scholarly journals The RNA Chaperone Hfq Is Required for Virulence of Bordetella pertussis

2013 ◽  
Vol 81 (11) ◽  
pp. 4081-4090 ◽  
Author(s):  
Ilona Bibova ◽  
Karolina Skopova ◽  
Jiri Masin ◽  
Ondrej Cerny ◽  
David Hot ◽  
...  
Keyword(s):  
Bordetella Pertussis ◽  
Posttranscriptional Regulation ◽  
Noncoding Rna ◽  
Whooping Cough ◽  
Lethal Dose ◽  
Rna Chaperone ◽  
Content Type ◽  
Small Noncoding Rna ◽  
Mrna Targets

ABSTRACTBordetella pertussisis a Gram-negative pathogen causing the human respiratory disease called pertussis or whooping cough. Here we examined the role of the RNA chaperone Hfq inB. pertussisvirulence. Hfq mediates interactions between small regulatory RNAs and their mRNA targets and thus plays an important role in posttranscriptional regulation of many cellular processes in bacteria, including production of virulence factors. We characterized anhfqdeletion mutant (Δhfq) ofB. pertussis18323 and show that the Δhfqstrain produces decreased amounts of the adenylate cyclase toxin that plays a central role inB. pertussisvirulence. Production of pertussis toxin and filamentous hemagglutinin was affected to a lesser extent.In vitro, the ability of the Δhfqstrain to survive within macrophages was significantly reduced compared to that of the wild-type (wt) strain. The virulence of the Δhfqstrain in the mouse respiratory model of infection was attenuated, with its capacity to colonize mouse lungs being strongly reduced and its 50% lethal dose value being increased by one order of magnitude over that of the wt strain. In mixed-infection experiments, the Δhfqstrain was then clearly outcompeted by the wt strain. This requirement for Hfq suggests involvement of small noncoding RNA regulation inB. pertussisvirulence.

scholarly journals Cyclic AMP-Elevating Capacity of Adenylate Cyclase Toxin-Hemolysin Is Sufficient for Lung Infection but Not for Full Virulence of Bordetella pertussis

2017 ◽  
Vol 85 (6) ◽  
Author(s):  
Karolina Skopova ◽  
Barbora Tomalova ◽  
Ivan Kanchev ◽  
Pavel Rossmann ◽  
Martina Svedova ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Bordetella Pertussis ◽  
Whooping Cough ◽  
Lung Parenchyma ◽  
Lung Pathology ◽  
Phagocytic Cells ◽  
Content Type ◽  
Adenylate Cyclase Toxin

ABSTRACT The adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) of Bordetella pertussis targets phagocytic cells expressing the complement receptor 3 (CR3, Mac-1, αMβ2 integrin, or CD11b/CD18). CyaA delivers into cells an N-terminal adenylyl cyclase (AC) enzyme domain that is activated by cytosolic calmodulin and catalyzes unregulated conversion of cellular ATP into cyclic AMP (cAMP), a key second messenger subverting bactericidal activities of phagocytes. In parallel, the hemolysin (Hly) moiety of CyaA forms cation-selective hemolytic pores that permeabilize target cell membranes. We constructed the first B. pertussis mutant secreting a CyaA toxin having an intact capacity to deliver the AC enzyme into CD11b-expressing (CD11b+) host phagocytes but impaired in formation of cell-permeabilizing pores and defective in cAMP elevation in CD11b− cells. The nonhemolytic AC+ Hly− bacteria inhibited the antigen-presenting capacities of coincubated mouse dendritic cells in vitro and skewed their Toll-like receptor (TLR)-triggered maturation toward a tolerogenic phenotype. The AC+ Hly− mutant also infected mouse lungs as efficiently as the parental AC+ Hly+ strain. Hence, elevation of cAMP in CD11b− cells and/or the pore-forming capacity of CyaA were not required for infection of mouse airways. The latter activities were, however, involved in bacterial penetration across the epithelial layer, enhanced neutrophil influx into lung parenchyma during sublethal infections, and the exacerbated lung pathology and lethality of B. pertussis infections at higher inoculation doses (>107 CFU/mouse). The pore-forming activity of CyaA further synergized with the cAMP-elevating activity in downregulation of major histocompatibility complex class II (MHC-II) molecules on infiltrating myeloid cells, likely contributing to immune subversion of host defenses by the whooping cough agent.

