ABSTRACT
We compared the immunogenicity of recombinant S. pneumoniae pneumolysin (rPly) when administered with and without Al(OH)3 adjuvant, and evaluated the protective properties of recombinant protein in the active defense experiment. It was shown that double immunization with rPly+Al(OH)3 increases the levels of IgG antibodies in comparison with the control (p<0.01), while triple immunization results in a more significant increase in IgG antibody levels (p<0.001). Double immunization with rPly without Al(OH)3 does not induce a significant increase in antibody levels in comparison with the control, while triple immunization results in a slight but significant increase in antibody levels (p<0.05). The active defense test proved the protective activity of rPly against S. pneumoniae serotype 3 at intranasal infection.
Subject(s)
Antibodies, Bacterial , Bacterial Proteins , Immunoglobulin G , Recombinant Proteins , Streptococcus pneumoniae , Streptolysins , Streptolysins/immunology , Streptolysins/genetics , Bacterial Proteins/immunology , Bacterial Proteins/genetics , Streptococcus pneumoniae/immunology , Streptococcus pneumoniae/genetics , Animals , Recombinant Proteins/immunology , Recombinant Proteins/genetics , Antibodies, Bacterial/immunology , Antibodies, Bacterial/blood , Immunoglobulin G/immunology , Immunoglobulin G/blood , Mice , Pneumococcal Infections/immunology , Pneumococcal Infections/prevention & control , Pneumococcal Infections/microbiology , Adjuvants, Immunologic , Aluminum Hydroxide/immunology , Aluminum Hydroxide/administration & dosage , Pneumococcal Vaccines/immunology , Pneumococcal Vaccines/administration & dosage , FemaleABSTRACT
At the end of 2022 into early 2023, the UK Health Security Agency reported unusually high levels of scarlet fever and invasive disease caused by Streptococcus pyogenes (StrepA or group A Streptococcus). During this time, we collected and genome-sequenced 341 non-invasive throat and skin S. pyogenes isolates identified during routine clinical diagnostic testing in Sheffield, a large UK city. We compared the data with that obtained from a similar collection of 165 isolates from 2016 to 2017. Numbers of throat-associated isolates collected peaked in early December 2022, reflecting the national scarlet fever upsurge, while skin infections peaked later in December. The most common emm-types in 2022-2023 were emm1 (28.7â%), emm12 (24.9â%) and emm22 (7.7â%) in throat and emm1 (22â%), emm12 (10â%), emm76 (18â%) and emm49 (7â%) in skin. While all emm1 isolates were the M1UK lineage, the comparison with 2016-2017 revealed diverse lineages in other emm-types, including emm12, and emergent lineages within other types including a new acapsular emm75 lineage, demonstrating that the upsurge was not completely driven by a single genotype. The analysis of the capsule locus predicted that only 51â% of throat isolates would produce capsule compared with 78% of skin isolates. Ninety per cent of throat isolates were also predicted to have high NADase and streptolysin O (SLO) expression, based on the promoter sequence, compared with only 56% of skin isolates. Our study has highlighted the value in analysis of non-invasive isolates to characterize tissue tropisms, as well as changing strain diversity and emerging genomic features which may have implications for spillover into invasive disease and future S. pyogenes upsurges.
Subject(s)
Streptococcal Infections , Streptococcus pyogenes , Streptococcus pyogenes/genetics , Streptococcus pyogenes/classification , Streptococcus pyogenes/isolation & purification , Humans , United Kingdom , Streptococcal Infections/microbiology , Bacterial Outer Membrane Proteins/genetics , Antigens, Bacterial/genetics , Pharynx/microbiology , Scarlet Fever/microbiology , Scarlet Fever/epidemiology , Carrier Proteins/genetics , Streptolysins/genetics , Whole Genome Sequencing/methods , Bacterial Proteins/genetics , Phylogeny , Child , Adult , NAD+ Nucleosidase/genetics , NAD+ Nucleosidase/metabolism , Skin/microbiology , Child, Preschool , MaleABSTRACT
We studied toxicity of recombinant Streptococcus pneumoniae pneumolysin protein in experiments on mice and its cytopathogenic effect on cultures of Vero green monkey kidney cells and human lung carcinoma A549 cells in vitro. In vivo and in vitro experiments proved the absence of compromised toxicity and direct cytopathogenic action of the recombinant protein.
