Tibody from eBioscience (San Diego, CA, USA) was used, and an irrelevant isotype-matchedCryptococcus gattii Induced Cytokine PatternSupporting InformationIL-1b induction by Pam3cys and E. coli LPS after blocking of TLR2 and TLR4 respectively. IL-1b production by human PBMCs is shown (A) induced by pam3cys [10 mg/ml] after preincubated for one hour with anti-TLR2 or control antibody [10 mg/ml] and (B) by E. coli LPS [10 ng/ml] after preincubation for one hour with TLR4 antagonist Bartonella quintana LPS [200 ng/ml] or culture medium. Mean values (n = 10) 6 SE of five independent experiments are presented. (TIF)Figure SAcknowledgmentsThe authors thank Ferry Hagen for providing cryptococcal strains.Author ContributionsConceived and designed the experiments: T. Schoffelen LABJ MGN JFM T. Sprong. Performed the experiments: T. Schoffelen LABJ MGN T. Sprong. Analyzed the data: T. Schoffelen LABJ MGN JFM T. Sprong. Contributed reagents/materials/analysis tools: MTIZ TB JFM. Wrote the paper: T. Schoffelen MTIZ LABJ MGN TB JFM 
T. Sprong.
Studies on the sub-cellular localization 
of bacterial proteins have changed our view on the organization of bacterial cells. Initially, these studies were essentially restricted to the model organisms Escherichia coli and 1655472 Bacillus subtilis. Despite their importance, these organisms do not eliminate the need for specific studies using different clinically important bacterial species, given the diversity in terms of morphology, physiology and metabolism among bacteria. Therefore, we have recently witnessed the development of new tools to allow cell biology studies in different pathogenic bacteria [1,2]. Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. This Gram-positive bacterium is associated with a range of infections, which can vary from simple otitis media to more complicated ones, such as pneumonia or meningitis. Infection by this important human pathogen is of particular concern in developing countries, in which MedChemExpress Eledoisin K162 web pneumococcal septicemia causes 25 of all preventable deaths in children under the age of five [3]. In order to design new and more efficient strategies to fight pneumococcal infections it is essential to understand how these bacteria divide or perform specific tasks important for their survival inside the host, such as the synthesis of peptidoglycan, the target of beta-lactam antibiotics which are widely used against S. pneumoniae, or the synthesis of the capsular polysaccharide, the target of several successful anti-pneumococcal vaccines. An important step to accomplish this goal is the study of the localization of proteins involved in these processes. However, fora long time, cell biology studies in S. pneumoniae were limited by the lack of appropriate tools. Localization of pneumococcal proteins involved in cell wall synthesis [4,5] and cell division [6,7,8] was initially accomplished using immunofluorescence techniques, which require cell fixation and lysis to allow access of the antibodies to the target proteins. Therefore, immunofluorescence can not be used with live cells and is prone to generate artifacts [9]. It was only recently that the first studies on the localization of proteins in live pneumococcal cells, using fluorescent protein fusions, tagged to Green Fluorescent Protein (GFP), was reported [1]. Since then, other proteins involved in processes such as cell division [10,11], cell wall synthesis [12] and capsular polysaccharide synthesis [13,14] have bee.Tibody from eBioscience (San Diego, CA, USA) was used, and an irrelevant isotype-matchedCryptococcus gattii Induced Cytokine PatternSupporting InformationIL-1b induction by Pam3cys and E. coli LPS after blocking of TLR2 and TLR4 respectively. IL-1b production by human PBMCs is shown (A) induced by pam3cys [10 mg/ml] after preincubated for one hour with anti-TLR2 or control antibody [10 mg/ml] and (B) by E. coli LPS [10 ng/ml] after preincubation for one hour with TLR4 antagonist Bartonella quintana LPS [200 ng/ml] or culture medium. Mean values (n = 10) 6 SE of five independent experiments are presented. (TIF)Figure SAcknowledgmentsThe authors thank Ferry Hagen for providing cryptococcal strains.Author ContributionsConceived and designed the experiments: T. Schoffelen LABJ MGN JFM T. Sprong. Performed the experiments: T. Schoffelen LABJ MGN T. Sprong. Analyzed the data: T. Schoffelen LABJ MGN JFM T. Sprong. Contributed reagents/materials/analysis tools: MTIZ TB JFM. Wrote the paper: T. Schoffelen MTIZ LABJ MGN TB JFM T. Sprong.
Studies on the sub-cellular localization of bacterial proteins have changed our view on the organization of bacterial cells. Initially, these studies were essentially restricted to the model organisms Escherichia coli and 1655472 Bacillus subtilis. Despite their importance, these organisms do not eliminate the need for specific studies using different clinically important bacterial species, given the diversity in terms of morphology, physiology and metabolism among bacteria. Therefore, we have recently witnessed the development of new tools to allow cell biology studies in different pathogenic bacteria [1,2]. Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. This Gram-positive bacterium is associated with a range of infections, which can vary from simple otitis media to more complicated ones, such as pneumonia or meningitis. Infection by this important human pathogen is of particular concern in developing countries, in which pneumococcal septicemia causes 25 of all preventable deaths in children under the age of five [3]. In order to design new and more efficient strategies to fight pneumococcal infections it is essential to understand how these bacteria divide or perform specific tasks important for their survival inside the host, such as the synthesis of peptidoglycan, the target of beta-lactam antibiotics which are widely used against S. pneumoniae, or the synthesis of the capsular polysaccharide, the target of several successful anti-pneumococcal vaccines. An important step to accomplish this goal is the study of the localization of proteins involved in these processes. However, fora long time, cell biology studies in S. pneumoniae were limited by the lack of appropriate tools. Localization of pneumococcal proteins involved in cell wall synthesis [4,5] and cell division [6,7,8] was initially accomplished using immunofluorescence techniques, which require cell fixation and lysis to allow access of the antibodies to the target proteins. Therefore, immunofluorescence can not be used with live cells and is prone to generate artifacts [9]. It was only recently that the first studies on the localization of proteins in live pneumococcal cells, using fluorescent protein fusions, tagged to Green Fluorescent Protein (GFP), was reported [1]. Since then, other proteins involved in processes such as cell division [10,11], cell wall synthesis [12] and capsular polysaccharide synthesis [13,14] have bee.
