The essentiality and involvement of <i>Streptococcus intermedius</i> histone‐like DNA‐binding protein in bacterial viability and normal growth

Liu, Dali; Yumoto, Hiromichi; Murakami, Keiji; Hirota, Kaoru; Ono, Tsuneko; Nagamune, Hideaki; Kayama, Shizuo; Matsuo, Takashi; Miyake, Yoichiro

doi:10.1111/j.1365-2958.2008.06232.x

Cited by 27 publications

(41 citation statements)

References 75 publications

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“…In general, untagged GFP is difficult to image because the fluorescence signal spreads through the entire cytoplasm by fast diffusion during the image acquisition time and is overwhelmed by cellular autofluorescence (37,38). To overcome this problem, we fused sfGFP(Bs) and mKate2 (here called gfp and rfp, respectively) to the 3= end of hlpA (SPD_0997), encoding the only known nucleoid binding protein in Streptococcus (39), and stably integrated the fusions by double crossover in the pneumococcal chromosome at the hlpA locus (Fig. 1A).…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2015

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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...

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Section: Resultsmentioning

confidence: 99%

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

et al. 2015

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“…S.i.-HLP was found to bind host cells and induce cytokine production. 28 It was reported that stimulation with recombinant S.i.-HLP (rS.i.-HLP) induced the secretion of IL-8, IL-1b, and tumor necrosis factor-a in human monocytic cell line THP-1. The stimulatory effect of rS.i.-HLP was cooperative by costimulation with LTA.…”

Section: Discussionmentioning

confidence: 99%

Long-term bacterial exposure can trigger nonsuppurative destructive cholangitis associated with multifocal epithelial inflammation

Haruta

Kikuchi

Hashimoto

et al. 2010

Laboratory Investigation

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“…Electrophoretic mobility shift assay was carried out as described by Liu et al (23). Briefly, a supercoiled plasmid, pGEMT (6 nM), was incubated with increasing amounts (60, 600, and 6,000 nM) of purified HP119 protein or bovine serum albumin (BSA) in reaction buffer (10 mM Tris [pH 8.0], and 75 mM KCl) at 25°C for 25 min.…”

Section: Methodsmentioning

confidence: 99%

A Novel DNA-Binding Protein Plays an Important Role in Helicobacter pylori Stress Tolerance and Survival in the Host

Wang

Maier

2015

J Bacteriol

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The gastric pathogen Helicobacter pylori must combat chronic acid and oxidative stress. It does so via many mechanisms, including macromolecule repair and gene regulation. Mitomycin C-sensitive clones from a transposon mutagenesis library were screened. One sensitive strain contained the insertion element at the locus of hp119, a hypothetical gene. No homologous gene exists in any (non-H. pylori) organism. Nevertheless, the predicted protein has some features characteristic of histone-like proteins, and we showed that purified HP119 protein is a DNA-binding protein. A ⌬hp119 strain was markedly more sensitive (viability loss) to acid or to air exposure, and these phenotypes were restored to wild-type (WT) attributes upon complementation of the mutant with the wild-type version of hp119 at a separate chromosomal locus. The mutant strain was approximately10-fold more sensitive to macrophage-mediated killing than the parent or the complemented strain. Of 12 mice inoculated with the wild type, all contained H. pylori, whereas 5 of 12 mice contained the mutant strain; the mean colonization numbers were 158-fold less for the mutant strain. A proteomic (two-dimensional PAGE with mass spectrometric analysis) comparison between the ⌬hp119 mutant and the WT strain under oxidative stress conditions revealed a number of important antioxidant protein differences; SodB, Tpx, TrxR, and NapA, as well as the peptidoglycan deacetylase PgdA, were significantly less expressed in the ⌬hp119 mutant than in the WT strain. This study identified HP119 as a putative histone-like DNA-binding protein and showed that it plays an important role in Helicobacter pylori stress tolerance and survival in the host. Helicobacter pylori infects the stomachs of approximately 50% of humans and results in a series of human gastric diseases, including gastritis, peptic ulcers, and gastric cancer (1-4). The pathogenesis of H. pylori relies on its persistence in surviving a harsh environment, including acidity, peristalsis, and attack by phagocyte cells and their released reactive oxygen species (5). H. pylori survives on the surface of the stomach lining, often for the lifetime of its host, and causes a chronic inflammatory response. Under physiological conditions, H. pylori is thought to frequently suffer oxidative and acid stress (6, 7). H. pylori is equipped with diverse oxidant detoxification enzymes (e.g., superoxide dismutase, catalase, and peroxiredoxins) (8) and potent acid avoidance mechanisms (mainly urease) (9). To survive the harsh conditions, H. pylori regulates its gene expression in response to the stress signals; however, the bacterium lacks many response regulators known to occur in model microorganisms, such as the SOS response, OxyR/SoxR, and RpoS. Our current knowledge of the stress tolerance mechanisms cannot account for the well-described dynamic survival abilities of H. pylori.Studies in recent years have indicated that DNA recombination and repair play a significant role in H. pylori's persistent colonization of the host (10-14). ...

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The essentiality and involvement of Streptococcus intermedius histone‐like DNA‐binding protein in bacterial viability and normal growth

Cited by 27 publications

References 75 publications

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

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

Long-term bacterial exposure can trigger nonsuppurative destructive cholangitis associated with multifocal epithelial inflammation

A Novel DNA-Binding Protein Plays an Important Role in Helicobacter pylori Stress Tolerance and Survival in the Host

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