scholarly journals Expression of BfrH, a Putative Siderophore Receptor of Bordetella bronchiseptica, Is Regulated by Iron, Fur1, and the Extracellular Function Sigma Factor EcfI

2009 ◽  
Vol 78 (3) ◽  
pp. 1147-1162 ◽  
Author(s):  
Jonathan M. Burgos ◽  
Natalie D. King-Lyons ◽  
Terry D. Connell
Keyword(s):  
Sigma Factor ◽  
Pathogenic Bacteria ◽  
Respiratory Infections ◽  
Etiologic Agent ◽  
Bordetella Bronchiseptica ◽  
Infection Model ◽  
Gene Encoding ◽  
Ecf Sigma Factor ◽  
Wide Range ◽  
Upper Respiratory

ABSTRACT Iron (Fe) in soluble elemental form is found in the tissues and fluids of animals at concentrations insufficient for sustaining growth of bacteria. Consequently, to promote colonization and persistence, pathogenic bacteria evolved a myriad of scavenging mechanisms to acquire Fe from the host. Bordetella bronchiseptica, the etiologic agent of upper respiratory infections in a wide range of mammalian hosts, expresses a number of proteins for acquisition of Fe. Using proteomic and genomic approaches, three Fe-regulated genes were identified in the bordetellae: bfrH, a gene encoding a putative siderophore receptor; ecfI, a gene encoding a putative extracellular function (ECF) sigma factor; and ecfR, a gene encoding a putative EcfI modulator. All three genes are highly conserved in B. pertussis, B. parapertussis, and B. avium. Genetic analysis revealed that transcription of bfrH was coregulated by ecfI, ecfR, and fur1, one of two fur homologues carried by B. bronchiseptica. Overexpression of ecfI decoupled bfrH from Fe-dependent regulation. In contrast, expression of bfrH was significantly reduced in an ecfI deletion mutant. Deletion of ecfR, however, was correlated with a significant increase in expression of bfrH, due in part to a cis-acting nucleotide sequence within ecfR which likely reduces the frequency of readthrough transcription of bfrH from the Fe-dependent ecfIR promoter. Using a murine competition infection model, bfrH was shown to be required for optimal virulence of B. bronchiseptica. These experiments revealed ecfIR-bfrH as a locus encoding a new member of the growing family of Fe and ECF sigma factor-modulated regulons in the bordetellae.

scholarly journals Modulating Pathogenesis with Mobile-CRISPRi

2019 ◽  
Vol 201 (22) ◽  
Author(s):  
Jiuxin Qu ◽  
Neha K. Prasad ◽  
Michelle A. Yu ◽  
Shuyan Chen ◽  
Amy Lyden ◽  
...  
Keyword(s):  
Gene Expression ◽  
Pathogenic Bacteria ◽  
Bacterial Species ◽  
Effector Proteins ◽  
Infection Model ◽  
Secretion Systems ◽  
Bacterial Genomes ◽  
Gene Encoding ◽  
Content Type ◽  
Animal Infection

ABSTRACT Conditionally essential (CE) genes are required by pathogenic bacteria to establish and maintain infections. CE genes encode virulence factors, such as secretion systems and effector proteins, as well as biosynthetic enzymes that produce metabolites not found in the host environment. Due to their outsized importance in pathogenesis, CE gene products are attractive targets for the next generation of antimicrobials. However, the precise manipulation of CE gene expression in the context of infection is technically challenging, limiting our ability to understand the roles of CE genes in pathogenesis and accordingly design effective inhibitors. We previously developed a suite of CRISPR interference-based gene knockdown tools that are transferred by conjugation and stably integrate into bacterial genomes that we call Mobile-CRISPRi. Here, we show the efficacy of Mobile-CRISPRi in controlling CE gene expression in an animal infection model. We optimize Mobile-CRISPRi in Pseudomonas aeruginosa for use in a murine model of pneumonia by tuning the expression of CRISPRi components to avoid nonspecific toxicity. As a proof of principle, we demonstrate that knock down of a CE gene encoding the type III secretion system (T3SS) activator ExsA blocks effector protein secretion in culture and attenuates virulence in mice. We anticipate that Mobile-CRISPRi will be a valuable tool to probe the function of CE genes across many bacterial species and pathogenesis models. IMPORTANCE Antibiotic resistance is a growing threat to global health. To optimize the use of our existing antibiotics and identify new targets for future inhibitors, understanding the fundamental drivers of bacterial growth in the context of the host immune response is paramount. Historically, these genetic drivers have been difficult to manipulate precisely, as they are requisite for pathogen survival. Here, we provide the first application of Mobile-CRISPRi to study conditionally essential virulence genes in mouse models of lung infection through partial gene perturbation. We envision the use of Mobile-CRISPRi in future pathogenesis models and antibiotic target discovery efforts.

