radiation therapy would benefit from patient-specific predictions of radiologically undetectable BMs. Here, we propose a mathematical modeling approach and use it to analyze clinical data of BM from NSCLC.\n\nPrimary tumor growth was best described by a gompertzian model for the pre-diagnosis history, followed by a tumor growth inhibition model during treatment. Growth parameters were estimated only from the size at diagnosis and histology, but predicted plausible individual estimates of the tumor age (2.1-5.3 years). Multiple metastatic models were assessed from fitting either literature data of BM probability (n = 183 patients) or longitudinal measurements of visible BMs in two patients. Among the tested models, the one featuring dormancy was best able to describe the data. It predicted latency phases of 4.4 - 5.7 months and onset of BMs 14 - 19 months before diagnosis. This quantitative model paves the way for a computational tool of potential help during therapeutic management.

cancer biology↗

bioRxiv2018

Predicting the effects of deep brain stimulation using a reduced coupled oscillator model

Deep brain stimulation (DBS) is known to be an effective treatment for a variety of neurological disorders, including Parkinsons disease and essential tremor (ET). At present, it involves administering a train of pulses with constant frequency via electrodes implanted into the brain. New closed-loop approaches involve delivering stimulation according to the ongoing symptoms or brain activity and have the potential to provide improvements in terms of efficiency, efficacy and reduction of side effects. The success of closed-loop DBS depends on being able to devise a stimulation strategy that minimizes oscillations in neural activity associated with symptoms of motor disorders. A useful stepping stone towards this is to construct a mathematical model, which can describe how the brain oscillations should change when stimulation is applied at a particular state of the system. Our work focuses on the use of coupled oscillators to represent neurons in areas generating pathological oscillations. Using a reduced form of the Kuramoto model, we analyse how a patient should respond to stimulation when neural oscillations have a given phase and amplitude. We predict that, provided certain conditions are satisfied, the best stimulation strategy should be phase specific but also that stimulation should have a greater effect if applied when the amplitude of brain oscillations is lower. We compare this surprising prediction with data obtained from ET patients. In light of our predictions, we also propose a new hybrid strategy which effectively combines two of the strategies found in the literature, namely phase-locked and adaptive DBS.\n\nAuthor summaryDeep brain stimulation (DBS) involves delivering electrical impulses to target sites within the brain and is a proven therapy for a variety of neurological disorders. Closed loop DBS is a promising new approach where stimulation is applied according to the state of a patient. Crucial to the success of this approach is being able to predict how a patient should respond to stimulation. Our work focusses on DBS as applied to patients with essential tremor (ET). On the basis of a theoretical model, which describes neurons as oscillators that respond to stimulation and have a certain tendency to synchronize, we provide predictions for how a patient should respond when stimulation is applied at a particular phase and amplitude of the ongoing tremor oscillations. Previous experimental studies of closed loop DBS provided stimulation either on the basis of ongoing phase or amplitude of pathological oscillations. Our study suggests how both of these measurements can be used to control stimulation. As part of this work, we also look for evidence for our theories in experimental data and find our predictions to be satisfied in one patient. The insights obtained from this work should lead to a better understanding of how to optimise closed loop DBS strategies.

neuroscience↗

bioRxiv2018

Analysis of an HIV model with post-treatment control

Recent investigation indicated that latent reservoir and immune impairment are responsible for the post-treatment control of HIV infection. In this paper, we simplify the disease model with latent reservoir and immune impairment and perform a series of mathematical analysis. We obtain the basic infection reproductive number R0 to characterize the viral dynamics. We prove that when R0 < 1, the uninfected equilibrium of the proposed model is globally asymptotically stable. When R0 > 1, we obtain two thresholds, the post-treatment immune control threshold and the elite control threshold. The model has bistable behaviors in the interval between the two thresholds. If the proliferation rate of CTLs is less than the post-treatment immune control threshold, the model does not have positive equilibria. In this case, the immune free equilibrium is stable and the system will have virus rebound. On the other hand, when the proliferation rate of CTLs is greater than the elite control threshold, the system has stable positive immune equilibrium and unstable immune free equilibrium. Thus, the system is under elite control.\n\nAuthor summaryIn this article, we use mathematical model to investigate the combined effect of latent reservoir and immune impairment on the post-treatment control of HIV infection. By simplifying an HIV model with latent reservoir and immune impairment, and performing mathematical analysis, we obtain the post-treatment immune control threshold and the elite control threshold for the HIV dynamics when R0 > 1. The HIV model displays bistable behaviors in the interval between the two thresholds. We illustrate our results using both mathematical analysis and numerical simulation. Our result is consistent with recent medical experiment. We show that patient with low proliferation rate of CTLs may undergo virus rebound, and patient with high proliferation rate of CTLs may obtain elite control of HIV infection. We perform bifurcation analysis to illustrate the infection status of patient with the variation of proliferation rate of CTLs, which potentially explain the reason behind different outcomes among HIV patients.

