Periplasmic Acid Stress Increases Cell Division Asymmetry (Polar Aging) of Escherichia coli

Under certain kinds of cytoplasmic stress, Escherichia coli selectively reproduce by distributing the newer cytoplasmic components to new-pole cells while sequestering older, damaged components in cells inheriting the old pole. This phenomenon is termed polar aging or cell division asymmetry. It is unknown whether cell division asymmetry can arise from a periplasmic stress, such as the stress of extracellular acid, which is mediated by the periplasm. We tested the effect of periplasmic acid stress on growth and division of adherent single cells. We tracked individual cell lineages over five or more generations, using fluorescence microscopy with ratiometric pHluorin to measure cytoplasmic pH. Adherent colonies were perfused continually with LBK medium buffered at pH 6.00 or at pH 7.50; the external pH determines periplasmic pH. In each experiment, cell lineages were mapped to correlate division time, pole age and cell generation number. In colonies perfused at pH 6.0, the cells inheriting the oldest pole divided significantly more slowly than the cells inheriting the newest pole. In colonies perfused at pH 7.50 (near or above cytoplasmic pH), no significant cell division asymmetry was observed. Under both conditions (periplasmic pH 6.0 or pH 7.5) the cells maintained cytoplasmic pH values at 7.2–7.3. No evidence of cytoplasmic protein aggregation was seen. Thus, periplasmic acid stress leads to cell division asymmetry with minimal cytoplasmic stress.     

Inhibition of Influenza Virus Replication by Targeting Broad Host Cell Pathways

Antivirals that are currently used to treat influenza virus infections target components of the virus which can mutate rapidly. Consequently, there has been an increase in the number of resistant strains to one or many antivirals in recent years. Here we compared the antiviral effects of lysosomotropic alkalinizing agents (LAAs) and calcium modulators (CMs), which interfere with crucial events in the influenza virus replication cycle, against avian, swine, and human viruses of different subtypes in MDCK cells. We observed that treatment with LAAs, CMs, or a combination of both, significantly inhibited viral replication. Moreover, the drugs were effective even when they were administered 8 h after infection. Finally, analysis of the expression of viral acidic polymerase (PA) revealed that both drugs classes interfered with early events in the viral replication cycle. This study demonstrates that targeting broad host cellular pathways can be an efficient strategy to inhibit influenza replication. Furthermore, it provides an interesting avenue for drug development where resistance by the virus might be reduced since the virus is not targeted directly.     

MitoFates: Improved Prediction of Mitochondrial Targeting Sequences and Their Cleavage Sites

