A Gene Expression Platform for Synthetic Biology in the human pathogen Streptococcus pneumoniae.

ACS Synth Biol. 2014 May 20. [Epub ahead of print]

A Gene Expression Platform for Synthetic Biology in the human pathogen Streptococcus pneumoniae.

Sorg RA, Kuipers OP, Veening JW.

Abstract

The human pathogen Streptococcus pneumoniae (pneumococcus) is a bacterium that owes its success to complex gene expression regulation patterns on both the cellular and the population level. Expression of virulence factors enables a mostly hazard-free presence of the commensal, in balance with the host and niche competitors. Under specific circumstances, changes in this expression can result in a more aggressive behavior and the reversion to the invasive form as pathogen. These triggering conditions are very difficult to study due to the fact that environmental cues are often unknown or barely possible to simulate outside the host (in vitro). An alternative way of investigating expression patterns is found in synthetic biology approaches of reconstructing regulatory networks that mimic an observed behavior with orthogonal components. Here, we created a genetic platform suitable for synthetic biology approaches in S. pneumoniae and characterized a set of standardized promoters and reporters. We show that our system allows for fast and easy cloning with the BglBrick system and that reliable and robust gene expression after integration into the S. pneumoniae genome is achieved. In addition, a standard gene expression measuring protocol utilizing firefly luciferase was established. Finally, the cloning system was extended to allow for direct linker-based assembly of ribosome binding sites, peptide tags and fusion proteins and we called this new generally-applicable standard "BglFusion". The gene expression platform and the methods described in this study pave the way for employing synthetic biology approaches in S. pneumoniae.

PMID: 24845455 [PubMed - as supplied by publisher]

Outbreak of invasive Streptococcus pneumoniae Serotype 12F among a marginalized inner-city population in Winnipeg, Canada (2009-2011)

Clin Infect Dis. 2014 May 19. pii: ciu366. [Epub ahead of print]

Outbreak of invasive Streptococcus pneumoniae Serotype 12F among a marginalized inner-city population in Winnipeg, Canada (2009-2011).

Schillberg E1, Isaac M2, Deng X3, Peirano G4, Wylie JL5, Van Caeseele P5, Pillai DR4, Sinnock H2, Mahmud SM6.

Abstract

BACKGROUND:

 In 2010, Winnipeg, Canada experienced a doubling of invasive pneumococcal disease (IPD) rates, with a significant increase in the number of cases due to Streptococcus pneumoniae serotype 12F, which previously had accounted for very few cases each year.

METHODS:

 All serotype 12F IPD cases reported between September, 2009 and January, 2011 were reviewed. Pulsed-field gel electrophoresis (PFGE) and multiple-locus variable number tandem repeat analysis (MLVA) were conducted on all isolates. PFGE and MLVA patterns identified several possible clusters. Additional interviews were conducted to obtain information on risk factors and outcomes.

RESULTS:

 Between September, 2009 and January, 2011, 169 cases of IPD were identified. The number of IPD cases due to 12F serotype increased sharply from about 3-4 cases/year (6% of IPD cases) in 2007-09 to 28 (29%) in 2010. All 12F isolates belonged to a single sequence type ST218, and they were generally susceptible to penicillin and fluoroquinolones but not to erythromycin. Compared to other serotypes, 12F cases were more likely to be homeless, to reside in poor inner-city communities and engage in substance abuse, including intravenous and crack cocaine use. Subclusters identified using MLVA had even higher rates of homelessness and substance use.

CONCLUSIONS:

 An immunization campaign targeting high-risk groups was undertaken with pneumococcal polysaccharide vaccine, and subsequently rates of serotype 12F decreased. To our knowledge, this is the largest documented community outbreak of serotype 12F IPD and the first report of an outbreak of IPD serotype 12F in a marginalized urban population in Canada.

© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

PMID:  

24842908

[PubMed - as supplied by publisher]

Penicillin-Binding Protein 2x of Streptococcus pneumoniae: The Mutation Ala707Asp within the C-terminal PASTA2 Domain Leads to Destabilization

Microb Drug Resist. 2014 May 19. [Epub ahead of print]

Penicillin-Binding Protein 2x of Streptococcus pneumoniae: The Mutation Ala707Asp within the C-terminal PASTA2 Domain Leads to Destabilization.

Schweizer I1, Peters K, Stahlmann C, Hakenbeck R, Denapaite D.

Author information

Abstract

Streptococcus pneumoniae penicillin-binding protein 2x (PBP2x) is an enzyme involved in the last stages of peptidoglycan assembly and essential for bacterial growth and survival. PBP2x localizes to the division site, a process that depends on its Penicillin-Binding Protein And Serine-Threonine-kinase Associated (PASTA) domains, which was previously demonstrated via GFP-PBP2x in living cells. During this study a mutant strain was isolated in which the GFP-PBP2x fusion protein did not localize at division sites and it contained reduced amounts of the full-length GFP-PBP2x. We now show that this defect is due to a point mutation within the C-terminal PASTA2 domain of PBP2x. The mutant protein was analyzed in detail in terms of beta-lactam binding, functionality, and localization in live cells. We demonstrate that the mutation affects the GFP-tagged PBP2x variant severely and renders it susceptible to the protease/chaperone HtrA.

PMID: 24841912 [PubMed - as supplied by publisher]