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

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

Identification of a predicted trimeric autotransporter adhesin required for biofilm formation of Burkholderia pseudomallei.

The autotransporters are a large and diverse family of bacterial secreted and outer membrane proteins, which are present in many Gram-negative bacterial pathogens and play a role in numerous environmental and virulence-associated interactions. As part of a larger systematic study on the autotransporters of Burkholderia pseudomallei, the causative agent of the severe tropical disease melioidosis, we have constructed an insertion mutant in the bpss1439 gene encoding an unstudied predicted trimeric autotransporter adhesin. The bpss1439 mutant demonstrated a significant reduction in biofilm formation at 48 hours in comparison to its parent 10276 wild-type strain. This phenotype was complemented to wild-type levels by the introduction of a full-length copy of the bpss1439 gene in trans. Examination of the wild-type and bpss1439 mutant strains under biofilm-inducing conditions by microscopy after 48 hours confirmed that the bpss1439 mutant produced less biofilm compared to wild-type. Additionally, it was observed that this phenotype was due to low levels of bacterial adhesion to the abiotic surface as well as reduced microcolony formation. In a murine melioidosis model, the bpss1439 mutant strain demonstrated a moderate attenuation for virulence compared to the wild-type strain. This attenuation was abrogated by in trans complementation, suggesting that bpss1439 plays a subtle role in the pathogenesis of B. pseudomallei. Taken together, these studies indicate that BPSS1439 is a novel predicted autotransporter involved in biofilm formation of B. pseudomallei; hence, this factor was named BbfA, Burkholderia biofilm factor A

The Salmonella Translocated Effector SopA Is Targeted to the Mitochondria of Infected Cells

This study investigates the Salmonella effector protein SopA. We show that in Salmonella enterica serovar Dublin-infected cells, SopA(1-347) fused to two carboxy-terminal hemagglutinin tags partially colocalized with mitochondria. Transfection of eukaryotic cells with a panel of constructs encoding truncated versions of SopA identified that amino acids 100 to 347 were sufficient to target SopA to the mitochondria