Bright Fluorescent Streptococcus pneumoniae for Live-Cell Imaging of Host-Pathogen Interactions

Abstract

c Streptococcus pneumoniae is a common nasopharyngeal resident in healthy people but, at the same time, one of the major causes of infectious diseases such as pneumonia, meningitis, and sepsis. The shift from commensal to pathogen and its interaction with host cells are poorly understood. One of the major limitations for research on pneumococcal-host interactions is the lack of suitable tools for live-cell imaging. To address this issue, we developed a generally applicable strategy to create genetically stable, highly fluorescent bacteria. Our strategy relies on fusing superfolder green fluorescent protein (GFP) or a far-red fluorescent protein (RFP) to the abundant histone-like protein HlpA. Due to efficient translation and limited cellular diffusion of these fusions, the cells are 25-fold brighter than those of the currently best available imaging S. pneumoniae strain. These novel bright pneumococcal strains are fully virulent, and the GFP reporter can be used for in situ imaging in mouse tissue. We used our reporter strains to study the effect of the polysaccharide capsule, a major pneumococcal virulence factor, on different stages of infection. By dual-color live-cell imaging experiments, we show that unencapsulated pneumococci adhere significantly better to human lung epithelial cells than encapsulated strains, in line with previous data obtained by classical approaches. We also confirm with live-cell imaging that the capsule protects pneumococci from neutrophil phagocytosis, demonstrating the versatility and usability of our reporters. The described imaging tools will pave the way for live-cell imaging of pneumococcal infection and help further understanding of the mechanisms of pneumococcal pathogenesis. Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide, and pneumococcal infections (e.g., pneumonia, septicemia, and meningitis) kill more than 1 million people every year (1). Pneumococci are also quiescent colonizers of the upper respiratory tract, particularly in children, but little is known about the mechanisms underlying the transition from commensal to pathogen. It is therefore of crucial importance to understand the entire pneumococcal pathogenesis cycle in detail.The polysaccharide capsule covering the cell surface is the most central virulence factor of S. pneumoniae. The involvement of the capsule in pneumococcal pathogenesis has been appreciated since Griffith in 1928 (2) published his famous transformation experiment with rough and smooth strains of S. pneumoniae. Today, it is known that the bulky capsule, which is either negatively charged or neutral, contributes to pathogenesis by protecting pneumococci against the human immune system. For example, the capsule hinders phagocytosis and inhibits complement activity (3-6). Over 90 different pneumococcal serotypes have been identified to date (7), and the different serotypes vary in how well they protect the bacteria against phagocytosis (6). Furthermore, the amount of capsule differs between bacteria, and it has been s...

Three new integration vectors and fluorescent proteins for use in the opportunistic human pathogenStreptococcus pneumoniae

“…A) A phase contrast image of VL2706. B) ComEA fusions produces strong foci around midcell as well as at the poles of the cell as previously shown (31), C) HlpA fusions produce distinct green signal that localizes at the chromosome(32,33), and D) SPV_1159 fusions produce distinct membrane associated signals in the bacterial cells. E) An overlay of the three simultaneously expressed fusion proteins and a yellow outline of F) containing an expanded view of the image.…”

“…We selected ComEA as this is a protein involved in DNA uptake and was previously shown to demonstrate a septal localization (31). HlpA (HU) was chosen as it binds to the nucleoid and is a good marker for the chromosome (32,33). SPV_1159 was selected as it is a predicted small membrane protein with two transmembrane domains with both the N-and C-terminus inside the cytoplasm and was previously shown to demonstrate homogenous membrane localization in our laboratory (Gallay and Veening, unpublished).…”

N -Acetyl- l -Cysteine and Cysteamine as New Strategies against Mixed Biofilms of Nonencapsulated Streptococcus pneumoniae and Nontypeable Haemophilus influenzae

“…Live-cell imaging of strains showing fluorescence or bioluminescence has been proposed as an alternative to FISH. For example, highly fluorescent S. pneumoniae has been constructed by using a superfolder green fluorescent protein as a reporter (34). Nevertheless, when we have used these constructs to produce NE S. pneumoniae and NE S. pneumoniae-NT H. influenzae in vitro biofilms, significant differences in fluorescence among individual bacteria were noted (unpublished observations).…”

Deep genome annotation of the opportunistic human pathogenStreptococcus pneumoniaeD39

“…Despite the differences observed with NCTC 7466, D39V is genetically stable under laboratory conditions, as both the hsdS locus and the ter configuration are identical in all analyzed derivative strains (not shown). Additionally and importantly, D39V is virulent in multiple infection models (78)(79)(80)(81), making it an ideal, stable genetic workhorse for pneumococcal research. Therefore, the strain will be made available to the community through the PHE National…”