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1.
Nature ; 572(7771): 665-669, 2019 08.
Article in English | MEDLINE | ID: mdl-31435014

ABSTRACT

Intestinal commensal bacteria can inhibit dense colonization of the gut by vancomycin-resistant Enterococcus faecium (VRE), a leading cause of hospital-acquired infections1,2. A four-strained consortium of commensal bacteria that contains Blautia producta BPSCSK can reverse antibiotic-induced susceptibility to VRE infection3. Here we show that BPSCSK reduces growth of VRE by secreting a lantibiotic that is similar to the nisin-A produced by Lactococcus lactis. Although the growth of VRE is inhibited by BPSCSK and L. lactis in vitro, only BPSCSK colonizes the colon and reduces VRE density in vivo. In comparison to nisin-A, the BPSCSK lantibiotic has reduced activity against intestinal commensal bacteria. In patients at high risk of VRE infection, high abundance of the lantibiotic gene is associated with reduced density of E. faecium. In germ-free mice transplanted with patient-derived faeces, resistance to VRE colonization correlates with abundance of the lantibiotic gene. Lantibiotic-producing commensal strains of the gastrointestinal tract reduce colonization by VRE and represent potential probiotic agents to re-establish resistance to VRE.


Subject(s)
Bacteriocins/metabolism , Bacteriocins/pharmacology , Enterococcus faecium/drug effects , Lactococcus lactis/metabolism , Probiotics , Vancomycin Resistance/drug effects , Vancomycin-Resistant Enterococci/drug effects , Animals , Anti-Bacterial Agents/biosynthesis , Anti-Bacterial Agents/isolation & purification , Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/pharmacology , Bacteriocins/genetics , Bacteriocins/isolation & purification , Enterococcus faecium/growth & development , Enterococcus faecium/isolation & purification , Feces/microbiology , Female , Gastrointestinal Tract/drug effects , Gastrointestinal Tract/microbiology , Germ-Free Life , Gram-Positive Bacteria/drug effects , Gram-Positive Bacteria/growth & development , Humans , Lactococcus lactis/chemistry , Lactococcus lactis/growth & development , Lactococcus lactis/physiology , Mice , Microbial Sensitivity Tests , Microbiota/genetics , Nisin/chemistry , Nisin/pharmacology , Symbiosis/drug effects , Vancomycin/pharmacology , Vancomycin-Resistant Enterococci/growth & development , Vancomycin-Resistant Enterococci/isolation & purification
2.
PLoS Pathog ; 17(2): e1009309, 2021 02.
Article in English | MEDLINE | ID: mdl-33556154

ABSTRACT

Gram-negative pathogens, such as Klebsiella pneumoniae, remodel their outer membrane (OM) in response to stress to maintain its integrity as an effective barrier and thus to promote their survival in the host. The emergence of carbapenem-resistant K. pneumoniae (CR-Kp) strains that are resistant to virtually all antibiotics is an increasing clinical problem and OM impermeability has limited development of antimicrobial agents because higher molecular weight antibiotics cannot access sites of activity. Here, we demonstrate that TAM (translocation and assembly module) deletion increases CR-Kp OM permeability under stress conditions and enhances sensitivity to high-molecular weight antimicrobials. SILAC-based proteomic analyses revealed mis-localization of membrane proteins in the TAM deficient strain. Stress-induced sensitization enhances clearance of TAM-deficient CR-Kp from the gut lumen following fecal microbiota transplantation and from infection sites following pulmonary or systemic infection. Our study suggests that TAM, as a regulator of OM permeability, represents a potential target for development of agents that enhance the effectiveness of existing antibiotics.


Subject(s)
Adaptation, Physiological , Anti-Bacterial Agents/pharmacology , Bacterial Outer Membrane Proteins/metabolism , Carbapenem-Resistant Enterobacteriaceae/drug effects , Klebsiella Infections/drug therapy , Klebsiella pneumoniae/drug effects , Proteome/metabolism , Animals , Bacterial Outer Membrane Proteins/genetics , Carbapenems/pharmacology , Cell Membrane Permeability , Female , Klebsiella Infections/genetics , Klebsiella Infections/metabolism , Klebsiella Infections/microbiology , Mice , Mice, Inbred C57BL , Stress, Physiological
3.
4.
Hum Mol Genet ; 24(10): 2826-40, 2015 May 15.
Article in English | MEDLINE | ID: mdl-25652409