Subject(s)
Bacterial Proteins , Recombinant Proteins , Streptococcus pneumoniae , Streptolysins , Streptolysins/toxicity , Streptolysins/genetics , Animals , Bacterial Proteins/toxicity , Bacterial Proteins/genetics , Chlorocebus aethiops , Mice , Vero Cells , Streptococcus pneumoniae/drug effects , Humans , Recombinant Proteins/toxicity , Recombinant Proteins/genetics , A549 CellsABSTRACT
The major gram-positive pathogen group A Streptococcus (GAS) is a model organism for studying microbial epidemics as it causes waves of infections. Since 1980, several GAS epidemics have been ascribed to the emergence of clones producing increased amounts of key virulence factors such as streptolysin O (SLO). Herein, we sought to identify mechanisms underlying our recently identified temporal clonal emergence among emm4 GAS, given that emergent strains did not produce augmented levels of virulence factors relative to historic isolates. By creating and analyzing isoallelic strains, we determined that a conserved mutation in a previously undescribed gene encoding a putative carbonic anhydrase was responsible for the defective in vitro growth observed in the emergent strains. We also identified that the emergent strains survived better inside macrophages and killed macrophages at lower rates than the historic strains. Via the creation of isogenic mutant strains, we linked the emergent strain "survival" phenotype to the downregulation of the SLO encoding gene and upregulation of the msrAB operon which encodes proteins involved in defense against extracellular oxidative stress. Our findings are in accord with recent surveillance studies which found a high ratio of mucosal (i.e., pharyngeal) relative to invasive infections among emm4 GAS. Since ever-increasing virulence is unlikely to be evolutionarily advantageous for a microbial pathogen, our data further understanding of the well-described oscillating patterns of virulent GAS infections by demonstrating mechanisms by which emergent strains adapt a "survival" strategy to outcompete previously circulating isolates.
Subject(s)
Bacterial Proteins , Macrophages , Streptococcal Infections , Streptococcus pyogenes , Streptolysins , Virulence Factors , Streptococcus pyogenes/genetics , Streptococcus pyogenes/pathogenicity , Streptococcus pyogenes/immunology , Streptococcal Infections/microbiology , Streptococcal Infections/immunology , Streptococcal Infections/mortality , Humans , Macrophages/microbiology , Macrophages/immunology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Streptolysins/genetics , Streptolysins/metabolism , Virulence Factors/genetics , Mutation , Host-Pathogen Interactions/immunology , Virulence/genetics , Animals , Antigens, Bacterial/genetics , Antigens, Bacterial/metabolism , Antigens, Bacterial/immunology , Microbial Viability , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/metabolism , Mice , Gene Expression Regulation, Bacterial , Carrier ProteinsABSTRACT
Streptococcus pyogenes is a human-specific pathogen that commonly colonizes the upper respiratory tract and skin, causing a wide variety of diseases ranging from pharyngitis to necrotizing fasciitis and toxic shock syndrome. S. pyogenes has a repertoire of secreted virulence factors that promote infection and evasion of the host immune system including the cytolysins streptolysin O (SLO) and streptolysin S (SLS). S. pyogenes does not naturally infect the upper respiratory tract of mice although mice transgenic for MHC class II human leukocyte antigens (HLA) become highly susceptible. Here we used HLA-transgenic mice to assess the role of both SLO and SLS during both nasopharyngeal and skin infection. Using S. pyogenes MGAS8232 as a model strain, we found that an SLS-deficient strain exhibited a 100-fold reduction in bacterial recovery from the nasopharynx and a 10-fold reduction in bacterial burden in the skin, whereas an SLO-deficient strain did not exhibit any infection defects in these models. Furthermore, depletion of neutrophils significantly restored the bacterial burden of the SLS-deficient bacteria in skin, but not in the nasopharynx. In mice nasally infected with the wildtype S. pyogenes, there was a marked change in localization of the tight junction protein ZO-1 at the site of infection, demonstrating damage to the nasal epithelia that was absent in mice infected with the SLS-deficient strain. Overall, we conclude that SLS is required for the establishment of nasopharyngeal infection and skin infection in HLA-transgenic mice by S. pyogenes MGAS8232 and provide evidence that SLS contributes to nasopharyngeal infection through the localized destruction of nasal epithelia.
Subject(s)
Streptococcal Infections , Streptococcus pyogenes , Humans , Mice , Animals , Streptococcus pyogenes/metabolism , Streptolysins/genetics , Streptolysins/metabolism , Mice, Transgenic , Streptococcal Infections/metabolism , Bacterial Proteins/metabolism , NasopharynxABSTRACT
IMPORTANCE: S. pneumoniae is a major human pathogen that undergoes a spontaneous and reversible phase variation that allows it to survive in different host environments. Interestingly, we found hsdSA , a gene that manipulated the phase variation, promoted the survival and replication of S. pneumoniae in macrophages by regulating EV production and EV-associated PLY. More importantly, here we provided the first evidence that higher EV-associated PLY (produced by D39) could form LAPosomes that were single membrane compartments containing S. pneumoniae, which are induced by integrin ß1/NOX2/ROS pathway. At the same time, EV-associated PLY increased the permeability of lysosome membrane and induced an insufficient acidification to escape the host killing, and ultimately prolonged the survival of S. pneumoniae in macrophages. In contrast, lower EV-associated PLY (produced by D39ΔhsdSA ) activated ULK1 recruitment to form double-layered autophagosomes to eliminate bacteria.
