Capsule carbohydrate structure determines virulence in Acinetobacter baumannii

Acinetobacter baumannii is a highly antibiotic-resistant bacterial pathogen for which novel therapeutic approaches are needed. Unfortunately, the drivers of virulence in A. baumannii remain uncertain. By comparing genomes among a panel of A. baumannii strains we identified a specific gene variation in the capsule locus that correlated with altered virulence. While less virulent strains possessed the intact gene gtr6, a hypervirulent clinical isolate contained a spontaneous transposon insertion in the same gene, resulting in the loss of a branchpoint in capsular carbohydrate structure. By constructing isogenic gtr6 mutants, we confirmed that gtr6-disrupted strains were protected from phagocytosis in vitro and displayed higher bacterial burden and lethality in vivo. Gtr6+ strains were phagocytized more readily and caused lower bacterial burden and no clinical illness in vivo. We found that the CR3 receptor mediated phagocytosis of gtr6+, but not gtr6-, strains in a complement-dependent manner. Furthermore, hypovirulent gtr6+ strains demonstrated increased virulence in vivo when CR3 function was abrogated. In summary, loss-of-function in a single capsule assembly gene dramatically altered virulence by inhibiting complement deposition and recognition by phagocytes across multiple A. baumannii strains. Thus, capsular structure can determine virulence among A. baumannii strains by altering bacterial interactions with host complement-mediated opsonophagocytosis.

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GFI1 Is a Tumor Suppressor in Myeloid Progenitors

Blood ◽

10.1182/blood.v112.11.2942.2942 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2942-2942

Author(s):

Aditya Chaubey ◽

Shane Hormon ◽

Chinavenmeni S. Velu ◽

Tristan Bourdeau ◽

Jinfang Zhu ◽

...

Keyword(s):

Dna Sequences ◽

Myeloid Leukemia ◽

Dependent Manner ◽

Loss Of Function ◽

Increased Risk ◽

Myeloid Leukemias ◽

Dose Dependent ◽

Anterior Posterior

Abstract In severe congenital neutropenia (SCN) patients and mice with Growth factor independent-1 (Gfi1) loss of function, arrested progenitors are suspended in a hyperproliferative state while terminal granulpoiesis is blocked. SCN patients are at increased risk for the development of acute myeloid leukemia. We demonstrate that Gfi1 directly targets HoxA9, Pbx1 and Meis1 during normal myelopoiesis. Gfi1−/− progenitors exhibit elevated levels of HoxA9, Pbx1 and Meis1, exaggerated HoxA9-Pbx1-Meis1 activity, and increased persistence in vivo and in vitro. Limiting HoxA9 alleles corrects, in a dose dependent manner, in vivo and in vitro phenotypes observed with loss of Gfi1. Moreover, in a manner conserved in Drosophila anterior/posterior patterning, we demonstrate that these factors can compete for occupancy of DNA sequences encoding composite Gfi1-HoxA9-Pbx1-Meis1 binding sites. Finally, the expression of Gfi1 and HoxA9 are inverse and stratify human myeloid leukemias, suggesting a role for HoxA9- Gfi1 antagonism in human AML. In agreement with this, a myeloproliferative disorder progresses into a rapid, lethal and transplantable myeloid leukemia in a Gfi1−/− setting. We conclude that the lifespan and oncogenic transformation of hematopoietic progenitor cells is regulated through a conserved competition between Gfi1 and HoxA9-Pbx1-Meis1.

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Therapeutic Effects of Inhibitor of ompA Expression against Carbapenem-Resistant Acinetobacter baumannii Strains

International Journal of Molecular Sciences ◽

10.3390/ijms222212257 ◽

2021 ◽

Vol 22 (22) ◽

pp. 12257

Author(s):