ABSTRACT

Neurons in the brain produce lamin C but almost no lamin A, a consequence of the removal of prelamin A transcripts by miR-9, a brain-specific microRNA. We have proposed that miR-9-mediated regulation of prelamin A in the brain could explain the absence of primary neurological disease in Hutchinson-Gilford progeria syndrome, a genetic disease caused by the synthesis of an internally truncated form of farnesyl-prelamin A (progerin). This explanation makes sense, but it is not entirely satisfying because it is unclear whether progerin-even if were expressed in neurons-would be capable of eliciting neuropathology. To address that issue, we created a new Lmna knock-in allele, Lmna(HG-C), which produces progerin transcripts lacking an miR-9 binding site. Mice harboring the Lmna(HG-C) allele produced progerin in neurons, but they had no pathology in the central nervous system. However, these mice invariably developed esophageal achalasia, and the enteric neurons and nerve fibers in gastrointestinal tract were markedly abnormal. The same disorder, achalasia, was observed in genetically modified mice that express full-length farnesyl-prelamin A in neurons (Zmpste24-deficient mice carrying two copies of a Lmna knock-in allele yielding full-length prelamin A transcripts lacking a miR-9 binding site). Our findings indicate that progerin and full-length farnesyl-prelamin A are toxic to neurons of the enteric nervous system.


Subject(s)
Enteric Nervous System/pathology , Esophageal Achalasia/genetics , Lamin Type A/genetics , Neurons/metabolism , Protein Prenylation , Animals , Esophageal Achalasia/pathology , Female , Gene Knock-In Techniques , Lamin Type A/metabolism , Male , Mice , Mice, Transgenic , MicroRNAs/metabolism , Mutation , Neurons/pathology , RNA Interference
5.
Ann Neurol ; 80(1): 59-70, 2016 07.
Article in English | MEDLINE | ID: mdl-27130255

ABSTRACT

OBJECTIVE: A study was undertaken to characterize the clinical features of the newly described hypomyelinating leukodystrophy type 10 with microcephaly. This is an autosomal recessive disorder mapped to chromosome 1q42.12 due to mutations in the PYCR2 gene, encoding an enzyme involved in proline synthesis in mitochondria. METHODS: From several international clinics, 11 consanguineous families were identified with PYCR2 mutations by whole exome or targeted sequencing, with detailed clinical and radiological phenotyping. Selective mutations from patients were tested for effect on protein function. RESULTS: The characteristic clinical presentation of patients with PYCR2 mutations included failure to thrive, microcephaly, craniofacial dysmorphism, progressive psychomotor disability, hyperkinetic movements, and axial hypotonia with variable appendicular spasticity. Patients did not survive beyond the first decade of life. Brain magnetic resonance imaging showed global brain atrophy and white matter T2 hyperintensities. Routine serum metabolic profiles were unremarkable. Both nonsense and missense mutations were identified, which impaired protein multimerization. INTERPRETATION: PYCR2-related syndrome represents a clinically recognizable condition in which PYCR2 mutations lead to protein dysfunction, not detectable on routine biochemical assessments. Mutations predict a poor outcome, probably as a result of impaired mitochondrial function. Ann Neurol 2016;80:59-70.


Subject(s)
Failure to Thrive/complications , Failure to Thrive/genetics , Microcephaly/complications , Microcephaly/genetics , Pyrroline Carboxylate Reductases/genetics , Adolescent , Child , Child, Preschool , Codon, Nonsense , Exome/genetics , Female , Fibroblasts , Gene Expression , Genetic Predisposition to Disease/genetics , Genotype , Humans , Infant , Male , Microcephaly/diagnosis , Mutation, Missense , Phenotype , Primary Cell Culture , Pyrroline Carboxylate Reductases/biosynthesis , Syndrome , Transfection , Young Adult
6.
Hum Mol Genet ; 23(6): 1506-15, 2014 Mar 15.
Article in English | MEDLINE | ID: mdl-24203701