immunology↗

bioRxiv2018

Selective advantages favour high genomic AT-contents in intracellular elements

Extrachromosomal genetic elements generally exhibit increased AT-contents relative to their hosts DNA. The AT-bias of endosymbiotic genomes is commonly explained by neutral evolutionary processes. Here we show experimentally that an increased AT-content of host-dependent elements can be selectively favoured on the host level. Manipulating the nucleotide composition of bacterial cells by introducing A+T-or G+C-rich plasmids, we demonstrate that cells containing GC-rich plasmids are less fit than cells containing AT-rich plasmids. Moreover, the cost of GC-rich elements could be compensated by providing G+C-, but not A+T-precursors, thus linking the observed fitness effects to the cytoplasmic availability of nucleotides. Our work identifies selection as a strong evolutionary force that drives the genomes of intracellular genetic elements toward higher A+T contents.\n\nAuthor SummaryGenomes of endosymbiotic bacteria are commonly more AT-rich than the ones of their free-living relatives. Interestingly, genomes of other intracellular elements like plasmids or bacteriophages also tend to be richer in AT than the genomes of their hosts. The AT-bias of endosymbiotic genomes is commonly explained by neutral evolutionary processes. However, since A+T nucleotides are both more abundant and energetically less expensive than G+C nucleotides, an alternative explanation is that selective advantages drive the nucleotide composition of intracellular elements. Here we provide strong experimental evidence that intracellular elements, whose genome is more AT-rich than the genome of the host, are selectively favored on the host level. Thus, our results emphasize the importance of selection for shaping the DNA base composition of extrachromosomal genetic elements.

evolutionary biology↗

bioRxiv2018

Chlamydia muridarum infection differentially changes smooth muscle contractility and responses to prostaglandins in uterus and cervix

Chlamydia trachomatis infection is a primary cause of reproductive tract diseases including chronic pelvic pain and infertility. Previous studies showed that this infection alters physiological activities in mouse oviducts. Whether this occurs in the uterus and cervix has never been investigated. This study characterized the physiological activity of the uterus and the cervix in a Chlamydia muridarum (Cmu) mouse model of reproductive tract infection. Uterine or cervix smooth muscle contractility, responses to oxytocin or prostaglandins (PGF2 and PGE2) and mRNA expression of oxytocin and PG receptors were assessed 14 days post infection. Cmu infection did not affect the contractions of the uterine horn but significantly decreased the contraction amplitude of the cervix. Cmu infection did not alter the responses of uterine horn or cervix to oxytocin, however PGF2 induced contractions of the uterine horn, but not the cervix, were significantly increased following Cmu infection. PGE2 contraction amplitude in both the uterine horn and cervix was unaffected by Cmu infection. An upregulation of Ptgfr and a down-regulation of Ptegr4 mRNA expression was observed in the uterine horn following Cmu infection. These results indicate that Cmu infection alters contractility and prostaglandin signalling in the female reproductive tract but the effects are localised to specific regions.

physiology↗

bioRxiv2018

Differential induction of interferon stimulated genes between type I and type III interferons is independent of interferon receptor abundance

It is currently believed that type I and III interferons (IFNs) have redundant functions. However, the preferential distribution of type III IFN receptor on epithelial cells suggests functional differences at epithelial surfaces. Here, using human intestinal epithelial cells we could show that although both type I and type III IFNs confer an antiviral state to the cells, they do so with distinct kinetics. Type I IFN signaling is characterized by an acute strong induction of interferon stimulated genes (ISGs) and confers fast antiviral protection. On the contrary, the slow acting type III IFN mediated antiviral protection is characterized by a weaker induction of ISGs in a delayed manner compared to type I IFN. Moreover, while transcript profiling revealed that both IFNs induced a similar set of ISGs, their temporal expression strictly depended on the IFNs, thereby leading to unique antiviral environments. Using a combination of data-driven mathematical modeling and experimental validation, we addressed the molecular reason for this differential kinetic of ISG expression. We could demonstrate that these kinetic differences are intrinsic to each signaling pathway and not due to different expression levels of the corresponding IFN receptors. We report that type III IFN is specifically tailored to act in specific cell types not only due to the restriction of its receptor but also by providing target cells with a distinct antiviral environment compared to type I IFN. We propose that this specific environment is key at surfaces that are often challenged with the extracellular environment.\n\nAuthor summaryThe human intestinal tract plays two important roles in the body: first it is responsible for nutrient absorption and second it is the primary barrier which protects the human body from the outside environment. This complex tissue is constantly exposed to commensal bacteria and is often exposed to both bacterial and viral pathogens. To protect itself, the gut produces, among others, secreted agents called interferons which help to fight against pathogen attacks. There are several varieties (type I, II, and III) of interferons and our work aims at understanding how type I and III interferon act to protect human intestinal epithelial cells (hIECs) during viral infection. In this study, we confirmed that both interferons can protect hIECs against viral infection but with different kinetics. We determined that type I confer an antiviral state to hIECs faster than type III interferons. We uncovered that these differences were intrinsic to each pathway and not the result of differential abundance of the respective interferon receptors. The results of this study suggest that type III interferon may provide a different antiviral environment to the epithelium target cells which is likely critical for maintaining gut homeostasis. Our findings will also help us to design therapies to aid in controlling and eliminating viral infections of the gut.

