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Identification of genes essential for Moraxella catarrhalis growth under iron-limiting conditions using Genomic Array Footprinting (GAF)

ABSTRACT: Iron-sequestration by the human host is a first line defense against respiratory pathogens like Moraxella catarrhalis, which consequently experiences a period of iron-starvation during colonization and infection. We determined the genetic requirements for M. catarrhalis growth during iron-starvation using the high-throughput genome-wide screening technology genomic array footprinting (GAF). To this end, a large marinerT7 transposon mutant library (~28,000 independent transposon mutants) was grown under iron-limiting conditions, achieved by sequestration of iron by 30 µM Desferal (DF30), and under control growth conditions (brain heart infusion broth, DF0). Mutants were recovered at exponential- and the early-stationary growth phase and used for the generation of mutant-specific cDNA probes that were hybridized to custom-designed NimbleGen GAF microarrays. The results described in this study are further discussed in Stefan P.W. de Vries, Peter Burghout, Jeroen D. Langereis, Aldert Zomer, Peter W.M. Hermans, Hester J. Bootsma: Genetic requirements for Moraxella catarrhalis growth under iron-limiting conditions, Molecular Microbiology.

ORGANISM(S): Moraxella catarrhalis BBH18

PROVIDER: GSE41546 | GEO | 2013/06/06

SECONDARY ACCESSION(S): PRJNA177406

REPOSITORIES: GEO

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Identification of genes essential for Moraxella catarrhalis growth under iron-limiting conditions using Genomic Array Footprinting (GAF)

Project description:Iron-sequestration by the human host is a first line defense against respiratory pathogens like Moraxella catarrhalis, which consequently experiences a period of iron-starvation during colonization and infection. We determined the genetic requirements for M. catarrhalis growth during iron-starvation using the high-throughput genome-wide screening technology genomic array footprinting (GAF). To this end, a large marinerT7 transposon mutant library (~28,000 independent transposon mutants) was grown under iron-limiting conditions, achieved by sequestration of iron by 30 M-BM-5M Desferal (DF30), and under control growth conditions (brain heart infusion broth, DF0). Mutants were recovered at exponential- and the early-stationary growth phase and used for the generation of mutant-specific cDNA probes that were hybridized to custom-designed NimbleGen GAF microarrays. The results described in this study are further discussed in Stefan P.W. de Vries, Peter Burghout, Jeroen D. Langereis, Aldert Zomer, Peter W.M. Hermans, Hester J. Bootsma: Genetic requirements for Moraxella catarrhalis growth under iron-limiting conditions, Molecular Microbiology. cDNA probes generated from mutants recovered during the exponential growth phase, or early-stationary phase under challenge (iron-limiting, DF30) or control conditions (DF0). Hybridization on GAF 4x72K custom design Nimblegen GAF arrays for read-out.

2013-06-06 | E-GEOD-41546 | biostudies-arrayexpress

Identification of genes essential for Moraxella catarrhalis growth under iron-limiting conditions using Genomic Array Footprinting (GAF): Expression analysis of directed gene deletion mutants.

2013-06-06 | GSE41547 | GEO

Identification of genes essential for Moraxella catarrhalis growth under iron-limiting conditions using Genomic Array Footprinting (GAF): Expression analysis of directed gene deletion mutants.

Project description:Iron-sequestration by the human host is a first line defense against respiratory pathogens like Moraxella catarrhalis, which consequently experiences a period of iron-starvation during colonization and infection. We determined the genetic requirements for M. catarrhalis growth during iron-starvation using the high-throughput genome-wide screening technology genomic array footprinting (GAF). To further characterize the functional consequences of the gene deletions (GAF identified genes), we determined transcriptome profiles of the wild-type and directed mutant strains during exponential growth in BHI medium. The results described in this study are further discussed in Stefan P.W. de Vries, Peter Burghout, Jeroen D. Langereis, Aldert Zomer, Peter W.M. Hermans, Hester J. Bootsma: Genetic requirements for Moraxella catarrhalis growth under iron-limiting conditions, Molecular Microbiology. M. catarrhalis BBH18 directed mutants (n = 3) and their parental wild-type BBH18 strain (n = 4) were expanded to mid-log phase (OD620nm, 1.2 to 1.4) and RNA was isolated as previously described (de Vries et al., 2010 J Bacteriol 192: 3574-3583). Total RNA was labeled according to standard Nimblegen gene expression array protocols and hybridized to 4x72K custom design Nimblegen arrays for read-out.

2013-06-06 | E-GEOD-41547 | biostudies-arrayexpress

SWATH-MS proteome of Moraxella catarrhalis under iron restricted conditions

Project description:Moraxella catarrhalis is a human-restricted bacterial pathogen that causes otitis media in children and exacerbations of chronic obstructive pulmonary disease in adults. Iron is a co-factor required for the function of proteins involved in respiration and DNA replication, and thus is essential to life for most bacterial species. As iron is sequestered by host tissues, all host-adapted pathogens encounter periods of iron starvation. Previous studies have defined the repertoire of genes required for growth of M. catarrhalis under iron restricted conditions, and described differential expression at the transcriptional level. However, global changes in protein expression in response to iron starvation have not been investigated. Here we provide a SWATH-MS dataset describing differential expression of the M. catarrhalis CCRI-195ME proteome under iron restricted versus iron replete conditions.