ABSTRACT

Lamins A and C (products of the LMNA gene) are found in roughly equal amounts in peripheral tissues, but the brain produces mainly lamin C and little lamin A. In HeLa cells and fibroblasts, the expression of prelamin A (the precursor to lamin A) can be reduced by miR-9, but the relevance of those cell culture studies to lamin A regulation in the brain was unclear. To address this issue, we created two new Lmna knock-in alleles, one (Lmna(PLAO-5NT)) with a 5-bp mutation in a predicted miR-9 binding site in prelamin A's 3' UTR, and a second (Lmna(PLAO-UTR)) in which prelamin A's 3' UTR was replaced with lamin C's 3' UTR. Neither allele had significant effects on lamin A levels in peripheral tissues; however, both substantially increased prelamin A transcript levels and lamin A protein levels in the cerebral cortex and the cerebellum. The increase in lamin A expression in the brain was more pronounced with the Lmna(PLAO-UTR) allele than with the Lmna(PLAO-5NT) allele. With both alleles, the increased expression of prelamin A transcripts and lamin A protein was greater in the cerebral cortex than in the cerebellum. Our studies demonstrate the in vivo importance of prelamin A's 3' UTR and its miR-9 binding site in regulating lamin A expression in the brain. The reduced expression of prelamin A in the brain likely explains why children with Hutchinson-Gilford progeria syndrome (a progeroid syndrome caused by a mutant form of prelamin A) are spared from neurodegenerative disease.


Subject(s)
Cerebellum/metabolism , Cerebral Cortex/metabolism , Lamin Type A/metabolism , MicroRNAs/metabolism , Nuclear Proteins/genetics , Progeria/genetics , Protein Precursors/genetics , 3' Untranslated Regions , Alleles , Animals , Disease Models, Animal , Female , Gene Expression Regulation , Gene Knock-In Techniques , Lamin Type A/genetics , Mice , Mice, Inbred C57BL , MicroRNAs/genetics , Mutation , Progeria/metabolism , Progeria/pathology
7.
Proc Natl Acad Sci U S A ; 110(21): E1923-32, 2013 May 21.
Article in English | MEDLINE | ID: mdl-23650370

ABSTRACT

The role of protein farnesylation in lamin A biogenesis and the pathogenesis of progeria has been studied in considerable detail, but the importance of farnesylation for the B-type lamins, lamin B1 and lamin B2, has received little attention. Lamins B1 and B2 are expressed in nearly every cell type from the earliest stages of development, and they have been implicated in a variety of functions within the cell nucleus. To assess the importance of protein farnesylation for B-type lamins, we created knock-in mice expressing nonfarnesylated versions of lamin B1 and lamin B2. Mice expressing nonfarnesylated lamin B2 developed normally and were free of disease. In contrast, mice expressing nonfarnesylated lamin B1 died soon after birth, with severe neurodevelopmental defects and striking nuclear abnormalities in neurons. The nuclear lamina in migrating neurons was pulled away from the chromatin so that the chromatin was left "naked" (free from the nuclear lamina). Thus, farnesylation of lamin B1--but not lamin B2--is crucial for brain development and for retaining chromatin within the bounds of the nuclear lamina during neuronal migration.


Subject(s)
Brain/embryology , Cell Movement/physiology , Chromatin/metabolism , Lamin Type B/metabolism , Nuclear Lamina/metabolism , Protein Prenylation/physiology , Animals , Chromatin/genetics , Lamin Type B/genetics , Mice , Mice, Transgenic , Nuclear Lamina/genetics
9.
EMBO J ; 30(24): 4860-73, 2011 Nov 15.
Article in English | MEDLINE | ID: mdl-22085932

ABSTRACT

It has been assumed, based largely on morphologic evidence, that human pluripotent stem cells (hPSCs) contain underdeveloped, bioenergetically inactive mitochondria. In contrast, differentiated cells harbour a branched mitochondrial network with oxidative phosphorylation as the main energy source. A role for mitochondria in hPSC bioenergetics and in cell differentiation therefore remains uncertain. Here, we show that hPSCs have functional respiratory complexes that are able to consume O(2) at maximal capacity. Despite this, ATP generation in hPSCs is mainly by glycolysis and ATP is consumed by the F(1)F(0) ATP synthase to partially maintain hPSC mitochondrial membrane potential and cell viability. Uncoupling protein 2 (UCP2) plays a regulating role in hPSC energy metabolism by preventing mitochondrial glucose oxidation and facilitating glycolysis via a substrate shunting mechanism. With early differentiation, hPSC proliferation slows, energy metabolism decreases, and UCP2 is repressed, resulting in decreased glycolysis and maintained or increased mitochondrial glucose oxidation. Ectopic UCP2 expression perturbs this metabolic transition and impairs hPSC differentiation. Overall, hPSCs contain active mitochondria and require UCP2 repression for full differentiation potential.


