Characterization of novel beta-galactosidase activity that contributes to glycoprotein degradation and virulence in Streptococcus pneumoniae.

The pneumococcus obtains its energy from the metabolism of host glycosides. Therefore, efficient degradation of host glycoproteins is integral to pneumococcal virulence. In search of novel pneumococcal glycosidases, we characterized the Streptococcus pneumoniae strain D39 protein encoded by SPD_0065 and found that this gene encodes a beta-galactosidase. The SPD_0065 recombinant protein released galactose from desialylated fetuin, which was used here as a model of glycoproteins found in vivo. A pneumococcal mutant with a mutation in SPD_0065 showed diminished beta-galactosidase activity, exhibited an extended lag period in mucin-containing defined medium, and cleaved significantly less galactose than the parental strain during growth on mucin. As pneumococcal beta-galactosidase activity had been previously attributed solely to SPD_0562 (bgaA), we evaluated the contribution of SPD_0065 and SPD_0562 to total beta-galactosidase activity. Mutation of either gene significantly reduced enzymatic activity, but beta-galactosidase activity in the double mutant, although significantly less than that in either of the single mutants, was not completely abolished. The expression of SPD_0065 in S. pneumoniae grown in mucin-containing medium or tissues harvested from infected animals was significantly upregulated compared to that in pneumococci from glucose-containing medium. The SPD_0065 mutant strain was found to be attenuated in virulence in a manner specific to the host tissue

Duodenal perforation in a patient with heroin use

A number of vascular pathologies are attributed to the opiate exposure such as myocardial infarction, ischaemic stroke, and hypoperfusion of the gastrointestinal tract. Here we presented a 30-year-old male patient with no history of any ischemic or gastrointestinal disease known to have 10-year opioid use and had suffered a duodenal perforation. Prolonged exposure of opioids may lead to hypersensitivity reactions, ischemia and hypoperfusion and combination of these entities with opioid-related gastrointestinal motility deficits could contribute to the epithelial damage and perforation as a consequence. The presence of ischemic events should be kept in mind in the presence of gastrointestinal symptoms in people using intensive heroin

RitR is an archetype for a novel family of redox sensors in the streptococci that has evolved from two-component response regulators and is required for pneumococcal colonization

To survive diverse host environments, the human pathogen Streptococcus pneumoniae must prevent its self-produced, extremely high levels of peroxide from reacting with intracellular iron. However, the regulatory mechanism(s) by which the pneumococcus accomplishes this balance remains largely enigmatic, as this pathogen and other related streptococci lack all known redox-sensing transcription factors. Here we describe a two-component-derived response regulator, RitR, as the archetype for a novel family of redox sensors in a subset of streptococcal species. We show that RitR works to both repress iron transport and enable nasopharyngeal colonization through a mechanism that exploits a single cysteine (Cys128) redox switch located within its linker domain. Biochemical experiments and phylogenetics reveal that RitR has diverged from the canonical two-component virulence regulator CovR to instead dimerize and bind DNA only upon Cys128 oxidation in air-rich environments. Atomic structures show that Cys128 oxidation initiates a "helical unravelling" of the RitR linker region, suggesting a mechanism by which the DNA-binding domain is then released to interact with its cognate regulatory DNA. Expanded computational studies indicate this mechanism could be shared by many microbial species outside the streptococcus genus

Structure-function analysis for the development of peptide inhibitors for a Gram-positive quorum sensing system

open access articleThe Streptococcus pneumoniae Rgg144/SHP144 regulator-peptide quorum sensing (QS) system is critical for nutrient utilization, oxidative stress response, and virulence. Here, we characterized this system by assessing the importance of each residue within the active short hydrophobic peptide (SHP) by alanine-scanning mutagenesis and testing the resulting peptides for receptor binding and activation of the receptor. Interestingly, several of the mutations had little effect on binding to Rgg144 but reduced transcriptional activation appreciably. In particular, a proline substitution (P21A) reduced transcriptional activation by 29-fold but bound with a 3-fold higher affinity than the wild-type SHP. Consistent with the function of Rgg144, the mutant peptide led to decreased utilization of mannose and increased susceptibility to superoxide generator paraquat. Pangenome comparison showed full conservation of P21 across SHP144 allelic variants. Crystallization of Rgg144 in the absence of peptide revealed a comparable structure to the DNA bound and free forms of its homologs suggesting similar mechanisms of activation. Together, these analyses identify key interactions in a critical pneumococcal QS system. Further manipulation of the SHP has the potential to facilitate the development of inhibitors that are functional across strains. The approach described here is likely to be effective across QS systems in multiple species

