scholarly journals Carbonic Anhydrase Is Essential for Streptococcus pneumoniae Growth in Environmental Ambient Air

2010 ◽  
Vol 192 (15) ◽  
pp. 4054-4062 ◽  
Author(s):  
Peter Burghout ◽  
Lorelei E. Cron ◽  
Henrik Gradstedt ◽  
Beatriz Quintero ◽  
Elles Simonetti ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Carbonic Anhydrase ◽  
Unsaturated Fatty Acids ◽  
De Novo ◽  
Ambient Air ◽  
Host Cells ◽  
Ambient Conditions ◽  
Growth And Survival ◽  
Cellular Processes

ABSTRACT The respiratory tract pathogen Streptococcus pneumoniae needs to adapt to the different levels of carbon dioxide (CO2) it encounters during transmission, colonization, and infection. Since CO2 is important for various cellular processes, factors that allow optimal CO2 sequestering are likely to be important for pneumococcal growth and survival. In this study, we showed that the putative pneumococcal carbonic anhydrase (PCA) is essential for in vitro growth of S. pneumoniae under the CO2-poor conditions found in environmental ambient air. Enzymatic analysis showed that PCA catalyzes the reversible hydration of CO2 to bicarbonate (HCO3 −), an essential step to prevent the cellular release of CO2. The addition of unsaturated fatty acids (UFAs) reversed the CO2-dependent in vitro growth inhibition of S. pneumoniae strains lacking the pca gene (Δpca), indicating that PCA-mediated CO2 fixation is at least associated with HCO3 −-dependent de novo biosynthesis of UFAs. Besides being necessary for growth in environmental ambient conditions, PCA-mediated CO2 fixation pathways appear to be required for intracellular survival in host cells. This effect was especially pronounced during invasion of human brain microvascular endothelial cells (HBMEC) and uptake by murine J774 macrophage cells but not during interaction of S. pneumoniae with Detroit 562 pharyngeal epithelial cells. Finally, the highly conserved pca gene was found to be invariably present in both CO2-independent and naturally circulating CO2-dependent strains, suggesting a conserved essential role for PCA and PCA-mediated CO2 fixation pathways for pneumococcal growth and survival.

scholarly journals In Vitro Activities of ER-119884 and E5700, Two Potent Squalene Synthase Inhibitors, against Leishmania amazonensis: Antiproliferative, Biochemical, and Ultrastructural Effects

2008 ◽  
Vol 52 (11) ◽  
pp. 4098-4114 ◽  
Author(s):  
Juliany Cola Fernandes Rodrigues ◽  
Juan Luis Concepcion ◽  
Carlos Rodrigues ◽  
Aura Caldera ◽  
Julio A. Urbina ◽  
...  
Keyword(s):  
De Novo ◽  
Squalene Synthase ◽  
Leishmania Amazonensis ◽  
Host Cells ◽  
Fluorescent Label ◽  
Trypan Blue Exclusion ◽  
Cytoskeleton Organization ◽  
Cellular Processes ◽  
Intracellular Parasites

ABSTRACT ER-119884 and E5700, novel arylquinuclidine derivatives developed as cholesterol-lowering agents, were potent in vitro growth inhibitors of both proliferative stages of Leishmania amazonensis, the main causative agent of cutaneous leishmaniasis in South America, with the 50% inhibitory concentrations (IC50s) being in the low-nanomolar to subnanomolar range. The compounds were very potent noncompetitive inhibitors of native L. amazonensis squalene synthase (SQS), with inhibition constants also being in the nanomolar to subnanomolar range. Growth inhibition was strictly associated with the depletion of the parasite's main endogenous sterols and the concomitant accumulation of exogenous cholesterol. Using electron microscopy, we identified the intracellular structures affected by the compounds. A large number of lipid inclusions displaying different shapes and electron densities were observed after treatment with both SQS inhibitors, and these inclusions were associated with an intense disorganization of the membrane that surrounds the cell body and flagellum, as well as the endoplasmic reticulum and the Golgi complex. Cells treated with ER-119884 but not those treated with E5700 had an altered cytoskeleton organization due to an abnormal distribution of tubulin, and many were arrested at cytokinesis. A prominent contractile vacuole and a phenotype typical of programmed cell death were frequently found in drug-treated cells. The selectivity of the drugs was demonstrated with the JC-1 mitochondrial fluorescent label and by trypan blue exclusion tests with macrophages, which showed that the IC50s against the host cells were 4 to 5 orders of magnitude greater that those against the intracellular parasites. Taken together, our results show that ER-119884 and E5700 are unusually potent and selective inhibitors of the growth of Leishmania amazonensis, probably because of their inhibitory effects on de novo sterol biosynthesis at the level of SQS, but some of our observations indicate that ER-119884 may also interfere with other cellular processes.