Subject(s)
Cell Differentiation , Energy Metabolism , Ion Channels/metabolism , Mitochondria/metabolism , Mitochondrial Proteins/metabolism , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Adenosine Triphosphate , Cell Line , Glycolysis , Humans , Hydrolysis , Ion Channels/genetics , Mitochondrial Proteins/genetics , Oxygen Consumption , Pluripotent Stem Cells/ultrastructure , Reactive Oxygen Species/metabolism , Uncoupling Protein 2
10.
Proc Natl Acad Sci U S A ; 109(7): E423-31, 2012 Feb 14.
Article in English | MEDLINE | ID: mdl-22308344

ABSTRACT

Lamins A and C, alternatively spliced products of the LMNA gene, are key components of the nuclear lamina. The two isoforms are found in similar amounts in most tissues, but we observed an unexpected pattern of expression in the brain. Western blot and immunohistochemistry studies showed that lamin C is abundant in the mouse brain, whereas lamin A and its precursor prelamin A are restricted to endothelial cells and meningeal cells and are absent in neurons and glia. Prelamin A transcript levels were low in the brain, but this finding could not be explained by alternative splicing. In lamin A-only knockin mice, where alternative splicing is absent and all the output of the gene is channeled into prelamin A transcripts, large amounts of lamin A were found in peripheral tissues, but there was very little lamin A in the brain. Also, in knockin mice expressing exclusively progerin (a toxic form of prelamin A found in Hutchinson-Gilford progeria syndrome), the levels of progerin in the brain were extremely low. Further studies showed that prelamin A expression, but not lamin C expression, is down-regulated by a brain-specific microRNA, miR-9. Expression of miR-9 in cultured cells reduced lamin A expression, and this effect was abolished when the miR-9-binding site in the prelamin A 3' UTR was mutated. The down-regulation of prelamin A expression in the brain could explain why mouse models of Hutchinson-Gilford progeria syndrome are free of central nervous system pathology.


Subject(s)
Brain/metabolism , Lamin Type A/metabolism , MicroRNAs/metabolism , Animals , Blotting, Western , Mice
11.
Gut Microbes ; 16(1): 2340486, 2024.
Article in English | MEDLINE | ID: mdl-38659243

ABSTRACT

Carbapenem-resistant Klebsiella pneumoniae (CR-Kp) is a significant threat to public health worldwide. The primary reservoir for CR-Kp is the intestinal tract. There, the bacterium is usually present at low density but can bloom following antibiotic treatment, mostly in hospital settings. The impact of disturbances in the intestinal environment on the fitness, survival, expansion, and drug susceptibility of this pathogen is not well-understood, yet it may be relevant to devise strategies to tackle CR-Kp colonization and infection. Here, we adopted an in vivo model to examine the transcriptional adaptation of a CR-Kp clinical isolate to immune activation in the intestine. We report that as early as 6 hours following host treatment with anti-CD3 antibody, CR-Kp underwent rapid transcriptional changes including downregulation of genes involved in sugar utilization and amino acid biosynthesis and upregulation of genes involved in amino acid uptake and catabolism, antibiotic resistance, and stress response. In agreement with these findings, treatment increased the concentration of oxidative species and amino acids in the mouse intestine. Genes encoding for proteins containing the domain of unknown function (DUF) 1471 were strongly upregulated, however their deletion did not impair CR-Kp fitness in vivo upon immune activation. Transcription factor enrichment analysis identified the global regulator cAMP-Receptor Protein, CRP, as a potential orchestrator of the observed transcriptional signature. In keeping with the recognized role of CRP in regulating utilization of alternative carbon sources, crp deletion in CR-Kp resulted in strongly impaired gut colonization, although this effect was not amplified by immune activation. Thus, following intestinal colonization, which occurs in a CRP-dependent manner, CR-Kp can rapidly respond to immune cues by implementing a well-defined and complex transcriptional program whose direct relevance toward bacterial fitness warrants further investigation. Additional analyses utilizing this model may identify key factors to tackle CR-Kp colonization of the intestine.