scholarly journals Mouse Cytomegalovirus MicroRNAs Dominate the Cellular Small RNA Profile during Lytic Infection and Show Features of Posttranscriptional Regulation

2007 ◽  
Vol 81 (24) ◽  
pp. 13771-13782 ◽  
Author(s):  
Lars Dölken ◽  
Jonathan Perot ◽  
Valérie Cognat ◽  
Abdelmalek Alioua ◽  
Matthias John ◽  
...  
Keyword(s):  
Gene Expression ◽  
Posttranscriptional Regulation ◽  
Noncoding Rna ◽  
Murine Cytomegalovirus ◽  
Knockout Mutant ◽  
Double Knockout ◽  
Viral Mirnas ◽  
Small Noncoding Rna ◽  
Human Cmv

ABSTRACT MicroRNAs (miRNAs) are small, noncoding RNA molecules that regulate gene expression at the posttranscriptional level. Originally identified in a variety of organisms ranging from plants to mammals, miRNAs have recently been identified in several viruses. Viral miRNAs may play a role in modulating both viral and host gene expression. Here, we report on the identification and characterization of 18 viral miRNAs from mouse fibroblasts lytically infected with the murine cytomegalovirus (MCMV). The MCMV miRNAs are expressed at early times of infection and are scattered in small clusters throughout the genome with up to four distinct miRNAs processed from a single transcript. No significant homologies to human CMV-encoded miRNAs were found. Remarkably, as soon as 24 h after infection, MCMV miRNAs constituted about 35% of the total miRNA pool, and at 72 h postinfection, this proportion was increased to more than 60%. However, despite the abundance of viral miRNAs during the early phase of infection, the expression of some MCMV miRNAs appeared to be regulated. Hence, for three miRNAs we observed polyuridylation of their 3′ end, coupled to subsequent degradation. Individual knockout mutants of two of the most abundant MCMV miRNAs, miR-m01-4 and miR-M44-1, or a double knockout mutant of miR-m21-1 and miR-M23-2, incurred no or only a very mild growth deficit in murine embryonic fibroblasts in vitro.

scholarly journals Bordetella pertussis Lipid A Glucosamine Modification Confers Resistance to Cationic Antimicrobial Peptides and Increases Resistance to Outer Membrane Perturbation

2014 ◽  
Vol 58 (8) ◽  
pp. 4931-4934 ◽  
Author(s):  
Nita R. Shah ◽  
Robert E. W. Hancock ◽  
Rachel C. Fernandez
Keyword(s):  
Immune System ◽  
Antimicrobial Peptides ◽  
Outer Membrane ◽  
Bordetella Pertussis ◽  
Lipid A ◽  
Whooping Cough ◽  
Human Immune System ◽  
Cationic Antimicrobial Peptides ◽  
Content Type ◽  
Membrane Perturbation

ABSTRACTBordetella pertussis, the causative agent of whooping cough, has many strategies for evading the human immune system. Lipopolysaccharide (LPS) is an important Gram-negative bacterial surface structure that activates the immune system via Toll-like receptor 4 and enables susceptibility to cationic antimicrobial peptides (CAMPs). We show modification of the lipid A region of LPS with glucosamine increased resistance to numerous CAMPs, including LL-37. Furthermore, we demonstrate that this glucosamine modification increased resistance to outer membrane perturbation.