Subject(s)
Streptococcus pneumoniae , Streptolysins , Humans , Streptococcus pneumoniae/genetics , Streptolysins/genetics , Bacterial Proteins/genetics , Macrophages/metabolismABSTRACT
Streptococcus pneumoniae is the leading cause of community-acquired pneumonia. The pore-forming cholesterol-dependent cytolysin (CDC) pneumolysin (PLY) and the physiological metabolite hydrogen peroxide (H2O2) can greatly increase the virulence of pneumococci. Although most studies have focused on the contribution of both virulence factors to the course of pneumococcal infection, it is unknown whether or how H2O2 can affect PLY activity. Of note, S. pneumoniae exploits endogenous H2O2 as an intracellular signalling molecule to modulate the activity of several proteins. Here, we demonstrate that H2O2 negatively affects the haemolytic activity of PLY in a concentration-dependent manner. Prevention of cysteine-dependent sulfenylation upon substitution of the unique and highly conserved cysteine residue to serine in PLY significantly reduces the toxin's susceptibility to H2O2 treatment and completely abolishes the ability of DTT to activate PLY. We also detect a clear gradual correlation between endogenous H2O2 generation and PLY release, with decreased H2O2 production causing a decline in the release of PLY. Comparative transcriptome sequencing analysis of the wild-type S. pneumoniae strain and three mutants impaired in H2O2 production indicates enhanced expression of several genes involved in peptidoglycan (PG) synthesis and in the production of choline-binding proteins (CPBs). One explanation for the impact of H2O2 on PLY release is the observed upregulation of the PG bridge formation alanyltransferases MurM and MurN, which evidentially negatively affect the PLY release. Our findings shed light on the significance of endogenous pneumococcal H2O2 in controlling PLY activity and release.
Subject(s)
Hydrogen Peroxide , Streptococcus pneumoniae , Streptococcus pneumoniae/metabolism , Hydrogen Peroxide/metabolism , Cysteine/metabolism , Streptolysins/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolismABSTRACT
A recombinant form of pneumolysin from Streptococcus pneumoniae was obtained. By using Vector NTI Advance 11.0 bioinformatic analysis software, specific primers were designed in order to amplify the genome fragment of strain No. 3358 S. pneumoniae serotype 19F containing the nucleotide sequence encoding the full-length pneumolysin protein. A PCR product with a molecular weight corresponding to the nucleotide sequence of the S. pneumoniae genome fragment encoding the full-length pneumolysin was obtained. An expression system for recombinant pneumolysin in E. coli was constructed. Sequencing confirmed the identity of the inserted nucleotide sequence encoding the full-length recombinant pneumolysin synthesized in E. coli M15 strain. Purification of the recombinant protein was performed by affinity chromatography using Ni-Sepharose in 8 M urea buffer solution. Confirmation of the recombinant protein was performed by immunoblotting with monoclonal antibodies to pneumolysin.
Subject(s)
Escherichia coli , Streptococcus pneumoniae , Streptococcus pneumoniae/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Streptolysins/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
Mutation within the Streptococcus pyogenes (Streptococcus group A; Strep A) covR/S regulatory system has been associated with a hypervirulent phenotype resulting from the upregulation of several virulence factors, including the pore-forming toxin, streptolysin O (SLO). In this study, we utilized a range of covR/S mutants, including M1T1 clonal strains (5448 and a covS mutant generated through mouse passage designated 5448AP), to investigate the contribution of SLO to the pathogenesis of covR/S mutant Strep A disease. Up-regulation of slo in 5448AP resulted in increased SLO-mediated hemolysis, decreased dendritic cell (DC) viability post coculture with Strep A, and increased production of tumor necrosis factor (TNF) and monocyte chemoattractant protein 1 (MCP-1) by DCs. Mouse passage of an isogenic 5448 slo-deletion mutant resulted in recovery of several covR/S mutants within the 5448Δslo background. Passage also introduced mutations in non-covR/S genes, but these were considered to have no impact on virulence. Although slo-deficient mutants exhibited the characteristic covR/S-controlled virulence factor upregulation, these mutants caused increased DC viability with reduced inflammatory cytokine production by infected DCs. In vivo, slo expression correlated with decreased DC numbers in infected murine skin and significant bacteremia by 3 days postinfection, with severe pathology at the infection site. Conversely, the absence of slo in the infecting strain (covR/S mutant or wild-type) resulted in detection of DCs in the skin and attenuated virulence in a murine model of pyoderma. slo-sufficient and -deficient covR/S mutants were susceptible to immune clearance mediated by a combination vaccine consisting of a conserved M protein peptide and a peptide from the CXC chemokine protease SpyCEP. IMPORTANCE Streptococcus pyogenes is responsible for significant numbers of invasive and noninvasive infections which cause significant morbidity and mortality globally. Strep A isolates with mutations in the covR/S system display greater propensity to cause severe invasive diseases, which are responsible for more than 163,000 deaths each year. This is due to the upregulation of virulence factors, including the pore-forming toxin streptolysin O. Utilizing covR/S and slo-knockout mutants, we investigated the role of SLO in virulence. We found that SLO alters interactions with host cell populations and increases Strep A viability at sterile sites of the host, such as the blood, and that its absence results in significantly less virulence. This work underscores the importance of SLO in Strep A virulence while highlighting the complex nature of Strep A pathogenesis. This improved insight into host-pathogen interactions will enable a better understanding of host immune evasion mechanisms and inform streptococcal vaccine development programs.
