Bactericidal activity of Lys49 and Asp49 myotoxic phospholipases A2 from Bothrops asper snake venom--synthetic Lys49 myotoxin II-(115-129)-peptide identifies its bactericidal region

Mammalian group-II phospholipases A2 (PLA2) of inflammatory fluids display bactericidal properties, which are dependent on their enzymatic activity. This study shows that myotoxins II (Lys49) and III (Asp49), two group-II PLA2 isoforms from the venom of Bothrops asper, are lethal to a broad spectrum of bacteria. Since the catalytically inactive Lys49 myotoxin II isoform has similar bactericidal effects to its catalytically active Asp49 counterpart, a bactericidal mechanism that is independent of an intrinsic PLA2 activity is demonstrated. Moreover, a synthetic 13-residue peptide of myotoxin II, comprising residues 115-129 (common numbering system) near the C-terminal loop, reproduced the bactericidal effect of the intact protein. Following exposure to the peptide or the protein, accelerated uptake of the hydrophobic probe N-phenyl-N-naphthylamine was observed in susceptible but not in resistant bacteria, indicating that the lethal effect was initiated on the bacterial membrane. The outer membrane, isolated lipopolysaccharide (LPS), and lipid A of susceptible bacteria showed higher binding to the myotoxin II-(115-129)-peptide than the corresponding moieties of resistant strains. Bacterial LPS chimeras indicated that LPS is a relevant target for myotoxin II-(115-129)-peptide. When heterologous LPS of the resistant strain was present in the context of susceptible bacteria, the chimera became resistant, and vice versa. Myotoxin II represents a group-II PLA2 with a direct bactericidal effect that is independent of an intrinsic enzymatic activity, but adscribed to the presence of a short cluster of basic/hydrophobic amino acids near its C-terminal loop.

Combined genomic and phenotype screening reveals secretory factor SPINK1 as an invasion and survival factor associated with patient prognosis in breast cancer

Secretory factors that drive cancer progression are attractive immunotherapeutic targets. We used a whole-genome data-mining approach on multiple cohorts of breast tumours annotated for clinical outcomes to discover such factors. We identified Serine protease inhibitor Kazal-type 1 (SPINK1) to be associated with poor survival in estrogen receptor-positive (ER+) cases. Immunohistochemistry showed that SPINK1 was absent in normal breast, present in early and advanced tumours, and its expression correlated with poor survival in ER+ tumours. In ER- cases, the prognostic effect did not reach statistical significance. Forced expression and/or exposure to recombinant SPINK1 induced invasiveness without affecting cell proliferation. However, down-regulation of SPINK1 resulted in cell death. Further, SPINK1 overexpressing cells were resistant to drug-induced apoptosis due to reduced caspase-3 levels and high expression of Bcl2 and phospho-Bcl2 proteins. Intriguingly, these anti-apoptotic effects of SPINK1 were abrogated by mutations of its protease inhibition domain. Thus, SPINK1 affects multiple aggressive properties in breast cancer: survival, invasiveness and chemoresistance. Because SPINK1 effects are abrogated by neutralizing antibodies, we suggest that SPINK1 is a viable potential therapeutic target in breast cancer.

The P/2010 A2 Asteroid Collision Confirmed by Rosetta/OSIRIS Observation

The discovery of P/2010 A2 by the LINEAR survey in January 2010 revealed an object displaying a large trail of material similar in shape to a cometary tail although no central condensation or coma could be detected. The appearance of this object in an asteroidal orbit in the inner main belt attracted attention as a potential new member of the Main Belt Comets class (MBCs) but the discovery of a nucleus, with an estimated diameter of 120 m, around 1500 km away from the trail implied that the extended object we were seeing could be the debris trail from a recent collision rather than the tail of a comet. Due to the low inclination of its orbit, it is difficult to conclude about the nature of P/2010 A2 from Earth-based data only, as different scenarios lead to the same appearance in the orbital configuration at the times of observations. We present here another set of images, acquired from the unique viewing geometry provided by ESA's Rosetta spacecraft en route to comet 67P/Churyumov-Gerasimenko. Albeit faint (22 magnitude), the object could be observed by the high-resolution camera OSIRIS. We used a Finson-Probstein model to simulate the shape of the trail, and estimate the time of emission and β parameter (ratio between solar radiation pressure and gravity) for the dust grains. Simulations were compared to the OSIRIS images and ground based observations acquired at NTT and Palomar telescopes. Thanks to the different phase angle provided by Rosetta, we could reduce the number of solutions to a unique model, leading to the conclusive demonstration that the trail is due to a single event rather than a period of cometary activity.

Comparative analysis of Klebsiella pneumoniae genomes identifies a phospholipase D family protein as a novel virulence factor

BACKGROUND: Klebsiella pneumoniae strains are pathogenic to animals and humans, in which they are both a frequent cause of nosocomial infections and a re-emerging cause of severe community-acquired infections. K. pneumoniae isolates of the capsular serotype K2 are among the most virulent. In order to identify novel putative virulence factors that may account for the severity of K2 infections, the genome sequence of the K2 reference strain Kp52.145 was determined and compared to two K1 and K2 strains of low virulence and to the reference strains MGH 78578 and NTUH-K2044.

