The Gram-positive human pathogen Staphylococcus aureus causes a variety of human diseases such as skin infections, pneumonia, and endocarditis. The micrococcal nuclease Nuc1 is one of the major S. aureus virulence factors and allows the bacterium to avoid neutrophil extracellular trap (NET)-mediated killing. We found that addition of the protein synthesis inhibitor clindamycin to S. aureus LAC cultures decreased nuc1 transcription and subsequently blunted nuclease activity in a molecular beacon-based fluorescence assay. We also observed reduced NET degradation through Nuc1 inhibition translating into increased NET-mediated clearance. Similarly, pooled human immunoglobulin specifically inhibited nuclease activity in a concentration-dependent manner. Inhibition of nuclease activity by clindamycin and immunoglobulin enhanced S. aureus clearance and should be considered in the treatment of S. aureus infections.
SpyCEP-mediated chemokine degradation translates into more efficient spreading and increased severity of invasive Group A Streptococcus (GAS) infections, due to impaired neutrophil recruitment to the site of infection. SpyCEP is markedly up-regulated in invasive as compared to colonizing GAS isolates raising the question whether SpyCEP expression hinders bacterial attachment and thus colonization of the host. To address this question we used a molecular approach involving the use of homologous GAS strains either expressing or not SpyCEP or expressing an enzymatically inactive variant of SpyCEP. We found that expression of enzymatically functional SpyCEP lowered GAS adherence and invasion potential toward various epithelial and endothelial cells. SpyCEP also blunted biofilm formation capacity. Our data indicate that expression of SpyCEP decreases colonization and thus might be detrimental for the spreading of GAS.
The endocytic Ashwell-Morell receptor (AMR) of hepatocytes detects pathogen remodeling of host glycoproteins by neuraminidase in the bloodstream and mitigates the lethal coagulopathy of sepsis. We have investigated the mechanism of host protection by the AMR during the onset of sepsis and in response to the desialylation of blood glycoproteins by the NanA neuraminidase of Streptococcus pneumoniae. We find that the AMR selects among potential glycoprotein ligands unmasked by microbial neuraminidase activity in pneumococcal sepsis to eliminate from blood circulation host factors that contribute to coagulation and thrombosis. This protection is attributable in large part to the rapid induction of a moderate thrombocytopenia by the AMR. We further show that neuraminidase activity in the blood can be manipulated to induce the clearance of AMR ligands including platelets, thereby preactivating a protective response in pneumococcal sepsis that moderates the severity of disseminated intravascular coagulation and enables host survival.
Epstein-Barr virus (EBV) infects >90% of the human population within the first 2 decades of life and establishes reversible latent infection in B cells. The stimuli that lead to switching from latent to lytic EBV infection in vivo are still elusive. Group A streptococci (GAS) are a common cause of bacterial pharyngotonsillitis in children and adolescents and colonize the tonsils and pharynx of up to 20% of healthy children. Thus, concomitant presence of EBV and GAS in the same individual is frequent. Here, we show that EBV carriers who are colonized with GAS shed EBV particles in higher numbers in their saliva, compared with EBV carriers not colonized with GAS. Messenger RNA levels of the master lytic regulatory EBV gene BZLF1 were more frequently detected in tonsils from EBV carriers colonized with GAS than from EBV carriers not colonized. Heat-killed GAS, potentially mimicking GAS colonization, elicited lytic EBV in latently infected lymphoblastoid cell lines (LCLs) partially via Toll-like receptor 2 triggering, as did purified GAS peptidoglycan. Thus, colonization by GAS might benefit EBV by increasing the EBV load in saliva and thereby enhancing the likelihood of EBV spread to other hosts.
Group A Streptococcus (GAS) is a human pathogen causing a wide range of mild to severe and life-threatening diseases. The GAS M1 protein is a major virulence factor promoting GAS invasiveness and resistance to host innate immune clearance. M1 displays an irregular coiled-coil structure, including the B-repeats that bind fibrinogen. Previously, we found that B-repeat stabilisation generates an idealised version of M1 (M1) characterised by decreased fibrinogen binding in vitro. To extend these findings based on a soluble truncated version of M1, we now studied the importance of the B-repeat coiled-coil irregularities in full length M1 and M1 expressed in live GAS and tested whether the modulation of M1-fibrinogen interactions would open up novel therapeutic approaches. We found that altering either the M1 structure on the GAS cell surface or removing its target host protein fibrinogen blunted GAS virulence. GAS expressing M1 showed an impaired ability to adhere to and to invade human endothelial cells, was more readily killed by whole blood or neutrophils and most importantly was less virulent in a murine necrotising fasciitis model. M1-mediated virulence of wild-type GAS was strictly dependent on the presence and concentration of fibrinogen complementing our finding that M1-fibrinogen interactions are crucial for GAS virulence. Consistently blocking M1-fibrinogen interactions by fragment D reduced GAS virulence in vitro and in vivo. This supports our conclusion that M1-fibrinogen interactions are crucial for GAS virulence and that interference may open up novel complementary treatment options for GAS infections caused by the leading invasive GAS strain M1.