scholarly journals Stereotypic neutralizing VH antibodies against SARS-CoV-2 spike protein receptor binding domain in COVID-19 patients and healthy individuals

2021 ◽  
pp. eabd6990
Author(s):  
Sang Il Kim ◽  
Jinsung Noh ◽  
Sujeong Kim ◽  
Younggeun Choi ◽  
Duck Kyun Yoo ◽  
...  
Keyword(s):  
B Cells ◽  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
De Novo ◽  
Binding Domain ◽  
Host Cells ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Healthy Individuals

Stereotypic antibody clonotypes exist in healthy individuals and may provide protective immunity against viral infections by neutralization. We observed that 13 out of 17 patients with COVID-19 had stereotypic variable heavy chain (VH) antibody clonotypes directed against the receptor-binding domain (RBD) of SARS-CoV-2 spike protein. These antibody clonotypes were comprised of immunoglobulin heavy variable (IGHV)3-53 or IGHV3-66 and immunoglobulin heavy joining (IGHJ)6 genes. These clonotypes included IgM, IgG3, IgG1, IgA1, IgG2, and IgA2 subtypes and had minimal somatic mutations, which suggested swift class switching after SARS-CoV-2 infection. The different immunoglobulin heavy variable chains were paired with diverse light chains resulting in binding to the RBD of SARS-CoV-2 spike protein. Human antibodies specific for the RBD can neutralize SARS-CoV-2 by inhibiting entry into host cells. We observed that one of these stereotypic neutralizing antibodies could inhibit viral replication in vitro using a clinical isolate of SARS-CoV-2. We also found that these VH clonotypes existed in six out of 10 healthy individuals, with IgM isotypes predominating. These findings suggest that stereotypic clonotypes can develop de novo from naïve B cells and not from memory B cells established from prior exposure to similar viruses. The expeditious and stereotypic expansion of these clonotypes may have occurred in patients infected with SARS-CoV-2 because they were already present.

scholarly journals A New MAP Kinase Protein Involved in Estradiol-Stimulated Reproduction of the Helminth ParasiteTaenia crassiceps

2010 ◽  
Vol 2010 ◽  
pp. 1-11 ◽  
Author(s):  
Galileo Escobedo ◽  
Gloria Soldevila ◽  
Guadalupe Ortega-Pierres ◽  
Jesús Ramsés Chávez-Ríos ◽  
Karen Nava ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Map Kinases ◽  
Host Cells ◽  
Cross Contamination ◽  
Flow Cytometry Analysis ◽  
Cellular Processes ◽  
17Β Estradiol ◽  
Activation Motif

MAP kinases (MAPK) are involved in the regulation of cellular processes such as reproduction and growth. In parasites, the role of MAPK has been scarcely studied. Here, we describe the participation of an ERK-like protein in estrogen-dependent reproduction of the helminth parasiteTaenia crassiceps. Our results show that 17β-estradiol induces a concentration-dependent increase in the bud number of in vitro cultured cysticerci. If parasites are also incubated in presence of an ERK-inhibitor, the stimulatory effect of estrogen is blocked. The expression of ERK-like mRNA and its corresponding protein was detected in the parasite. The ERK-like protein was over-expressed by all treatments. Nevertheless, a strong induction of phosphorylation of this protein was observed only in response to 17β-estradiol. Cross-contamination by host cells was discarded by flow cytometry analysis. Parasite cells expressing the ERK-like protein were exclusively located at the subtegument tissue by confocal microscopy. Finally, the ERK-like protein was separated by bidimensional electrophoresis and then sequenced, showing the conserved TEY activation motif, typical of all known ERK 1/2 proteins. Our results show that an ERK-like protein is involved in the molecular signalling during the interaction between the host andT. crassiceps, and may be considered as target for anti-helminth drugs design.

