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.
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.
Project description:M. catarrhalis strain O35E.rpsl was inoculated into the nasopharynx of healthy, male adult chinchillas. 24 hours later the nasopharyngeal tissues were extracted and homogenized. Total RNA was extracted from these tissue samples. Subsequently, M. catarrhalis genome directed primers were utilized to synthesize cDNA from the total RNA sample. As a control, M.catarrhalis strain O35E.rpsl was grown in BHI broth to a Klett density of 200 units and underwent RNA extraction per standard protocols. The genome directed primers mentioned above were utilized to synthesize cDNA. Both cDNA samples were subsequently labelled with either Cy3 or Cy5 and hybridized to a custom Microarrays, Inc. gene chip and scanned after 16 hours. Differential gene expression was measured utilizing the broth grown cells as the baseline and the chinchilla isolated cells as the experimental variable.
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.
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.
Project description:M. catarrhalis strain O35E.rpsl was inoculated into the nasopharynx of healthy, male adult chinchillas. 24 hours later the nasopharyngeal tissues were extracted and homogenized. Total RNA was extracted from these tissue samples. Subsequently, M. catarrhalis genome directed primers were utilized to synthesize cDNA from the total RNA sample. As a control, M.catarrhalis strain O35E.rpsl was grown in BHI broth to a Klett density of 200 units and underwent RNA extraction per standard protocols. The genome directed primers mentioned above were utilized to synthesize cDNA. Both cDNA samples were subsequently labelled with either Cy3 or Cy5 and hybridized to a custom Microarrays, Inc. gene chip and scanned after 16 hours. Differential gene expression was measured utilizing the broth grown cells as the baseline and the chinchilla isolated cells as the experimental variable. There are 5 individual sample results included in this series. These represent the data from four individual biological replicates (i.e. 4 different sets of inoculated animals). For each replicate the control samples are simultaneously grown broth samples. Three dye swap experiments were performed.
Project description:Comparison of the gene expression profile of Moraxella catarrhalis grown in the presence of 20% pooled human sputum in chemically-defined medium relative to Moraxella catarrhalis grown in chemically-defined medium alone.