Natural killer T cells (NKT cells) recognize glycolipid antigens presented by CD1d. These cells express an evolutionarily conserved, invariant T cell antigen receptor (TCR), but the forces that drive TCR conservation have remained uncertain. Here we show that NKT cells recognized diacylglycerol-containing glycolipids from Streptococcus pneumoniae, the leading cause of community-acquired pneumonia, and group B Streptococcus, which causes neonatal sepsis and meningitis. Furthermore, CD1d-dependent responses by NKT cells were required for activation and host protection. The glycolipid response was dependent on vaccenic acid, which is present in low concentrations in mammalian cells. Our results show how microbial lipids position the sugar for recognition by the invariant TCR and, most notably, extend the range of microbes recognized by this conserved TCR to several clinically important bacteria.
Timely recognition and elimination of invasive group A Streptococcus (GAS) by innate immunity is crucial to control infection. The intracellular pattern recognition receptor Toll-like receptor 9 (TLR9) promotes macrophage hypoxia-inducible factor-1? levels, oxidative burst and nitric oxide production in response to GAS. TLR9 contributes to GAS clearance in vivo in both localized cutaneous and systemic infection models.
To determine whether the mitochondria or cytoplasm produces superoxide during ischemia-reperfusion of the brain, we analyzed lucigenine-enhanced chemiluminescence emission in slices of brain tissue prepared from manganese-superoxide dismutase (Mn-SOD)-deficient (Sod2-deficient) and copper and zinc-superoxide dismutase (Cu,Zn-SOD)-deficient (Sod1-deficient) mice during oxygenation and hypoxia-reoxygenation. The steady-state level of chemiluminescence under oxygenated conditions was significantly enhanced by a lack of either Sod. We hypothesize that the enhanced chemiluminescence produced by Sod2 and Sod1 deficiency reflects in situ superoxide generation in the mitochondria and cytoplasm, respectively. Based on this hypothesis, the major site of intracellular superoxide generation was assumed to be the cytoplasm. However, mitochondria occupy less cellular space than the cytoplasm. In terms of volume, the superoxide concentration is assumed to be higher in mitochondria than in the cytoplasm. Mn-SOD activity was 18% of the Cu,Zn-SOD activity observed in the wild-type mouse brain. However, when mitochondrial SOD activity was expressed as per volume, it was assumed to be equal to that observed in the cytoplasm. This imbalance between superoxide and SOD activity is expected to cause mitochondrial oxidative damage. The chemiluminescence intensity increased significantly during reoxygenation and was enhanced by Sod2 deficiency but was not significantly affected by Sod1 deficiency. The superoxide concentration in the reoxygenated brain would be higher in the mitochondria than in the cytoplasm. The present study indicated that the major site of intracellular superoxide generation in the brain during oxygenation is the cytoplasm, whereas it is the mitochondria during reoxygenation.
Manganese superoxide dismutase (Mn-SOD) is a mitochondrial enzyme that converts toxic O(2)(-) to H(2)O(2). Previous studies have reported that a systemic deficiency in Mn-SOD causes neonatal lethality in mice. Therefore, no mouse model is available for the analysis of the pathological role of O(2)(-) injuries in adult tissues. To explore an adult-type mouse model, we generated tissue-specific Mn-SOD conditional knockout mice using a Cre-loxp system. First, we generated liver-specific Mn-SOD-deficient mice by crossbreeding with albumin-Cre transgenic mice. Mn-SOD proteins were significantly downregulated in the liver of liver-specific Mn-SOD knockout mice. Interestingly, the mutant mice showed no obvious morphological abnormalities or biochemical alterations in the liver, suggesting a redundant or less important physiological role for Mn-SOD in the liver than previously thought. Next, we generated heart/muscle-specific Mn-SOD-deficient mice by crossbreeding muscle creatine kinase-Cre transgenic mice. The mutant mice developed progressive dilated cardiomyopathy with specific molecular defects in mitochondrial respiration. Furthermore, brain-specific Mn-SOD-deficient mice that had been developed by crossbreeding with nestin-Cre transgenic mice developed a spongiform encephalopathy-like pathology associated with gliosis and died within 3 weeks of birth. These results imply that the superoxide generated in mitochondria plays a pivotal role in the development and progression of pathologies in the heart and brain, but not in the liver. In conclusion, we successfully generated various tissue-specific Mn-SOD conditional knockout mice that provide useful tools for the analysis of various oxidative stress-associated diseases.