MicroRNAs in domestic livestock

2013 ◽  
Vol 45 (16) ◽  
pp. 685-696 ◽  
Author(s):  
Attia Fatima ◽  
Dermot G. Morris
Keyword(s):  
Posttranscriptional Regulation ◽  
Noncoding Rna ◽  
Expression Patterns ◽  
Regulation Of Gene Expression ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Small Noncoding Rna ◽  
Domestic Livestock ◽  
Temporal Expression Pattern ◽  
Mrna Binding

microRNAs (miRNAs) are a class of small noncoding RNA that bind to complementary sequences in the untranslated regions of multiple target mRNAs resulting in posttranscriptional regulation of gene expression. The recent discovery and expression-profiling studies of miRNAs in domestic livestock have revealed both their tissue-specific and temporal expression pattern. In addition, breed-dependent expression patterns as well as single nucleotide polymorphisms in either the miRNA or in the target mRNA binding site have revealed associations with traits of economic importance and highlight the potential use of miRNAs in future genomic selection programs.

scholarly journals Parvovirus Expresses a Small Noncoding RNA That Plays an Essential Role in Virus Replication

2017 ◽  
Vol 91 (8) ◽  
Author(s):  
Zekun Wang ◽  
Weiran Shen ◽  
Fang Cheng ◽  
Xuefeng Deng ◽  
John F. Engelhardt ◽  
...  
Keyword(s):  
Dna Replication ◽  
Noncoding Rna ◽  
Rna Polymerase Iii ◽  
Human Bocavirus ◽  
Nonstructural Proteins ◽  
Viral Dna ◽  
Content Type ◽  
Small Noncoding Rna ◽  
Pol Iii ◽  
Tract Infections

ABSTRACT Human bocavirus 1 (HBoV1) belongs to the species Primate bocaparvovirus of the genus Bocaparvovirus of the Parvoviridae family. HBoV1 causes acute respiratory tract infections in young children and has a selective tropism for the apical surface of well-differentiated human airway epithelia (HAE). In this study, we identified an additional HBoV1 gene, bocavirus-transcribed small noncoding RNA (BocaSR), within the 3′ noncoding region (nucleotides [nt] 5199 to 5338) of the viral genome of positive sense. BocaSR is transcribed by RNA polymerase III (Pol III) from an intragenic promoter at levels similar to that of the capsid protein-coding mRNA and is essential for replication of the viral DNA in both transfected HEK293 and infected HAE cells. Mechanistically, we showed that BocaSR regulates the expression of HBoV1-encoded nonstructural proteins NS1, NS2, NS3, and NP1 but not NS4. BocaSR is similar to the adenovirus-associated type I (VAI) RNA in terms of both nucleotide sequence and secondary structure but differs from it in that its regulation of viral protein expression is independent of RNA-activated protein kinase (PKR) regulation. Notably, BocaSR accumulates in the viral DNA replication centers within the nucleus and likely plays a direct role in replication of the viral DNA. Our findings reveal BocaSR to be a novel viral noncoding RNA that coordinates the expression of viral proteins and regulates replication of viral DNA within the nucleus. Thus, BocaSR may be a target for antiviral therapies for HBoV and may also have utility in the production of recombinant HBoV vectors. IMPORTANCE Human bocavirus 1 (HBoV1) is pathogenic to humans, causing acute respiratory tract infections in young children. In this study, we identified a novel HBoV1 gene that lies in the 3′ noncoding region of the viral positive-sense genome and is transcribed by RNA polymerase III into a noncoding RNA of 140 nt. This bocavirus-transcribed small RNA (BocaSR) diverges from both adenovirus-associated (VA) RNAs and Epstein-Barr virus-encoded small RNAs (EBERs) with respect to RNA sequence, representing a third species of this kind of Pol III-dependent viral noncoding RNA and the first noncoding RNA identified in autonomous parvoviruses. Unlike the VA RNAs, BocaSR localizes to the viral DNA replication centers of the nucleus and is essential for expression of viral nonstructural proteins independent of RNA-activated protein kinase R and replication of HBoV1 genomes. The identification of BocaSR and its role in virus DNA replication reveals potential avenues for developing antiviral therapies.