Subject(s)
Streptococcal Infections , Streptococcus pyogenes , Animals , Mice , Virulence/genetics , Streptolysins/genetics , Streptolysins/metabolism , Bacterial Proteins/metabolism , Virulence Factors/metabolismABSTRACT
Pneumolysin (PLY) is a bacterial pore forming toxin and primary virulence factor of Streptococcus pneumonia, a major cause of pneumonia. PLY binds cholesterol-rich domains of the endothelial cell (EC) plasma membrane resulting in pore assembly and increased intracellular (IC) Ca2+ levels that compromise endothelial barrier integrity. Caveolae are specialized plasmalemma microdomains of ECs enriched in cholesterol. We hypothesized that the abundance of cholesterol-rich domains in EC plasma membranes confers cellular susceptibility to PLY. Contrary to this hypothesis, we found increased PLY-induced IC Ca2+ following membrane cholesterol depletion. Caveolin-1 (Cav-1) is an essential structural protein of caveolae and its regulation by cholesterol levels suggested a possible role in EC barrier function. Indeed, Cav-1 and its scaffolding domain peptide protected the endothelial barrier from PLY-induced disruption. In loss of function experiments, Cav-1 was knocked-out using CRISPR-Cas9 or silenced in human lung microvascular ECs. Loss of Cav-1 significantly enhanced the ability of PLY to disrupt endothelial barrier integrity. Rescue experiments with re-expression of Cav-1 or its scaffolding domain peptide protected the EC barrier against PLY-induced barrier disruption. Dynamin-2 (DNM2) is known to regulate caveolar membrane endocytosis. Inhibition of endocytosis, with dynamin inhibitors or siDNM2 amplified PLY induced EC barrier dysfunction. These results suggest that Cav-1 protects the endothelial barrier against PLY by promoting endocytosis of damaged membrane, thus reducing calcium entry and PLY-dependent signaling.
Subject(s)
Bacterial Proteins , Caveolin 1 , Lung , Pneumonia, Pneumococcal , Pneumonia , Streptolysins , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Caveolin 1/genetics , Caveolin 1/metabolism , Cholesterol/metabolism , Endothelium, Vascular/metabolism , Humans , Lung/blood supply , Lung/metabolism , Microvessels/metabolism , Pneumonia/genetics , Pneumonia/metabolism , Pneumonia/microbiology , Pneumonia, Pneumococcal/genetics , Pneumonia, Pneumococcal/metabolism , Pneumonia, Pneumococcal/microbiology , Streptococcus pneumoniae/metabolism , Streptococcus pneumoniae/pathogenicity , Streptolysins/genetics , Streptolysins/metabolism , Vascular Diseases/genetics , Vascular Diseases/metabolism , Vascular Diseases/microbiologyABSTRACT
Published data for the Streptococcus pneumoniae virulence factor Pneumolysin (Ply) show contradictory effects on the host inflammatory response to infection. Ply has been shown to activate the inflammasome, but also can bind to MRC-1 resulting in suppression of dendritic cell inflammatory responses. We have used an in vitro infection model of human monocyte-derived macrophages (MDM), and a mouse model of pneumonia to clarify whether pro- or anti-inflammatory effects dominate the effects of Ply on the initial macrophage inflammatory response to S. pneumoniae, and the consequences during early lung infection. We found that infection with S. pneumoniae expressing Ply suppressed tumour necrosis factor (TNF) and interleukin-6 production by MDMs compared to cells infected with ply-deficient S. pneumoniae. This effect was independent of bacterial effects on cell death. Transcriptional analysis demonstrated S. pneumoniae expressing Ply caused a qualitatively similar but quantitatively lower MDM transcriptional response to S. pneumoniae compared to ply-deficient S. pneumoniae, with reduced expression of TNF and type I IFN inducible genes. Reduction of the MDM inflammatory response was prevented by inhibition of SOCS1. In the early lung infection mouse model, the TNF response to ply-deficient S. pneumoniae was enhanced and bacterial clearance increased compared to infection with wild-type S. pneumoniae. Overall, these data show Ply inhibits the initial macrophage inflammatory response to S. pneumoniae, probably mediated through SOCS1, and this was associated with improved immune evasion during early lung infection.
Subject(s)
Inflammasomes , Streptococcus pneumoniae , Animals , Anti-Inflammatory Agents , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Humans , Interleukin-6 , Macrophages/metabolism , Mice , Streptolysins/genetics , Streptolysins/metabolism , Streptolysins/pharmacology , Tumor Necrosis Factors , Virulence FactorsABSTRACT
BACKGROUND: Streptococcus dysgalactiae subspecies equisimilis (SDSE) has emerged as an important cause of severe invasive infections including streptococcal toxic shock syndrome (STSS). The present study aimed to identify genes involved in differences in invasiveness between STSS and non-invasive SDSE isolates. METHODS: STSS and non-invasive SDSE isolates were analysed to identify csrS/csrR mutations, followed by a comparative analysis of genomic sequences to identify mutations in other genes. Mutant strains were generated to examine changes in gene expression profiles and altered pathogenicity in mice. FINDINGS: Of the 79 STSS-SDSE clinical isolates, 15 (19.0%) harboured csrS/csrR mutations, while none were found in the non-invasive SDSE isolates. We identified a small RNA (sRNA) that comprised three direct repeats along with an inverted repeat and was transcribed in the same direction as the sagA gene. The sRNA was referred to as srrG (streptolysin S regulatory RNA in GGS). srrG mutations were identified in the STSS-SDSE strains and were found to be associated with elevated expression of the streptolysin S (SLS) gene cluster and enhanced pathogenicity in mice. INTERPRETATION: The csrS/csrR and srrG mutations that increased virulence gene expression in STSS-SDSE isolates were identified, and strains carrying these mutations caused increased lethality in mice. A significantly higher frequency of mutations was observed in STSS-SDSE isolates, thereby highlighting their importance in STSS. FUNDING: Japan Agency for Medical Research and Development, the Japan Society for the Promotion of Science (JSPS), and the Ministry of Health, Labor, and Welfare of Japan.