Streptococcus pneumoniae (SPN), the leading cause of meningitis in children and adults worldwide, is associated with an overwhelming host inflammatory response and subsequent brain injury. Here we examine the global response of the blood-brain barrier to SPN infection and the role of neuraminidase A (NanA), an SPN surface anchored protein recently described to promote central nervous system tropism. Microarray analysis of human brain microvascular endothelial cells (hBMEC) during infection with SPN or an isogenic NanA-deficient (?nanA) mutant revealed differentially activated genes, including neutrophil chemoattractants IL-8, CXCL-1, CXCL-2. Studies using bacterial mutants, purified recombinant NanA proteins and in vivo neutrophil chemotaxis assays indicated that pneumococcal NanA is necessary and sufficient to activate host chemokine expression and neutrophil recruitment during infection. Chemokine induction was mapped to the NanA N-terminal lectin-binding domain with a limited contribution of the sialidase catalytic activity, and was not dependent on the invasive capability of the organism. Furthermore, pretreatment of hBMEC with recombinant NanA protein significantly increased bacterial invasion, suggesting that NanA-mediated activation of hBMEC is a prerequisite for efficient SPN invasion. These findings were corroborated in an acute murine infection model where we observed less inflammatory infiltrate and decreased chemokine expression following infection with the ?nanA mutant.
GBS (Group B Streptococcus) requires capsular Sia (sialic acid) for virulence and partially modifies this sugar by O-acetylation. In the present paper we describe serotype-specific patterns of GBS Sia O-acetylation that can be manipulated by genetic and biochemical means. In vitro and in vivo assays demonstrate that this subtle modification attenuates GBS Sia-mediated neutrophil suppression and animal virulence.
The effects of certain tea components on the prevention of obesity in humans have recently been reported, although it is still unclear whether black tea consumption is beneficial. We obtained black tea extract (BTPE) consisting of polyphenols specific to black tea, and from it, prepared a polymerized polyphenol fraction (BTP). The effectiveness of oral administration of the BTPE was examined in in vitro and in vivo experiments.
In humans, Streptococcus pneumoniae (SPN) is the leading cause of bacterial meningitis, a disease with high attributable mortality and frequent permanent neurological sequelae. The molecular mechanisms underlying the central nervous system tropism of SPN are incompletely understood, but include a primary interaction of the pathogen with the blood-brain barrier (BBB) endothelium. All SPN strains possess a gene encoding the surface-anchored sialidase (neuraminidase) NanA, which cleaves sialic acid on host cells and proteins. Here, we use an isogenic SPN NanA-deficient mutant and heterologous expression of the protein to show that NanA is both necessary and sufficient to promote SPN adherence to and invasion of human brain microvascular endothelial cells (hBMECs). NanA-mediated hBMEC invasion depends only partially on sialidase activity, whereas the N-terminal lectinlike domain of the protein plays a critical role. NanA promotes SPN-BBB interaction in a murine infection model, identifying the protein as proximal mediator of CNS entry by the pathogen.
Human neutrophil Siglec-9 is a lectin that recognizes sialic acids (Sias) via an amino-terminal V-set Ig domain and possesses tyrosine-based inhibitory motifs in its cytoplasmic tail. We hypothesized that Siglec-9 recognizes host Sias as "self," including in cis interactions with Sias on the neutrophils own surface, thereby dampening unwanted neutrophil reactivity. Here we show that neutrophils presented with immobilized multimerized Siaalpha2-3Galbeta1-4GlcNAc units engage them in trans via Siglec-9. The sialylated capsular polysaccharide of group B Streptococcus (GBS) also presents terminal Siaalpha2-3Galbeta1-4GlcNAc units, and similarly engages neutrophil Siglec-9, dampening neutrophil responses in a Sia- and Siglec-9-dependent manner. Reduction in the neutrophil oxidative burst, diminished formation of neutrophil extracellular DNA traps, and increased bacterial survival are also facilitated by GBS sialylated capsular polysaccharide interactions with Siglec-9. Thus, GBS can impair neutrophil defense functions by coopting a host inhibitory receptor via sialoglycan molecular mimicry, a novel mechanism of bacterial immune evasion.
Group A Streptococcus (GAS) has developed a broad arsenal of virulence factors that serve to circumvent host defense mechanisms. The virulence factor DNase Sda1 of the hyperinvasive M1T1 GAS clone degrades DNA-based neutrophil extracellular traps allowing GAS to escape extracellular killing. TLR9 is activated by unmethylated CpG-rich bacterial DNA and enhances innate immune resistance. We hypothesized that Sda1 degradation of bacterial DNA could alter TLR9-mediated recognition of GAS by host innate immune cells. We tested this hypothesis using a dual approach: loss and gain of function of DNase in isogenic GAS strains and presence and absence of TLR9 in the host. Either DNA degradation by Sda1 or host deficiency of TLR9 prevented GAS induced IFN-? and TNF-? secretion from murine macrophages and contributed to bacterial survival. Similarly, in a murine necrotizing fasciitis model, IFN-? and TNF-? levels were significantly decreased in wild type mice infected with GAS expressing Sda1, whereas no such Sda1-dependent effect was seen in a TLR9-deficient background. Thus GAS Sda1 suppressed both the TLR9-mediated innate immune response and macrophage bactericidal activity. Our results demonstrate a novel mechanism of bacterial innate immune evasion based on autodegradation of CpG-rich DNA by a bacterial DNase.
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