Subject(s)
Anti-Bacterial Agents , Intestines , Klebsiella Infections , Klebsiella pneumoniae , Klebsiella pneumoniae/genetics , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/immunology , Animals , Mice , Klebsiella Infections/microbiology , Klebsiella Infections/immunology , Intestines/microbiology , Intestines/immunology , Anti-Bacterial Agents/pharmacology , Carbapenem-Resistant Enterobacteriaceae/genetics , Gene Expression Regulation, Bacterial , Carbapenems/pharmacology , Mice, Inbred C57BL , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Female , Humans
12.
J Biol Chem ; 287(20): 16103-10, 2012 May 11.
Article in English | MEDLINE | ID: mdl-22416132

ABSTRACT

The nuclear lamina is composed mainly of lamins A and C (A-type lamins) and lamins B1 and B2 (B-type lamins). Dogma has held that lamins B1 and B2 play unique and essential roles in the nucleus of every eukaryotic cell. Recent studies have raised doubts about that view but have uncovered crucial roles for lamins B1 and B2 in neuronal migration during the development of the brain. The relevance of lamins A and C in the brain remains unclear, but it is intriguing that prelamin A expression in the brain is low and is regulated by miR-9, a brain-specific microRNA.


Subject(s)
Brain/embryology , Cell Movement/physiology , Lamin Type A/biosynthesis , Lamin Type B/biosynthesis , Neurons/metabolism , Animals , Brain/cytology , Cell Nucleus/metabolism , Gene Expression Regulation, Developmental/physiology , Humans , MicroRNAs/metabolism , Neurons/cytology , Nuclear Proteins/metabolism , Protein Precursors/metabolism
13.
J Lipid Res ; 53(6): 1176-82, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22448028

ABSTRACT

Protein farnesyltransferase (FTase) inhibitors, generally called "FTIs," block the farnesylation of prelamin A, inhibiting the biogenesis of mature lamin A and leading to an accumulation of prelamin A within cells. A recent report found that a GGTI, an inhibitor of protein geranylgeranyltransferase-I (GGTase-I), caused an exaggerated accumulation of prelamin A in the presence of low amounts of an FTI. This finding was interpreted as indicating that prelamin A can be alternately prenylated by GGTase-I and that inhibiting both protein prenyltransferases leads to more prelamin A accumulation than blocking FTase alone. Here, we tested an alternative hypothesis-GGTIs are not specific for GGTase-I, and they lead to prelamin A accumulation by inhibiting ZMPSTE24 (a zinc metalloprotease that converts farnesyl-prelamin A to mature lamin A). In our studies, commonly used GGTIs caused prelamin A accumulation in human fibroblasts, but the prelamin A in GGTI-treated cells exhibited a more rapid electrophoretic mobility than prelamin A from FTI-treated cells. The latter finding suggested that the prelamin A in GGTI-treated cells might be farnesylated (which would be consistent with the notion that GGTIs inhibit ZMPSTE24). Indeed, metabolic labeling studies revealed that the prelamin A in GGTI-treated fibroblasts is farnesylated. Moreover, biochemical assays of ZMPSTE24 activity showed that ZMPSTE24 is potently inhibited by a GGTI. Our studies show that GGTIs inhibit ZMPSTE24, leading to an accumulation of farnesyl-prelamin A. Thus, caution is required when interpreting the effects of GGTIs on prelamin A processing.


Subject(s)
Alkyl and Aryl Transferases/antagonists & inhibitors , Membrane Proteins/antagonists & inhibitors , Metalloendopeptidases/antagonists & inhibitors , Nuclear Proteins/metabolism , Peptidomimetics/pharmacology , Protease Inhibitors/pharmacology , Protein Precursors/metabolism , Animals , Fibroblasts/drug effects , Fibroblasts/metabolism , Humans , Lamin Type A , Mice
14.
J Biol Chem ; 285(7): 5066-75, 2010 Feb 12.
Article in English | MEDLINE | ID: mdl-20007711