Seok-Hyeon Na ◽

Hyejin Jeon ◽

Man-Hwan Oh ◽

Yoo-Jeong Kim ◽

Mingi Chu ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Survival Rates ◽

Clinical Settings ◽

Therapeutic Effects ◽

Dependent Manner ◽

Bacteriostatic Activity ◽

Carbapenem Resistant ◽

Eskape Pathogens

The widespread of carbapenem-resistant Acinetobacter baumannii (CRAB) is of great concern in clinical settings worldwide. It is urgent to develop new therapeutic agents against this pathogen. This study aimed to evaluate the therapeutic potentials of compound 62520, which has been previously identified as an inhibitor of the ompA promoter activity of A. baumannii, against CRAB isolates, both in vitro and in vivo. Compound 62520 was found to inhibit the ompA expression and biofilm formation in A. baumannii ATCC 17978 at sub-inhibitory concentrations in a dose-dependent manner. These inhibitory properties were also observed in clinical CRAB isolates belonging to sequence type (ST) 191. Additionally, compound 62520 exhibited a bacteriostatic activity against clinical clonal complex (CC) 208 CRAB isolates, including ST191, and ESKAPE pathogens. This bacteriostatic activity was not different between STs of CRAB isolates. Bacterial clearance was observed in mice infected with bioimaging A. baumannii strain 24 h after treatment with compound 62520. Compound 62520 was shown to significantly increase the survival rates of both immunocompetent and neutropenic mice infected with A. baumannii ATCC 17978. This compound also increased the survival rates of mice infected with clinical CRAB isolate. These results suggest that compound 62520 is a promising scaffold to develop a novel therapeutic agent against CRAB infections.

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Pelle kinase is activated by autophosphorylation during Toll signaling in Drosophila

Development ◽

10.1242/dev.129.8.1925 ◽

2002 ◽

Vol 129 (8) ◽

pp. 1925-1933 ◽

Cited By ~ 1

Author(s):

Baohe Shen ◽

James L. Manley

Keyword(s):

Dependent Manner ◽

Loss Of Function ◽

Concentration Dependent Manner ◽

Downstream Target ◽

Toll Signaling ◽

Early Embryos ◽

High Concentrations ◽

L2 Cells

The Drosophila Pelle kinase plays a key role in the evolutionarily conserved Toll signaling pathway, but the mechanism responsible for its activation has been unknown. We present in vivo and in vitro evidence establishing an important role for concentration-dependent autophosphorylation in the signaling process. We first show that Pelle phosphorylation can be detected transiently in early embryos, concomitant with activation of signaling. Importantly, Pelle phosphorylation is enhanced in a gain-of-function Toll mutant (Toll10b), but decreased by loss-of-function Toll alleles. Next we found that Pelle is phosphorylated in transfected Schneider L2 cells in a concentration-dependent manner such that significant modification is observed only at high Pelle concentrations, which coincide with levels required for phosphorylation and activation of the downstream target, Dorsal. Pelle phosphorylation is also enhanced in L2 cells co-expressing Toll10b, and is dependent on Pelle kinase activity. In vitro kinase assays revealed that recombinant, autophosphorylated Pelle is far more active than unphosphorylated Pelle. Importantly, unphosphorylated Pelle becomes autophosphorylated, and activated, by incubation at high concentrations. We discuss these results in the context of Toll-like receptor mediated signaling in both flies and mammals.

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Rapid Generation of Long Noncoding RNA Knockout Mice Using CRISPR/Cas9 Technology

Non-Coding RNA ◽

10.3390/ncrna5010012 ◽

2019 ◽

Vol 5 (1) ◽

pp. 12 ◽

Cited By ~ 7

Author(s):

Nils R. Hansmeier ◽

Pia J. M. Widdershooven ◽

Sajjad Khani ◽

Jan-Wilhelm Kornfeld

Keyword(s):

Gene Targeting ◽

Mouse Models ◽

Noncoding Rna ◽

Genome Engineering ◽

Model Organisms ◽

Specific Gene ◽

Loss Of Function ◽

Mouse Lines

In recent years, long noncoding RNAs (lncRNAs) have emerged as multifaceted regulators of gene expression, controlling key developmental and disease pathogenesis processes. However, due to the paucity of lncRNA loss-of-function mouse models, key questions regarding the involvement of lncRNAs in organism homeostasis and (patho)-physiology remain difficult to address experimentally in vivo. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 platform provides a powerful genome-editing tool and has been successfully applied across model organisms to facilitate targeted genetic mutations, including Caenorhabditis elegans, Drosophila melanogaster, Danio rerio and Mus musculus. However, just a few lncRNA-deficient mouse lines have been created using CRISPR/Cas9-mediated genome engineering, presumably due to the need for lncRNA-specific gene targeting strategies considering the absence of open-reading frames in these loci. Here, we describe a step-wise procedure for the generation and validation of lncRNA loss-of-function mouse models using CRISPR/Cas9-mediated genome engineering. In a proof-of-principle approach, we generated mice deficient for the liver-enriched lncRNA Gm15441, which we found downregulated during development of metabolic disease and induced during the feeding/fasting transition. Further, we discuss guidelines for the selection of lncRNA targets and provide protocols for in vitro single guide RNA (sgRNA) validation, assessment of in vivo gene-targeting efficiency and knockout confirmation. The procedure from target selection to validation of lncRNA knockout mouse lines can be completed in 18–20 weeks, of which <10 days hands-on working time is required.