Mitochondria provide numerous essential functions for cells and their dysfunction leads to a variety of diseases. Thus, obtaining a complete mitochondrial proteome should be a crucial step toward understanding the roles of mitochondria. Many mitochondrial proteins have been identified experimentally but a complete list is not yet available. To fill this gap, methods to computationally predict mitochondrial proteins from amino acid sequence have been developed and are widely used, but unfortunately, their accuracy is far from perfect. Here we describe MitoFates, an improved prediction method for cleavable N-terminal mitochondrial targeting signals (presequences) and their cleavage sites. MitoFates introduces novel sequence features including positively charged amphiphilicity, presequence motifs, and position weight matrices modeling the presequence cleavage sites. These features are combined with classical ones such as amino acid composition and physico-chemical properties as input to a standard support vector machine classifier. On independent test data, MitoFates attains better performance than existing predictors in both detection of presequences and in predicting their cleavage sites. We used MitoFates to look for undiscovered mitochondrial proteins from 42,217 human proteins (including isoforms such as alternative splicing or translation initiation variants). MitoFates predicts 1167 genes to have at least one isoform with a presequence. Five-hundred and eighty of these genes were not annotated as mitochondrial in either UniProt or Gene Ontology. Interestingly, these include candidate regulators of parkin translocation to damaged mitochondria, and also many genes with known disease mutations, suggesting that careful investigation of MitoFates predictions may be helpful in elucidating the role of mitochondria in health and disease. MitoFates is open source with a convenient web server publicly available.Mitochondria not only function as the provider of ATP but also play crucial roles in the metabolism of amino acids and lipids, the biosynthesis of iron-sulfur clusters, cell signaling pathways, and apoptosis in eukaryotic cells. Moreover, mitochondrial dysfunction has been implicated in a wide variety of medical conditions such as muscle and neurodegenerative disease, cardiovascular disease, diabetes, and cancer (1).Obtaining the complete proteome of mitochondria is an essential step toward fully understanding its role in health and disease. To this end, ∼900 (in yeast) and 1100 (in mouse) mitochondrial proteins have been identified by large-scale proteomics analyses (2, 3); and compiled with other relevant mitochondrial proteomics data in useful databases such as MitoCarta (3) and MitoMiner (4). However, these lists are probably not yet complete, and indeed fungi and animal mitochondria have been estimated to host ∼1000 and ∼1500 distinct proteins, respectively (5). Thus, many mitochondrial proteins seem to remain undiscovered even in model organisms. If high accuracy can be achieved, prediction of mitochondrial proteins from primary sequence can save time and effort by identifying promising novel candidate mitochondrial proteins.The vast majority of mitochondrial proteins are encoded in the nuclear genome and imported by translocator complexes in the mitochondrial membranes. These mitochondrial proteins can be classified into two groups based on the type of targeting signal they contain: an N-terminal cleavable targeting signal (presequence); or a noncleavable, internal targeting signal (6). A recent proteomic analysis of yeast estimated that ∼70% of mitochondrial proteins possess a presequence (7). Thus, improved prediction of presequences should contribute to detecting undiscovered mitochondrial proteins.Presequences reside in the first 10–90 N-terminal residues, exhibit a high composition of arginine and near absence of negatively charged residues (8, 9). Proteins containing such presequences are translocated by the TOM and TIM protein complexes in the outer and the inner membranes, respectively (6, 10, 11). Tom20 and Tom22 in the TOM complex are reported to initiate import of these proteins by recognizing presequence segments capable of forming a local amphiphilic α-helical structure with hydrophobic residues on one face and positively charged residues on the opposite face (6, 12, 13). Widely used prediction tools such as MitoProt, TargetP, Predotar, and TPpred2 were developed with these properties of presequences in mind (14–17).The cleavage of mitochondrial protein presequences is an important event implicated in efficient protein import (18) and disease (19). Upon import into mitochondria, most presequences are cleaved off by the heterodimer mitochondrial processing peptidase (MPP)¹ in the matrix, and some of them subsequently further cleaved by intermediate peptidases such as Oct1 (20) and the recently discovered Icp55 (7). Although methods exist to predict these cleavage sites, their accuracy leaves much room for improvement (7, 21). Because the correct primary sequence of mature proteins is a prerequisite for precise structural modeling, improving the accuracy of cleavage site prediction should be useful for planning protein crystallography experiments or other structural studies of mitochondrial proteins. Also, accurate in silico prediction of the mature N-termini of mitochondrial proteins could in principle be used to improve the identification of N-terminal peptides in shotgun proteomics.In this study, we describe MitoFates, a novel method for mitochondrial presequence and cleavage site prediction. MitoFates formulates presequence prediction as a binary classification problem, employing a standard support vector machine (SVM) classifier. Our contribution is the preparation of an updated data set incorporating some recent proteomic data; and the selection of classical and novel sequence features such as amino acid composition, physicochemical properties, a novel positive amphiphilicity score, novel presequence motifs, and refined position weight matrices (PWMs) modeling peptidase cleavage sites. On the task of discriminating between presequences and nonpresequences, MitoFates achieves a true positive rate of 76% at a false positive rate of only 1.7%, improving significantly on previous methods. Moreover, MitoFates predicts the position of cleavage sites with an error rate of only ∼29% versus ∼47% for the best previous method.To investigate the potential of MitoFates to reveal interesting candidate mitochondrial proteins, we looked for undiscovered mitochondrial proteins among 42,217 human proteins (including isoforms such as alternative splicing or translation initiation variants), and obtained 580 candidate undiscovered mitochondrial proteins. Open source software downloads and a convenient MitoFates web server is available at http://mitf.cbrc.jp/MitoFates/.     