RESULTS: In addition to diverse functions related to host colonization and virulence encoded in genomic regions common to the four strains, four genomic islands specific for Kp52.145 were identified. These regions encoded genes for the synthesis of colibactin toxin, a putative cytotoxin outer membrane protein, secretion systems, nucleases and eukaryotic-like proteins. In addition, an insertion within a type VI secretion system locus included sel1 domain containing proteins and a phospholipase D family protein (PLD1). The pld1 mutant was avirulent in a pneumonia model in mouse. The pld1 mRNA was expressed in vivo and the pld1 gene was associated with K. pneumoniae isolates from severe infections. Analysis of lipid composition of a defective E. coli strain complemented with pld1 suggests an involvement of PLD1 in cardiolipin metabolism.

CONCLUSIONS: Determination of the complete genome of the K2 reference strain identified several genomic islands comprising putative elements of pathogenicity. The role of PLD1 in pathogenesis was demonstrated for the first time and suggests that lipid metabolism is a novel virulence mechanism of K. pneumoniae.

The Ability of Secretory Leukocyte Protease Inhibitor to Inhibit Apoptosis in Monocytes Is Independent of Its Antiprotease Activity

Secretory Leukocyte Protease Inhibitor (SLPI) is a serine protease inhibitor produced by epithelial and myeloid cells with anti-inflammatory properties. Research has shown that SLPI exerts its anti-inflammatory activity by directly binding to NF-κB DNA binding sites and, in so doing, prevents binding and subsequent transcription of proinflammatory gene expression. In the current study, we demonstrate that SLPI can inhibit TNF-α-induced apoptosis in U937 cells and peripheral blood monocytes. Specifically, SLPI inhibits TNF-α-induced caspase-3 activation and DNA degradation associated with apoptosis. We go on to show that this ability of SLPI to inhibit apoptosis is not dependent on its antiprotease activity as antiprotease deficient variants of SLPI can also inhibit TNF-α-induced apoptosis. This reduction in monocyte apoptosis may preserve monocyte function during inflammation resolution and promote infection clearance at mucosal sites.

A Secretory Leukocyte Protease Inhibitor Variant with Improved Activity against Lung Infection

Secretory leukocyte protease inhibitor (SLPI) is an important respiratory tract host defense protein, which is proteolytically inactivated by excessive neutrophil elastase (NE) during chronic Pseudomonas infection in the cystic fibrosis (CF) lung. We generated two putative NE-resistant variants of SLPI by site-directed mutagenesis, SLPI-A16G and SLPI-S15G-A16G, with a view to improving SLPI’s proteolytic stability. Both variants showed enhanced resistance to degradation in the presence of excess NE as well as CF patient sputum compared with SLPI-wild type (SLPI-WT). The ability of both variants to bind bacterial lipopolysaccharides and interact with nuclear factor-κB DNA binding sites was also preserved. Finally, we demonstrate increased anti-inflammatory activity of the SLPI-A16G protein compared with SLPI-WT in a murine model of pulmonary Pseudomonas infection. This study demonstrates the increased stability of these SLPI variants compared with SLPI-WT and their therapeutic potential as a putative anti-inflammatory treatment for CF lung disease.

Ultrastructure of head organs (anterior adhesive apparatus) and posterior secretory systems of Caballeria liewi Lim, 1995 (Monogenea, Ancyrocephalidae)

Caballeria liewi Lim, 1995, uses adhesive secretions from the head organs and posterior secretory systems to assist in locomotion and attachment. Ultrastructural investigations show that the head organs of C. liewi consist of three pairs of antero-lateral pit-like openings bearing microvilli and ducts leading from two types of uninucleated gland cells (located lateral to the pharynx), one type producing rod-like (S1) bodies with an electron-dense matrix containing less electron-dense vesicles and the second type producing oval (S2) bodies with a homogeneous electron-dense matrix. Interlinking band-like structures are observed between S1 bodies and between S2 bodies. S1 body is synthesised in the granular endoplasmic reticulum, transported to a Golgi complex to be packaged into vesicles and routed into ducts for exudation. The synthesis of the S2 body is unresolved. Haptoral secretions manifested externally as net-like structures are derived from dual electron-dense (DED) secretory body produced in the peduncular gland cells. The DED body consists of a less electron-dense oval core in a homogeneous electron-dense matrix. On exocytosis into the pyriform haptoral reservoir, DED bodies are transformed into a secretion with two types of inclusions (less electron-dense oval and electron-dense spherical inclusions) in an electron-dense matrix. The secretions are further transformed (as small, oval, electron-dense bodies) when transported to the superficial anchor grooves, and on exudation into the gill tissues, the secretions become an electron-dense matrix. Secretory bodies associated with uniciliated structures, anchor sleeves and marginal hooks are also observed.