ABSTRACT

Store-operated Ca(2+) entry (SOCE) due to activation of Ca(2+) release-activated Ca(2+) (CRAC) channels leads to sustained elevation of cytoplasmic Ca(2+) and activation of lymphocytes. CRAC channels consisting of four pore-forming Orai1 subunits are activated by STIM1, an endoplasmic reticulum Ca(2+) sensor that senses intracellular store depletion and migrates to plasma membrane proximal regions to mediate SOCE. One of the fundamental properties of CRAC channels is their Ca(2+)-dependent fast inactivation. To identify the domains of Orai1 involved in fast inactivation, we have mutated residues in the Orai1 intracellular loop linking transmembrane segment II to III. Mutation of four residues, V(151)SNV(154), at the center of the loop (MutA) abrogated fast inactivation, leading to increased SOCE as well as higher CRAC currents. Point mutation analysis identified five key amino acids, N(153)VHNL(157), that increased SOCE in Orai1 null murine embryonic fibroblasts. Expression or direct application of a peptide comprising the entire intracellular loop or the sequence N(153)VHNL(157) blocked CRAC currents from both wild type (WT) and MutA Orai1. A peptide incorporating the MutA mutations had no blocking effect. Concatenated Orai1 constructs with four MutA monomers exhibited high CRAC currents lacking fast inactivation. Reintroduction of a single WT monomer (MutA-MutA-MutA-WT) was sufficient to fully restore fast inactivation, suggesting that only a single intracellular loop can block the channel. These data suggest that the intracellular loop of Orai1 acts as an inactivation particle, which is stabilized in the ion permeation pathway by the N(153)VHNL(157) residues. These results along with recent reports support a model in which the N terminus and the selectivity filter of Orai1 as well as STIM1 act in concert to regulate the movement of the intracellular loop and evoke fast inactivation.


Subject(s)
Calcium Channels/metabolism , Animals , Calcium Channels/chemistry , Calcium Channels/drug effects , Calcium Channels/genetics , Calcium Channels/physiology , Cell Line , Cells, Cultured , HeLa Cells , Humans , Mice , Mutagenesis , ORAI1 Protein , Patch-Clamp Techniques , Peptides/chemistry , Peptides/genetics , Peptides/pharmacology , Structure-Activity Relationship
15.
J Biol Chem ; 285(27): 20818-26, 2010 Jul 02.
Article in English | MEDLINE | ID: mdl-20439468

ABSTRACT

Lamin A, a key component of the nuclear lamina, is generated from prelamin A by four post-translational processing steps: farnesylation, endoproteolytic release of the last three amino acids of the protein, methylation of the C-terminal farnesylcysteine, and finally, endoproteolytic release of the last 15 amino acids of the protein (including the farnesylcysteine methyl ester). The last cleavage step, mediated by ZMPSTE24, releases mature lamin A. This processing scheme has been conserved through vertebrate evolution and is widely assumed to be crucial for targeting lamin A to the nuclear envelope. However, its physiologic importance has never been tested. To address this issue, we created mice with a "mature lamin A-only" allele (Lmna(LAO)), which contains a stop codon immediately after the last codon of mature lamin A. Thus, Lmna(LAO/LAO) mice synthesize mature lamin A directly, bypassing prelamin A synthesis and processing. The levels of mature lamin A in Lmna(LAO/LAO) mice were indistinguishable from those in "prelamin A-only" mice (Lmna(PLAO/PLAO)), where all of the lamin A is produced from prelamin A. Lmna(LAO/LAO) exhibited normal body weights and had no detectable disease phenotypes. A higher frequency of nuclear blebs was observed in Lmna(LAO/LAO) embryonic fibroblasts; however, the mature lamin A in the tissues of Lmna(LAO/LAO) mice was positioned normally at the nuclear rim. We conclude that prelamin A processing is dispensable in mice and that direct synthesis of mature lamin A has little if any effect on the targeting of lamin A to the nuclear rim in mouse tissues.


Subject(s)
Cell Nucleus/pathology , Fibroblasts/pathology , Lamin Type A/biosynthesis , Animals , Blotting, Western , Conserved Sequence , Crosses, Genetic , Embryo, Mammalian , Fibroblasts/cytology , Fibroblasts/metabolism , Introns , Lamin Type A/genetics , Methylation , Mice , Mice, Knockout , Microscopy, Fluorescence , Mutagenesis, Site-Directed , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Phenotype , Protein Modification, Translational , Protein Precursors/genetics , Protein Precursors/metabolism , Vertebrates
16.
mBio ; 10(2)2019 03 12.
Article in English | MEDLINE | ID: mdl-30862751