Host Cell Autophagy Is Induced by Toxoplasma gondii and Contributes to Parasite Growth

Autophagy has been shown to contribute to defense against intracellular bacteria and parasites. In comparison, the ability of such pathogens to manipulate host cell autophagy to their advantage has not been examined. Here we present evidence that infection by Toxoplasma gondii, an intracellular protozoan parasite, induces host cell autophagy in both HeLa cells and primary fibroblasts, via a mechanism dependent on host Atg5 but independent of host mammalian target of rapamycin suppression. Infection led to the conversion of LC3 to the autophagosome-associated form LC3-II, to the accumulation of LC3-containing vesicles near the parasitophorous vacuole, and to the relocalization toward the vacuole of structures labeled by the phosphatidylinositol 3-phosphate indicator YFP-2×FYVE. The autophagy regulator beclin 1 was concentrated in the vicinity of the parasitophorous vacuole in infected cells. Inhibitor studies indicated that parasite-induced autophagy is dependent on calcium signaling and on abscisic acid. At physiologically relevant amino acid levels, parasite growth became defective in Atg5-deficient cells, indicating a role for host cell autophagy in parasite recovery of host cell nutrients. A flow cytometric analysis of cell size as a function of parasite content revealed that autophagy-dependent parasite growth correlates with autophagy-dependent consumption of host cell mass that is dependent on parasite progression. These findings indicate a new role for autophagy as a pathway by which parasites may effectively compete with the host cell for limiting anabolic resources.Macroautophagy (hereafter referred to as autophagy) is a major catabolic process in which cytosolic constituents are sequestered within double-membraned vesicles (autophagosomes) and subsequently delivered to lysosomes for degradation. Current evidence indicates at least two distinct functions for this process. On the one hand, autophagy can be up-regulated under nutrient-limiting conditions to increase nutrient supply via recycling of the products of autophagic degradation, which may be exported from the lysosome (1). The up-regulation of autophagy upon starvation is thought to be mediated by the suppression of signaling through the mTOR pathway (2). On the other hand, autophagy can serve to maintain cellular homeostasis by facilitating the removal of damaged or deleterious elements, such as misfolded protein aggregates (3). An important example of the latter function is the role of autophagy in restricting the growth of intracellular pathogens, including both free bacteria that have escaped into host cytosol, such as group A Streptococcus, and pathogens, such as Mycobacterium tuberculosis, that reside in parasitophorous vacuoles in macrophages (4, 5). In macrophages infected with Toxoplasma gondii, fusion of the parasitophorous vacuole with lysosomes can be induced in an autophagy-dependent manner when host cell anti-parasitic function is activated via CD40 (6). Autophagy as a component of host defense may be up-regulated by inflammatory agents such as lipopolysaccharide (7) and interferon-γ (8).Although the clearance function of autophagy may enhance pathogen killing in host cells that have been activated to generate antimicrobial or antiparasitic function, in permissive host cells, in which the pathogen is less susceptible to sequestration by the autophagosome, autophagy may conceivably play a quite different role. Modulation of the balance between anabolic and catabolic processes may affect the outcome of competition between pathogen and host cell for limiting nutrients. In particular, the nutritive function of autophagy could favor pathogen expansion by providing greater access to host cell biomass. The intracellular apicomplexan parasite, T. gondii, is a suitable agent for the investigation of this hypothesis, because it has been shown to be highly dependent on its host cell for the supply of several nutrients, including amino acids (9), lipids (10), and purines (11). T. gondii replicates within a parasitophorous vacuole that, in permissive host cells, is protected from lysosomal fusion. Recent evidence indicates that in such permissive cells, in which the parasite can differentiate into bradyzoites associated with chronic infection, the pathogen is able to actively sequester host cell lysosome-derived vesicles, thereby potentially gaining access to their contents (12).The ability of intracellular parasites to regulate host cell autophagy has been little examined, and there is also little information with respect to the impact of these pathogens on host cell signals that potentially affect the autophagic pathway. In addition to mTOR, these include calcium ions, which have been implicated in autophagy induced by endoplasmic reticulum stress (13). In this study, we provide evidence that T. gondii induces host cell autophagy by a mechanism dependent on calcium but independent of mTOR and that it exploits the nutritive function of host autophagy to enhance its proliferation.     

