Hyaluronan Breakdown Contributes to Immune Defense against Group A Streptococcus

Group A Streptococcus (GAS) commonly infects human skin and occasionally causes severe and life-threatening invasive diseases. The hyaluronan (HA) capsule of GAS has been proposed to protect GAS from host defense by mimicking endogenous HA, a large and abundant glycosaminoglycan in the skin. However, HA is degraded during tissue injury, and the functions of short-chain HA that is generated during infection have not been studied. To examine the impact of the molecular mass of HA on GAS infection, we established infection models in vitro and in vivo in which the size of HA was defined by enzymatic digestion or custom synthesis. We discovered that conversion of high molecular mass HA to low molecular mass HA facilitated GAS phagocytosis by macrophages and limited the severity of infection in mice. In contrast, native high molecular mass HA significantly impaired internalization by macrophages and increased GAS survival in murine blood. Thus, our data demonstrate that GAS virulence can be influenced by the size of HA derived from both the bacterium and host and suggest that high molecular mass HA facilitates GAS deep tissue infections, whereas the generation of short-chain HA can be protective.     

Conformational Analysis of the Streptococcus pneumoniae Hyaluronate Lyase and Characterization of Its Hyaluronan-specific Carbohydrate-binding Module

For a subset of pathogenic microorganisms, including Streptococcus pneumoniae, the recognition and degradation of host hyaluronan contributes to bacterial spreading through the extracellular matrix and enhancing access to host cell surfaces. The hyaluronate lyase (Hyl) presented on the surface of S. pneumoniae performs this role. Using glycan microarray screening, affinity electrophoresis, and isothermal titration calorimetry we show that the N-terminal module of Hyl is a hyaluronan-specific carbohydrate-binding module (CBM) and the founding member of CBM family 70. The 1.2 Å resolution x-ray crystal structure of CBM70 revealed it to have a β-sandwich fold, similar to other CBMs. The electrostatic properties of the binding site, which was identified by site-directed mutagenesis, are distinct from other CBMs and complementary to its acidic ligand, hyaluronan. Dynamic light scattering and solution small angle x-ray scattering revealed the full-length Hyl protein to exist as a monomer/dimer mixture in solution. Through a detailed analysis of the small angle x-ray scattering data, we report the pseudoatomic solution structures of the monomer and dimer forms of the full-length multimodular Hyl.     

Functional and Structural Properties of a Novel Protein and Virulence Factor (Protein sHIP) in Streptococcus pyogenes

Streptococcus pyogenes is a significant bacterial pathogen in the human population. The importance of virulence factors for the survival and colonization of S. pyogenes is well established, and many of these factors are exposed to the extracellular environment, enabling bacterial interactions with the host. In the present study, we quantitatively analyzed and compared S. pyogenes proteins in the growth medium of a strain that is virulent to mice with a non-virulent strain. Particularly, one of these proteins was present at significantly higher levels in stationary growth medium from the virulent strain. We determined the three-dimensional structure of the protein that showed a unique tetrameric organization composed of four helix-loop-helix motifs. Affinity pull-down mass spectrometry analysis in human plasma demonstrated that the protein interacts with histidine-rich glycoprotein (HRG), and the name sHIP (streptococcal histidine-rich glycoprotein-interacting protein) is therefore proposed. HRG has antibacterial activity, and when challenged by HRG, sHIP was found to rescue S. pyogenes bacteria. This and the finding that patients with invasive S. pyogenes infection respond with antibody production against sHIP suggest a role for the protein in S. pyogenes pathogenesis.     

Structural characterization of Streptococcus pneumoniae serotype 9A capsule polysaccharide reveals role of glycosyl 6-O-acetyltransferase wcjE in serotype 9V capsule biosynthesis and immunogenicity

The putative capsule O-acetyltransferase gene wcjE is highly conserved across various Streptococcus pneumoniae serotypes, but the role of the gene in capsule biosynthesis and bacterial fitness remains largely unclear. Isolates expressing pneumococcal serotype 9A arise from precursors expressing wcjE-associated serotype 9V through loss-of-function mutation to wcjE. To define the biosynthetic role of 9V wcjE, we characterized the structure and serological properties of serotype 9V and 9A capsule polysaccharide (PS). NMR data revealed that both 9V and 9A PS are composed of an identical pentasaccharide repeat unit, as reported previously. However, in sharp contrast to previous studies on 9A PS being devoid of any O-acetylation, we identified O-acetylation of α-glucuronic acid and α-glucose in 9A PS. In addition, 9V PS also contained -CH(2) O-acetylation of β-N-acetylmannosamine, a modification that disappeared following in vitro recombinatorial deletion of wcjE. We also show that serotyping sera and monoclonal antibodies specific for 9V and 9A bound capsule PS in an O-acetate-dependent manner. Furthermore, IgG and to a lesser extent IgM from human donors immunized with serotype 9V PS displayed stronger binding to 9V compared with 9A PS. We conclude that serotype 9V wcjE mediates 6-O-acetylation of β-N-acetylmannosamine. This PS modification can be selectively targeted by antibodies in immunized individuals, identifying a potential selective advantage for wcjE inactivation and serotype 9A emergence.     