Inactivation of the Complement Lectin Pathway by Candida tropicalis Secreted Aspartyl Protease-1

open access articleCandida tropicalis is an opportunistic fungal pathogen and is one of the most frequently isolated non-albicans species. It can cause localised as well as invasive systemic infections particularly in immunocompromised patients. Increased resistance to common anti-fungal drugs is an emerging problem. In order to establish disseminated infections, Candida has evolved several strategies to escape the host immune system. A detailed understanding of how C. tropicalis escapes the host immune attack is needed as it can help develop novel anti-fungal therapies. Secreted aspartyl proteinases (Saps) of C. albicans have been shown to be determinants of virulence and immune evasion. However, the immune evasion properties of C. tropicalis Saps have been poorly characterised. This study investigated the immune evasion properties of C. tropicalis secreted aspartic protease 1 (Sapt1). Sapt1 was recombinantly produced using a Kluyveromyces lactis yeast expression system. A range of complement proteins and immunogloublins were screened to test if Sapt1 had any proteolytic activity. Sapt1 efficiently cleaved human mannose-binding lectin (MBL) and collectin-11, which are the initiating molecules of the lectin pathway of the complement system, but not l-ficolin. In addition, Sapt1 cleaved DC-SIGN, the receptor on antigen presenting dendritic cells. Proteolysis was prominent in acidic condition (pH 5.2), a characteristic of aspartyl protease. No proteolytic activity was detected against complement proteins C1q, C3, C3b, IgG and IgA. In view of the ability of Sapt1 to cleave MBL and collectin-11, we found that Sapt1 could prevent activation of the complement lectin pathway. RT-qPCR analysis using three different C. tropicalis clinical isolates (oral, blood and peritoneal dialysis fluid) revealed relatively higher levels of mRNA expression of Sapt1 gene when compared to a reference strain; Sapt1 protein was found to be secreted by all the tested strains. Lectin pathway and its initiating components are crucial to provide front line defence against Candida infections. For the first time, we have shown that a Candida protease can proteolytically degrade the key initiating components of lectin pathway and inhibit complement activation. Findings from this study highlight the importance of exploring Sapt1 as a potential therapeutic target. We conclude that C. tropicalis secretes Sapt1 to target the complement lectin pathway, a key pattern recognition and clearance mechanism, for its survival and pathogenesis

Activation of invariant natural killer T cells stimulated with microbial α-mannosyl glycolipids

Some synthetic and bacterial glycolipids presented by CD1d specifically activate invariant NKT (iNKT) cells bearing an invariant Vα14-Jα18 (mouse) or Vα24-Jα18 (human) TCR. The antigenic glycolipids identified to date consist of two hydrophobic chains and an α-glycoside in which the 2′-OH group is in the cis orientation toward the anomeric group, namely, either an α-galactoside or an α-glucoside. Several microbial α-mannosyl glycolipids, in which the 2′-OH group is in the trans orientation, were herein examined to establish whether they have potential to activate iNKT cells. We found that α-mannnosyl1-3 (6′-O-acyl α-mannosyl)-1-1 monoacylglycerol and cholesteryl 6′-O-acyl α-mannoside, found in Saccharopolyspora and Candida albicans, respectively, induced the activation of iNKT cells, dependent on CD1d. In contrast, α-mannosyldiacylglycerol found in Streptococcus suis or α-mannosylceramide demonstrated markedly less antigenicity for iNKT cells. The potentially antigenic α-mannosyl glycolipids contributed to the protection of mice against infection with S. pneumoniae in which iNKT cells have previously been found to participate. Furthermore, these glycolipids induced the production of proinflammatory cytokines by macrophages, thereby suggesting their recognition by specific pattern recognition receptors (PRRs). Collectively, these results suggest that these microbial α-mannosyl glycolipids are capable of being recognized by both the invariant TCR and PRRs and inducing immune responses

Host Glycan Sugar-Specific Pathways in Streptococcus pneumonia:Galactose as a Key Sugar in Colonisation and Infection

The human pathogen Streptococcus pneumoniae is a strictly fermentative organism that relies on glycolytic metabolism to obtain energy. In the human nasopharynx S. pneumoniae encounters glycoconjugates composed of a variety of monosaccharides, which can potentially be used as nutrients once depolymerized by glycosidases. Therefore, it is reasonable to hypothesise that the pneumococcus would rely on these glycan-derived sugars to grow. Here, we identified the sugar-specific catabolic pathways used by S. pneumoniae during growth on mucin. Transcriptome analysis of cells grown on mucin showed specific upregulation of genes likely to be involved in deglycosylation, transport and catabolism of galactose, mannose and N acetylglucosamine. In contrast to growth on mannose and N-acetylglucosamine, S. pneumoniae grown on galactose re-route their metabolic pathway from homolactic fermentation to a truly mixed acid fermentation regime. By measuring intracellular metabolites, enzymatic activities and mutant analysis, we provide an accurate map of the biochemical pathways for galactose, mannose and N-acetylglucosamine catabolism in S. pneumoniae. Intranasal mouse infection models of pneumococcal colonisation and disease showed that only mutants in galactose catabolic genes were attenuated. Our data pinpoint galactose as a key nutrient for growth in the respiratory tract and highlights the importance of central carbon metabolism for pneumococcal pathogenesis