scholarly journals Identification of a Siderophore Receptor Required for Ferric Ornibactin Uptake in Burkholderia cepacia

2000 ◽  
Vol 68 (12) ◽  
pp. 6554-6560 ◽  
Author(s):  
P. A. Sokol ◽  
P. Darling ◽  
S. Lewenza ◽  
C. R. Corbett ◽  
C. D. Kooi
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Burkholderia Cepacia ◽  
Parent Strain ◽  
Respiratory Infections ◽  
Infection Model ◽  
Protein Profiles ◽  
Outer Membrane Receptor ◽  
Gene Encoding

ABSTRACT Ornibactins are linear hydroxamate siderophores produced byBurkholderia cepacia with peptide structures similar to that of pyoverdines produced by the fluorescent pseudomonads. The gene encoding the outer membrane receptor (orbA) was identified, sequenced, and demonstrated to have significant homology with hydroxamate receptors produced by other organisms. The orbAprecursor was predicted to be a protein with a molecular mass of 81 kDa. An orbA mutant was constructed and demonstrated to be unable to take up 59Fe-ornibactins or to grow in medium supplemented with ornibactins. Outer membrane protein profiles from the parent strain, K56-2, revealed an iron-regulated outer membrane protein of 78 kDa that was not detectable in the K56orbA::tp mutant. When this mutant harbored a plasmid containing the orbA gene, the 78-kDa protein was present in the outer membrane protein profiles and the mutant was able to utilize ornibactin to acquire iron. The orbA mutant was less virulent in a chronic respiratory infection model than the parent strain, indicating that ornibactin uptake and utilization are important in the pathogenesis of B. cepacia respiratory infections.

scholarly journals Vitamin D supplementation –Is it the time to stop wasting money on vitamin D?

JMS SKIMS ◽  
2019 ◽  
Vol 22 (1) ◽  
Author(s):  
Shariq R Masoodi
Keyword(s):  
Vitamin D ◽  
Bone Health ◽  
Respiratory Infections ◽  
Vitamin D Supplementation ◽  
Increased Risk ◽  
Wide Range ◽  
Upper Respiratory ◽  
The Relationship

In recent years, vitamin D has received increased attention, as a number of studies have shown its link to the pathogenesis of various diseases. Apart from its benefits on bone health, vitamin D supplementation has been shown beneficial in reducing risk for many chronic diseases including autoimmune diseases, type 2 diabetes, heart disease, many cancers and infectious diseases. Vitamin deficiency has been associated with increased risk of elevated blood pressure, heart attack and stroke in studies. The relationship between vitamin D deficiency and the increased incidence of upper respiratory infections, asthma and eczema, among children, has been observed in several studies. Recent meta-analyzes showed a relationship between vitamin D administration during the early months of life and a lower incidence of type 1 diabetes later in life. Therefore, it is not surprising that Vitamin D supplementation is used to prevent and treat a wide range of diseases, and the use has increased considerably in the last decade.

scholarly journals Burkholderia cenocepacia Requires the RpoN Sigma Factor for Biofilm Formation and Intracellular Trafficking within Macrophages

2008 ◽  
Vol 76 (3) ◽  
pp. 1059-1067 ◽  
Author(s):  
M. Soledad Saldías ◽  
Julie Lamothe ◽  
Robert Wu ◽  
Miguel A. Valvano
Keyword(s):  
Biofilm Formation ◽  
Pulmonary Infection ◽  
Fluorescent Protein ◽  
Respiratory Infections ◽  
Cell Envelope ◽  
Burkholderia Cenocepacia ◽  
Infection Model ◽  
Wild Type ◽  
Gene Encoding ◽  
Flagellar Biosynthesis