Group A streptococcus cell-associated pathogenic proteins as revealed by growth in hyaluronic acid-enriched media

Group A streptococcus (GAS), also know as Streptococcus pyogenes, is a human pathogen and can cause several fatal invasive diseases such as necrotising fasciitis, the so-called flesh-eating disease, and toxic shock syndrome. The destruction of connective tissue and the hyaluronic acid (HA) therein, is a key element of GAS pathogenesis. We therefore propagated GAS in HA-enriched growth media in an attempt to create a simple biological system that could reflect some elements of GAS pathogenesis. Our results show that several recognised virulence factors were up-regulated in HA-enriched media, including the M1 protein, a collagen-like surface protein and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, which has been shown to play important roles in streptococcal pathogenesis. Interestingly, two hypothetical proteins of unknown function were also up-regulated and detailed bioinformatics analysis showed that at least one of these hypothetical proteins is likely to be involved in pathogenesis. It was therefore concluded that this simple biological system provided a valuable tool for the identification of potential GAS virulence factors.     

The Fibrinogen-binding M1 Protein Reduces Pharyngeal Cell Adherence and Colonization Phenotypes of M1T1 Group A Streptococcus

Group A Streptococcus (GAS) is a leading human pathogen producing a diverse array of infections from simple pharyngitis (“strep throat”) to invasive conditions, including necrotizing fasciitis and toxic shock syndrome. The surface-anchored GAS M1 protein is a classical virulence factor that promotes phagocyte resistance and exaggerated inflammation by binding host fibrinogen (Fg) to form supramolecular networks. In this study, we used a virulent WT M1T1 GAS strain and its isogenic M1-deficient mutant to examine the role of M1-Fg binding in a proximal step in GAS infection-interaction with the pharyngeal epithelium. Expression of the M1 protein reduced GAS adherence to human pharyngeal keratinocytes by 2-fold, and this difference was increased to 4-fold in the presence of Fg. In stationary phase, surface M1 protein cleavage by the GAS cysteine protease SpeB eliminated Fg binding and relieved its inhibitory effect on GAS pharyngeal cell adherence. In a mouse model of GAS colonization of nasal-associated lymphoid tissue, M1 protein expression was associated with an average 6-fold decreased GAS recovery in isogenic strain competition assays. Thus, GAS M1 protein-Fg binding reduces GAS pharyngeal cell adherence and colonization in a fashion that is counterbalanced by SpeB. Inactivation of SpeB during the shift to invasive GAS disease allows M1-Fg binding, increasing pathogen phagocyte resistance and proinflammatory activities.     

Prevalence and genomic characterization of Group A Streptococcus dysgalactiae subsp. equisimilis isolated from patients with invasive infections in Toyama prefecture,Japan

Streptococcus dysgalactiae subsp. equisimilis (SDSE) causes severe invasive streptococcal infections, especially in elderly people. Between 2013 and 2018, 88 streptococci were isolated from clinical blood culture in a hospital in Toyama prefecture, Japan. The collection included six Group A SDSE (ASD) strains, which are rarely isolated. Multilocus sequence typing categorized five of the six strains into ST128 and the remaining strain into a new type. Maximum-likelihood phylogenetic analysis revealed that the six ASD strains had highly similar genome sequences. Bayesian analysis indicated that the most recent common ancestor of the strains appeared 39 years ago. The ASD strains possessed carbohydrate synthase genes that are conserved in Streptococcus pyogenes strains, whereas one strain featured a different arrangement of the gene cluster. The carbohydrate synthase genes varied by Lancefield type (A, C, and G).     