scholarly journals Prostaglandin E2Receptor Antagonist with Antimicrobial Activity against Methicillin-ResistantStaphylococcus aureus

2017 ◽  
Vol 62 (3) ◽  
Author(s):  
Mélanie A. C. Ikeh ◽  
Paul L. Fidel ◽  
Mairi C. Noverr
Keyword(s):  
Antimicrobial Activity ◽  
Receptor Antagonist ◽  
Enzyme Inhibitors ◽  
Lethal Dose ◽  
Prostaglandin E ◽  
Protective Effects ◽  
Physiological Concentration ◽  
Content Type ◽  
Abdominal Infections

ABSTRACTPolymicrobial intra-abdominal infections (IAI) involvingCandida albicansandStaphylococcus aureusare associated with severe morbidity and mortality (∼80%). Our laboratory discovered that the immunomodulatory eicosanoid prostaglandin E2(PGE2) plays a key role in the lethal inflammatory response during polymicrobial IAI using a mouse model of infection. In studies designed to uncover key PGE2biosynthesis/signaling components involved in the response, selective eicosanoid enzyme inhibitors and receptor antagonists were selected and prescreened for antimicrobial activity againstC. albicansorS. aureus. Unexpectedly, we found that the EP4receptor antagonist L-161,982 had direct growth-inhibitory effects onS. aureusin vitroat the physiological concentration required to block the PGE2interaction with EP4. This antimicrobial activity was observed with methicillin-sensitiveS. aureusand methicillin-resistantS. aureus(MRSA) strains, with the MIC and minimum bactericidal concentration values for planktonic cells being 50 μg/ml and 100 μg/ml, respectively. In addition, L-161,982 inhibitedS. aureusbiofilm formation and had activity against preformed mature biofilms. More importantly, treatment of mice with L-161,982 following intraperitoneal inoculation with a lethal dose of MRSA significantly reduced the bioburden and enhanced survival. Furthermore, L-161,982 protected mice against the synergistic lethality induced by coinfection withC. albicansandS. aureus. The antimicrobial activity of L-161,982 is independent of EP4receptor inhibitory activity; an alternative EP4receptor antagonist exerted no antimicrobial or protective effects. Taken together, these findings demonstrate that L-161,982 has potent antimicrobial activity against MRSA and may represent a significant therapeutic alternative in improving the prognosis of mono- or polymicrobial infections involving MRSA.

scholarly journals BordetellaAdenylate Cyclase Toxin Inhibits Monocyte-to-Macrophage Transition and Dedifferentiates Human Alveolar Macrophages into Monocyte-like Cells

mBio ◽  
2019 ◽  
Vol 10 (5) ◽  
Author(s):  
Jawid Nazir Ahmad ◽  
Jana Holubova ◽  
Oldrich Benada ◽  
Olga Kofronova ◽  
Ludek Stehlik ◽  
...  
Keyword(s):  
Adenylate Cyclase ◽  
Immune Evasion ◽  
Bordetella Pertussis ◽  
Alveolar Macrophages ◽  
Human Monocyte ◽  
Content Type ◽  
Macrophage Cells ◽  
Human Alveolar Macrophages ◽  
Adenylate Cyclase Toxin

