Quercetin protects rats from catheter‐related Staphylococcus aureus infections by inhibiting coagulase activity

Coagulase (Coa) activity is essential for the virulence of Staphylococcus aureus (S aureus), one of the most important pathogenic bacteria leading to catheter‐related bloodstream infections (CRBSI). We have demonstrated that the mutation of coagulase improved outcomes in disease models of S aureus CRBSI, suggesting that targeting Coa may represent a novel antiinfective strategy for CRBSI. Here, we found that quercetin, a natural compound that does not affect S aureus viability, could inhibit Coa activity. Chemical biological analysis revealed that the direct engagement of quercetin with the active site (residues Tyr187, Leu221 and His228) of Coa inhibited its activity. Furthermore, treatment with quercetin reduced the retention of bacteria on catheter surfaces, decreased the bacterial load in the kidneys and alleviated kidney abscesses in vivo. These data suggest that antiinfective therapy targeting Coa with quercetin may represent a novel strategy and provide a new leading compound with which to combat bacterial infections.     

Overall picture of an emerging neonatal infectious disease induced by a superantigenic exotoxin mainly produced by methicillin‐resistant Staphylococcus aureus

Since 1992, many neonates in neonatal intensive care units in Japan have been developing fever and systemic exanthema. Immunological analyses of neonates with these symptoms has revealed that the bacterial superantigen, toxic shock syndrome toxin‐1 (TSST‐1) is the cause. The name neonatal TSS‐like exanthematous disease (NTED) has been applied to this condition. The most striking clinical finding has been that none of the term neonates have developed shock or died of NTED. The timing of NTED epidemics has coincided with the spread of emerging TSST‐1‐producing methicillin‐resistant Staphylococcus aureus clones in Japan. The low frequency of pregnant women with positive anti‐TSST‐1 antibody titers could be one reason for the spread of NTED in Japan. Neonates have immune tolerance against TSST‐1 and may actively suppress the immune response to NTED with interleukin‐10. According to the T cell responses in infants or young children with diseases induced by TSST‐1, the pathophysiology of TSST‐1‐related diseases may be age‐dependent. The precise mechanism of anergy and deletion of specific T cells stimulated with TSST‐1 should be investigated in neonates infected with NTED. Both NTED and TSS might provide good models for analyzing the mechanism(s) of neonatal immune tolerance and the age‐dependence of human immunity. This disease has not only become representative of diseases caused by superantigens, but has also yielded a considerable amount of evidence about human immune reactions against superantigens.     

Staphylococcus aureus virulence attenuation and immune clearance mediated by a phage lysin‐derived protein

New anti‐infective approaches are much needed to control multi‐drug‐resistant (MDR) pathogens, such as methicillin‐resistant Staphylococcus aureus (MRSA). Here, we found for the first time that a recombinant protein derived from the cell wall binding domain (CBD) of the bacteriophage lysin PlyV12, designated as V12CBD, could attenuate S. aureus virulence and enhance host immune defenses via multiple manners. After binding with V12CBD, S. aureus became less invasive to epithelial cells and more susceptible to macrophage killing. The expressions of multiple important virulence genes of S. aureus were reduced 2.4‐ to 23.4‐fold as response to V12CBD. More significantly, V12CBD could activate macrophages through NF‐κB pathway and enhance phagocytosis against S. aureus. As a result, good protections of the mice from MRSA infections were achieved in therapeutic and prophylactic models. These unique functions of V12CBD would render it a novel alternative molecule to control MDRS. aureus infections.     

Evidence for multiple modes of neutrophil serine protease recognition by the EAP family of Staphylococcal innate immune evasion proteins

Neutrophils contain high levels of chymotrypsin‐like serine proteases (NSPs) within their azurophilic granules that have a multitude of functions within the immune system. In response, the pathogen Staphylococcus aureus has evolved three potent inhibitors (Eap, EapH1, and EapH2) that protect the bacterium as well as several of its secreted virulence factors from the degradative action of NSPs. We previously showed that these so‐called EAP domain proteins represent a novel class of NSP inhibitors characterized by a non‐covalent inhibitory mechanism and a distinct target specificity profile. Based upon high levels of structural homology amongst the EAP proteins and the NSPs, as well as supporting biochemical data, we predicted that the inhibited complex would be similar for all EAP/NSP pairs. However, we present here evidence that EapH1 and EapH2 bind the canonical NSP, Neutrophil Elastase (NE), in distinct orientations. We discovered that alteration of EapH1 residues at the EapH1/NE interface caused a dramatic loss of affinity and inhibition of NE, while mutation of equivalent positions in EapH2 had no effect on NE binding or inhibition. Surprisingly, mutation of residues in an altogether different region of EapH2 severely impacted both the NE binding and inhibitory properties of EapH2. Even though EapH1 and EapH2 bind and inhibit NE and a second NSP, Cathepsin G, equally well, neither of these proteins interacts with the structurally related, but non‐proteolytic granule protein, azurocidin. These studies expand our understanding of EAP/NSP interactions and suggest that members of this immune evasion protein family are capable of diverse target recognition modes.     