Analysis of Polymorphic Residues Reveals Distinct Enzymatic and Cytotoxic Activities of the Streptococcus pyogenes NAD+ Glycohydrolase

The Streptococcus pyogenes NAD⁺ glycohydrolase (SPN) is secreted from the bacterial cell and translocated into the host cell cytosol where it contributes to cell death. Recent studies suggest that SPN is evolving and has diverged into NAD⁺ glycohydrolase-inactive variants that correlate with tissue tropism. However, the role of SPN in both cytotoxicity and niche selection are unknown. To gain insight into the forces driving the adaptation of SPN, a detailed comparison of representative glycohydrolase activity-proficient and -deficient variants was conducted. Of a total 454 amino acids, the activity-deficient variants differed at only nine highly conserved positions. Exchanging residues between variants revealed that no one single residue could account for the inability of the deficient variants to cleave the glycosidic bond of β-NAD⁺ into nicotinamide and ADP-ribose; rather, reciprocal changes at 3 specific residues were required to both abolish activity of the proficient version and restore full activity to the deficient variant. Changing any combination of 1 or 2 residues resulted in intermediate activity. However, a change to any 1 residue resulted in a significant decrease in enzyme efficiency. A similar pattern involving multiple residues was observed for comparison with a second highly conserved activity-deficient variant class. Remarkably, despite differences in glycohydrolase activity, all versions of SPN were equally cytotoxic to cultured epithelial cells. These data indicate that the glycohydrolase activity of SPN may not be the only contribution the toxin has to the pathogenesis of S. pyogenes and that both versions of SPN play an important role during infection.     

猪链球菌2型烯醇化酶的分子克隆与免疫学特性

对新近测定的猪链球菌2型(S. suis 2) 05ZYH33全基因序列进行生物信息学分析, 并与相关家族蛋白进行同源性比较, 设计合成引物, PCR法扩增出约1.3 kb的烯醇化酶编码基因 (enolase, eno), 将其克隆入pMD-18T载体中, 进一步亚克隆入表达载体pET32a。将重组表达质粒pET32a::eno转化E. coli BL21 (DE3), 经IPTG诱导表达后, SDS-PAGE初步检测到分子量约为75kD的蛋白带。通过His-Tag亲和层析纯化, 获得融合蛋白His-ENO。Western-blot表明该表达产物具有免疫原性。基于ELISA进行的细胞定位实验证实了Enolase可以部分存在S. suis 2 05ZYH33细菌的表面。这提示了Enolase作为一种新发现的抗原对于引发猪链球菌相关疾病可能发挥着重要的作用。     

猪链球菌2型烯醇化酶的分子克隆与免疫学特性

对新近测定的猪链球菌2型(S.suis 2)05ZYH33全基因序列进行生物信息学分析,并与相关家族蛋白进行同源性比较,设计合成引物,PCR法扩增出约1.3 kb的烯醇化酶编码基因(enolase,eno),将其克隆入pMD-18T载体中,进一步亚克隆入表达载体pET32a.将重组表达质粒pET32a::eno转化E.coli BL21(DE3),经IPTG诱导表达后,SDS-PAGE初步检测到分子量约为75kD的蛋白带.通过His-Tag亲和层析纯化,获得融合蛋白His-ENO.Western-blot表明该表达产物具有免疫原性.基于ELISA进行的细胞定位实验证实了Enolase可以部分存在S.suis 2 05ZYH33细菌的表面.这提示了Enolase作为一种新发现的抗原对于引发猪链球菌相关疾病可能发挥着重要的作用.     

The Human Antimicrobial Peptide LL-37 Binds Directly to CsrS,a Sensor Histidine Kinase of Group A Streptococcus,to Activate Expression of Virulence Factors

Group A Streptococcus (GAS) responds to subinhibitory concentrations of LL-37 by up-regulation of virulence factors through the CsrRS (CovRS) two-component system. The signaling mechanism, however, is unclear. To determine whether LL-37 signaling reflects specific binding to CsrS or rather a nonspecific response to LL-37-mediated membrane damage, we tested LL-37 fragments for CsrRS signaling and for GAS antimicrobial activity. We identified a 10-residue fragment (RI-10) of LL-37 as the minimal peptide that retains the ability to signal increased expression of GAS virulence factors, yet it has no detectable antimicrobial activity against GAS. Substitution of individual key amino acids in RI-10 reduced or abrogated signaling. These data do not support the hypothesis that CsrS detects LL-37-induced damage to the bacterial cell membrane but rather suggest that LL-37 signaling is mediated by a direct interaction with CsrS. To test whether LL-37 binds to CsrS, we used the purified CsrS extracellular domain to pull down LL-37 in vitro, a result that provides further evidence that LL-37 binds to CsrS. The dissociation of CsrS-mediated signaling from membrane damage by LL-37 fragments together with in vitro evidence for a direct LL-37-CsrS binding interaction constitute compelling evidence that signal transduction by LL-37 through CsrS reflects a direct ligand/receptor interaction.     