ABSTRACTMonocytes arriving at the site of infection differentiate into functional effector macrophages to replenish the resident sentinel cells.Bordetella pertussis, the pertussis agent, secretes an adenylate cyclase toxin-hemolysin (CyaA) that binds myeloid phagocytes through complement receptor 3 (CD11b/CD18) and swiftly delivers its adenylyl cyclase enzyme domain into phagocytes. This ablates the bactericidal capacities of phagocytes through massive and unregulated conversion of cytosolic ATP into the key signaling molecule cAMP. We show that exposure of primary human monocytes to as low a concentration as 22.5 pM CyaA, or a low (2:1) multiplicity of infection by CyaA-producingB. pertussisbacteria, blocks macrophage colony-stimulating factor (M-CSF)-driven differentiation of monocytes. CyaA-induced cAMP signaling mediated through the activity of protein kinase A (PKA) efficiently blocked expression of macrophage markers, and the monocytes exposed to 22.5 pM CyaA failed to acquire the characteristic intracellular complexity of mature macrophage cells. Neither M-CSF-induced endoplasmic reticulum (ER) expansion nor accumulation of Golgi bodies, mitochondria, or lysosomes was observed in toxin-exposed monocytes, which remained small and poorly phagocytic and lacked pseudopodia. Exposure to 22.5 pM CyaA toxin provoked loss of macrophage marker expression onin vitrodifferentiated macrophages, as well as on primary human alveolar macrophages, which appeared to dedifferentiate into monocyte-like cells with upregulated CD14 levels. This is the first report that terminally differentiated tissue-resident macrophage cells can be dedifferentiatedin vitro. The results suggest that blocking of monocyte-to-macrophage transition and/or dedifferentiation of the sentinel cells of innate immunity through cAMP-elevating toxin action may represent a novel immune evasion strategy of bacterial pathogens.IMPORTANCEMacrophages are key sentinel cells of the immune system, and, as such, they are targeted by the toxins produced by the pertussis agentBordetella pertussis. The adenylate cyclase toxin (CyaA) mediates immune evasion ofB. pertussisby suspending the bactericidal activities of myeloid phagocytes. We reveal a novel mechanism of potential subversion of host immunity, where CyaA at very low (22 pM) concentrations could inhibit maturation of human monocyte precursors into the more phagocytic macrophage cells. Furthermore, exposure to low CyaA amounts has been shown to trigger dedifferentiation of mature primary human alveolar macrophages back into monocyte-like cells. This unprecedented capacity is likely to promote survival of the pathogen in the airways, both by preventing maturation of monocytes attracted to the site of infection into phagocytic macrophages and by dedifferentiation of the already airway-resident sentinel cells.

scholarly journals Shortening the Lipid A Acyl Chains of Bordetella pertussis Enables Depletion of Lipopolysaccharide Endotoxic Activity

Vaccines ◽  
2020 ◽  
Vol 8 (4) ◽  
pp. 594
Author(s):  
Jesús Arenas ◽  
Elder Pupo ◽  
Coen Phielix ◽  
Dionne David ◽  
Afshin Zariri ◽  
...  
Keyword(s):  
Bordetella Pertussis ◽  
Lipid A ◽  
Whooping Cough ◽  
Acyl Chain ◽  
In Vitro Assays ◽  
Whole Cell ◽  
Whole Cells ◽  
Acyl Chain Length ◽  
Acyl Chains

Whooping cough, or pertussis, is an acute respiratory infectious disease caused by the Gram-negative bacterium Bordetella pertussis. Whole-cell vaccines, which were introduced in the fifties of the previous century and proved to be effective, showed considerable reactogenicity and were replaced by subunit vaccines around the turn of the century. However, there is a considerable increase in the number of cases in industrialized countries. A possible strategy to improve vaccine-induced protection is the development of new, non-toxic, whole-cell pertussis vaccines. The reactogenicity of whole-cell pertussis vaccines is, to a large extent, derived from the lipid A moiety of the lipopolysaccharides (LPS) of the bacteria. Here, we engineered B. pertussis strains with altered lipid A structures by expressing genes for the acyltransferases LpxA, LpxD, and LpxL from other bacteria resulting in altered acyl-chain length at various positions. Whole cells and extracted LPS from the strains with shorter acyl chains showed reduced or no activation of the human Toll-like receptor 4 in HEK-Blue reporter cells, whilst a longer acyl chain increased activation. Pyrogenicity studies in rabbits confirmed the in vitro assays. These findings pave the way for the development of a new generation of whole-cell pertussis vaccines with acceptable side effects.

