Porphyromonas gingivalis is a member of the human oral microbiome abundant in dysbiosis and implicated in the pathogenesis of periodontal (gum) disease. It employs a newly described type-IX secretion system (T9SS) for secretion of virulence factors. Cargo proteins destined for secretion through T9SS carry a recognition signal in the conserved C-terminal domain (CTD), which is removed by sortase PorU during translocation. Here, we identified a novel component of T9SS, PorZ, which is essential for surface exposure of PorU and posttranslational modification of T9SS cargo proteins. These include maturation of enzyme precursors, CTD removal and attachment of anionic lipopolysaccharide for anchorage in the outer membrane. The crystal structure of PorZ revealed two β-propeller domains and a C-terminal β-sandwich domain, which conforms to the canonical CTD architecture. We further documented that PorZ is itself transported to the cell surface via T9SS as a full-length protein with its CTD intact, independently of the presence or activity of PorU. Taken together, our results shed light on the architecture and possible function of a novel component of the T9SS. Knowledge of how T9SS operates will contribute to our understanding of protein secretion as part of host-microbiome interactions by dysbiotic members of the human oral cavity.

In the recently characterized Type IX Secretion System (T9SS), the conserved C-terminal domain (CTD) in secreted proteins functions as an outer membrane translocation signal for export of virulence factors to the cell surface in the Gram-negative Bacteroidetes phylum. In the periodontal pathogen Porphyromonas gingivalis, the CTD is cleaved off by PorU sortase in a sequence-independent manner, and anionic lipopolysaccharide (A-LPS) is attached to many translocated proteins, thus anchoring them to the bacterial surface. Here, we solved the atomic structure of the CTD of gingipain B (RgpB) from P. gingivalis, alone and together with a preceding immunoglobulin-superfamily domain (IgSF). The CTD was found to possess a typical Ig-like fold encompassing seven antiparallel β-strands organized in two β-sheets, packed into a β-sandwich structure that can spontaneously dimerise through C-terminal strand swapping. Small angle X-ray scattering (SAXS) revealed no fixed orientation of the CTD with respect to the IgSF. By introducing insertion or substitution of residues within the inter-domain linker in the native protein, we were able to show that despite the region being unstructured, it nevertheless is resistant to general proteolysis. These data suggest structural motifs located in the two adjacent Ig-like domains dictate the processing of CTDs by the T9SS secretion pathway.

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Staphylococcus aureus is the most frequently isolated pathogen in Gram-positive sepsis often complicated by a blood clotting disorder, and is the leading cause of infective endocarditis induced by bacterial destruction of endocardial tissues. The bacterium secretes cysteine proteases referred to as staphopain A (ScpA) and staphopain B (SspB). To investigate virulence activities of staphopains pertinent to clotting disorders and tissue destruction, we examined their effects on collagen, one of the major tissue components, and on plasma clotting. Both staphopains prolonged the partial thromboplastin time of plasma in a dose-and activitydependent manner, with SspB being threefold more potent than ScpA. Staphopains also prolonged the thrombin time of both plasma and fibrinogen, indicating that these enzymes can cause impaired plasma clotting through fibrinogen degradation. Whereas SspB cleaved the fibrinogen Aa-chain at the C-terminal region very efficiently, ScpA degraded it rather slowly. This explains the superior ability of the former enzyme to impair fibrinogen clottability. Enzymically active staphopains, at concentrations as low as 10 nM, degraded collagen with comparable efficiency. These results show novel virulence activities of staphopains in degrading fibrinogen and collagen, and suggest an involvement of staphopains in the clotting impairment and tissue destruction caused by staphylococcal infection. INTRODUCTIONSepsis is a serious medical condition, in which living bacteria are present in the bloodstream. Shock and disseminated intravascular coagulation (DIC) are common and potentially fatal consequences of sepsis. DIC occurs in as many as 40 % of sepsis patients, often leading to multiple organ failure (Levi & ten Cate, 1999), and is directly linked to a high mortality rate. Clinical studies have shown that Gram-positive micro-organisms are as common as Gram-negative bacteria in causing sepsis (Ahmed et al., 1991;Kieft et al., 1993).Staphylococcus aureus is the most frequently isolated pathogen in Gram-positive sepsis (Ahmed et al., 1991;Bone, 1993). In addition, this bacterium is the leading cause of infective endocarditis in many regions of the world (Fowler et al., 2005). The development of endocarditis increases the mortality rate of patients with bacteraemia (Chang et al., 2003) et al., 2005). These findings suggest that staphopains contribute to S. aureus septic shock, one of the major fatal complications of sepsis.Clotting induction and a subsequent tendency to bleeding are prominent clinical features of DIC, another lethal outcome of sepsis. Staphopains may participate in the onset of the clotting disorder through activation or inactivation of clotting factors in plasma by proteolytic cleavage. However, the ability of staphopains to affect plasma clotting has not been studied. Invasion of S. aureus into the endocardium to cause infective endocarditis requires the degradation of connective tissue including extracellular matrix proteins, and is facilitated by proteases. In this context, staphy...

Abstract. In this paper we study the Hutchinson-Barnsley theory of fractals in the setting of multimetric spaces (which are sets endowed with point separating families of pseudometrics) and in the setting of topological spaces. We find natural connections between these two approaches.

Objectives: Maternal pre-pregnancy body mass index (BMI) and gestational weight gain (GWG) have a meaningful impact on pregnancy and perinatal outcomes. The first aim of the study was to analyze the association between pre-pregnancy BMI and the prevalence of small for gestational age (SGA) and large for gestational age (LGA) outcomes. The second aim was to assess the relationship between pre-pregnancy BMI combined with gestational weight gain (GWG) and the prevalence of SGA and LGA measurements. Material and methods: The retrospective cohort study was conducted at Jagiellonian University Hospital in Cracow, Poland from 2016 to 2017. During this time there were 2,123 deliveries. Patients with chronic diseases, multiple pregnancies, fetal defects and incomplete data were excluded. Finally, 474 cases were enrolled. Patients were divided into BMI groups (underweight, normal, overweight and obese) and into GWG groups (inadequate, adequate, excessive). Relationships between maternal BMI, GWG and newborn weight were examined. Results: There was no statistically significant association between maternal pre-pregnancy BMI and prevalence of SGA measurements. However, underweight women with inadequate GWG showed a higher risk to bear SGA babies (OR 5.2, 95%

BackgroundWe have previously reported the use of PCR Melting Profile (PCR MP) technique based on using low denaturation temperatures during ligation mediated PCR (LM PCR) for bacterial strain differentiation. The aim of the current study was to evaluate this method for intra-species differentiation of Candida albicans strains.MethodsIn total 123 Candida albicans strains (including 7 reference, 11 clinical unrelated, and 105 isolates from patients of two hospitals in Poland) were examined using three genotyping methods: PCR MP, macrorestriction analysis of the chromosomal DNA by pulsed-field gel electrophoresis (REA-PFGE) and RAPD techniques.ResultsThe genotyping results of the PCR MP were compared with results from REA-PFGE and RAPD techniques giving 27, 26 and 25 unique types, respectively. The results showed that the PCR MP technique has at least the same discriminatory power as REA-PFGE and RAPD.ConclusionData presented here show for the first time the evaluation of PCR MP technique for candidial strains differentiation and we propose that this can be used as a relatively simple and cheap technique for epidemiological studies in short period of time in hospital.

Answering an old question of M.Hata, we construct an example of a 1-dimensional Peano continuum which is not homeomorphic to an attractor of IFS.Comment: 4 pages, 2 figure