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Effect of Chromatin-Remodeling Agents in Hepatic Differentiation of Rat Bone Marrow-Derived Mesenchymal Stem CellsIn VitroandIn Vivo

Stem Cells International ◽

10.1155/2016/3038764 ◽

2016 ◽

Vol 2016 ◽

pp. 1-11 ◽

Cited By ~ 7

Author(s):

Danna Ye ◽

Tong Li ◽

Philip Heraud ◽

Rangsun Parnpai

Keyword(s):

Bone Marrow ◽

Liver Damage ◽

Dna Methyltransferase ◽

Trichostatin A ◽

Specific Gene ◽

Dependent Manner ◽

Hepatic Differentiation ◽

Rat Bone

Epigenetic events, including covalent histone modifications and DNA methylation, play fundamental roles in the determination of lineage-specific gene expression and cell fates. The aim of this study was to determine whether the DNA methyltransferase inhibitor (DNMTi) 5-aza-2′-deoxycytidine (5-aza-dC) and the histone deacetylase inhibitor (HDACi) trichostatin A (TSA) promote the hepatic differentiation of rat bone marrow-derived mesenchymal stem cells (rBM-MSCs) and their therapeutic effect on liver damage. 1 μM TSA and 20 μM 5-aza-dC were added to standard hepatogenic medium especially at differentiation and maturation steps and their potential function on hepatic differentiationin vitroandin vivowas determined. Exposure of rBM-MSCs to 1 μM TSA at both the differentiation and maturation steps considerably improved hepatic differentiation. TSA enhanced the development of the hepatocyte shape, promoted the chronological expression of hepatocyte-specific markers, and improved hepatic functions. In contrast, treatment of rBM-MSCs with 20 μM 5-aza-dC alone or in combination with TSA was ineffective in improving hepatic differentiationin vitro. TSA and/or 5-aza-dC derived hepatocytes-like cells failed to improve the therapeutic potential in liver damage. We conclude that HDACis enhance hepatic differentiation in a time-dependent manner, while DNMTis do not induce the hepatic differentiation of rBM-MSCsin vitro. Theirin vivofunction needs further investigation.

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Gfi1 integrates progenitor versus granulocytic transcriptional programming

Blood ◽

10.1182/blood-2008-09-179747 ◽

2009 ◽

Vol 113 (22) ◽

pp. 5466-5475 ◽

Cited By ~ 48

Author(s):

Shane R. Horman ◽

Chinavenmeni S. Velu ◽

Aditya Chaubey ◽

Tristan Bourdeau ◽

Jinfang Zhu ◽

...

Keyword(s):

Dependent Manner ◽

Loss Of Function ◽

Lineage Specification ◽

Myeloid Progenitor ◽

Myeloid Progenitor Cells ◽

Dose Dependent ◽

Transcription Program ◽

Myeloid Progenitors

AbstractIn patients with severe congenital neutropenia (SCN) and mice with growth factor independent-1 (Gfi1) loss of function, arrested myeloid progenitors accumulate, whereas terminal granulopoiesis is blocked. One might assume that Gfi-null progenitors accumulate because they lack the ability to differentiate. Instead, our data indicate that Gfi1 loss of function deregulates 2 separable transcriptional programs, one of which controls the accumulation and lineage specification of myeloid progenitors, but not terminal granulopoiesis. We demonstrate that Gfi1 directly represses HoxA9, Pbx1, and Meis1 during normal myelopoiesis. Gfi1−/− progenitors exhibit elevated levels of HoxA9, Pbx1 and Meis1, exaggerated HoxA9-Pbx1-Meis1 activity, and progenitor transformation in collaboration with oncogenic K-Ras. Limiting HoxA9 alleles corrects, in a dose-dependent manner, in vivo and in vitro phenotypes observed with loss of Gfi1 in myeloid progenitor cells but did not rescue Gfi1−/− blocked granulopoiesis. Thus, Gfi1 integrates 2 events during normal myeloid differentiation; the suppression of a HoxA9-Pbx1-Meis1 progenitor program and the induction of a granulopoietic transcription program.