Biochemical, Genetic, and Serological Characterization of Two Capsule Subtypes among Streptococcus pneumoniae Serotype 20 Strains: DISCOVERY OF A NEW PNEUMOCOCCAL SEROTYPE

The bacterial pathogen Streptococcus pneumoniae expresses one of over 90 structurally distinct polysaccharide (PS) capsule serotypes. Prior PS structural analyses of the vaccine-associated serotype 20 do not agree with reports describing the genes that mediate capsule synthesis. Furthermore, using immunized human sera-based assays, serological differences were recently noted among strains typed as serotype 20. We examined the capsule structures of two serologically dissimilar serotype 20 strains, 20α and 20β, by extensive biochemical analysis. 20α PS was composed of the previously described serotype 20 hexasaccharide repeat unit, whereas the 20β PS was composed of a novel heptasaccharide repeat unit containing an extra branching α-glucose residue. Genetic analysis of the subtypes revealed that 20α may have arisen from a 20β progenitor following loss of function mutation to the glycosyltransferase gene whaF. Conventional serotyping methods using rabbit polyclonal or mouse monoclonal antibodies were unable to distinguish the subtypes. However, genetic analysis of multiple "serotype 20" clinical isolates revealed that all strains contain the 20β genotype. We propose naming bacteria that express the previously described 20α capsule structure 20A and bacteria that express the novel 20β capsule structure 20B, a new pneumococcal serotype.     

Epidemiological characteristics and disease burden of infectious mononucleosis in hospitalized children in China: A nationwide retrospective study

Epstein-Barr virus (EBV) is very common, with the infection rate in adults over 90% worldwide. Infectious mononucleosis (IM) is caused by primary infection with EBV. Most IM patients are generally considered to have a favorable prognosis, but a few patients will also develop complications. Children with severe symptoms will require hospitalization. However, the disease burden of children hospitalized with IM in China has been rarely described. In this study, we included the Face sheets of discharge medical records from 27 member children's hospitals of Futang Research Center of Pediatric Development from Jan 1st, 2016 to Dec 31st, 2020, and medical information such as gender, age, region, time of admission, length of stay and expenditure were extracted. There were 24,120 IM cases, which accounted for 0.42% (24,120/5,693,262) of all hospitalized cases during this period. The ratio of male to female was 1.48:1. Hospitalization for IM in the 4–6 years age group was the highest among inpatients of all age groups. Case numbers increased year by year between 2016 and 2020, and the monthly hospitalization was generally high from Jul to Sep but reduced from Jan to Feb per year. Bronchitis/pneumonia and hepatic dysfunction were two common complications in hospitalized IM patients. The median length of stay was 8 days, and the median cost of hospitalization was 970.59 US dollars. This study will help understand the epidemiological characteristics and disease burden of hospitalized children with IM in China.     

Transactivation of the Receptor-tyrosine Kinase Ephrin Receptor A2 Is Required for the Low Molecular Weight Hyaluronan-mediated Angiogenesis That Is implicated in Tumor Progression

Angiogenesis or the formation of new blood vessels is important in the growth and metastatic potential of various cancers. Therefore, understanding the mechanism(s) by which angiogenesis occurs can have important therapeutic implications in numerous malignancies. We and others have demonstrated that low molecular weight hyaluronan (LMW-HA, ∼2500 Da) promotes endothelial cell (EC) barrier disruption and angiogenesis. However, the mechanism(s) by which this occurs is poorly defined. Our data indicate that treatment of human EC with LMW-HA induced CD44v10 association with the receptor-tyrosine kinase, EphA2, transactivation (tyrosine phosphorylation) of EphA2, and recruitment of the PDZ domain scaffolding protein, PATJ, to the cell periphery. Silencing (siRNA) CD44, EphA2, PATJ, or Dbs (RhoGEF) expression blocked LMW-HA-mediated angiogenesis (EC proliferation, migration, and tubule formation). In addition, silencing EphA2, PATJ, Src, or Dbs expression blocked LMW-HA-mediated RhoA activation. To translate our in vitro findings, we utilized a novel anginex/liposomal targeting of murine angiogenic endothelium with either CD44 or EphA2 siRNA and observed inhibition of LMW-HA-induced angiogenesis in implanted Matrigel plugs. Taken together, these results indicate LMW-HA-mediated transactivation of EphA2 is required for PATJ and Dbs membrane recruitment and subsequent RhoA activation required for angiogenesis. These results suggest that targeting downstream effectors of LMW-HA could be a useful therapeutic intervention for angiogenesis-associated diseases including tumor progression.     

