Structural analysis of Salmonella enterica effector protein SopD

Salmonella outer protein D (SopD) is a type III secreted virulence effector protein from Salmonella enterica. Full-length SopD and SopD lacking 16 amino acids at the N-terminus (SopDDeltaN) have been expressed as fusions with GST in Escherichia coli, purified with a typical yield of 20-30 mg per litre of cell culture and crystallized. Biophysical characterization has been carried out mainly on SopDDeltaN. Analytical size exclusion chromatography shows that SopDDeltaN is monomeric and probably globular in aqueous solution. The secondary structure composition, calculated from the CD spectrum, is mixed (38% alpha-helix and 26% beta-strand). Sequence analysis indicates that SopD contains a coiled coil motif, as found in numerous other type III secretion system-associated proteins. This suggests that SopD has the potential for one or more heterotypic protein-protein interactions. Limited trypsin digestion of SopDDeltaN, monitored by both one-dimensional proton NMR spectroscopy and SDS-PAGE, shows that the protein has a large, protease-resistant core domain of 286 amino acid residues. This single-domain architecture suggests that SopD lacks a cognate chaperone. In crystallization trials, SopDDeltaN produced better crystals than either full-length SopD or trypsin-digested SopDDeltaN. Diffraction to 3.0 Angstrom resolution has so far been obtained from crystals of SopDDeltaN

Autotransporters and Their Role in the Virulence of Burkholderia pseudomallei and Burkholderia mallei

Burkholderia pseudomallei and Burkholderia mallei are closely related Gram-negative bacteria responsible for the infectious diseases melioidosis and glanders, respectively. Autotransporters (ATs) comprise a large and diverse family of secreted and outer membrane proteins that includes virulence-associated invasins, adhesins, proteases, and actin-nucleating factors. The B. pseudomallei K96243 genome contains 11 predicted ATs, eight of which share homologs in the B. mallei ATCC 23344 genome. This review distils key findings from in silico, in vitro, and in vivo studies on the ATs of B. pseudomallei and B. mallei. To date, the best characterized of the predicted ATs of B. pseudomallei and B. mallei is BimA, a predicted trimeric AT mediating actin-based motility which varies in sequence and mode of action between Burkholderia species. Of the remaining eight predicted B. pseudomallei trimeric autotransporters, five of which are also present in B. mallei, two (BoaA and BoaB), have been implicated in bacterial adhesion to epithelial cells. Several predicted Burkholderia ATs are recognized by human humoral and cell-mediated immunity, indicating that they are expressed during infection and may be useful for diagnosis and vaccine-mediated protection. Further studies on the mode of secretion and functions of Burkholderia ATs will facilitate the rational design of control strategies

Expression, purification, crystallization and preliminary crystallographic analysis of BipD, a component of the Burkholderia pseudomallei type III secretion system

A construct consisting of residues 10–310 of mature BipD, a component of the B. pseudomallei type III secretion system, has been crystallized. Native BipD crystals and SeMet and K2PtCl4 derivative crystals have undergone preliminary crystallographic analysis

Isolation and characterization of a novel podovirus which infects burkholderia pseudomallei

Burkholderia pseudomallei is a saprophytic soil bacterium and the etiological agent that causes melioidosis. It is naturally resistant to many antibiotics and therefore is difficult to treat. Bacteriophages may provide an alternative source of treatment. We have isolated and characterised the bacteriophage ΦBp-AMP1. The phage is a member of the Podoviridae family and has a genome size of ~ 45 Kb. Molecular data based on the gene which encodes for the phage tail tubular protein suggests that the phage is distinct from known phages but related to phages which infect B. thailandensis and Ralstonia spp. The phage ΦBp-AMP1 is the first B. pseudomallei podovirus to be isolated from the environment rather than being induced from a bacterial culture. It has a broad host range within B. pseudomallei and can infect all 11 strains that we tested it on but not related Burkholderia species. It is heat stable for 8 h at 50°C but not stable at 60°C. It may potentially be a useful tool to treat or diagnose B. pseudomallei infections as it can lyse several strains of clinical relevance

Therapeutic effects of oral administration of lytic Salmonella phages in a mouse model of non-typhoidal salmonellosis

Acute non-typhoidal salmonellosis (NTS) caused by a Gram-negative bacterium Salmonella enterica serovar Typhimurium (S. Tm) is one of the most common bacterial foodborne diseases worldwide. Bacteriophages (phages) can specifically target and lyse their host bacteria, including the multidrug-resistant strains, without collateral damage to other bacteria in the community. However, the therapeutic use of Salmonella phages in vivo is still poorly investigated. Salmonella phages ST-W77 and SE-W109 have previously been shown by our group to be useful for biocontrol properties. Here, we tested whether phages ST-W77 and SE-W109 can reduce Salmonella invasion into cultured human cells and confer a therapeutic benefit for acute NTS in a mammalian host. Human colonocytes, T84 cells, were treated with phages ST-W77, SE-W109, and its combination for 5 min before S. Tm infection. Gentamicin protection assays demonstrated that ST-W77 and SE-W109 significantly reduced S. Tm invasion and inflammatory response in human colonocytes. Next, streptomycin-pretreated mice were orally infected with S. Tm (10(8) CFU/mouse) and treated with a single or a combination of ST-W77 and SE-W109 (10(10) PFU/mouse for 4 days) by oral feeding. Our data showed that phage-treated mice had lower S. Tm numbers and tissue inflammation compared to the untreated mice. Our study also revealed that ST-W77 and SE-W109 persist in the mouse gut lumen, but not in systemic sites. Together, these data suggested that Salmonella phages ST-W77 and SE-W109 could be further developed as an alternative approach for treating an acute NTS in mammalian hosts

Induced Burkholderia prophages detected from the hemoculture: a biomarker for Burkholderia pseudomallei infection.