immunology↗

bioRxiv2018

Wolbachia endosymbionts subvert the endoplasmic reticulum to acquire host membranes without triggering ER stress

The reproductive parasite Wolbachia are the most common endosymbionts on earth, present in a plethora of arthropod species. They have been introduced into mosquitos to successfully prevent the spread of vector-borne diseases, yet the strategies of host cell subversion underlying their obligate intracellular lifestyle remain to be explored in depth in order to gain insights into the mechanisms of pathogen-blocking. Like some other intracellular bacteria, Wolbachia reside in a host-derived vacuole in order to replicate and escape the immune surveillance. Using here the pathogen-blocking Wolbachia strain from Drosophila melanogaster, introduced into two different Drosophila cell lines, we show that Wolbachia subvert the endoplasmic reticulum to acquire their vacuolar membrane and colonize the host cell at high density. Wolbachia redistribute the endoplasmic reticulum to increase contact sites, and time lapse experiments reveal tight coupled dynamics suggesting important signalling events or nutrient uptake. They however do not affect the tubular or cisternal morphologies. A fraction of endoplasmic reticulum becomes clustered, allowing the endosymbionts to reside in between the endoplasmic reticulum and the Golgi apparatus, possibly modulating the traffic between these two organelles. Gene expression analyses and immunostaining studies suggest that Wolbachia achieve persistent infections at very high titers without triggering endoplasmic reticulum stress or enhanced ERAD-driven proteolysis, suggesting that amino acid salvage is achieved through modulation of other signalling pathways.\n\nAuthor summaryWolbachia are a genus of intracellular bacteria living in symbiosis with millions of arthropod species. They have the ability to block the transmission of arboviruses when introduced into mosquito vectors, by interfering with the cellular resources exploited by these viruses. Despite the biomedical interest of this symbiosis, little is known about the mechanisms by which Wolbachia survive and replicate in the host cell. We show here that the membrane composing the Wolbachia vacuole is acquired from the endoplasmic reticulum, a central organelle required for protein and lipid synthesis, and from which originates a vesicular trafficking toward the Golgi apparatus and the secretory pathway. Wolbachia modify the distribution of this organelle to increase their interactions with this source of membrane and likely of nutrients as well. In contrast to some intracellular pathogenic bacteria, the effect of Wolbachia on the cell homeostasis does not induce a stress on the endoplasmic reticulum. One of the consequences of such a stress would be an increased proteolysis used to relieve the cell from an excess of misfolded proteins. Incidentally, this shows that Wolbachia do not acquire amino acids from the host cell through this strategy.

cell biology↗

bioRxiv2018

Hexokinase and glucokinases are essential for fitness and virulence in the pathogenic yeast Candida albicans

Metabolic flexibility promotes infection and commensal colonization by the opportunistic pathogen Candida albicans. Yeast cell survival depends upon assimilation of fermentable and non-fermentable locally available carbon sources. Physiologically relevant sugars like glucose and fructose are present at low level in host niches. However, because glucose is the preferred substrate for energy and biosynthesis of structural components, its efficient metabolization is fundamental for the metabolic adaptation of the pathogen. We explored and characterized the C. albicans hexose kinase system composed of one hexokinase (CaHxk2) and two glucokinases (CaGlk1 and CaGlk4). Using a set of mutant strains, we found that hexose phosphorylation is mostly assured by CaHxk2, which sustains growth on hexoses. Our data on hexokinase and glucokinase expression point out an absence of cross regulation mechanisms at the transcription level and different regulatory pathways. In the presence of glucose, CaHxk2 migrates in the nucleus and contributes to the glucose repression signaling pathway. In addition, CaHxk2 participates to oxidative, osmotic and cell wall stress responses, while glucokinases are overexpressed under hypoxia. Hexose phosphorylation is a key step necessary for filamentation, that is affected in the hexokinase mutant. Virulence of this mutant is clearly impacted in the Galleria mellonella and macrophage models. Filamentation, glucose phosphorylation and stress response defects of the hexokinase mutant prevent host killing by C. albicans. By contributing to metabolic flexibility, stress answer response and morphogenesis, hexose kinase enzymes play an essential role in the virulence of C. albicans.\n\nAuthor summaryThe pathogenic yeast C. albicans is both a powerful commensal and pathogen of humans that can infect wide range of organs and body sites. To grow in its host and establish an infection, the pathogen must assimilate carbon from these heterogenous environments. C. albicans regulates central carbon metabolism in a niche-specific manner, activating alternatively gluconeogenesis, glyoxylate cycle and the glycolytic metabolism. For yeast and other microorganisms, glucose is the preferred carbon and energy source and its accurate detection and metabolism is essential. However, the glycolytic hexose kinase system has not been investigated yet in C. albicans. In this report, we showed that hexokinase and glucokinases contribute to the fitness and virulence of C. albicans. We revealed the main metabolic role of the hexokinase CaHxk2 which impacts on growth, glucose signalling, morphological transition and virulence. However, glucokinases contribute to the anoxic response and their implication in regulation processes is suggested.

microbiology↗