ABSTRACT Chronic respiratory infections by Burkholderia cenocepacia in cystic fibrosis patients are associated with increased morbidity and mortality, but virulence factors determining the persistence of the infection in the airways are not well characterized. Using a chronic pulmonary infection model, we previously identified an attenuated mutant with an insertion in a gene encoding an RpoN activator protein, suggesting that RpoN and/or components of the RpoN regulon play a role in B. cenocepacia virulence. In this study, we demonstrate that a functional rpoN gene is required for bacterial motility and biofilm formation in B. cenocepacia K56-2. Unlike other bacteria, RpoN does not control flagellar biosynthesis, as evidenced by the presence of flagella in the rpoN mutant. We also demonstrate that, in macrophages, the rpoN mutant is rapidly trafficked to lysosomes while intracellular wild-type B. cenocepacia localizes in bacterium-containing vacuoles that exhibit a pronounced delay in phagolysosomal fusion. Rapid trafficking to the lysosomes is also associated with the release of red fluorescent protein into the vacuolar lumen, indicating loss of bacterial cell envelope integrity. Although a role for RpoN in motility and biofilm formation has been previously established, this study is the first demonstration that the RpoN regulon in B. cenocepacia is involved in delaying phagolysosomal fusion, thereby prolonging bacterial intracellular survival within macrophages.

scholarly journals The ECF sigma factor PvdS regulates the type I-F CRISPR-Cas system in Pseudomonas aeruginosa

2020 ◽  
Author(s):  
Stephen Dela Ahator ◽  
Wang Jianhe ◽  
Lian-Hui Zhang
Keyword(s):  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Sigma Factor ◽  
Regulatory System ◽  
Stress Factors ◽  
Iron Limitation ◽  
Transport Systems ◽  
Type I ◽  
Ecf Sigma Factor ◽  
Wide Range

AbstractDuring infection, successful colonization of bacteria requires a fine-tuned supply of iron acquired via iron transport systems. However, the transport systems serve as phage attachment sites and entry portals for foreign nucleic acid. Most bacteria possess the CRISPR-Cas system, which targets and destroys foreign nucleic acids and prevents deleterious effects of horizontal gene transfer. To understand the regulation of the CRISPR-Cas system, we performed genome-wide random transposon mutagenesis which led to the identification of the Extracytoplasmic Function (ECF) Sigma factor, PvdS as a regulator of the Type I-F CRISPR-Cas system in P. aeruginosa. We show that under iron-depleted conditions PvdS induces the expression of the type I-F CRISPR-Cas system. This regulatory mechanism involves direct interaction of PvdS with specific binding sites in the promoter region of cas1. Furthermore, activation of the CRISPR-Cas system under iron-depleted conditions increases horizontal gene transfer (HGT) interference and adaptation. The PvdS activation of the CRISPR-Cas system under iron limitation highlights the versatility of the P. aeruginosa in multitasking its regulatory machinery to integrate multiple stress factors.ImportanceP. aeruginosa infects a wide range of host organisms and adapts to various environmental stress factors such as iron limitation due to its elaborate regulatory system. P aeruginosa possesses the type I-F CRISPR-Cas system as a defense mechanism against phages infection and HGT. This work highlights the ability of P. aeruginosa to multitask its iron regulatory system to control the CRISPR-Cas system under a physiologically relevant stress factor such as iron limitation where the bacteria are vulnerable to phage infection. It also adds to the knowledge of the regulation of the CRISPR-Cas system in bacteria and presents a possible target that could prevent the emergence of phage resistance via the CRISPR-Cas system during the development of phage therapy.

scholarly journals Expression of hurP, a Gene Encoding a Prospective Site 2 Protease, Is Essential for Heme-Dependent Induction of bhuR in Bordetella bronchiseptica

2007 ◽  
Vol 189 (17) ◽  
pp. 6266-6275 ◽  
Author(s):  
Natalie D. King-Lyons ◽  
Kelsy F. Smith ◽  
Terry D. Connell
Keyword(s):  
Membrane Protein ◽  
Sigma Factor ◽  
Cytoplasmic Membrane ◽  
Regulatory System ◽  
Sole Source ◽  
Bordetella Bronchiseptica ◽  
Dependent Manner ◽  
Gene Encoding ◽  
Reading Frame ◽  
Regulatory Cascade