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KIAA1429 and ALKBH5 Oppositely Influence Aortic Dissection Progression via Regulating the Maturation of Pri-miR-143-3p in an m6A-Dependent Manner

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.668377 ◽

2021 ◽

Vol 9 ◽

Author(s):

Peng Wang ◽

Zhiwei Wang ◽

Min Zhang ◽

Qi Wu ◽

Feng Shi ◽

...

Keyword(s):

Aortic Dissection ◽

Dependent Manner ◽

Loss Of Function ◽

Therapeutic Approaches ◽

Downstream Target ◽

Downstream Target Gene ◽

Molecular Mechanism Of Action ◽

New Research

Despite decades of study into aortic dissection (AD), a lethal cardiovascular emergency due to a tear in the aorta intima or bleeding within the aortic wall, leading to the separation of the different layers of it, the factors that influence its progression and the deeper regulatory mechanisms remain poorly understood. Nowadays, with the maturity of N6-methyladenosine (m6A) sequence technology, m6A modification, one type of RNA epigenesis, has gradually become a new research hotspot for epigenetic molecular regulation. Especially recently, increasing evidence has revealed that m6A modification functions as a pivotal post-transcriptional modification to influence the progression of multiple diseases. Based on these findings, it is reasonable to speculate that m6A modification may affect the onset and progression of AD. To explore the validity of our conjecture and to elucidate its underlying molecular mechanism of action, we conducted the present study. In this study, we found that KIAA1429 is downregulated while ALKBH5 is upregulated in aortic tissues from AD patients. Furthermore, gain- and loss-of-function studies showed that KIAA1429 and ALKBH5 can oppositely regulate HASMC proliferation, HAEC apoptosis, and AD progression in AngII-infused mice. Mechanistically, we demonstrated that KIAA1429/ALKBH5-mediated m6A modifications can regulate the processing of pri-miR-143-3p through interacting with the microprocessor protein DGCR8, thus indirectly regulating the downstream target gene of mature miR-143-3p, DDX6, to perform their biological functions in vitro and in vivo. Our findings have revealed a novel connection between m6A modification and AD progression and may provide a novel molecular basis for subsequent researchers to search for novel therapeutic approaches to improve the health of patients struggling with AD.

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Caspase-1 and Caspase-11 Mediate Pyroptosis, Inflammation, and Control ofBrucellaJoint Infection

Infection and Immunity ◽

10.1128/iai.00361-18 ◽

2018 ◽

Vol 86 (9) ◽

Cited By ~ 21

Author(s):

Carolyn A. Lacey ◽

William J. Mitchell ◽

Alexis S. Dadelahi ◽

Jerod A. Skyberg

Keyword(s):

Interleukin 1 ◽

Joint Infection ◽

Bacterial Pathogen ◽

Joint Inflammation ◽

Dependent Manner ◽

Content Type ◽

Caspase 1 ◽

And Control

ABSTRACTBrucellosis, caused by the intracellular bacterial pathogenBrucella, is a zoonotic disease for which arthritis is the most common focal complication in humans. Here we investigated the role of inflammasomes and their effectors, including interleukin-1 (IL-1), IL-18, and pyroptosis, on inflammation and control of infection duringBrucella-induced arthritis. Early in infection, both caspase-1 and caspase-11 were found to initiate joint inflammation and proinflammatory cytokine production. However, by 1 week postinfection, caspase-1 and caspase-11 also contributed to control ofBrucellajoint infection. Inflammasome-dependent restriction ofBrucellajoint burdens did not require AIM2 (absent in melanoma 2) or NLRP3 (NLR family, pyrin domain containing 3). IL-1R had a modest effect onBrucella-induced joint swelling, but mice lacking IL-1R were not impaired in their ability to control infection of the joint byBrucella. In contrast, IL-18 contributed to the initiation of joint swelling and control of jointBrucellainfection. Caspase1/11-dependent cell death was observedin vivo, andin vitrostudies demonstrated that both caspase-1 and caspase-11 induce pyroptosis, which limitedBrucellainfection in macrophages.Brucellalipopolysaccharide alone was also able to induce caspase-11-dependent pyroptosis. Collectively, these data demonstrate that inflammasomes induce inflammation in an IL-18-dependent manner and that inflammasome-dependent IL-18 and pyroptosis restrictBrucellainfection.