Subject(s)
RNA, Small Untranslated , Shock, Septic , Streptococcal Infections , Animals , Genes, Regulator , Mice , Mutation , Shock, Septic/genetics , Streptococcal Infections/genetics , Streptococcus , Streptolysins/genetics , Virulence/geneticsABSTRACT
Streptococcus pneumoniae, the most common cause of community-acquired pneumonia, causes severe invasive infections, including meningitis and bacteremia. The widespread use of macrolides has been reported to increase the prevalence of macrolide-resistant S. pneumoniae (MRSP), thereby leading to treatment failure in patients with pneumococcal pneumonia. However, previous studies have demonstrated that several macrolides and lincosamides have beneficial effects on MRSP infection since they inhibit the production and release of pneumolysin, a pneumococcal pore-forming toxin released during autolysis. In this regard, we previously demonstrated that the mechanisms underlying the inhibition of pneumolysin release by erythromycin involved both the transcriptional downregulation of the gene encoding pneumolysin and the impairment of autolysis in MRSP. Here, using a cell supernatant of the culture, we have shown that clarithromycin inhibits pneumolysin release in MRSP. However, contrary to previous observations in erythromycin-treated MRSP, clarithromycin upregulated the transcription of the pneumococcal autolysis-related lytA gene and enhanced autolysis, leading to the leakage of pneumococcal DNA. On the other hand, compared to erythromycin, clarithromycin significantly downregulated the gene encoding pneumolysin. In a mouse model of MRSP pneumonia, the administration of both clarithromycin and erythromycin significantly decreased the pneumolysin protein level in bronchoalveolar lavage fluid and improved lung injury and arterial oxygen saturation without affecting bacterial load. Collectively, these in vitro and in vivo data reinforce the benefits of macrolides on the clinical outcomes of patients with pneumococcal pneumonia. IMPORTANCE Pneumolysin is a potent intracellular toxin possessing multiple functions that augment pneumococcal virulence. For over 10 years, sub-MICs of macrolides, including clarithromycin, have been recognized to decrease pneumolysin production and release from pneumococcal cells. However, this study indicates that macrolides significantly slowed pneumococcal growth, which may be related to decreased pneumolysin release recorded by previous studies. In this study, we demonstrated that clarithromycin decreases pneumolysin production through downregulation of ply gene transcription, regardless of its inhibitory activity against bacterial growth. Additionally, administration of clarithromycin resulted in the amelioration of lung injury in a mouse model of pneumonia induced by macrolide-resistant pneumococci. Therefore, therapeutic targeting of pneumolysin offers a good strategy to treat pneumococcal pneumonia.
Subject(s)
Clarithromycin/pharmacology , Erythromycin/pharmacology , Protein Synthesis Inhibitors/pharmacology , Streptococcus pneumoniae/metabolism , Streptolysins/biosynthesis , Animals , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Disease Models, Animal , Down-Regulation/drug effects , Humans , Lincosamides/pharmacology , Male , Mice , Mice, Inbred BALB C , Pneumonia/drug therapy , Pneumonia/microbiology , Streptococcus pneumoniae/genetics , Streptolysins/genetics , Transcription, Genetic/drug effectsABSTRACT
Epigenetic modifications regulate gene expression in the host response to a diverse range of pathogens. The extent and consequences of epigenetic modification during macrophage responses to Streptococcus pneumoniae, and the role of pneumolysin, a key Streptococcus pneumoniae virulence factor, in influencing these responses, are currently unknown. To investigate this, we infected human monocyte derived macrophages (MDMs) with Streptococcus pneumoniae and addressed whether pneumolysin altered the epigenetic landscape and the associated acute macrophage transcriptional response using a combined transcriptomic and proteomic approach. Transcriptomic analysis identified 503 genes that were differentially expressed in a pneumolysin-dependent manner in these samples. Pathway analysis highlighted the involvement of transcriptional responses to core innate responses to pneumococci including modules associated with metabolic pathways activated in response to infection, oxidative stress responses and NFκB, NOD-like receptor and TNF signalling pathways. Quantitative proteomic analysis confirmed pneumolysin-regulated protein expression, early after bacterial challenge, in representative transcriptional modules associated with innate immune responses. In parallel, quantitative mass spectrometry identified global changes in the relative abundance of histone post translational modifications (PTMs) upon pneumococcal challenge. We identified an increase in the relative abundance of H3K4me1, H4K16ac and a decrease in H3K9me2 and H3K79me2 in a PLY-dependent fashion. We confirmed that pneumolysin blunted early transcriptional responses involving TNF-α and IL-6 expression. Vorinostat, a histone deacetylase inhibitor, similarly downregulated TNF-α production, reprising the pattern observed with pneumolysin. In conclusion, widespread changes in the macrophage transcriptional response are regulated by pneumolysin and are associated with global changes in histone PTMs. Modulating histone PTMs can reverse pneumolysin-associated transcriptional changes influencing innate immune responses, suggesting that epigenetic modification by pneumolysin plays a role in dampening the innate responses to pneumococci.