Proteolysis during long‐term glucose starvation in Staphylococcus aureus COL

A combination of pulse‐chase experiments and 2‐D PAGE revealed that protein degradation appears to play a crucial role for the cell physiology of Staphylococcus aureus COL during extended periods of glucose starvation. The synthesis rate of virtually all cytosolic and radioactively labeled proteins from growing cells seemed dramatically reduced in the first 3.5 h of glucose starvation. The stability of proteins synthesized in growing cells was monitored by a pulse‐chase approach on a proteome wide scale. Especially, enzymes involved in nucleic acid and amino acid biosyntheses, energy metabolism and biosynthesis of cofactors were found rather rapidly degraded within the onset of the stationary phase, whereas the majority of glycolytic and tricarboxylic acid cycle enzymes remained more stable. Furthermore, single enzymes of biosynthetic pathways were differentially degraded. A metabolite analysis revealed that glucose completely depleted from the medium in the transient phase, and amino acids such as alanine and glycine were taken up by the cells in the stationary phase. We suggest that vegetative proteins no longer required in non‐growing cells and thus no longer protected by integration into functional complexes were degraded. Proteolysis of putative non‐substrate‐bound or “unemployed” proteins appears to be a characteristic feature of S. aureus in order to access nutrients as an important survival strategy under starvation conditions.     

The polyphenol (-)-epicatechin gallate disrupts the secretion of virulence-related proteins by Staphylococcus aureus

Aim: (?)‐Epicatechin gallate (ECg) modifies the morphology, cell wall architecture and β‐lactam antibiotic susceptibility of Staphylococcus aureus. As these effects result primarily from intercalation into the bacterial cytoplasmic membrane, the capacity of ECg to modulate the secretion of two key staphylococcal virulence factors, coagulase and α‐toxin, was examined. Methods and Results: Bioassays were used to determine coagulase and haemolysin activity in culture supernatants of a number of S. aureus isolates grown in the presence and absence of ECg; α‐toxin secretion was also evaluated by immunoblotting. Growth in ECg reduced the levels of activity of both proteins in culture supernatants; the effects could only be partly explained by ECg‐mediated inhibition of bioactivity and by induction of secreted proteases. Conclusion: ECg suppresses the secretion of coagulase and α‐toxin by clinical isolates of S. aureus. Significance and Impact of the Study: The observation that secretion of key components of staphylococcal virulence can be compromised by a naturally occurring polyphenol supports the notion that ECg and related compounds may have therapeutic utility for the control of infections that are currently difficult to treat due to the propensity of methicillin‐resistant S. aureus to accumulate antibiotic resistance genes.     

Targeting the R domain of coagulase by active vaccination protects mice against lethal Staphylococcus aureus infection

Coagulase (Coa) secreted by Staphylococcus aureus is associated with the establishment of staphylococcal disease, which activates host prothrombin and generates fibrin shields. The R domain of Coa, consisting of several conserved repeats, is important in immune evasion during S. aureus infection. However, previous research showed that the Coa R domain induced very weak specific antibody responses. In this study, we constructed a new R domain, CoaR6, consisting of 6 repeats that occur most frequently in clinical isolates. By fusing CoaR6 with Hc, the C-terminal fragment of the heavy chain of tetanus neurotoxin, we successfully increased anti-CoaR6 IgG levels in immunized mice which were hardly detected in mice immunized with CoaR6 plus alum. To further improve anti-CoaR6 responses, the combination adjuvants alum plus CpG were formulated with the antigen and exhibited a significantly higher specific antibody response. Moreover, active Th1/Th17 immune responses were observed in Hc-CoaR6 immunized group rather than CoaR6. Active immunization of Hc-CoaR6 with alum plus CpG showed protective effects in a peritonitis model induced by two S. aureus strains with different coagulase types. Our results provided strategies to improve the immunogenicity of R domain and supporting evidences for R domain to be an S. aureus vaccine candidate.     

The C‐type lectin receptor MGL senses N‐acetylgalactosamine on the unique Staphylococcus aureus ST395 wall teichoic acid

Staphylococcus aureus is a common skin commensal but is also associated with various skin and soft tissue pathologies. Upon invasion, S. aureus is detected by resident innate immune cells through pattern‐recognition receptors (PRRs), although a comprehensive understanding of the specific molecular interactions is lacking. Recently, we demonstrated that the PRR langerin (CD207) on epidermal Langerhans cells senses the conserved β‐1,4‐linked N‐acetylglucosamine (GlcNAc) modification on S. aureus wall teichoic acid (WTA), thereby increasing skin inflammation. Interestingly, the S. aureus ST395 lineage as well as certain species of coagulase‐negative staphylococci (CoNS) produce a structurally different WTA molecule, consisting of poly‐glycerolphosphate with α‐O‐N‐acetylgalactosamine (GalNAc) residues, which are attached by the glycosyltransferase TagN. Here, we demonstrate that S. aureus ST395 strains interact with the human Macrophage galactose‐type lectin (MGL; CD301) receptor, which is expressed by dendritic cells and macrophages in the dermis. MGL bound S. aureus ST395 in a tagN‐ and GalNAc‐dependent manner but did not interact with different tagN‐positive CoNS species. However, heterologous expression of Staphylococcus lugdunensis tagN in S. aureus conferred phage infection and MGL binding, confirming the role of this CoNS enzyme as GalNAc‐transferase. Functionally, the detection of GalNAc on S. aureus ST395 WTA by human monocyte‐derived dendritic cells significantly enhanced cytokine production. Together, our findings highlight differential recognition of S. aureus glycoprofiles by specific human innate receptors, which may affect downstream adaptive immune responses and pathogen clearance.     