ABSTRACT

A diverse, antibiotic-naive microbiota prevents highly antibiotic-resistant microbes, including carbapenem-resistant Klebsiella pneumoniae (CR-Kp), from achieving dense colonization of the intestinal lumen. Antibiotic-mediated destruction of the microbiota leads to expansion of CR-Kp in the gut, markedly increasing the risk of bacteremia in vulnerable patients. While preventing dense colonization represents a rational approach to reduce intra- and interpatient dissemination of CR-Kp, little is known about pathogen-associated factors that enable dense growth and persistence in the intestinal lumen. To identify genetic factors essential for dense colonization of the gut by CR-Kp, we constructed a highly saturated transposon mutant library with >150,000 unique mutations in an ST258 strain of CR-Kp and screened for in vitro growth and in vivo intestinal colonization in antibiotic-treated mice. Stochastic and partially reversible fluctuations in the representation of different mutations during dense colonization revealed the dynamic nature of intestinal microbial populations. We identified genes that are crucial for early and late stages of dense gut colonization and confirmed their role by testing isogenic mutants in in vivo competition assays with wild-type CR-Kp Screening of the transposon library also identified mutations that enhanced in vivo CR-Kp growth. These newly identified colonization factors may provide novel therapeutic opportunities to reduce intestinal colonization by CR-KpIMPORTANCEKlebsiella pneumoniae is a common cause of bloodstream infections in immunocompromised and hospitalized patients, and over the last 2 decades, some strains have acquired resistance to nearly all available antibiotics, including broad-spectrum carbapenems. The U.S. Centers for Disease Control and Prevention has listed carbapenem-resistant K. pneumoniae (CR-Kp) as an urgent public health threat. Dense colonization of the intestine by CR-Kp and other antibiotic-resistant bacteria is associated with an increased risk of bacteremia. Reducing the density of gut colonization by CR-Kp is likely to reduce their transmission from patient to patient in health care facilities as well as systemic infections. How CR-Kp expands and persists in the gut lumen, however, is poorly understood. Herein, we generated a highly saturated mutant library in a multidrug-resistant K. pneumoniae strain and identified genetic factors that are associated with dense gut colonization by K. pneumoniae This study sheds light on host colonization by K. pneumoniae and identifies potential colonization factors that contribute to high-density persistence of K. pneumoniae in the intestine.


Subject(s)
Anti-Bacterial Agents/administration & dosage , Carbapenems/administration & dosage , Genome, Bacterial , Klebsiella Infections/microbiology , Klebsiella pneumoniae/growth & development , Virulence Factors/genetics , beta-Lactam Resistance , Animals , Anti-Bacterial Agents/pharmacology , Carbapenems/pharmacology , DNA Transposable Elements , Disease Models, Animal , Genetic Testing , Klebsiella pneumoniae/genetics , Mice , Mutagenesis, Insertional
17.
J Clin Invest ; 126(4): 1592-602, 2016 Apr 01.
Article in English | MEDLINE | ID: mdl-26999604

ABSTRACT

The alternatively spliced products of LMNA, lamin C and prelamin A (the precursor to lamin A), are produced in similar amounts in most tissues and have largely redundant functions. This redundancy suggests that diseases, such as Hutchinson-Gilford progeria syndrome (HGPS), that are caused by prelamin A-specific mutations could be treated by shifting the output of LMNA more toward lamin C. Here, we investigated mechanisms that regulate LMNA mRNA alternative splicing and assessed the feasibility of reducing prelamin A expression in vivo. We identified an exon 11 antisense oligonucleotide (ASO) that increased lamin C production at the expense of prelamin A when transfected into mouse and human fibroblasts. The same ASO also reduced the expression of progerin, the mutant prelamin A protein in HGPS, in fibroblasts derived from patients with HGPS. Mechanistic studies revealed that the exon 11 sequences contain binding sites for serine/arginine-rich splicing factor 2 (SRSF2), and SRSF2 knockdown lowered lamin A production in cells and in murine tissues. Moreover, administration of the exon 11 ASO reduced lamin A expression in wild-type mice and progerin expression in an HGPS mouse model. Together, these studies identify ASO-mediated reduction of prelamin A as a potential strategy to treat prelamin A-specific diseases.