Subject(s)
Bacterial Proteins/metabolism , Epigenesis, Genetic , Gene Expression Profiling , Macrophages/metabolism , Streptococcus pneumoniae/metabolism , Streptolysins/metabolism , Bacterial Proteins/genetics , Cells, Cultured , Cytokines/genetics , Cytokines/metabolism , Histones/metabolism , Host-Pathogen Interactions , Humans , Macrophages/microbiology , Methylation , Protein Processing, Post-Translational , Proteome/metabolism , Proteomics/methods , Streptococcus pneumoniae/genetics , Streptococcus pneumoniae/physiology , Streptolysins/geneticsABSTRACT
Group A Streptococcus (GAS; Streptococcus pyogenes) is a major human pathogen that causes streptococcal pharyngitis, skin and soft tissue infections, and life-threatening conditions such as streptococcal toxic-shock syndrome. During infection, GAS not only invades diverse host cells but also injects effector proteins such as NAD-glycohydrolase (Nga) into the host cells through a streptolysin O (SLO)-dependent mechanism without invading the cells; Nga and SLO are two major virulence factors that are associated with increased bacterial virulence. Here, we have shown that the invading GAS induces fragmentation of the Golgi complex and inhibits anterograde transport in the infected host cells through the secreted toxins SLO and Nga. GAS infection-induced Golgi fragmentation required both bacterial invasion and SLO-mediated Nga translocation into the host cytosol. The cellular Golgi network is critical for the sorting of surface molecules and is thus essential for the integrity of the epithelial barrier and for the immune response of macrophages to pathogens. In epithelial cells, inhibition of anterograde trafficking by invading GAS and Nga resulted in the redistribution of E-cadherin to the cytosol and an increase in bacterial translocation across the epithelial barrier. Moreover, in macrophages, interleukin-8 secretion in response to GAS infection was found to be suppressed by intracellular GAS and Nga. Our findings reveal a previously undescribed bacterial invasion-dependent function of Nga as well as a previously unrecognized GAS-host interaction that is associated with GAS pathogenesis.IMPORTANCE Two prominent virulence factors of group A Streptococcus (GAS), streptolysin O (SLO) and NAD-glycohydrolase (Nga), are linked to enhanced pathogenicity of the prevalent GAS strains. Recent advances show that SLO and Nga are important for intracellular survival of GAS in epithelial cells and macrophages. Here, we found that invading GAS disrupts the Golgi complex in host cells through SLO and Nga. We show that GAS-induced Golgi fragmentation requires bacterial invasion into host cells, SLO pore formation activity, and Nga NADase activity. GAS-induced Golgi fragmentation results in the impairment of the epithelial barrier and chemokine secretion in macrophages. This immune inhibition property of SLO and Nga by intracellular GAS indicates that the invasion of GAS is associated with virulence exerted by SLO and Nga.
Subject(s)
Epithelial Cells/microbiology , Golgi Apparatus/pathology , Host-Pathogen Interactions/genetics , NAD+ Nucleosidase/genetics , Streptococcus pyogenes/pathogenicity , Streptolysins/genetics , A549 Cells , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cytoplasm/microbiology , Golgi Apparatus/genetics , Golgi Apparatus/microbiology , HeLa Cells , Host-Pathogen Interactions/immunology , Humans , Interleukin-8/immunology , NAD+ Nucleosidase/metabolism , Streptococcal Infections/microbiology , Streptococcus pyogenes/immunology , Streptolysins/metabolism , THP-1 Cells , Virulence FactorsABSTRACT
Significance:Streptococcus pneumoniae (Spn), a facultative anaerobic Gram-positive human pathogen with increasing rates of penicillin and macrolide resistance, is a major cause of lower respiratory tract infections worldwide. Pneumococci are a primary agent of severe pneumonia in children younger than 5 years and of community-acquired pneumonia in adults. A major defense mechanism toward Spn is the generation of reactive oxygen species, including hydrogen peroxide (H2O2), during the oxidative burst of neutrophils and macrophages. Paradoxically, Spn produces high endogenous levels of H2O2 as a strategy to promote colonization. Recent Advances: Pneumococci, which express neither catalase nor common regulators of peroxide stress resistance, have developed unique mechanisms to protect themselves from H2O2. Spn generates high levels of H2O2 as a strategy to promote colonization. Production of H2O2 moreover constitutes an important virulence phenotype and its cellular activities overlap and complement those of other virulence factors, such as pneumolysin, in modulating host immune responses and promoting organ injury. Critical Issues: This review examines the dual role of H2O2 in pneumococcal pneumonia, from the viewpoint of both the pathogen (defense mechanisms, lytic activity toward competing pathogens, and virulence) and the resulting host-response (inflammasome activation, endoplasmic reticulum stress, and damage to the alveolar-capillary barrier in the lungs). Future Directions: An understanding of the complexity of H2O2-mediated host-pathogen interactions is necessary to develop novel strategies that target these processes to enhance lung function during severe pneumonia.