ABSTRACT Expression of the hurIR bhuRSTUV heme utilization locus in Bordetella bronchiseptica is coordinately controlled by the global iron-dependent regulator Fur and the extracytoplasmic function sigma factor HurI. Activation of HurI requires transduction of a heme-dependent signal via HurI, HurR, and BhuR, a three-component heme-dependent regulatory system. In silico searches of the B. bronchiseptica genome to identify other genes that encode additional participants in this heme-dependent regulatory cascade revealed hurP, an open reading frame encoding a polypeptide with homology to (i) RseP, a site 2 protease (S2P) of Escherichia coli required for modifying the cytoplasmic membrane protein RseA, and (ii) YaeL, an S2P of Vibrio cholerae required for modification of the cytoplasmic membrane protein TcpP. A mutant of B. bronchiseptica defective for hurP was incapable of regulating expression of BhuR in a heme-dependent manner. Furthermore, the hurP mutant was unable to utilize hemin as a sole source of nutrient Fe. These defects in hemin utilization and heme-dependent induction of BhuR were restored when recombinant hurP (or recombinant rseP) was introduced into the mutant. Introduction of hurP into a yaeL mutant of V. cholerae also complemented its S2P defect. These data provided strong evidence that protease activity and cleavage site recognition was conserved in HurP, RseP, and YaeL. The data are consistent with a model in which HurP functionally modifies HurR, a sigma factor regulator that is essential for heme-dependent induction of bhuR.

scholarly journals Otitis media in guineapigs: pathology and bacteriology

1986 ◽  
Vol 20 (3) ◽  
pp. 242-248 ◽  
Author(s):  
R. Boot ◽  
H. C. Walvoort
Keyword(s):  
Otitis Media ◽  
Pathogenic Bacteria ◽  
Bacterial Species ◽  
Clinical Signs ◽  
Bordetella Bronchiseptica ◽  
Simultaneous Occurrence ◽  
Streptococcus Zooepidemicus ◽  
Post Mortem ◽  
Upper Respiratory Tract ◽  
Upper Respiratory

In the course of post-mortem examination of conventional random-bred and inbred (immunosuppressed) strain 2/N guinea pigs kept in separate quarters, otitis media was diagnosed in 62 of 462 animals (13·4%) and 18 of 66 animals (27·3%) respectively. Clinical signs of otitis media were seen in only two randombred animals but in nearly 50% of affected inbred animals. In random-bred guineapigs, purulent, often bilateral, otitis media was associated mainly with the isolation of Streptococcus zooepidemicus, Bordetella bronchiseptica, Pasteurella and Actinobacillus spp. and micrococci. In strain 2/N guineapigs serous or purulent often bilateral otitis media was associated mainly with the isolation of B. bronchiseptica and Pseudomonas aeruginosa serotypes 10 and 11. The simultaneous occurrence of similar pathogenic bacteria in both ears of bilaterally affected animals and in pneumonic lung tissue (in random-bred animals) suggested ascending and descending infection originating from the upper respiratory tract. It is concluded that otitis media, associated with the isolation of a variety of respiratory bacterial species, must be considered a major disease problem in guineapigs.

scholarly journals Identification of AlcR, an AraC-Type Regulator of Alcaligin Siderophore Synthesis in Bordetella bronchiseptica and Bordetella pertussis

1998 ◽  
Vol 180 (4) ◽  
pp. 871-880 ◽  
Author(s):  
Elizabeth Pradel ◽  
Nicole Guiso ◽  
Camille Locht
Keyword(s):  
Bordetella Pertussis ◽  
Growth Conditions ◽  
Bordetella Bronchiseptica ◽  
Open Reading Frames ◽  
Infection Model ◽  
Gene Encoding ◽  
Sequence Comparisons ◽  
New Gene ◽  
Type Gene ◽  
Titration Assay

ABSTRACT A Fur titration assay was used to isolate DNA fragments bearing putative Fur binding sites (FBS) from a partial Bordetella bronchiseptica genomic DNA library. A recombinant plasmid bearing a 3.5-kb DNA insert was further studied. Successive deletions in the cloned fragment enabled us to map a putative FBS at about 2 kb from one end. Sequence analysis revealed the presence of an FBS upstream from a new gene encoding an AraC-type transcriptional regulator. The deduced protein displays similarity to PchR, an activator of pyochelin siderophore and ferripyochelin receptor synthesis in Pseudomonas aeruginosa. Homologous genes in Bordetella pertussisand Bordetella parapertussis were PCR amplified, and sequence comparisons indicated a very high conservation in the three species. The B. pertussis and B. bronchisepticachromosomal genes were inactivated by allelic exchange. Under low-iron growth conditions, the mutants did not secrete the alcaligin siderophore and lacked AlcC, an alcaligin biosynthetic enzyme. Alcaligin production was restored after transformation with a plasmid bearing the wild-type gene. On the basis of its role in regulation of alcaligin biosynthesis, the new gene was designated alcR. Additional sequence determination showed that alcR is located about 2 kb downstream from the alcABC operon and is transcribed in the same orientation. Two tightly linked open reading frames, alcD and alcE, were identified betweenalcC and alcR. AlcE is a putative iron-sulfur protein; AlcD shows no homology with the proteins in the database. The production of major virulence factors and colonization in the mouse respiratory infection model are AlcR independent.