Bulk RNA degradation by nitrogen starvation‐induced autophagy in yeast

Autophagy is a catabolic process conserved among eukaryotes. Under nutrient starvation, a portion of the cytoplasm is non‐selectively sequestered into autophagosomes. Consequently, ribosomes are delivered to the vacuole/lysosome for destruction, but the precise mechanism of autophagic RNA degradation and its physiological implications for cellular metabolism remain unknown. We characterized autophagy‐dependent RNA catabolism using a combination of metabolome and molecular biological analyses in yeast. RNA delivered to the vacuole was processed by Rny1, a T2‐type ribonuclease, generating 3′‐NMPs that were immediately converted to nucleosides by the vacuolar non‐specific phosphatase Pho8. In the cytoplasm, these nucleosides were broken down by the nucleosidases Pnp1 and Urh1. Most of the resultant bases were not re‐assimilated, but excreted from the cell. Bulk non‐selective autophagy causes drastic perturbation of metabolism, which must be minimized to maintain intracellular homeostasis.     

Microbiome precision editing: Using PEG as a selective fermentation initiator against methicillin‐resistant Staphylococcus aureus

Recent creation of a Unified Microbiome Initiative (UMI) has the aim of understanding how microbes interact with each other and with us. When pathogenic Staphylococcus aureus infects the skin, the interplay between S. aureus and skin commensal bacteria occurs. Our previous data revealed that skin commensal bacteria can mediate fermentation against the growth of USA300, a community‐acquired methicillin‐resistant S. aureus MRSA. By using a fermentation process with solid media on a small scale, we define poly(ethylene glycol) dimethacrylate (PEG‐DMA) as a selective fermentation initiator which can specifically intensify the probiotic ability of skin commensal Staphylococcus epidermidis bacteria. At least five short‐chain fatty acids including acetic, butyric and propionic acids with anti‐USA300 activities are produced by PEG‐DMA fermentation of S. epidermidis. Furthermore, the S. epidermidis‐laden PEG‐DMA hydrogels effectively decolonized USA300 in skin wounds in mice. The PEG‐DMA and its derivatives may become novel biomaterials to specifically tailor the human skin microbiome against invading pathogens.     

SIRT6 protects against pathological damage caused by diet‐induced obesity

The NAD+‐dependent SIRT6 deacetylase is a therapeutic candidate against the emerging metabolic syndrome epidemic. SIRT6, whose deficiency in mice results in premature aging phenotypes and metabolic defects, was implicated in a calorie restriction response that showed an opposite set of phenotypes from the metabolic syndrome. To explore the role of SIRT6 in metabolic stress, wild type and transgenic (TG) mice overexpressing SIRT6 were fed a high fat diet. In comparison to their wild‐type littermates, SIRT6 TG mice accumulated significantly less visceral fat, LDL‐cholesterol, and triglycerides. TG mice displayed enhanced glucose tolerance along with increased glucose‐stimulated insulin secretion. Gene expression analysis of adipose tissue revealed that the positive effect of SIRT6 overexpression is associated with down regulation of a selective set of peroxisome proliferator‐activated receptor‐responsive genes, and genes associated with lipid storage, such as angiopoietin‐like protein 4, adipocyte fatty acid‐binding protein, and diacylglycerol acyltransferase 1, which were suggested as potential targets for drugs to control metabolic syndrome. These results demonstrate a protective role for SIRT6 against the metabolic consequences of diet‐induced obesity and suggest a potentially beneficial effect of SIRT6 activation on age‐related metabolic diseases.     

Human CST complex protects stalled replication forks by directly blocking MRE11 degradation of nascent‐strand DNA

Degradation and collapse of stalled replication forks are main sources of genomic instability, yet the molecular mechanisms for protecting forks from degradation/collapse are not well understood. Here, we report that human CST (CTC1‐STN1‐TEN1) proteins, which form a single‐stranded DNA‐binding complex, localize at stalled forks and protect stalled forks from degradation by the MRE11 nuclease. CST deficiency increases MRE11 binding to stalled forks, leading to nascent‐strand degradation at reversed forks and ssDNA accumulation. In addition, purified CST complex binds to 5’ DNA overhangs and directly blocks MRE11 degradation in vitro, and the DNA‐binding ability of CST is required for blocking MRE11‐mediated nascent‐strand degradation. Our results suggest that CST inhibits MRE11 binding to reversed forks, thus antagonizing excessive nascent‐strand degradation. Finally, we uncover that CST complex inactivation exacerbates genome instability in BRCA2 deficient cells. Collectively, our findings identify the CST complex as an important fork protector that preserves genome integrity under replication perturbation.