Project description:We performed transcriptome sequencing to profile gene expressions upon the treatment of vancomycin in two Streptococcus anginosus strains.
Project description:Streptococcus anginosus is an important cause of brain and liver abscesses, meningitis, appendicitis, female genital tract infection, neonatal sepsis, and bacteremia. Recent studies show that S. anginosus is responsible for around 30% of streptococci associated purulent infections of internal organs. Genome sequencing of S. anginosus identified a phage-like chromosomal island (SanCI) integrated into the DNA mismatch repair operon. In S. pyogenes, related chromosomal islands (SpyCI) integrated into the same operon confer a mutator phenotype as well as alter global gene regulation, including the increased expression of many virulence factors. We hypothesized that SanCI will also alter global transcription patterns and virulence in S. anginosus. The SanCI from strain F0211 was introduced into a SanCI-free S. anginosus strain (J4211) by natural transformation. To provide a selectable marker for transformation, gene strA, encoding a predicted transcriptional regulator, was replaced in F0211 with the gene conferring erythromycin resistance (ermB), which after transfer created strain OKSan3. A functional copy of strA was then returned to this derivative of J4211, creating strain OKSan4. RNA sequencing (RNA-seq) of strains J4211, OKSan4 and OKSan3 confirmed that the addition of the SanCI altered S. anginosus global transcription patterns with increased expression of virulence, stress response and competence factors in a growth phase dependent manner. Virulence studies using Galleria mellonella as an acute infection model showed significantly increased inflammation and mortality in larvae challenged with strains OKSan4 and OKSan3 as compare to SanCI-free strain J4211. qRT-PCR analysis supported these results, revealing that antimicrobial factors of G. mellonella were more highly expressed after infection with OKSan4 compare to J4211 infection. Further, RNA-seq suggested that SanCI-encoded gene strA may encode one of the factors responsible for altered global transcriptional changes. The result of this study along with our previous studies in S. pyogenes demonstrate that streptococcal chromosomal islands are a unique class of virulence factors that improve the fitness of their host cell by altering global transcription patterns and increasing virulence.
Project description:S. anginosus, S. aureus LMG 10147 and P. aeruginosa DK2 are often co-isolated in sputum samples from cystic fibrosis patients. We found that S. anginosus LMG 14502 becomes less suceptible towards treatment with several antibiotics when it's grown together with S. aureus LMG 10147 and P. aeruginosa DK2, compared to when it's grown alone. In order to elucidate the molecular mechanisms responsible, we performed RNA-seq of an S. anginosus monospecies biofilm and of a multispecies bioiflm of S. anginosus, S. aureus and P. aeruginosa. First, biofilms of S. anginosus alone or in combination with S. aureus and P. aeruginosa were grown. Next, RNA was isolated. Subsequently, a Truseq stranded RNA library preparation kit (Illumina) was used to create strand specific libraries. After a quality and concentration control, the libraries were equimolarly pooled and sequenced using an Illumina NextSeq 500, generating 75bp unpaired reads.
