Project description:Antibiotic resistance in Streptococcus pneumoniae is often the result of horizontal gene transfer events involving closely related streptococcal species. Laboratory experiments confirmed that S. mitis DNA functions as donor in transformation experiments, using the laboratory strain S. pneumoniae R6 as recipient and chromosomal DNA of a high level penicillin resistant S. mitis B6 strain. After four transformation steps, alterations in five penicillin-binding proteins (PBP) were observed, and sequence analysis confirmed recombination events in the corresponding PBP genes. In order to detect regions where recombination with S. mitis DNA has occurred we analyzed the S. pneumoniae transformants by microarray analyses, using oligonucleotide microarrays designed for the S. pneumoniae genome and the S. mitis B6 genome as well.
Project description:Streptococcus pneumoniae (S. pneumoniae) is a major human pathogen causing morbidity and mortality worldwide. Efficiently acquiring iron from the environment is critical for S. pneumoniae to sustain growth and cause infection. There are only three known iron-uptake systems in Streptococcal species responsible for iron acquisition from the host, including ABC transporters PiaABC, PiuABC and PitABC. Besides, no other iron-transporting system has been suggested. In this work, we employed our newly established translating mRNA analysis integrated with proteomics to evaluate the possible existence of novel iron transporters in the bacterium. We simultaneously deleted the iron-binding protein genes of the three iron-uptake systems to construct a piaA/piuA/pitA triple mutant (Tri-Mut) of S. pneumoniae D39, in which genes and proteins related to iron transport should be regulated in response to the deletion. With ribosome associated mRNA sequencing-based translatomics focusing on translating mRNA and iTRAQ quantitative proteomics based on the covalent labeling of peptides with tags of varying mass, we indeed observed a large number of genes and proteins representing various coordinated biological pathways with significantly altered expression levels in the Tri-Mut mutant. Highlighted in this observation is the identification of several new potential iron-uptake ABC transporters for Streptococcal iron metabolism. In particular, putative protein SPD_1609 in operon 804 was verified to be a novel iron-binding protein with similar function to PitA in S. pneumoniae. These data derived from the integrative translatomics and proteomics analyses provided rich information and insightful clues for further investigations on iron-transporting mechanism in bacteria and the interplay between Streptococcal iron availability and the biological metabolic pathways.
Project description:In a subclinical infection such as bovine streptococcal mastitis, early recognition is a great challenge, and miRNAs profiling could potentially assist in the diagnosis and contribute to the understanding of pathogenicity and defense mechanisms. We have examined the miRNA repertoire during the early phase response of bovine macrophages to in vitro infection with live Streptococcus agalactiae. Next generation sequencing of 20 small RNA libraries from blood monocyte-derived macrophages exposed to two sequence types of S. agalactiae (ST103 and ST12) for 6 hours in vitro was performed. Analyzes of over 356 million of high quality sequence reads, revealed that 17 and 44 miRNAs were differentially expressed (P < 0.05) between the control unchallenged macrophages and the macrophages infected with ST103 and ST12, respectively. We also identified the expression of 31 potentially novel bovine miRNAs.
Project description:Recent whole-genome sequencing of large populations of the same bacterial species has revealed significant disparity among genes in the frequency of single nucleotide polymorphisms (SNPs). For example, a previous analysis of invasive serotype M3 group A streptococci (GAS) found the highest frequency of SNPs in the gene (ropB) encoding the regulator of proteinase B (RopB). This finding led us to hypothesize that RopB polymorphisms contribute to altered GAS host-pathogen interactions. Sequencing of ropB in 171 invasive serotype M3 GAS strains from a surveillance study identified 19 distinct ropB alleles. Inactivation of the ropB gene in strains producing distinct RopB variants had dramatically different effects on GAS global gene expression. Further, analysis of laboratory-generated isoallelic GAS strains differing only by a single amino acid replacement in RopB confirmed that the variant protein affected the transcript level of the gene encoding streptococcal proteinase B, a major RopB-regulated virulence factor. Comparison of parental, RopB-inactivated, and RopB isoallelic strains in mouse infection models demonstrated that RopB polymorphisms significantly influence GAS virulence and disease manifestations. These studies detail a paradigm in which unbiased, whole-genome sequence analysis of populations of clinical bacterial isolates creates new avenues of productive investigation into the pathogenesis of common human infections. This study examined the effects of RopB inactivaiton on two distinct serotype M3 group A streptococcal strains with distinct forms of the RopB protein. RopB was inactivated in a strain with a wild-type RopB allele (strain MGAS10870) and in a strain with a RopB allele containing a C85Y polymorphism (strain MGAS9937). The wild-type and RopB inactivated strains were grown in duplicate to the early stationary growth phase in standard laboratory medium (THY). Total RNA was isolated, converted to cDNA, and hybridized to a custom-made Affymetrix GeneChip.
Project description:we used high-throughput Illumina Genome Analyzer IIx (GAIIx) technology to sequence the small RNA transcriptomes of the mangrove species, Avicennia marina. Based on sequence similarity or the secondary structure of precursors, we have identified 193 conserved miRNAs and 26 novel miRNAs in the small RNA transcriptome of Avicennia marina.