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A novel extracellular vesicle-associated endodeoxyribonuclease helps Streptococcus pneumoniae evade neutrophil extracellular traps and is required for full virulence.

ABSTRACT: Streptococcus pneumoniae (pneumococcus) is a major bacterial pathogen that causes pneumonia and septicemia in humans. Pneumococci are cleared from the host primarily by antibody dependent opsonophagocytosis by phagocytes like neutrophils. Neutrophils release neutrophil extracellular traps (NETs) on contacting pneumococci. NETs immobilize pneumococci and restrict its dissemination in the host. One of the strategies utilized by pneumococci to evade the host immune response involves use of DNase(s) to degrade NETs. We screened the secretome of autolysin deficient S. pneumoniae to identify novel DNase(s). Zymogram analysis revealed 3 bands indicative of DNase activity. Mass spectrometric analysis led to the identification of TatD as a potential extracellular DNase. Recombinant TatD showed nucleotide sequence-independent endodeoxyribonuclease activity. TatD was associated with extracellular vesicles. Pneumococcal secretome degraded NETs from human neutrophils. Extracellular vesicle fraction from tatD deficient strain showed little NET degrading activity. Recombinant TatD efficiently degraded NETs. tatD deficient pneumococci showed lower bacterial load in lungs, blood and spleen in a murine sepsis model compared to wildtype strain, and showed less severe lung pathology and compromised virulence. This study provides insights into the role of a novel extracellular DNase in evasion of the innate immune system.

SUBMITTER: Jhelum H

PROVIDER: S-EPMC5964101 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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A novel extracellular vesicle-associated endodeoxyribonuclease helps Streptococcus pneumoniae evade neutrophil extracellular traps and is required for full virulence.

Jhelum Hina H Sori Hema H Sehgal Devinder D

Scientific reports 20180522 1

Streptococcus pneumoniae (pneumococcus) is a major bacterial pathogen that causes pneumonia and septicemia in humans. Pneumococci are cleared from the host primarily by antibody dependent opsonophagocytosis by phagocytes like neutrophils. Neutrophils release neutrophil extracellular traps (NETs) on contacting pneumococci. NETs immobilize pneumococci and restrict its dissemination in the host. One of the strategies utilized by pneumococci to evade the host immune response involves use of DNase(s) ...[more]

PMID: 29789571

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Project description:The pathogen Streptococcus equi subsp. zooepidemicus is associated with a wide range of animals, including humans, and outbreaks frequently occur in pigs, equines, and goats. Thus far, few studies have assessed interactions between the host immune system and S. equi subsp. zooepidemicus and how these interactions explain the wide host spectrum of S. equi subsp. zooepidemicus Neutrophils, the first line of innate immunity, possess a defense mechanism called neutrophil extracellular traps (NETs), which primarily consist of DNA and granule proteins that trap bacteria via charge interactions. Extracellular nucleases play important roles in the degradation of the DNA backbone of NETs. Here, two related extracellular nucleases, nuclease and 5'-nucleotidase (named ENuc and 5Nuc, respectively, in this study), were identified as being encoded by the SESEC_RS04165 gene and the SESEC_RS05720 gene (named ENuc and 5Nuc, respectively), and three related gene deletion mutant strains, specifically, the single-mutant ΔENuc and Δ5Nuc strains and the double-mutant ΔENuc Δ5Nuc strain, were constructed. The ΔENuc and Δ5Nuc single-mutant strains and the ΔENuc Δ5Nuc double-mutant strain demonstrated lower virulence than wild-type S. equi subsp. zooepidemicus when the mouse survival rate was evaluated postinfection. Furthermore, wild-type S. equi subsp. zooepidemicus more frequently traversed the bloodstream and transferred to other organs. Wild-type S. equi subsp. zooepidemicus induced fewer NETs and was able to survive in NETs, whereas only 40% of the ΔENuc Δ5Nuc double-mutant cells survived. S. equi subsp. zooepidemicus degraded the NET DNA backbone and produced deoxyadenosine, primarily through the action of ENuc and/or 5Nuc. However, the double-mutant ΔENuc Δ5Nuc strain lost the ability to degrade NETs into deoxyadenosine. Deoxyadenosine decreased RAW 264.7 cell phagocytosis to 40% of that of normal macrophages. Streptococcus equi subsp. zooepidemicus causes serious bacteremia in its hosts. However, little is known about how S. equi subsp. zooepidemicus interacts with the host innate immune system, particularly innate cells found in the blood. S. equi subsp. zooepidemicus is capable of evading NET-mediated killing via the actions of its potent extracellular nucleases, ENuc and 5Nuc, which directly degrade the NET DNA backbone to deoxyadenosine. In previous studies, other pathogens have required the synergism of nuclease and 5'-nucleotidase to engage in this self-protective process; however, ENuc and 5Nuc both possess nuclease activity and 5'-nucleotidase activity, highlighting the novelty of this discovery. Furthermore, deoxyadenosine impairs phagocytosis but not the intracellular bactericidal activity of macrophages. Here we describe a novel mechanism for S. equi subsp. zooepidemicus extracellular nucleases in NET degradation, which may provide new insights into the pathogen immune evasion mechanism and the prevention and treatment of bacterial disease.

Project description:An important determinant of disease following Streptococcus pneumoniae (pneumococcus) lung infection is pulmonary inflammation mediated by polymorphonuclear leukocytes (PMNs). We found that upon intratracheal challenge of mice, recruitment of PMNs into the lungs within the first 3 hours coincided with decreased pulmonary pneumococci, whereas large numbers of pulmonary PMNs beyond 12 hours correlated with a greater bacterial burden. Indeed, mice that survived infection largely resolved inflammation by 72 hours, and PMN depletion at peak infiltration, i.e. 18 hours post-infection, lowered bacterial numbers and enhanced survival. We investigated host signaling pathways that influence both pneumococcus clearance and pulmonary inflammation. Pharmacologic inhibition and/or genetic ablation of enzymes that generate extracellular adenosine (EAD) (e.g. the ectoenzyme CD73) or degrade EAD (e.g. adenosine deaminase) revealed that EAD dramatically increases murine resistance to S. pneumoniae lung infection. Moreover, adenosine diminished PMN movement across endothelial monolayers in vitro, and although inhibition or deficiency of CD73 had no discernible impact on PMN recruitment within the first 6 hours after intratracheal inoculation of mice, these measures enhanced PMN numbers in the pulmonary interstitium after 18 hours of infection, culminating in dramatically elevated numbers of pulmonary PMNs at three days post-infection. When assessed at this time point, CD73-/- mice displayed increased levels of cellular factors that promote leukocyte migration, such as CXCL2 chemokine in the murine lung, as well as CXCR2 and β-2 integrin on the surface of pulmonary PMNs. The enhanced pneumococcal susceptibility of CD73-/- mice was significantly reversed by PMN depletion following infection, suggesting that EAD-mediated resistance is largely mediated by its effects on PMNs. Finally, CD73-inhibition diminished the ability of PMNs to kill pneumococci in vitro, suggesting that EAD alters both the recruitment and bacteriocidal function of PMNs. The EAD-pathway may provide a therapeutic target for regulating potentially harmful inflammatory host responses during Gram-positive bacterial pneumonia.

Dataset Information

A novel extracellular vesicle-associated endodeoxyribonuclease helps Streptococcus pneumoniae evade neutrophil extracellular traps and is required for full virulence.

Publications

A novel extracellular vesicle-associated endodeoxyribonuclease helps Streptococcus pneumoniae evade neutrophil extracellular traps and is required for full virulence.

Similar Datasets

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