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Synthetic Lethality Screening Highlights Colorectal Cancer Vulnerability to Concomitant Blockade of NEDD8 and EGFR Pathways

Cancers ◽

10.3390/cancers13153805 ◽

2021 ◽

Vol 13 (15) ◽

pp. 3805

Author(s):

Federica Invrea ◽

Simona Punzi ◽

Consalvo Petti ◽

Rosalba Minelli ◽

Michael D. Peoples ◽

...

Keyword(s):

Colorectal Cancer ◽

Target Genes ◽

Enzyme Inhibitor ◽

Synthetic Lethality ◽

Drop Out ◽

Specific Gene ◽

Loss Of Function ◽

Egfr Signaling Pathway

Colorectal cancer (CRC) is a heterogeneous disease showing significant variability in clinical aggressiveness. Primary and acquired resistance limits the efficacy of available treatments, and identification of effective drug combinations is needed to further improve patients’ outcomes. We previously found that the NEDD8-activating enzyme inhibitor pevonedistat induced tumor stabilization in preclinical models of poorly differentiated, clinically aggressive CRC resistant to available therapies. To identify drugs that can be effectively combined with pevonedistat, we performed a “drop-out” loss-of-function synthetic lethality screening with an shRNA library covering 200 drug-target genes in four different CRC cell lines. Multiple screening hits were found to be involved in the EGFR signaling pathway, suggesting that, rather than inhibition of a specific gene, interference with the EGFR pathway at any level could be effectively leveraged for combination therapies based on pevonedistat. Exploiting both BRAF-mutant and RAS/RAF wild-type CRC models, we validated the therapeutic relevance of our findings by showing that combined blockade of NEDD8 and EGFR pathways led to increased growth arrest and apoptosis both in vitro and in vivo. Pathway modulation analysis showed that compensatory feedback loops induced by single treatments were blunted by the combinations. These results unveil possible therapeutic opportunities in specific CRC clinical settings.

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Colistin Dependence in Extensively Drug-Resistant Acinetobacter baumannii Strain Is Associated with ISAjo2 and ISAba13 Insertions and Multiple Cellular Responses

International Journal of Molecular Sciences ◽

10.3390/ijms22020576 ◽

2021 ◽

Vol 22 (2) ◽

pp. 576

Author(s):

Sherley Chamoun ◽

Jenny Welander ◽

Mihaela-Maria Martis-Thiele ◽

Maria Ntzouni ◽

Carina Claesson ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Lipid A ◽

Antimicrobial Agents ◽

Bacterial Pathogen ◽

Lipid Profiling ◽

Membrane Asymmetry ◽

Resistance Nodulation Division ◽

Lipid A Biosynthesis

The nosocomial opportunistic Gram-negative bacterial pathogen Acinetobacter baumannii is resistant to multiple antimicrobial agents and an emerging global health problem. The polymyxin antibiotic colistin, targeting the negatively charged lipid A component of the lipopolysaccharide on the bacterial cell surface, is often considered as the last-resort treatment, but resistance to colistin is unfortunately increasing worldwide. Notably, colistin-susceptible A. baumannii can also develop a colistin dependence after exposure to this drug in vitro. Colistin dependence might represent a stepping stone to resistance also in vivo. However, the mechanisms are far from clear. To address this issue, we combined proteogenomics, high-resolution microscopy, and lipid profiling to characterize and compare A. baumannii colistin-susceptible clinical isolate (Ab-S) of to its colistin-dependent subpopulation (Ab-D) obtained after subsequent passages in moderate colistin concentrations. Incidentally, in the colistin-dependent subpopulation the lpxA gene was disrupted by insertion of ISAjo2, the lipid A biosynthesis terminated, and Ab-D cells displayed a lipooligosaccharide (LOS)-deficient phenotype. Moreover, both mlaD and pldA genes were perturbed by insertions of ISAjo2 and ISAba13, and LOS-deficient bacteria displayed a capsule with decreased thickness as well as other surface imperfections. The major changes in relative protein abundance levels were detected in type 6 secretion system (T6SS) components, the resistance-nodulation-division (RND)-type efflux pumps, and in proteins involved in maintenance of outer membrane asymmetry. These findings suggest that colistin dependence in A. baumannii involves an ensemble of mechanisms seen in resistance development and accompanied by complex cellular events related to insertional sequences (ISs)-triggered LOS-deficiency. To our knowledge, this is the first study demonstrating the involvement of ISAjo2 and ISAba13 IS elements in the modulation of the lipid A biosynthesis and associated development of dependence on colistin.

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