Ultraviolet light and ozone stimulate accumulation of salicylic acid,pathogenesis-related proteins and virus resistance in tobacco

In tobacco (Nicotiana tabacum L. cv. Xanthinc), salicylic acid (SA) levels increase in leaves inoculated by necrotizing pathogens and in healthy leaves located above the inoculated site. Systemic SA increase may trigger disease resistance and synthesis of pathogenesis-related proteins (PR proteins). Here we report that ultraviolet (UV)-C light or ozone induced biochemical responses similar to those induced by necrotizing pathogens. Exposure of leaves to UV-C light or ozone resulted in a transient ninefold increase in SA compared to controls. In addition, in UV-light-irradiated plants, SA increased nearly fourfold to 0.77 g·g^–1 fresh weight in leaves that were shielded from UV light. Increased SA levels were accompanied by accumulation of an SA conjugate and by an increase in the activity of benzoic acid 2-hydroxylase which catalyzes SA biosynthesis. In irradiated and in unirradiated leaves of plants treated with UV light, as well as in plants fumigated with ozone, PR proteins 1a and 1b accumulated. This was paralleled by the appearance of induced resistance to a subsequent challenge with tobacco mosaic virus. The results suggest that UV light, ozone fumigation and tobacco mosaic virus can activate a common signal-transduction pathway that leads to SA and PR-protein accumulation and increased disease resistance.Abbreviations PR protein pathogenesis-related protein - SA salicylic acid - TMV tobacco mosaic virus - UV ultraviolet This work was financed by grants from the U.S. Department of Agriculture (Competitive Research Grants Office), Division of Energy Biosciences of U.S. Department of Energy, the Rockefeller Foundation, the New Jersey Commission for Science and Technology, and the New Jersey Agricultural Experiment Station.     

中国山姜属一新种和一新组合

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2017年湖北省襄阳市手足口病患者柯萨奇病毒A2型分离株的突变和重组分析

目的通过对2016-2017年襄阳市手足口病(hand, foot and mouth disease, HFMD)样品的分离与鉴定,了解主要病原之一的柯萨奇病毒A2型(Coxsackievirus, CV-A2)的分子生物学特征。方法收集2016年9月-2017年12月襄阳市HFMD患儿肛拭子样品,用人类横纹肌肉瘤细胞(RD细胞)培养,分离病毒。RT-PCR扩增CV-A2 VP1基因,Megalign软件分析VP1基因同源性,MEGA6软件构建系统发育树。比较3种疾病(急性迟缓性麻痹、手足口病和急性呼吸道感染)CV-A2分离株VP1氨基酸序列,分析可能的致病位点。扩增CV-A2代表株基因组全长序列,用SimPlot软件分析可能的重组事件。结果 2016-2017年CV-A2襄阳株之间VP1核苷酸及氨基酸同源性分别为96.4%~99.8%,98.0%~100.0%;襄阳株与CV-A2原型株(Fleetwood株)之间的核苷酸及氨基酸同源性分别为80.8%~81.9%,95.3%~95.9%。与襄阳株同源性最高的为2017年江西株(GenBank:MG926784),核苷酸及氨基酸同源性分别为96.7%~98.8%,98.0%~98.6%。襄阳市HFMD主要病原之一的CV-A2 VP1基因系统发育树显示,襄阳株与国内主要流行株同属于基因型D。3种疾病分离株的VP1氨基酸比对发现,急性迟缓性麻痹分离株和HFMD分离株在第21、60、82和215位存在差异;而HFMD分离株与呼吸道感染分离株之间只在167位存在差异,由谷氨酸变成天冬氨酸。CV-A2重组分析提示,襄阳株在P1区域与Fleetwood株同属一分支,在P2区域与CV-A5 Swartz株亲缘性最高,但在P3区域与CV-A16 G-10株有较高的相似度。结论虽然CV-A2襄阳株与其他流行株在VP1区序列上亲缘性较高,但其VP1氨基酸突变或与其他肠道病毒的型间重组可能导致其致病特性与流行病学特点发生变化。