Differential Inhibition of Cell Division in Euglena gracilis by Cysteamine and Cystamine

Cysteamine (2-aminoethanethiol) inhibited cell division in synchronously dividing cultures of Euglena gracilis at relatively low concentrations (0.005 M), Cystamine (2,2′-dithiobis(ethylamine). however, was only partially inhibitory at high concentrations (0.1 M). This differential inhibition may reflect certain unique features of nuclear division in euglenoid flagellates.     

Trypanosoma cruzi Cell Death Induced by the Morita-Baylis-Hillman Adduct 3-Hydroxy-2-Methylene-3-(4-Nitrophenylpropanenitrile)

Chagas disease, caused by the protozoan Trypanosoma cruzi, remains a serious health concern due to the lack of effective vaccines or satisfactory treatment. In the search for new compounds against this neglected disease, we have previously demonstrated that the compound 3-Hydroxy-2-methylene-3-(4-nitrophenylpropanenitrile) (MBHA3), derived from the Morita-Baylis-Hillman reaction, effectively caused a loss of viability in both the epimastigote and trypomastigote forms. However, the mechanisms of parasite death elicited by MBHA3 remain unknown. The aim of this study was to better understand the morphophysiological changes and the mechanism of cell death induced by MBHA3 treatment on T. cruzi. To perform this analysis, we used confocal microscopy and flow cytometry to monitor the fluorescent probes such as annexin-V/propidium iodide (AV/PI), calcein-AM/ethidium homodimer (CA/EH), acridine orange (AO) and rhodamine 123 (Rho 123). Lower concentrations of MBHA3 led to alterations in the mitochondrial membrane potential and AO labeling, but did not decrease the viability of the epimastiogote forms, as determined by the CA/EH and AV/PI assays. Conversely, treatment with higher concentrations of MBHA3 led to extensive plasma membrane damage, loss of mitochondrion membrane potential, DNA fragmentation and acidification of the cytoplasm. Our findings suggest that at higher concentrations, MBHA3 induces T. cruzi epimastigote death by necrosis in a mitochondrion-dependent manner.     

Temporal Controls of the Asymmetric Cell Division Cycle in Caulobacter crescentus

The asymmetric cell division cycle of Caulobacter crescentus is orchestrated by an elaborate gene-protein regulatory network, centered on three major control proteins, DnaA, GcrA and CtrA. The regulatory network is cast into a quantitative computational model to investigate in a systematic fashion how these three proteins control the relevant genetic, biochemical and physiological properties of proliferating bacteria. Different controls for both swarmer and stalked cell cycles are represented in the mathematical scheme. The model is validated against observed phenotypes of wild-type cells and relevant mutants, and it predicts the phenotypes of novel mutants and of known mutants under novel experimental conditions. Because the cell cycle control proteins of Caulobacter are conserved across many species of alpha-proteobacteria, the model we are proposing here may be applicable to other genera of importance to agriculture and medicine (e.g., Rhizobium, Brucella).     

