Pneumococcal DNA-binding proteins released through autolysis induce the production of proinflammatory cytokines via toll-like receptor 4

doi:10.1016/j.cellimm.2018.01.006

Cellular Immunology

Volume 325, March 2018, Pages 14-22

https://doi.org/10.1016/j.cellimm.2018.01.006 Get rights and content

Highlights

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Pneumococcal autolysis causes release of DnaK, EF-Tu, and GAPDH associated with its DNA.
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DnaK, EF-Tu, and GAPDH induced the production of proinflammatory cytokines in mouse peritoneal macrophages and THP-1-derived macrophage-like cells via TLR4.

Abstract

Streptococcus pneumoniae is a leading cause of bacterial pneumonia. Our previous study suggested that S. pneumoniae autolysis-dependently releases intracellular pneumolysin, which subsequently leads to lung injury. In this study, we hypothesized that pneumococcal autolysis induces the leakage of additional intracellular molecules that could increase the pathogenicity of S. pneumoniae. Liquid chromatography tandem-mass spectrometry analysis identified that chaperone protein DnaK, elongation factor Tu (EF-Tu), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were released with pneumococcal DNA by autolysis. We demonstrated that recombinant (r) DnaK, rEF-Tu, and rGAPDH induced significantly higher levels of interleukin-6 and tumor necrosis factor production in peritoneal macrophages and THP-1-derived macrophage-like cells via toll-like receptor 4. Furthermore, the DNA-binding activity of these proteins was confirmed by surface plasmon resonance assay. We demonstrated that pneumococcal DnaK, EF-Tu, and GAPDH induced the production of proinflammatory cytokines in macrophages, and might cause host tissue damage and affect the development of pneumococcal diseases.

Introduction

Streptococcus pneumoniae, also known as pneumococcus, is a Gram-positive diplococcus and major human pathogen. This bacterium asymptomatically colonizes the upper respiratory airway and causes common clinical syndromes, such as otitis media, sinusitis, bronchitis, and empyema, or even severe life-threatening diseases, including pneumonia, meningitis, and septicemia [1], [2]. Pneumococcal infections have led to significant morbidity and mortality worldwide especially in children under 5 years old and adults over 65 years old in developing countries [3].

A variety of pneumococcal virulence factors, including the autolytic enzyme LytA, contribute to the development of pneumococcal diseases [4]. LytA is responsible for the characteristic autolytic behavior associated with pneumococcus. It has been reported that LytA potentially contributes to pneumococcal pathogenesis by catalyzing the release of intracellular toxins and generating proinflammatory cell wall fragments [5]. Our previous study suggested that S. pneumoniae autolysis-dependently releases pneumolysin (PLY), which is a cholesterol-dependent cytolytic pore-forming toxin, that induces the disruption of pulmonary immune defenses [6]. Therefore, autolysis plays a central role in the pathogenesis of pneumococcal diseases.

The innate immune response is the first line of defense against any bacterial infection. In this regard, macrophages respond immediately to diverse microbial pathogens and control the replication of invading pathogens [7], [8]. Macrophages express various pattern recognition receptors, such as toll-like receptors (TLRs), which activate downstream signaling and induce the production of proinflammatory cytokines. TLRs play a crucial role against microbial infections, initiating and activating both host innate and adaptive immunity [9]. They recognize the presence of microbial pathogens via detection of conserved structures. It has been shown that the cell wall components of pneumococci, such as lipoteichoic acid and peptidoglycan are recognized by TLR2 [10]. TLR4 is a key component of the innate response to Gram-negative infections through the recognition of lipopolysaccharide (LPS). In addition, PLY is also considered to be a TLR4 ligand. TLR4-mutant mice are more susceptible to lethal infection after intranasal infection with pneumococcus [11]. It has also been reported that TLR9 plays an important role in the recognition of pneumococcus [12]. However, the possibility that other pneumococcal virulent factors interact with the host innate immune system and participate in pneumococcal pathogenesis remains to be explored.

In this study, we hypothesized that pneumococcal autolysis induces the leakage of additional intracellular virulent factors, which increase the pathogenicity of S. pneumoniae. Here, we demonstrated that chaperone protein DnaK, elongation factor Tu (EF-Tu), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were released into the pneumococcal culture supernatant by autolysis. In addition, we examined their activity of inducing the production of proinflammatory cytokines in mouse peritoneal macrophages and THP-1-derived macrophage-like cells.

Section snippets

Bacterial strains and reagents

S. pneumoniae D39 (NCTC 7466) strain was purchased from the National Collection of Type Cultures (Salisbury, UK). Inactivation of lytA gene in S. pneumoniae D39 strain was performed as described previously [13]. S. pneumoniae was grown in tryptic soy broth (TSB; Becton Dickinson, Franklin Lakes, NJ, USA), with 100 µg/mL spectinomycin (Sigma-Aldrich, St. Louis, MO, USA) added to the medium to allow for lytA-negative mutant-strain (ΔlytA) selection. Recombinant (r) LytA protein was kindly

S. pneumoniae autolysis causes DNA leakage into the bacterial supernatant

We first investigated whether pneumococcal autolysis can cause DNA leakage into the bacterial supernatant. Fig. 1A shows that both S. pneumoniae D39 and ΔlytA (autolytic enzyme gene lytA-negative mutant) strains reached a stationary growth phase after a 12-h incubation. The optical density of S. pneumoniae D39 strain was significantly lower than that of ΔlytA, which was due to S. pneumoniae D39 autolysis. Fig. 1B shows that significantly higher level of eDNA (extracellular DNA) was released

Discussion

In this study, we showed that pneumococcal autolysis induces DNA leakage. Although eDNA itself did not induce the production of proinflammatory cytokines in mouse peritoneal macrophages, DnaK, EF-Tu, and GAPDH were released as DNA-binding proteins and induced the production of proinflammatory cytokines in mouse peritoneal macrophages and THP-1 macrophages via TLR4.

LytA is an enzyme that degrades the peptidoglycan backbone of bacterial organisms [21], leading to bacterial autolysis. This enzyme

Acknowledgments

We acknowledge Ms. Chisato Jimbo (Niigata university) for providing technical assistance. This work was supported by JSPS KAKENHI grants 26305034, 17H04367, 16K15785, and 16K11439.

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