Bacteriophages (phages), viruses that infect bacteria, are found in abundance not only in the environment but also in the human body. The use of phages for the diagnosis of melioidosis, a tropical infectious disease caused by Burkholderia pseudomallei, is emerging as a promising novel approach, but our understanding of conditions under which Burkholderia prophages can be induced remains limited. Here, we first demonstrated the isolation of Burkholderia phages from the hemocultures of melioidosis patients. The B. pseudomallei-positive hemoculture bottles were filtered to remove bacteria, and then phages were isolated and purified by spot and double agar overlay plaque assays. Forty blood samples (hemoculture-confirmed melioidosis) were tested, and phages were found in 30% of the samples. Transmission electron microscopy and genome analysis of the isolated phages, vB_HM387 and vB_HM795, showed that both phages are Myoviruses. These two phages were stable at a pH of 5-7 and temperatures of 25-37°C, suggesting their ability to survive in human blood. The genome sizes of vB_HM387 and vB_HM795 are 36.3 and 44.0 kb, respectively. A phylogenetic analysis indicated that vB_HM387 has homologs, but vB_HM795 is a novel Myovirus, suggesting the heterogeneity of Burkholderia phages in melioidosis patients. The key finding that Burkholderia phages could be isolated from the blood of melioidosis patients highlights the potential application of phage-based assays by detecting phages in blood as a pathogen-derived biomarker of infection

Expression of the envelope antigen F1 of Yersinia pestis is mediated by the product of caf1M gene having homology with the chaperone protein PapD of Escherichia coli.

The effective synthesis of the envelope antigen F1 of Y. pestis in E. coli HB101 is mediated by the expression of the cuf1M gene. This gene was sequenced, and the protein encoded was found to have a significant homology with the chaperone protein PapD of uropathogenic E. coli. The data presented allow one to suppose Caf1M and PapD proteins perform similar functions in the biogenesis of the Y. pestis capsule and E. coli P-pili, respectively. When you are citing the document, use the following link http://essuir.sumdu.edu.ua/handle/123456789/905

The Tripartite Type III Secreton of Shigella flexneri Inserts Ipab and Ipac into Host Membranes

Bacterial type III secretion systems serve to translocate proteins into eukaryotic cells, requiring a secreton and a translocator for proteins to pass the bacterial and host membranes. We used the contact hemolytic activity of Shigella flexneri to investigate its putative translocator. Hemolysis was caused by formation of a 25-Å pore within the red blood cell (RBC) membrane. Of the five proteins secreted by Shigella upon activation of its type III secretion system, only the hydrophobic IpaB and IpaC were tightly associated with RBC membranes isolated after hemolysis. Ipa protein secretion and hemolysis were kinetically coupled processes. However, Ipa protein secretion in the immediate vicinity of RBCs was not sufficient to cause hemolysis in the absence of centrifugation. Centrifugation reduced the distance between bacterial and RBC membranes beyond a critical threshold. Electron microscopy analysis indicated that secretons were constitutively assembled at 37°C before any host contact. They were composed of three parts: (a) an external needle, (b) a neck domain, and (c) a large proximal bulb. Secreton morphology did not change upon activation of secretion. In mutants of some genes encoding the secretion machinery the organelle was absent, whereas ipaB and ipaC mutants displayed normal secretons

The Role of the County Professional Council in Advanced Training and Professional Development of Class Teachers

Stručno usvršavanje je obvezan dio učiteljskog posla koji se provodi na četiri osnovne razine: individualnoj, školskoj, županijskoj i državnoj. Brojne su prednosti i nedostaci svakog oblika usavršavanja, a svi su oni na putu profesionalnog razvoja učitelja jednako važni i korisni. U radu je prikazan model stručnog usvršavanja učitelja na županijskoj razini (Županijsko stručno vijeće učitelja razredne nastave – Grad Sisak) pri čemu veliku ulogu imaju upravo županijski voditelji koji su poveznica između školske i državne razine stručnog usavršavanja učitelja.Advanced training is a mandatory part of teacher’s job and it is being carried out at four basic levels: individual, school, county and state level. There are numerous advantages and disadvantages of any form of training, and they are all equally important and useful in the course of professional development of teachers. This study presents the model of advanced training of class teachers at county level (County professional council of class teachers – the Town of Sisak), where the very county leaders, who are the link between the school and state level of advanced training of teachers, have a great role