scholarly journals Thiol Peroxidase Is an Important Component of Streptococcus pneumoniae in Oxygenated Environments

2012 ◽  
Vol 80 (12) ◽  
pp. 4333-4343 ◽  
Author(s):  
Barak Hajaj ◽  
Hasan Yesilkaya ◽  
Rachel Benisty ◽  
Maayan David ◽  
Peter W. Andrew ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Mass Spectrometry Analysis ◽  
Activity Levels ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Cellular Processes ◽  
Thiol Peroxidase ◽  
Fine Tune

ABSTRACTStreptococcus pneumoniaeis an aerotolerant Gram-positive bacterium that causes an array of diseases, including pneumonia, otitis media, and meningitis. During aerobic growth,S. pneumoniaeproduces high levels of H2O2. SinceS. pneumoniaelacks catalase, the question of how it controls H2O2levels is of critical importance. Thepsalocus encodes an ABC Mn2+-permease complex (psaBCA) and a putative thiol peroxidase,tpxD. This study shows thattpxDencodes a functional thiol peroxidase involved in the adjustment of H2O2homeostasis in the cell. Kinetic experiments showed that recombinant TpxD removed H2O2efficiently. However,in vivoexperiments revealed that TpxD detoxifies only a fraction of the H2O2generated by the pneumococcus. Mass spectrometry analysis demonstrated that TpxD Cys58undergoes selective oxidationin vivo, under conditions where H2O2is formed, confirming the thiol peroxidase activity. Levels of TpxD expression and synthesisin vitrowere significantly increased in cells grown under aerobic versus anaerobic conditions. The challenge with D39 and TIGR4 with H2O2resulted intpxDupregulation, whilepsaBCAexpression was oppositely affected. However, the challenge of ΔtpxDmutants with H2O2did not affectpsaBCA, implying that TpxD is involved in the regulation of thepsaoperon, in addition to its scavenging activity. Virulence studies demonstrated a notable difference in the survival time of mice infected intranasally with D39 compared to that of mice infected intranasally with D39ΔtpxD. However, when bacteria were administered directly into the blood, this difference disappeared. The findings of this study suggest that TpxD constitutes a component of the organism's fundamental strategy to fine-tune cellular processes in response to H2O2.

scholarly journals Francisella tularensis ΔpyrF Mutants Show that Replication in Nonmacrophages Is Sufficient for Pathogenesis In Vivo

2010 ◽  
Vol 78 (6) ◽  
pp. 2607-2619 ◽  
Author(s):  
Joseph Horzempa ◽  
Dawn M. O'Dee ◽  
Robert M. Q. Shanks ◽  
Gerard J. Nau
Keyword(s):  
Francisella Tularensis ◽  
De Novo ◽  
Human Fibroblasts ◽  
Host Cells ◽  
Hek 293 ◽  
293 Cells ◽  
Animal Infection ◽  
Schu S4

ABSTRACT The pathogenesis of Francisella tularensis has been associated with this bacterium's ability to replicate within macrophages. F. tularensis can also invade and replicate in a variety of nonphagocytic host cells, including lung and kidney epithelial cells and hepatocytes. As uracil biosynthesis is a central metabolic pathway usually necessary for pathogens, we characterized ΔpyrF mutants of both F. tularensis LVS and Schu S4 to investigate the role of these mutants in intracellular growth. As expected, these mutant strains were deficient in de novo pyrimidine biosynthesis and were resistant to 5-fluoroorotic acid, which is converted to a toxic product by functional PyrF. The F. tularensis ΔpyrF mutants could not replicate in primary human macrophages. The inability to replicate in macrophages suggested that the F. tularensis ΔpyrF strains would be attenuated in animal infection models. Surprisingly, these mutants retained virulence during infection of chicken embryos and in the murine model of pneumonic tularemia. We hypothesized that the F. tularensis ΔpyrF strains may replicate in cells other than macrophages to account for their virulence. In support of this, F. tularensis ΔpyrF mutants replicated in HEK-293 cells and normal human fibroblasts in vitro. Moreover, immunofluorescence microscopy showed abundant staining of wild-type and mutant bacteria in nonmacrophage cells in the lungs of infected mice. These findings indicate that replication in nonmacrophages contributes to the pathogenesis of F. tularensis.