Subject(s)
Drug Resistance, Bacterial/genetics , Hydrogen Peroxide/metabolism , Pneumonia, Pneumococcal/drug therapy , Streptococcus pneumoniae/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Host-Pathogen Interactions/genetics , Humans , Lung/metabolism , Lung/microbiology , Lung/pathology , Neutrophils/metabolism , Neutrophils/microbiology , Oxidants/metabolism , Pneumonia, Pneumococcal/genetics , Pneumonia, Pneumococcal/microbiology , Pneumonia, Pneumococcal/pathology , Streptococcus pneumoniae/drug effects , Streptococcus pneumoniae/pathogenicity , Streptolysins/genetics , Streptolysins/metabolismABSTRACT
Catabolite control protein A (CcpA) is a master regulator of carbon source utilization and contributes to the virulence of numerous medically important Gram-positive bacteria. Most functional assessments of CcpA, including interaction with its key co-factor HPr, have been performed in nonpathogenic bacteria. In this study we aimed to identify the in vivo DNA binding profile of CcpA and assess the extent to which HPr is required for CcpA-mediated regulation and DNA binding in the major human pathogen group A Streptococcus (GAS). Using a combination RNAseq/ChIP-seq approach, we found that CcpA affects transcript levels of 514 of 1667 GAS genes (31%) whereas direct DNA binding was identified for 105 GAS genes. Three of the directly regulated genes encode the key GAS virulence factors Streptolysin S, PrtS (IL-8 degrading proteinase), and SpeB (cysteine protease). Mutating CcpA Val301 to Ala (strain 2221-CcpA-V301A) abolished interaction between CcpA and HPr and impacted the transcript levels of 205 genes (40%) in the total CcpA regulon. By ChIP-seq analysis, CcpAV301A bound to DNA from 74% of genes bound by wild-type CcpA, but generally with lower affinity. These data delineate the direct CcpA regulon and clarify the HPr-dependent and independent activities of CcpA in a key pathogenic bacterium.
Subject(s)
Bacterial Proteins/metabolism , DNA, Bacterial/metabolism , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Bacterial/genetics , Streptococcus pyogenes/metabolism , Bacterial Proteins/genetics , Carrier Proteins/metabolism , Chromatin/genetics , DNA, Bacterial/genetics , DNA-Binding Proteins/genetics , Exotoxins/genetics , Genome, Bacterial/genetics , Protein Binding/physiology , RNA-Seq , Repressor Proteins/metabolism , Serine Endopeptidases/genetics , Streptococcus pyogenes/genetics , Streptococcus pyogenes/pathogenicity , Streptolysins/genetics , Virulence/genetics , Virulence Factors/geneticsABSTRACT
Streptococcus pneumoniae and other streptococci produce a greenish halo on blood agar plates referred to as alpha-hemolysis. This phenotype is utilized by clinical microbiology laboratories to report culture findings of alpha-hemolytic streptococci, including S. pneumoniae, and other bacteria. The alpha-hemolysis halo on blood agar plates has been related to the hemolytic activity of pneumococcal pneumolysin (Ply) or, to a lesser extent, to lysis of erythrocytes by S. pneumoniae-produced hydrogen peroxide. We investigated the molecular basis of the alpha-hemolysis halo produced by S. pneumoniae Wild-type strains TIGR4, D39, R6, and EF3030 and isogenic derivative Δply mutants produced similar alpha-hemolytic halos on blood agar plates, while cultures of hydrogen peroxide knockout ΔspxB ΔlctO mutants lacked this characteristic halo. Moreover, in the presence of catalase, the alpha-hemolysis halo was absent in cultures of the wild-type (wt) and Δply mutant strains. Spectroscopic studies demonstrated that culture supernatants of TIGR4 released hemoglobin-bound heme (heme-hemoglobin) from erythrocytes and oxidized oxy-hemoglobin to met-hemoglobin within 30 min of incubation. As expected, given Ply hemolytic activity and that hydrogen peroxide contributes to the release of Ply, TIGR4Δply and ΔspxB ΔlctO isogenic mutants had significantly decreased release of heme-hemoglobin from erythrocytes. However, TIGR4Δply that produces hydrogen peroxide oxidized oxy-hemoglobin to met-hemoglobin, whereas TIGR4ΔspxB ΔlctO failed to produce oxidation of oxy-hemoglobin. Studies conducted with all other wt strains and isogenic mutants resulted in similar findings. We demonstrated that the so-called alpha-hemolysis halo is caused by the oxidation of oxy-hemoglobin (Fe+2) to a non-oxygen-binding met-hemoglobin (Fe+3) by S. pneumoniae-produced hydrogen peroxide.IMPORTANCE There is a misconception that alpha-hemolysis observed on blood agar plate cultures of Streptococcus pneumoniae and other alpha-hemolytic streptococci is produced by a hemolysin or, alternatively, by lysis of erythrocytes caused by hydrogen peroxide. We noticed in the course of our investigations that wild-type S. pneumoniae strains and hemolysin (e.g., pneumolysin) knockout mutants produced the alpha-hemolytic halo on blood agar plates. In contrast, hydrogen peroxide-defective mutants prepared in four different strains lacked the characteristic alpha-hemolysis halo. We also demonstrated that wild-type strains and pneumolysin mutants oxidized oxy-hemoglobin to met-hemoglobin. Hydrogen peroxide knockout mutants, however, failed to oxidize oxy-hemoglobin. Therefore, the greenish halo formed on cultures of S. pneumoniae and other so-called alpha-hemolytic streptococci is caused by the oxidation of oxy-hemoglobin produced by hydrogen peroxide. Oxidation of oxy-hemoglobin to the nonbinding oxygen form, met-hemoglobin, might occur in the lungs during pneumococcal pneumonia.