Inhibition of Pseudomonas aeruginosa LPS‐Induced airway inflammation by RIPK3 in human airway

Although the physiological function of receptor‐interacting protein kinase (RIPK) 3 has emerged as a critical mediator of programmed necrosis/necroptosis, the intracellular role it plays as an attenuator in human lungs and human bronchial epithelia remains unclear. Here, we show that the expression of RIPK3 dramatically decreased in the inflamed tissues of human lungs, and moved from the nucleus to the cytoplasm. The overexpression of RIPK3 dramatically increased F‐actin formation and decreased the expression of genes for pro‐inflammatory cytokines (IL‐6 and IL‐1β), but not siRNA‐RIPK3. Interestingly, whereas RIPK3 was bound to histone 1b without LPS stimulation, the interaction between them was disrupted after 15 min of LPS treatment. Histone methylation could not maintain the binding of RIPK3 and activated movement towards the cytoplasm. In the cytoplasm, overexpressed RIPK3 continuously attenuated pro‐inflammatory cytokine gene expression by inhibiting NF‐κB activation, preventing the progression of inflammation during Pseudomonas aeruginosa infection. Our data indicated that RIPK3 is critical for the regulation of the LPS‐induced inflammatory microenvironment. Therefore, we suggest that RIPK3 is a potential therapeutic candidate for bacterial infection‐induced pulmonary inflammation.     

Morphology of Nurse Cell Nuclei Induced by Meloidodera floridensis: A Graphics Application

The highly irregular distribution of nuclear material in the host nurse cell induced by Meloidodera floridensis has made it difficult to interpret the number of nuclei from two-dimensional micrographs alone. The primary goal of this investigation was to determine the distribution of nuclear material from a three-dimensional solid surface model of the nurse cell nucleus. This model demonstrated the continuity of nuclear material as a single highly irregular nucleus. Custom computer graphics programs were written to accept digitized tracings of nuclear material. From these digitized tracings, a wireframe or polygonalized mesh model was generated. The model was shaded, colored, rotated, and analyzed. This technique provides controlled transparency of the model to display nucleoli within the nucleus. Photographs of the computer screen, color printouts, and video recordings were used to record final results. These refined computer graphic tools have a range of applications in nematode host-parasite relationships, ontogeny, and morphology.     

Inhibition by Liposomal Cholesterol of Streptococcus Pneumoniae Induced Lung Epithelial Cell Damage

Abstract

Streptococcus pneumoniae was shown to be capable of lysing A549 cells in culture. Membrane damage to cells as assessed by trypan blue exclusion increased with increasing concentration of bacteria. After 45 min of incubation with 7.5 × 10⁸ bacteria/ml less than 20% of A549 cells excluded trypan blue. The lytic activity of S. pneumoniae was inhibited by phosphatidylcholine liposomes containing cholesterol. Using an haemolysis assay and S. pneumoniae's culture filtrates, the efficiency of the anti-lytic activity of liposomes was found to be distearoylphosphatidylcholine (DSPC) > dipalmitoylphosphatidylcho-line (DPPC) > dimyristoylphosphatidylcholine (DMPC). Furthermore, the anti-lytic activity also depended on the cholesterol content in a non-trivial manner. There was no protection against haemolytic activity at cholesterol content of less than 20% for DSPC and 35 mole% for DPPC and DMPC liposomes respectively. Above these threshold values inhibition of lytic activity increased sharply. In agreement with the haemolysis results, A549 cells were protected by liposomes against the lytic activity of S. pneumoniae with the efficiency also being DSPC > DPPC > DMPC. Clearly the efficiency of liposomal cholesterol is increased with increasing gel to liquid crystalline phase transition temperature of the lipid matrix. The results suggest that liposomal cholesterol may be used to protect the host against cell damage caused by S. pneumoniae.     

