Project description:Streptococcus pneumoniae is the dominant cause of community-acquired pneumonia world-wide. Invasion of the pleural space is common and results in increased mortality. We set out to determine the bacterial and host factors that influence invasion of the pleural space. In a murine model of pneumococcal infection, we isolated neutrophil-dominated samples of bronchoalveolar and pleural fluid containing bacteria 48 hours after infection. Using dual RNA-seq, we characterised bacterial and host transcripts that were differentially regulated between these compartments and bacteria in broth and resting neutrophils respectively. Pleural and lung samples showed upregulation of genes involved in positive regulation of neutrophil extravasation but down-regulation of genes mediating bacterial killing. Compared to the lung samples, cells within the pleural space showed marked upregulation of many genes induced by type I interferons, cytokines implicated in preventing bacterial transmigration across epithelial barriers. Differences in the bacterial transcripts between the infected samples and bacteria grown in broth showed upregulation of genes in the bacteriocin locus, the pneumococcal surface adhesin PsaA, and the glycopeptide resistance gene, vanZ; the gene encoding the ClpP protease was downregulated in infection. 169 intergenic putative small bacterial RNAs were also identified, of which 43 (25.4%) had been previously described. 42 of the small RNAs were upregulated in pleura compared to broth, including many previously identified as important in virulence. Our results have identified key host and bacterial responses to invasion of the pleural space that can be potentially exploited to develop alternative antimicrobial strategies for prevention and treatment of pneumococcal pleural disease.
Project description:Zebrafish embryo has been emerging as an interesting model of infection due to their fecundity, transparency and availability of genetic tools. Streptococcus pneumoniae, the pneumococcus, is the main etiological agent of pneumonia, sepsis and meningitis. The bacteria expresses a very important virulence factor, pneumolysin able to form pores on cholesterol-based membranes and to activate innate immune system. Here, we exploited the recently described dual RNA-seq to simultaneously measure genome-wide expression of host and pathogen eight hours into infection. Functional enrichment analysis showed certain pathways such as autophagy and apoptosis being activated in the host while stress responses including pneumococcal competence. The study is the first to describe dual RNA-seq application in whole organism sequencing in infection model.
Project description:Tropism is a very important factor in pneumococcal infection. Previous study has shown that clinical isolate showed niche preference in model organism. In particular, ear isolate showed particular tropism in nasopharyngeal carriage while blood isolate survives better in the murine lung. We have detected a SNP located inside rafR, repressor of sugar metabolism regulon, that contributes to strain tropism. We then perform a swap mutation between the rafR ear and blood isolates and perform intranasal infection using the four strains: original ear and blood isolates, ear isolate with rafR blood isolate and blood isolate with rafR ear isolate. We harvested the lung six hours into infection and exploited the recently described dual RNA-seq to reconstruct transcriptomics landscape during host-pathogen interaction.
Project description:BACKGROUND: Streptococcus pneumoniae, the pneumococcus, is the main etiological agent of pneumonia. Pneumococcal infection is initiated by bacterial adherence to lung epithelial cells. The exact transcriptional changes occurring in both host and microbe during infection are unknown. Here, we developed a time-resolved infection model of human lung alveolar epithelial cells by S. pneumoniae and assess the resulting transcriptome changes in both organisms simultaneously by using dual RNA-seq. RESULTS: Functional analysis of the time-resolved dual RNA-seq data identifies several features of pneumococcal infection. For instance, we show that the glutathione-dependent reactive oxygen detoxification pathway in epithelial cells is activated by reactive oxygen species produced by S. pneumoniae. Addition of the antioxidant resveratrol during infection abates this response. At the same time, pneumococci activate the competence regulon during co-incubation with lung epithelial cells. By comparing transcriptional changes between wild-type encapsulated and mutant unencapsulated pneumococci, we demonstrate that adherent pneumococci, but not free-floating bacteria, repress innate immune responses in epithelial cells including expression of the chemokine IL-8 and the production of antimicrobial peptides. We also show that pneumococci activate several sugar transporters in response to adherence to epithelial cells and demonstrate that this activation depends on host-derived mucins. CONCLUSIONS: We provide a dual-transcriptomics overview of early pneumococcal infection in a time-resolved manner, providing new insights into host-microbe interactions. To allow easy access to the data by the community, a web-based platform was developed ( http://dualrnaseq.molgenrug.nl ). Further database exploration may expand our understanding of epithelial-pneumococcal interaction, leading to novel antimicrobial strategies.
Project description:Secondary bacterial pneumonia following influenza infection is a significant cause of mortality worldwide. Upper respiratory tract pneumococcal carriage is important as both determinants of disease and population transmission. The immunological mechanisms that contain pneumococcal carriage are well-studied in mice but remain unclear in humans. Loss of this control of carriage following influenza infection is associated with secondary bacterial pneumonia during seasonal and pandemic outbreaks. We used a human type 6B pneumococcal challenge model to show that carriage acquisition induces early degranulation of resident neutrophils and recruitment of monocytes to the nose. Monocyte function associated with clearance of pneumococcal carriage. Prior nasal infection with live attenuated influenza virus induced inflammation, impaired innate function and altered genome-wide nasal gene responses to pneumococcal carriage. Levels of the cytokine IP-10 promoted by viral infection at the time of pneumococcal encounter was positively associated with bacterial density. These findings provide novel insights in nasal immunity to pneumococcus and viral-bacterial interactions during co-infection.
Project description:Analysis of pulmonary gene expression in two mouse strains, resistant (BALB/c) and susceptible (CBA/Ca) to Streptococcus pneumoniae infection. Data collected at 6h post-infection and for control animals (PBS-treated). The list of differentially expressed genes was created by comparisons of infected versus PBS-treated animals and PBS-treated BALB/c versus CBA/Ca. The hypothesis tested in the present study was that pulmonary transcriptomes of both mouse strains differ during pneumococcal infection and in non-disease conditions. Results provided important information on differences in immune responses between both mouse strains. The results identified genes and pathways uniquely regulated by only one of the tested mouse strains helping to understand molecular mechanism behind resistance or susceptibility to pneumococcal infections.
Project description:Analysis of pulmonary gene expression in two mouse strains, resistant (BALB/c) and susceptible (CBA/Ca) to Streptococcus pneumoniae infection. Data collected at 6h post-infection and for control animals (PBS-treated). The list of differentially expressed genes was created by comparisons of infected versus PBS-treated animals and PBS-treated BALB/c versus CBA/Ca. The hypothesis tested in the present study was that pulmonary transcriptomes of both mouse strains differ during pneumococcal infection and in non-disease conditions. Results provided important information on differences in immune responses between both mouse strains. The results identified genes and pathways uniquely regulated by only one of the tested mouse strains helping to understand molecular mechanism behind resistance or susceptibility to pneumococcal infections. Total RNA obtained from lung tissue from BALB/cOlaHsd and CBA/CaOlaHsd mouse strains (Harlan) 6 hours post intranasal infection with Streptococcus pneumoniae serotype 2 strain D39 dose 5.0E06 or PBS-treated animals