scholarly journals Transcriptional Regulation and Signature Patterns Revealed by Microarray Analyses of Streptococcus pneumoniae R6 Challenged with Sublethal Concentrations of Translation Inhibitors

2003 ◽  
Vol 185 (1) ◽  
pp. 359-370 ◽  
Author(s):  
Wai-Leung Ng ◽  
Krystyna M. Kazmierczak ◽  
Gregory T. Robertson ◽  
Raymond Gilmour ◽  
Malcolm E. Winkler
Keyword(s):  
Streptococcus Pneumoniae ◽  
Amino Acid ◽  
Gene Cluster ◽  
Mode Of Action ◽  
De Novo ◽  
Regulatory Protein ◽  
Purine Nucleotide ◽  
Cellular Processes ◽  
Microarray Analyses ◽  
Sublethal Concentrations

ABSTRACT The effects of sublethal concentrations of four different classes of translation inhibitors (puromycin, tetracycline, chloramphenicol, and erythromycin) on global transcription patterns of Streptococcus pneumoniae R6 were determined by microarray analyses. Consistent with the general mode of action of these inhibitors, relative transcript levels of genes that encode ribosomal proteins and translation factors or that mediate tRNA charging and amino acid biosynthesis increased or decreased, respectively. Transcription of the heat shock regulon was induced only by puromycin or streptomycin treatment, which lead to truncation or mistranslation, respectively, but not by other antibiotics that block translation, transcription, or amino acid charging of tRNA. In contrast, relative transcript amounts of certain genes involved in transport, cellular processes, energy metabolism, and purine nucleotide (pur) biosynthesis were changed by different translation inhibitors. In particular, transcript amounts from a pur gene cluster and from purine uptake and salvage genes were significantly elevated by several translation inhibitors, but not by antibiotics that target other cellular processes. Northern blotting confirmed increased transcript amounts from part of the pur gene cluster in cells challenged by translation inhibitors and revealed the presence of a 10-kb transcript. Purine metabolism genes were negatively regulated by a homologue of the PurR regulatory protein, and full derepression in a ΔpurR mutant depended on optimal translation. Unexpectedly, hierarchical clustering of the microarray data distinguished among the global transcription patterns caused by antibiotics that inhibit different steps in the translation cycle. Together, these results show that there is extensive control of transcript amounts by translation in S. pneumoniae, especially for de novo purine nucleotide biosynthesis. In addition, these global transcription patterns form a signature that can be used to classify the mode of action and potential mechanism of new translation inhibitors.

scholarly journals Deubiquitinating Function of Adenovirus Proteinase

2002 ◽  
Vol 76 (12) ◽  
pp. 6323-6331 ◽  
Author(s):  
Maxim Y. Balakirev ◽  
Michel Jaquinod ◽  
Arthur L. Haas ◽  
Jadwiga Chroboczek
Keyword(s):  
Structural Model ◽  
Proteolytic Processing ◽  
Host Cells ◽  
Cellular Proteins ◽  
Viral Gene ◽  
Cellular Processes ◽  
Infected Cells ◽  
Substrate Binding Sites

ABSTRACT The invasion strategy of many viruses involves the synthesis of viral gene products that mimic the functions of the cellular proteins and thus interfere with the key cellular processes. Here we show that adenovirus infection is accompanied by an increased ubiquitin-cleaving (deubiquitinating) activity in the host cells. Affinity chromatography on ubiquitin aldehyde (Ubal), which was designed to identify the deubiquitinating proteases, revealed the presence of adenovirus L3 23K proteinase (Avp) in the eluate from adenovirus-infected cells. This proteinase is known to be necessary for the processing of viral precursor proteins during virion maturation. We show here that in vivo Avp deubiquitinates a number of cellular proteins. Analysis of the substrate specificity of Avp in vitro demonstrated that the protein deubiquitination by this enzyme could be as efficient as proteolytic processing of viral proteins. The structural model of the Ubal-Avp interaction revealed some similarity between S1-S4 substrate binding sites of Avp and ubiquitin hydrolases. These results may reflect the acquisition of an advantageous property by adenovirus and may indicate the importance of ubiquitin pathways in viral infection.