2020-01-24 | PXD017206 | Pride

Global gene expression in Moraxella catarrhalis ATCC 43617 grown under iron-replete and iron-limiting conditions

Project description:Total RNA samples were isolated from M. catarrhalis grown in BHI (iron-replete condition, designated DF0) and in BHI containing 30 mM Desferal (iron-limiting condition, designated DF30). Total RNA and M. catarrhalis genome-directed primers (GDPs) were used for synthesis of Cy3- or Cy5-labeled cDNA samples. Three independent M. catarrhalis growth experiments and total RNA isolations were used for synthesis of cDNA samples, and a total of six DNA microarray hybridizations were performed in this study. Keywords: Effect of Growth Environment on Global Gene Expression by Moraxella catarrhalis

2007-12-07 | GSE4346 | GEO

Characterization of the molecular interplay between Moraxella catarrhalis and human respiratory tract epithelial cells: Expression Moraxella catarrhalis strain BBH18 during adherence to human phanryngeal epithelial Detroit 562 cells

2013-06-27 | GSE47870 | GEO

Characterization of the molecular interplay between Moraxella catarrhalis and human respiratory tract epithelial cells: Expression response of human phanryngeal epithelial Detroit 562 cells to adherent Moraxella catarrhalis strain BBH18

2013-06-27 | GSE47711 | GEO

Project description:During the course of infection, respiratory pathogens like Moraxella catarrhalis needs to adhere to epithelial cells of different host niches such as the nasopharynx and lungs. Consequently, efficient adhesion to epithelial cells is considered an important virulence trait of M. catarrhalis. We examined the interaction between human pharyngeal epithelial Detroit 562 cells and M. catarrhalis BBH18 during adherence using a combination of Tn-seq, a genome-wide negative selection screenings technology, and expression profiling of both host and pathogen. The results described in this study are further discussed in Stefan P.W. de Vries, Marc J. Eleveld, Peter W.M. Hermans, Hester J. Bootsma: Characterization of the molecular interplay between Moraxella catarrhalis and human respiratory tract epithelial cells, submitted. RNA was isolated from Detroit cells exposed to culture medium alone (n=6; control), and Detroits cells exposed to adherent M. catarrhalis BBH18 (n=6) using RNeasy columns. Total RNA was labeled according to standard Nimblegen gene expression array protocols and hybridized to a 12x135 Nimblegen human expression array for read-out.

2013-06-27 | E-GEOD-47711 | biostudies-arrayexpress

Project description:During the course of infection, respiratory pathogens like Moraxella catarrhalis needs to adhere to epithelial cells of different host niches such as the nasopharynx and lungs. Consequently, efficient adhesion to epithelial cells is considered an important virulence trait of M. catarrhalis. We examined the interaction between human pharyngeal epithelial Detroit 562 cells and M. catarrhalis BBH18 during adherence using a combination of Tn-seq, a genome-wide negative selection screenings technology, and expression profiling of both host and pathogen. The results described in this study are further discussed in Stefan P.W. de Vries, Marc J. Eleveld, Peter W.M. Hermans, Hester J. Bootsma: Characterization of the molecular interplay between Moraxella catarrhalis and human respiratory tract epithelial cells, submitted. After 1 hour adherence to Detroit 562 cells, RNA was isolated from adherent (n = 4) and non-adherent (planktonic) (n = 4) M. catarrhalis BBH18 as well as from bacteria incubated in the infection medium alone (n = 3). RNA obtained from the adherent fraction was enriched for bacterial RNA using the MICROBEnrich kit (Ambion). Total RNA was labeled according to standard Nimblegen gene expression array protocols and hybridized to a 4x72K Nimblegen M. catarrhalis expression array for read-out.

2013-06-27 | E-GEOD-47870 | biostudies-arrayexpress

RNA-seq-based analysis of the physiologic cold shock-induced changes in Moraxella catarrhalis gene expression

Project description:Background: Moraxella catarrhalis, a major nasopharyngeal pathogen of the human respiratory tract, is exposed to rapid downshifts of environmental temperature when humans breathe cold air. The prevalence of pharyngeal colonization and respiratory tract infections caused by M. catarrhalis is greatest in winter. We investigated how M. catarrhalis uses the physiologic exposure to cold air to regulate pivotal survival systems that may contribute to M. catarrhalis virulence. Results: In this study we used the RNA-seq techniques to quantitatively catalogue the transcriptome of M. catarrhalis exposed to a 26Â°C cold shock or to continuous growth at 37Â°C. Validation of RNA-seq data using quantitative RT-PCR analysis demonstrated the RNA-seq results to be highly reliable. We observed that a 26Â°C cold shock induces the expression of genes that in other bacteria have been related to virulence: a strong induction was observed for genes involved in high affinity phosphate transport and iron acquisition, indicating that M. catarrhalis makes a better use of both phosphate and iron resources after exposure to cold shock. We detected the induction of genes involved in nitrogen metabolism, as well as several outer membrane proteins, including ompA, m35-like porin and multidrug efflux pump (acrAB) indicating that M. catarrhalis remodels its membrane components in response to downshift of temperature. Furthermore, we demonstrate that a 26Â°C cold shock enhances the induction of genes encoding the type IV pili that are essential for natural transformation, and increases the genetic competence of M. catarrhalis, which may facilitate the rapid spread and acquisition of novel virulence-associated genes. mRNA profiles of Moraxella catarrhalis were generated by deep sequencing, in triplicate, using Illumina HiSeq 2000.

2013-07-22 | E-GEOD-46256 | biostudies-arrayexpress

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