Subject(s)
Hydrogen Peroxide/metabolism , Pneumococcal Infections/microbiology , Streptococcus pneumoniae/physiology , Streptolysins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Humans , Lung/microbiology , Oxyhemoglobins/genetics , Oxyhemoglobins/metabolism , Streptococcus pneumoniae/genetics , Streptococcus pneumoniae/growth & development , Streptolysins/geneticsABSTRACT
The opportunistic pathogen Streptococcus pneumoniae has dual lifestyles: one of an asymptomatic colonizer in the human nasopharynx and the other of a deadly pathogen invading sterile host compartments. The latter triggers an overwhelming inflammatory response, partly driven via pore forming activity of the cholesterol dependent cytolysin (CDC), pneumolysin. Although pneumolysin-induced inflammation drives person-to-person transmission from nasopharynx, the primary reservoir for pneumococcus, it also contributes to high mortality rates, creating a bottleneck that hampers widespread bacterial dissemination, thus acting as a double-edged sword. Serotype 1 ST306, a widespread pneumococcal clone, harbours a non-hemolytic variant of pneumolysin (Ply-NH). Performing crystal structure analysis of Ply-NH, we identified Y150H and T172I as key substitutions responsible for loss of its pore forming activity. We uncovered a novel inter-molecular cation-π interaction, governing formation of the transmembrane ß-hairpins (TMH) in the pore state of Ply, which can be extended to other CDCs. H150 in Ply-NH disrupts this interaction, while I172 provides structural rigidity to domain-3, through hydrophobic interactions, inhibiting TMH formation. Loss of pore forming activity enabled improved cellular invasion and autophagy evasion, promoting an atypical intracellular lifestyle for pneumococcus, a finding that was corroborated in in vivo infection models. Attenuation of inflammatory responses and tissue damage promoted tolerance of Ply-NH-expressing pneumococcus in the lower respiratory tract. Adoption of this altered lifestyle may be necessary for ST306 due to its limited nasopharyngeal carriage, with Ply-NH, aided partly by loss of its pore forming ability, facilitating a benign association of SPN in an alternative, intracellular host niche.
Subject(s)
Adaptation, Physiological , Inflammation/microbiology , Loss of Function Mutation , Pneumococcal Infections/microbiology , Streptococcus pneumoniae/physiology , Streptolysins/metabolism , Amino Acid Sequence , Animals , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cell Membrane/microbiology , Cholesterol/metabolism , Cytoplasm/microbiology , Female , Humans , Mice , Models, Structural , Perforin/genetics , Perforin/metabolism , Sequence Alignment , Streptococcus pneumoniae/genetics , Streptolysins/geneticsABSTRACT
Streptococcus pneumoniae serotype 1 remains a huge problem in low-and-middle income countries, particularly in sub-Saharan Africa. Despite its importance, studies in this serotype have been hindered by the lack of genetic tools to modify it. In this study, we describe a method to genetically modify a serotype 1 clinical isolate (strain 519/43). Interestingly, this was achieved by exploiting the Pneumococcus' ability to naturally acquire DNA. However, unlike most pneumococci, the use of linear DNA was not successful; to mutate this important strain, a suicide plasmid had to be used. This methodology has provided the means for a deeper understanding of this elusive serotype, both in terms of its biology and pathogenicity. To validate the method, the major known pneumococcal toxin, pneumolysin, was mutated because it has a well-known and easy to follow phenotype. We showed that the mutant, as expected, lost its ability to lyse red blood cells. By being able to mutate an important gene in the serotype of interest, we were able to observe different phenotypes for loss of function mutants upon intraperitoneal and intranasal infections from the ones observed for other serotypes. In summary, this study proves that strain 519/43 (serotype 1) can be genetically modified.