The Burkholderia cenocepacia iron starvation σ factor, OrbS, possesses an on-board iron sensor.

Burkholderia cenocepacia is an opportunistic pathogen that causes severe infections of the cystic fibrosis (CF) lung. To acquire iron, B. cenocepacia secretes the Fe(III)-binding compound, ornibactin. Genes for synthesis and utilisation of ornibactin are served by the iron starvation (IS) extracytoplasmic function (ECF) σ factor, OrbS. Transcription of orbS is regulated in response to the prevailing iron concentration by the ferric uptake regulator (Fur), such that orbS expression is repressed under iron-sufficient conditions. Here we show that, in addition to Fur-mediated regulation of orbS, the OrbS protein itself responds to intracellular iron availability. Substitution of cysteine residues in the C-terminal region of OrbS diminished the ability to respond to Fe(II) in vivo. Accordingly, whilst Fe(II) impaired transcription from and recognition of OrbS-dependent promoters in vitro by inhibiting the binding of OrbS to core RNA polymerase (RNAP), the cysteine-substituted OrbS variant was less responsive to Fe(II). Thus, the cysteine residues within the C-terminal region of OrbS contribute to an iron-sensing motif that serves as an on-board 'anti-σ factor' in the presence of Fe(II). A model to account for the presence two regulators (Fur and OrbS) that respond to the same intracellular Fe(II) signal to control ornibactin synthesis and utilisation is discussed.

Targeting tumour-initiating cells with TRAIL based combination therapy ensures complete and lasting eradication of multiple myeloma tumours in vivo.

Multiple myeloma (MM) remains an incurable disease despite improvements to available treatments and efforts to identify new drug targets. Consequently new approaches are urgently required. We have investigated the potential of native tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), in combination with doxorubicin, to induce apoptotic cell death in phenotypically distinct populations of myeloma cells in vitro and in vivo. The cytotoxic potential of TRAIL alone, and in combination with DOX, was assessed in vitro in purified CD138(+) and CD138(-) cells from the MM cell lines and samples from patients with MM. Mouse xenografts obtained by implanting CD138(-) MM cells were used to assess the efficacy of TRAIL, alone and in combination with DOX, in vivo. CD138(-) cells were shown to be more resistant to the cytotoxic activity of TRAIL than CD138(+) cells and have reduced expression of TRAIL death receptors. This resistance results in preferential killing of CD 138(+) cells during exposure of MM culture to TRAIL. Furthermore, prolonged exposure results in the appearance of TRAIL-resistant CD138(-) cells. However, when TRAIL is combined with doxorubicin, this results in complete eradication of MM cells in vivo. Most importantly, this treatment successfully eliminates CD138(-) cells implicated in tumour initiation and growth maintenance. These findings may explain the failure of current therapies and offer a promising new approach in the quest to cure MM and disseminated cancers.

Investigating the interaction between osteoprotegerin and receptor activator of NF-kappaB or tumor necrosis factor-related apoptosis-inducing ligand: evidence for a pivotal role for osteoprotegerin in regulating two distinct pathways.

Osteoprotegerin (OPG) binds the ligand for receptor activator of nuclear factor kappaB (RANKL) to prevent association with its receptor RANK and inhibit osteoclast-mediated bone resorption. OPG has been reported, recently, to inhibit tumor necrosis factor-related apoptosis-induced ligand (TRAIL)-induced tumor cell apoptosis. This raises the possibility that OPG may play a unique role in regulating these two signaling pathways. However, there are little data on the interactions between OPG, RANKL, and TRAIL, and the relative affinity of OPG for these two ligands is unknown. In the present study we examined the ability of OPG to bind native human TRAIL and RANKL under physiological conditions. Native TRAIL was expressed in Escherichia coli, purified to homogeneity, and shown to induce human myeloma cell apoptosis. OPG inhibited native TRAIL from binding the TRAILR1 at 37 degrees C in vitro. Similarly, OPG prevented RANKL from binding to RANK. TRAIL also prevented OPG-mediated inhibition of RANKL from binding RANK. The affinity of OPG for native TRAIL and RANKL at 37 degrees C was determined by plasmon surface resonance analysis. OPG had a binding affinity for TRAIL of 45 nM, whereas the affinity of OPG for RANKL was 23 nM. These data suggest that OPG can bind both RANKL and TRAIL and that the affinity of OPG for these two ligands is of a similar order of magnitude. Furthermore, OPG prevented TRAIL-mediated reductions in cell viability, whereas TRAIL inhibited OPG-mediated inhibition of osteoclastogenesis in vitro. This highlights the pivotal role of OPG in regulating the biology of both RANKL and TRAIL.