Inhibition of Cell Division and DNA Synthesis by Gibberellin in Isolated Zinnia Mesophyll Cells

A culture system of isolated mesophyll cells of Zinnia eleganswas used to examine the action of gibberellic acid (GA) on celldivision. Isolated Zinnia mesophyll cells cultured in a mediumcontaining auxin and cytokinin reinitiated cell division ina partly synchronized manner. When mesophyll cells isolatedfrom 21-day-old seedlings were used, GA added to the culturemedium at concentrations of 1 x 10^–6 M or higher suppressedthe initial rise in the number of divided cells. Tracer experimentswith [³H]-dThd revealed that GA treatment inhibited the incorporationof [³H]-dThd into DNA in the nucleus without inhibiting theuptake of [³H]-dThd into the cells, indicating that GA inhibitedDNA synthesis. GA applied at 48 h inhibited the incorporationof [³H]-dThd into DNA during the following 24 h, but GA appliedat 72 h did not inhibit the incorporation during the subsequent24 h. This suggests that GA affects the process of reinitiationof DNA synthesis, but does not affect DNA synthesis once cellshave become proliferative. (Received January 14, 1986; Accepted March 31, 1986)     

A Trisubstituted Benzimidazole Cell Division Inhibitor with Efficacy against Mycobacterium tuberculosis

Trisubstituted benzimidazoles have demonstrated potency against Gram-positive and Gram-negative bacterial pathogens. Previously, a library of novel trisubstituted benzimidazoles was constructed for high throughput screening, and compounds were identified that exhibited potency against M. tuberculosis H37Rv and clinical isolates, and were not toxic to Vero cells. A new series of 2-cyclohexyl-5-acylamino-6-N, N-dimethylaminobenzimidazoles derivatives has been developed based on SAR studies. Screening identified compounds with potency against M. tuberculosis. A lead compound from this series, SB-P17G-A20, was discovered to have an MIC of 0.16 µg/mL and demonstrated efficacy in the TB murine acute model of infection based on the reduction of bacterial load in the lungs and spleen by 1.73±0.24 Log₁₀ CFU and 2.68±Log₁₀ CFU, respectively, when delivered at 50 mg/kg by intraperitoneal injection (IP) twice daily (bid). The activity of SB-P17G-A20 was determined to be concentration dependent and to have excellent stability in mouse and human plasma, and liver microsomes. Together, these studies demonstrate that SB-P17G-A20 has potency against M. tuberculosis clinical strains with varying susceptibility and efficacy in animal models of infection, and that trisubstituted benzimidazoles continue to be a platform for the development of novel inhibitors with efficacy.     

Analysis of Cell Division and Elongation Underlying the Developmental Acceleration of Root Growth in Arabidopsis thaliana

To investigate the relation between cell division and expansion in the regulation of organ growth rate, we used Arabidopsis thaliana primary roots grown vertically at 20°C with an elongation rate that increased steadily during the first 14 d after germination. We measured spatial profiles of longitudinal velocity and cell length and calculated parameters of cell expansion and division, including rates of local cell production (cells mm⁻¹ h⁻¹) and cell division (cells cell⁻¹ h⁻¹). Data were obtained for the root cortex and also for the two types of epidermal cell, trichoblasts and atrichoblasts. Accelerating root elongation was caused by an increasingly longer growth zone, while maximal strain rates remained unchanged. The enlargement of the growth zone and, hence, the accelerating root elongation rate, were accompanied by a nearly proportionally increased cell production. This increased production was caused by increasingly numerous dividing cells, whereas their rates of division remained approximately constant. Additionally, the spatial profile of cell division rate was essentially constant. The meristem was longer than generally assumed, extending well into the region where cells elongated rapidly. In the two epidermal cell types, meristem length and cell division rate were both very similar to that of cortical cells, and differences in cell length between the two epidermal cell types originated at the apex of the meristem. These results highlight the importance of controlling the number of dividing cells, both to generate tissues with different cell lengths and to regulate the rate of organ enlargement.