Subject(s)
Alternative Splicing/drug effects , Lamin Type A/biosynthesis , Oligodeoxyribonucleotides, Antisense/pharmacology , Progeria/drug therapy , Progeria/metabolism , RNA, Messenger/metabolism , Animals , Disease Models, Animal , Exons , Gene Knockdown Techniques , Humans , Lamin Type A/genetics , Mice , Mice, Transgenic , Mutation , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Oligodeoxyribonucleotides, Antisense/genetics , Progeria/genetics , RNA, Messenger/genetics , Ribonucleoproteins/genetics , Ribonucleoproteins/metabolism , Serine-Arginine Splicing Factors
18.
Sci Rep ; 6: 37863, 2016 12 02.
Article in English | MEDLINE | ID: mdl-27910869

ABSTRACT

We introduce a label-free method to rapidly phenotype and classify cells purely based on physical properties. We extract 15 biophysical parameters from cells as they deform in a microfluidic stretching flow field via high-speed microscopy and apply machine-learning approaches to discriminate different cell types and states. When employing the full 15 dimensional dataset, the technique robustly classifies individual cells based on their pluripotency, with accuracy above 95%. Rheological and morphological properties of cells while deforming were critical for this classification. We also show the application of this method in accurate classifying cells based on their viability, drug screening and detecting populations of malignant cells in mixed samples. We show that some of the extracted parameters are not linearly independent, and in fact we reach maximum classification accuracy by using only a subset of parameters. However, the informative subsets could vary depending on cell types in the sample. This work shows the utility of an assay purely based on intrinsic biophysical properties of cells to identify changes in cell state. In addition to a label-free alternative to flow cytometry in certain applications, this work, also can provide novel intracellular metrics that would not be feasible with labeled approaches (i.e. flow cytometry).


Subject(s)
Embryonic Stem Cells/cytology , Flow Cytometry , Machine Learning , Animals , Biophysics , Cell Count , Fibroblasts/cytology , Humans , Hydrodynamics , Mice , Microfluidic Analytical Techniques , Microfluidics , Phenotype , Rheology
19.
Nucleus ; 5(4): 287-92, 2014.
Article in English | MEDLINE | ID: mdl-25482116

ABSTRACT

Lamins B1 and B2 have a high degree of sequence similarity and are widely expressed from the earliest stages of development. Studies of Lmnb1 and Lmnb2 knockout mice revealed that both of the B-type lamins are crucial for neuronal migration in the developing brain. These observations naturally posed the question of whether the two B-type lamins might play redundant functions in the development of the brain. To explore that issue, Lee and coworkers generated "reciprocal knock-in mice" (knock-in mice that produce lamin B1 from the Lmnb2 locus and knock-in mice that produce lamin B2 from the Lmnb1 locus). Both lines of knock-in mice manifested neurodevelopmental abnormalities similar to those in conventional knockout mice, indicating that lamins B1 and B2 have unique functions and that increased production of one B-type lamin cannot compensate for the loss of the other.


Subject(s)
Lamin Type B/metabolism , Animals , Brain/metabolism , Lamin Type B/genetics , Mice , Mice, Knockout , Nuclear Envelope/metabolism , Nuclear Lamina/metabolism
20.
Mol Cell Biol ; 34(15): 2776-85, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24842906

ABSTRACT

Much of the work on nuclear lamins during the past 15 years has focused on mutations in LMNA (the gene for prelamin A and lamin C) that cause particular muscular dystrophy, cardiomyopathy, partial lipodystrophy, and progeroid syndromes. These disorders, often called "laminopathies," mainly affect mesenchymal tissues (e.g., striated muscle, bone, and fibrous tissue). Recently, however, a series of papers have identified important roles for nuclear lamins in the central nervous system. Studies of knockout mice uncovered a key role for B-type lamins (lamins B1 and B2) in neuronal migration in the developing brain. Also, duplications of LMNB1 (the gene for lamin B1) have been shown to cause autosome-dominant leukodystrophy. Finally, recent studies have uncovered a peculiar pattern of nuclear lamin expression in the brain. Lamin C transcripts are present at high levels in the brain, but prelamin A expression levels are very low-due to regulation of prelamin A transcripts by microRNA 9. This form of prelamin A regulation likely explains why "prelamin A diseases" such as Hutchinson-Gilford progeria syndrome spare the central nervous system. In this review, we summarize recent progress in elucidating links between nuclear lamins and neurobiology.


Subject(s)
Lamins/genetics , Lamins/metabolism , Nuclear Lamina/genetics , Nuclear Lamina/metabolism , Animals , Humans , Neurobiology/methods
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