scholarly journals GC-072, a Novel Therapeutic Candidate for Oral Treatment of Melioidosis and Infections Caused by Select Biothreat Pathogens

2019 ◽  
Vol 63 (12) ◽  
Author(s):  
Jeffry D. Shearer ◽  
Michelle L. Saylor ◽  
Christine M. Butler ◽  
Anthony M. Treston ◽  
Henry S. Heine ◽  
...  
Keyword(s):  
Acute Phase ◽  
Oral Treatment ◽  
Lethal Dose ◽  
Current Therapy ◽  
Content Type ◽  
Development Of Resistance ◽  
Strong Candidate ◽  
Oral Therapeutic ◽  
Drug Resistant Strains

ABSTRACT Burkholderia pseudomallei, the etiological agent of melioidosis, is a Gram-negative bacterium with additional concern as a biothreat pathogen. The mortality rate from B. pseudomallei varies depending on the type of infection and extent of available health care; in the case of septicemia, left untreated, it can range from 50% to 90%. Current therapy for melioidosis is biphasic, consisting of parenteral acute-phase treatment for 2 weeks or longer, followed by oral eradication-phase treatment lasting several months. An effective oral therapeutic for outpatient treatment of acute-phase melioidosis is needed. GC-072 is a potent, 4-oxoquinolizine antibiotic with selective inhibitory activity against bacterial topoisomerases. GC-072 has demonstrated in vitro potency against susceptible and drug-resistant strains of B. pseudomallei and is also active against Burkholderia mallei, Bacillus anthracis, Yersinia pestis, and Francisella tularensis. GC-072 is bactericidal both extra- and intracellularly, with rapid killing noted within a few hours and reduced development of resistance compared to that for ceftazidime. GC-072, delivered intragastrically to mimic oral administration, promoted dose-dependent survival in mice using lethal inhalational models of B. pseudomallei infection following exposure to a 24- or 339-LD50 (50% lethal dose) challenge with B. pseudomallei strain 1026b. Overall, GC-072 appears to be a strong candidate for first-line oral treatment of melioidosis.

scholarly journals The Adenylate Cyclase (CyaA) Toxin from Bordetella pertussis Has No Detectable Phospholipase A (PLA) Activity In Vitro

Toxins ◽  
2019 ◽  
Vol 11 (2) ◽  
pp. 111 ◽  
Author(s):  
Alexis Voegele ◽  
Mirko Sadi ◽  
Dorothée Raoux-Barbot ◽  
Thibaut Douché ◽  
Mariette Matondo ◽  
...  
Keyword(s):  
Adenylate Cyclase ◽  
Bordetella Pertussis ◽  
Whooping Cough ◽  
Adenyl Cyclase ◽  
Phospholipase A ◽  
Eukaryotic Cells ◽  
Experimental Conditions ◽  
Cyclase Domain ◽  
Charged Membranes

The adenylate cyclase (CyaA) toxin produced in Bordetella pertussis is the causative agent of whooping cough. CyaA exhibits the remarkable capacity to translocate its N-terminal adenyl cyclase domain (ACD) directly across the plasma membrane into the cytosol of eukaryotic cells. Once translocated, calmodulin binds and activates ACD, leading to a burst of cAMP that intoxicates the target cell. Previously, Gonzalez-Bullon et al. reported that CyaA exhibits a phospholipase A activity that could destabilize the membrane to facilitate ACD membrane translocation. However, Bumba and collaborators lately reported that they could not replicate these results. To clarify this controversy, we assayed the putative PLA activity of two CyaA samples purified in two different laboratories by using two distinct fluorescent probes reporting either PLA2 or both PLA1 and PLA2 activities, as well as in various experimental conditions (i.e., neutral or negatively charged membranes in different buffers.) However, we could not detect any PLA activity in these CyaA batches. Thus, our data independently confirm that CyaA does not possess any PLA activity.

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