Relaxed cleavage specificity of an immunoglobulin A1 protease from Neisseria meningitidis.

Respiratory pathogens, such as Neisseria meningitidis, secrete site-specific proteases able to cleave human immunoglobulin A1 (IgA1), the first line of defense at mucosal membranes. Bacterial isolates show wide variability in IgA1 protease activity, and those isolated from patients with clinical infection possess the highest levels of activity. A feature of this enzyme is the self-cleavage required for secretion of the mature extracellular form. Known cleavage targets contain a proline-rich consensus recognition sequence, Pro-Pro-Ser-Pro, residing in the variable linker region that connects the protease and translocator domains. Here, we report the sequence of the NMB IgA1 protease and the unexpected self-cleavage and subsequent extracellular release of mature IgA1 protease from mutants lacking the previously defined consensus cleavage site. We investigated the possible link between enzyme secretion and variability in the linker sequence segment using site-directed mutagenesis and linker domain swapping to construct mutated and chimeric forms of the IgA1 protease from N. meningitidis strain NMB. The observed change in secreted activity levels compared to the wild-type clone indicated that the precise amino acid sequence of the intervening region, between mature IgA1 protease and the beta-core translocator domain, influences the efficacy of autoproteolytic processing. The broader specificity uncovered for the NMB IgA1 protease suggests that it could cleave a far wider range of human proteins than previously appreciated.

Nontypeable Haemophilus influenzae in carriage and disease: a difference in IgA1 protease activity levels.

CONTEXT: Nontypeable Haemophilus influenzae strains form part of the normal flora of the human upper respiratory tract but are also implicated in a wide range of diseases. Infections caused by nontypeable H influenzae are major health and socioeconomic burdens. No single bacterial trait has been associated with disease as opposed to colonization. OBJECTIVES: To compare IgA1 protease activity in nontypeable H influenzae strains isolated from patients with symptomatic Haemophilus infection (sputum, cerebrospinal fluid, blood, or normally sterile tissue) vs strains from throat swabs of asymptomatic carriers and to compare iga gene carriage and variability in nontypeable H influenzae strains. DESIGN AND SETTING: Retrospective study of 63 strains (44 clinical and 19 carriage) collected between 1991 and 2000 and maintained at the Public Health Laboratory, Gwynedd General Hospital, Bangor, Wales. MAIN OUTCOME MEASURES: Levels of IgA1 protease activity produced by carriage strains and clinical isolates from symptomatic patients; the determination of the size and sequence of a variable region of the iga gene. RESULTS: Bacterial IgA1 protease activity was significantly higher (P<.001) in strains isolated from sputum, blood, cerebrospinal fluid, or normally sterile tissue of symptomatic individuals (median, 155 mU; interquartile range [IQR], 80-220 mU; mean, 169 mU; 95% confidence interval [CI], 126-211 mU) than in those isolated from throat swabs of asymptomatic carriers (median, 30 mU; IQR, 15-90 mU; mean, 56 mU; 95% CI, 26-86 mU; assayed on secretory IgA). The iga gene was detected in 97% of all strains examined. Variations in the sizes and sequences of part of the iga genes were also apparent. This variable region encodes a polypeptide linker connecting the protease domain to the beta-core autotranslocator, a porelike structure required for secretion of the protease. CONCLUSIONS: These findings reveal the importance of iga gene variability and expression levels in the establishment of disease phenotype. They identify nontypeable H influenzae IgA1 protease as a virulence factor and as a potential target for the development of new strategies to fight these important pathogens.