Dataset Information

Interaction of phase variation, host and pressure/gas composition: pneumococcal gene expression of PsaA, SpxB, Ply and LytA in simulated middle ear environments.

ABSTRACT: Streptococcus pneumoniae, a leading cause of otitis media (OM), undergoes spontaneous intra-strain variations in colony morphology. Transparent (T) variants are more efficient in colonizing the nasopharynx while opaque (O) variants exhibit greater virulence during systemic infections. This study was intended to delineate the underlying molecular mechanisms by which the predominant S. pneumoniae variant efficiently infects the middle ear (ME) mucosa.Human ME epithelial cells were preconditioned for 24h under one of the three gas/pressure conditions designed to simulate those for (1) normal ME (NME), (2) ME with Eustachian tube obstruction (ETO) and (3) ME with tympanostomy tube placement (TT), and then were incubated with ? 10(7)CFU/ml of either T or O variants of S. pneumoniae (6A) for 3h. Relative expression levels of genes encoding virulence factors, PsaA (surface adhesion), SpxB (pyruvate oxidase), Ply (pneumolysin), and LytA (autolysin) were assessed separately in epithelium-attached and supernatant bacteria 3h post infection using real-time PCR.Basal levels of the virulence molecules in inocula were comparable between two variants. However, relative expression levels of the gene transcripts were significantly induced in epithelium-attached T variants 3h after infection. Comparing with NME and TT conditions, ETO environment produced the largest effect on the differential expression of the virulence genes in the infected ME epithelial cells between T (induced) and O (suppressed) phenotypic pneumococci.T variant is a predominant phenotype responsible for the pathogenesis of pneumococcal OM.

SUBMITTER: Li-Korotky HS

PROVIDER: S-EPMC2891361 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Interaction of phase variation, host and pressure/gas composition: pneumococcal gene expression of PsaA, SpxB, Ply and LytA in simulated middle ear environments.

Li-Korotky Ha-Sheng HS Lo Chia-Yee CY Zeng Fan-Rui FR Lo David D Banks Juliane M JM

International journal of pediatric otorhinolaryngology 20090813 10

<h4>Objective</h4>Streptococcus pneumoniae, a leading cause of otitis media (OM), undergoes spontaneous intra-strain variations in colony morphology. Transparent (T) variants are more efficient in colonizing the nasopharynx while opaque (O) variants exhibit greater virulence during systemic infections. This study was intended to delineate the underlying molecular mechanisms by which the predominant S. pneumoniae variant efficiently infects the middle ear (ME) mucosa.<h4>Methods</h4>Human ME epit ...[more]

PMID: 19682756

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Project description:The accurate diagnosis of pneumococcal disease has frequently been hampered not only by the difficulties in obtaining isolates of the organism from patient specimens but also by the misidentification of pneumococcus-like viridans group streptococci (P-LVS) as Streptococcus pneumoniae. This is especially critical when the specimen comes from the respiratory tract. In this study, three novel real-time PCR assays designed for the detection of specific sequence regions of the lytA, ply, and psaA genes were developed (lytA-CDC, ply-CDC, and psaA, respectively). These assays showed high sensitivity (<10 copies for lytA-CDC and ply-CDC and an approximately twofold less sensitivity for psaA). Two additional real-time PCR assays for lytA and ply described previously for pneumococcal DNA detection were also evaluated. A panel of isolates consisting of 67 S. pneumoniae isolates (44 different serotypes and 3 nonencapsulated S. pneumoniae isolates from conjunctivitis outbreaks) and 104 nonpneumococcal isolates was used. The 67 S. pneumoniae isolates were reactive in all five assays. The new real-time detection assays targeting the lytA and psaA genes were the most specific for the detection of isolates confirmed to be S. pneumoniae, with lytA-CDC showing the greatest specificity. Both ply PCRs were positive for all isolates of S. pseudopneumoniae, along with 13 other isolates of other P-LVS isolates confirmed to be non-S. pneumoniae by DNA-DNA reassociation. Thus, the use of the ply gene for the detection of pneumococci can lead to false-positive reactions in the presence of P-LVS. The five assays were applied to 15 culture-positive cerebrospinal fluid specimens with 100% sensitivity; and serum and ear fluid specimens were also evaluated. Both the lytA-CDC and psaA assays, particularly the lytA-CDC assay, have improved specificities compared with those of currently available assays and should therefore be considered the assays of choice for the detection of pneumococcal DNA, particularly when upper respiratory P-LVS might be present in the clinical specimen.

Project description:IntroductionAir pollutants such as Asian sand dust (ASD) and Streptococcus pneumoniae are risk factors for otitis media (OM). In this study, we evaluate the role of ASD in pneumococcal in vitro biofilm growth and colonization on human middle ear epithelium cells (HMEECs) and rat middle ear using the rat OM model.MethodsS. pneumoniae D39 in vitro biofilm growth in the presence of ASD (50-300 μg/ml) was evaluated in metal ion-free BHI medium using CV-microplate assay, colony-forming unit (cfu) counts, resazurin staining, scanning electron microscopy (SEM), and confocal microscopy (CF). Biofilm gene expression analysis was performed using real-time RT-PCR. The effects of ASD or S. pneumoniae individually or on co-treatment on HMEECs were evaluated by detecting HMEEC viability, apoptosis, and reactive oxygen species (ROS) production. In vivo colonization of S. pneumoniae in the presence of ASD was evaluated using the rat OM model, and RNA-Seq was used to evaluate the alterations in gene expression in rat middle ear mucosa.ResultsS. pneumoniae biofilm growth was significantly (P < 0.05) elevated in the presence of ASD. SEM and CF analysis revealed thick and organized pneumococcal biofilms in the presence of ASD (300 μg/ml). However, in the absence of ASD, bacteria were unable to form organized biofilms, the cell size was smaller than normal, and long chain-like structures were formed. Biofilms grown in the presence of ASD showed elevated expression levels of genes involved in biofilm formation (luxS), competence (comA, comB, ciaR), and toxin production (lytA and ply). Prior exposure of HMEECs to ASD, followed by treatment for pneumococci, significantly (P < 0.05) decreased cell viability and increased apoptosis, and ROS production. In vivo experiment results showed significantly (P < 0.05) more than 65% increased bacteria colonization in rat middle ear mucosa in the presence of ASD. The apoptosis, cell death, DNA repair, inflammation and immune response were differentially regulated in three treatments; however, number of genes expressed in co-treatments was higher than single treatment. In co-treatment, antimicrobial protein/peptide-related genes (S100A family, Np4, DEFB family, and RATNP-3B) and OM-related genes (CYLD, SMAD, FBXO11, and CD14) were down regulated, and inflammatory cytokines and interleukins, such as IL1β, and TNF-related gene expression were elevated.ConclusionASD presence increased the generation of pneumococcal biofilms and colonization.

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Dataset Information

Interaction of phase variation, host and pressure/gas composition: pneumococcal gene expression of PsaA, SpxB, Ply and LytA in simulated middle ear environments.

Publications

Interaction of phase variation, host and pressure/gas composition: pneumococcal gene expression of PsaA, SpxB, Ply and LytA in simulated middle ear environments.

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