scholarly journals 4-Methylcytosine DNA modification is critical for global epigenetic regulation and virulence in the human pathogen Leptospira interrogans

2020 ◽  
Vol 48 (21) ◽  
pp. 12102-12115
Author(s):  
Robert A Gaultney ◽  
Antony T Vincent ◽  
Céline Lorioux ◽  
Jean-Yves Coppée ◽  
Odile Sismeiro ◽  
...  
Keyword(s):  
Sigma Factor ◽  
Acute Infection ◽  
Dna Methyltransferases ◽  
Host Cells ◽  
Infection Model ◽  
Cellular Processes ◽  
Growth Defects ◽  
Ecf Sigma Factor ◽  
Epigenetic Marker ◽  
Genome Wide

Abstract In bacteria, DNA methylation can be facilitated by ‘orphan’ DNA methyltransferases lacking cognate restriction endonucleases, but whether and how these enzymes control key cellular processes are poorly understood. The effects of a specific modification, 4-methylcytosine (4mC), are even less clear, as this epigenetic marker is unique to bacteria and archaea, whereas the bulk of epigenetic research is currently performed on eukaryotes. Here, we characterize a 4mC methyltransferase from the understudied pathogen Leptospira spp. Inactivating this enzyme resulted in complete abrogation of CTAG motif methylation, leading to genome-wide dysregulation of gene expression. Mutants exhibited growth defects, decreased adhesion to host cells, higher susceptibility to LPS-targeting antibiotics, and, importantly, were no longer virulent in an acute infection model. Further investigation resulted in the discovery of at least one gene, that of an ECF sigma factor, whose transcription was altered in the methylase mutant and, subsequently, by mutation of the CTAG motifs in the promoter of the gene. The genes that comprise the regulon of this sigma factor were, accordingly, dysregulated in the methylase mutant and in a strain overexpressing the sigma factor. Our results highlight the importance of 4mC in Leptospira physiology, and suggest the same of other understudied species.

scholarly journals Passively Released Heme from Hemoglobin and Myoglobin Is a Potential Source of Nutrient Iron for Bordetella bronchiseptica

2007 ◽  
Vol 75 (10) ◽  
pp. 4857-4866 ◽  
Author(s):  
Jeffrey C. Mocny ◽  
John S. Olson ◽  
Terry D. Connell
Keyword(s):  
Direct Contact ◽  
Pathogenic Bacteria ◽  
Whooping Cough ◽  
Respiratory Infections ◽  
Bordetella Bronchiseptica ◽  
Common Mechanism ◽  
Atrophic Rhinitis ◽  
Dependent Manner ◽  
Potential Source ◽  
Successful Colonization

ABSTRACT Colonization by Bordetella bronchiseptica results in a variety of inflammatory respiratory infections, including canine kennel cough, porcine atrophic rhinitis, and a whooping cough-like disease in humans. For successful colonization, B. bronchiseptica must acquire iron (Fe) from the infected host. A vast amount of Fe within the host is sequestered within heme, a metalloporphyrin which is coordinately bound in hemoglobin and myoglobin. Utilization of hemoglobin and myoglobin as sources of nutrient Fe by B. bronchiseptica requires expression of BhuR, an outer membrane protein. We hypothesize that hemin is acquired by B. bronchiseptica in a BhuR-dependent manner after spontaneous loss of the metalloporphyrin from hemoglobin and/or myoglobin. Sequestration experiments demonstrated that direct contact with hemoglobin or myoglobin was not required to support growth of B. bronchiseptica in an Fe-limiting environment. Mutant myoglobins, each exhibiting a different affinity for heme, were employed to demonstrate that the rate of growth of B. bronchiseptica was directly correlated with the rate at which heme was lost from the hemoprotein. Finally, Escherichia coli cells expressing recombinant BhuR had the capacity to remove hemin from solution. Collectively, these experiments provided strong experimental support for the model that BhuR is a hemin receptor and B. bronchiseptica likely acquires heme during infection after passive loss of the metalloporphyrin from hemoglobin and/or myoglobin. These results also suggest that spontaneous hemin loss by hemoglobin and myoglobin may be a common mechanism by which many pathogenic bacteria acquire heme and heme-bound Fe.

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