Volume CXIV, Number 4, November 7, 1996

Objective: Turner syndrome (TS) is a chromosomal disorder caused by complete or partial X chromosome monosomy that manifests various clinical features depending on the karyotype and on the genetic background of affected girls. This study aimed to systematically investigate the key clinical features of TS in relationship to karyotype in a large pediatric Turkish patient population.Methods: Our retrospective study included 842 karyotype-proven TS patients aged 0-18 years who were evaluated in 35 different centers in Turkey in the years 2013-2014.Results: The most common karyotype was 45,X (50.7%), followed by 45,X/46,XX (10.8%), 46,X,i(Xq) (10.1%) and 45,X/46,X,i(Xq) (9.5%). Mean age at diagnosis was 10.2±4.4 years. The most common presenting complaints were short stature and delayed puberty. Among patients diagnosed before age one year, the ratio of karyotype 45,X was significantly higher than that of other karyotype groups. Cardiac defects (bicuspid aortic valve, coarctation of the aorta and aortic stenosis) were the most common congenital anomalies, occurring in 25% of the TS cases. This was followed by urinary system anomalies (horseshoe kidney, double collector duct system and renal rotation) detected in 16.3%. Hashimoto's thyroiditis was found in 11.1% of patients, gastrointestinal abnormalities in 8.9%, ear nose and throat problems in 22.6%, dermatologic problems in 21.8% and osteoporosis in 15.3%. Learning difficulties and/or psychosocial problems were encountered in 39.1%. Insulin resistance and impaired fasting glucose were detected in 3.4% and 2.2%, respectively. Dyslipidemia prevalence was 11.4%.Conclusion: This comprehensive study systematically evaluated the largest group of karyotype-proven TS girls to date. The karyotype distribution, congenital anomaly and comorbidity profile closely parallel that from other countries and support the need for close medical surveillance of these complex patients throughout their lifespa

Studies on the role of superoxide dismutase (SOD) in the virulence of Streptococcus pneumoniae and the effects of interferon gamma on sensitivity of phagocytes to the toxin pneumolyin

The major point of the study was into how the penumococcus copes with reactive derivatives of oxygen. Superoxide dismutase is one of the enzymes that provides protection against deleterious effects of superoxide radical by conversion of superoxide to hydrogen peroxide and molecular oxygen. In this study, it was shown that S. pneumoniae contains two types of superoxide dismutase, MnSOD and FeSOD. While the level of MnSOD increased during growth in an aerobic environment, the amount of FeSOD remained unchanged. An isogenic sodA insertion-mutant strain of S. pneumoniae was constructed to study the role for the enzyme in vitro and in vivo. Aerobically the mutant strain of S. pneumoniae, designated as D39HY1, had a lower growth rate than the wild type and exhibited susceptibility to the redox active compound, paraquat. Anaerobic growth of D39HY1 was identical to the wild-type. Virulence studies showed that the median survival time of mice infected intranasally with D39HY1 was significantly longer than mice infected with the wild type pneumococcus. In contrast to the wild-type, D39HY1 did not multiply in lungs during the first 24h but thereafter grew at the same rate as the wild-type. Appearance in the bloodstream also was delayed but growth in the blood was unimpaired by the sodA mutation.;The aim of the second project was to assess whether IFN- may enhance resistance of macrophages to the deleterious effects of bacterial products. To assess this possibility, the effects of interferon gamma on sensitivity of phagocytes to the toxin pneumolysin, an important virulence determinant of S. pneumoniae, was tested by employing respiratory burst as an assay of sublytic effects. It was observed that IFN- can alter the susceptibility of cells to the lytic effect of pneumolysin. Also the respiratory burst of IFN- treated cells was insensitive to sublytic concentrations of pneumolysin

The ability to utilize mucin affects the regulation of virulence gene expression in Streptococcus pneumoniae.

Streptococcus pneumoniae colonizes the mucin-rich environment of the nasopharynx. As colonization may be the first stage of infection, investigation was carried out as to whether the pneumococcus could utilize mucin as a source of nutrient and whether its virulence gene expression is influenced by this glycoprotein. It was found that when grown in Sicard's defined medium supplemented with 1% w/v mucin, the organism grew at a rate similar to that in rich medium. The presence of sialate O-acetylesterase activity, an enzyme implicated in glycoprotein degradation, in pneumococcal cell extracts was also reported. The authors hypothesized that neuraminidase A, which is linked to pneumococcal virulence, plays an important role in mucin utilization. Growth in mucin resulted in an increase in nanA transcription and a DeltananA-deficient strain of pneumococcus could not grow when mucin was used as the sole carbon source