scholarly journals SLC25A1 promotes tumor growth and survival by reprogramming energy metabolism in colorectal cancer

2021 ◽  
Vol 12 (12) ◽  
Author(s):  
Ying Yang ◽  
Jiaxing He ◽  
Bo Zhang ◽  
Zhansheng Zhang ◽  
Guozhan Jia ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Lipid Metabolism ◽  
Energy Metabolism ◽  
Cell Apoptosis ◽  
De Novo ◽  
Lipid Synthesis ◽  
Growth And Survival ◽  
Metabolism Regulation

AbstractAbnormal lipid metabolism has been commonly observed in various human cancers, including colorectal cancer (CRC). The mitochondrial citrate carrier SLC25A1 (also known as mitochondrial citrate/isocitrate carrier, CIC), has been shown to play an important role in lipid metabolism regulation. Our bioinformatics analysis indicated that SLC25A1 was markedly upregulated in CRC. However, the role of SLC25A1 in the pathogenesis and aberrant lipid metabolism in CRC remain unexplored. Here, we found that SLC25A1 expression was significantly increased in tumor samples of CRC as compared with paired normal samples, which is associated with poor survival in patients with CRC. Knockdown of SLC25A1 significantly inhibited the growth of CRC cells by suppressing the progression of the G1/S cell cycle and inducing cell apoptosis both in vitro and in vivo, whereas SLC25A1 overexpression suppressed the malignant phenotype. Additionally, we demonstrated that SLC25A1 reprogrammed energy metabolism to promote CRC progression through two mechanisms. Under normal conditions, SLC25A1 increased de novo lipid synthesis to promote CRC growth. During metabolic stress, SLC25A1 increased oxidative phosphorylation (OXPHOS) to protect protects CRC cells from energy stress-induced cell apoptosis. Collectively, SLC25A1 plays a pivotal role in the promotion of CRC growth and survival by reprogramming energy metabolism. It could be exploited as a novel diagnostic marker and therapeutic target in CRC.

scholarly journals Involvement of potD in Streptococcus pneumoniae Polyamine Transport and Pathogenesis

2006 ◽  
Vol 74 (1) ◽  
pp. 352-361 ◽  
Author(s):  
D. Ware ◽  
Y. Jiang ◽  
W. Lin ◽  
E. Swiatlo
Keyword(s):  
Streptococcus Pneumoniae ◽  
De Novo ◽  
Optimal Growth ◽  
Wild Type ◽  
Polyamine Biosynthesis ◽  
Transport Pathways ◽  
Rich Media ◽  
Polyamine Transport ◽  
Inoculation Route

ABSTRACT Polyamines such as putrescine, spermidine, and cadaverine are small, polycationic molecules that are required for optimal growth in all cells. The intracellular concentrations of these molecules are maintained by de novo synthesis and transport pathways. The human pathogen Streptococcus pneumoniae possesses a putative polyamine transporter (pot) operon that consists of the four pot-specific genes potABCD. The studies presented here examined the involvement of potD in polyamine transport and in pneumococcal pathogenesis. A potD-deficient mutant was created in the mouse-virulent serotype 3 strain WU2 by insertion duplication mutagenesis. The growth of the WU2ΔpotD mutant was identical to that of the wild-type strain WU2 in vitro in rich media. However, WU2ΔpotD possessed severely delayed growth compared to wild-type WU2 in the presence of the polyamine biosynthesis inhibitors DFMO (α-dimethyl-fluroornitithine) and MGBG [methylgloxal-bis (guanyl hydrazone)]. The mutant strain also showed a significant attenuation in virulence within murine models of systemic and pulmonary infection regardless of the inoculation route or location. These data suggest that potD is involved in pneumococcal polyamine transport and is important for pathogenesis within various infection models.

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