Coal seam gas industry methane emissions in the Surat Basin, Australia: comparing airborne measurements with inventories.

Coal seam gas (CSG) accounts for about one-quarter of natural gas production in Australia and rapidly increasing amounts globally. This is the first study worldwide using airborne measurement techniques to quantify methane (CH4) emissions from a producing CSG field: the Surat Basin, Queensland, Australia. Spatially resolved CH4 emissions were quantified from all major sources based on top-down (TD) and bottom-up (BU) approaches, the latter using Australia's UNFCCC reporting workflow. Based on our TD-validated BU inventory, CSG sources emit about 0.4% of the produced gas, comparable to onshore dry gas fields in the USA and The Netherlands, but substantially smaller than in other onshore regions, especially those where oil is co-produced (wet gas). The CSG CH4 emission per unit of gas production determined in this study is two to three times higher than existing inventories for the region. Our results indicate that the BU emission factors for feedlots and grazing cattle need review, possibly requiring an increase for Queensland's conditions. In some subregions, the BU estimate for gathering and boosting stations is potentially too high. The results from our iterative BU inventory process, which feeds into TD data, illustrate how global characterization of CH4 emissions could be improved by incorporating empirical TD verification surveys into national reporting. This article is part of a discussion meeting issue 'Rising methane: is warming feeding warming? (part 1)'.

Human mesenchymal stem cells: New sojourn of bacterial pathogens.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (M. tuberculosis), is the leading infectious disease which claims one human life every 15-20s globally. The persistence of this deadly disease in human population can be attributed to the ability of the bacterium to stay in latent form. M. tuberculosis possesses a plethora of mechanisms not only to survive latently under harsh conditions inside the host but also modulate the host immune cells in its favour. Various M. tuberculosis gene families have also been described to play a role in this process. Recently, human bone marrow derived mesenchymal stem cells (MSCs) have been reported as a niche for dormant M. tuberculosis. MSCs possess abilities to alter the host immune response. The bacterium finds this self-renewal and immune privileged nature of MSCs very favourable not only to modulate the host immune system, with some help from its own genes, but also to avoid the external drug pressure. We suggest that the MSCs not only provide a resting place for M. tuberculosis but could also, by virtue of their intrinsic ability to disseminate in the body, explain the genesis of extra-pulmonary TB. A similar exploitation of stem cells by other bacterial pathogens is a distinct possibility. It may be likely that other intracellular bacterial pathogens adopt this strategy to 'piggy-back' on to ovarian stem cells to ensure vertical transmission and successful propagation to the next generation.

Collagen IV-derived peptide binds hydrophobic cavity of Legionella pneumophila Mip and interferes with bacterial epithelial transmigration.

The Legionella virulence factor Mip (macrophage infectivity potentiator) contributes to bacterial dissemination within infected lung tissue. The Mip protein, which belongs to the enzyme family of FK506-binding proteins (FKBP), binds specifically to collagen IV. We identified a surface-exposed Mip-binding sequence in the NC1 domain of human collagen IV α1. The corresponding collagen IV-derived peptide (P290) co-precipitated with Mip and competitively inhibited the Mip-collagen IV binding. Transmigration of Legionella pneumophila across a barrier of NCI-H292 lung epithelial cells and extracellular matrix was efficiently inhibited by P290. This significantly reduced transmigration was comparable to the inefficient transmigration of PPIase-negative Mip mutant or rapamycin-treated L. pneumophila. Based on NMR data and docking studies a model for the mode of interaction of P290 and Mip was developed. The amino acids of the hydrophobic cavity of Mip, D142 and to a lesser extent Y185 were identified to be part of the interaction surface. In the complex structure of Mip(77-213) and P290, both amino acid residues form hydrogen bonds to P290. Utilizing modelling, molecular dynamics (MD) simulations and structural data of human PPIase FKBP12, the most related human orthologue of Mip, we were able to propose optimized P290 variants with increased binding specificity and selectivity for the putative bacterial drug target Mip.

Regulatory Interactions among adhesin gene systems of uropathogenic Escherichia coli.

Uropathogenic Escherichia coli strain J96 carries multiple determinants for fimbrial adhesins. The regulatory protein PapB of P fimbriae has previously been implicated in potential coregulatory events. The focB gene of the F1C fimbria determinant is highly homologous to papB; the translated sequences share 81% identity. In this study we investigated the role of PapB and FocB in regulation of the F1C fimbriae. By using gel mobility shift assays, we showed that FocB binds to sequences in both the pap and foc operons in a somewhat different manner than PapB. The results of both in vitro cross-linking and in vivo oligomerization tests indicated that FocB could function in an oligomeric fashion. Furthermore, our results suggest that PapB and FocB can form heterodimers and that these complexes can repress expression of the foc operon. The effect of FocB on expression of type 1 fimbriae was also tested. Taken together, the results that we present expand our knowledge about a regulatory network for different adhesin gene systems in uropathogenic E. coli and suggest a hierarchy for expression of the fimbrial adhesins.

Collagen binding protein Mip enables Legionella pneumophila to transmigrate through a barrier of NCI-H292 lung epithelial cells and extracellular matrix.

Guinea pigs are highly susceptible to Legionella pneumophila infection and therefore have been the preferred animal model for studies of legionellosis. In this study guinea pig infections revealed that the Legionella virulence factor Mip (macrophage infectivity potentiator) contributes to the bacterial dissemination within the lung tissue and the spread of Legionella to the spleen. Histopathology of infected animals, binding assays with components of the extracellular matrix (ECM), bacterial transmigration experiments across an artificial lung epithelium barrier, inhibitor studies and ECM degradation assays were used to elucidate the underlying mechanism of the in vivo observation. The Mip protein, which belongs to the enzyme family of FK506-binding proteins (FKBP), was shown to bind to the ECM protein collagen (type I, II, III, IV, V, VI). Transwell assays with L. pneumophila and recombinant Escherichia coli HB101 strains revealed that Mip enables these bacteria to transmigrate across a barrier of NCI-H292 lung epithelial cells and ECM (NCI-H292/ECM barrier). Mip-specific monoclonal antibodies and the immunosuppressants rapamycin and FK506, which inhibit the peptidyl prolyl cis/trans isomerase (PPIase) activity of Mip, were able to inhibit this transmigration. By using protease inhibitors we found that the penetration of the NCI-H292/ECM barrier additionally requires a serine protease activity. Degradation assays with (35)S-labelled ECM proteins supported the finding of a concerted action of Mip and a serine protease. The described synergism between the activity of the collagen binding Mip protein and the serine protease activity represents an entirely new mechanism for bacterial penetration of the lung epithelial barrier and has implications for other prokaryotic and eukaryotic pathogens.

Down-regulation of key virulence factors makes the Salmonella enterica serovar Typhimurium rfaH mutant a promising live-attenuated vaccine candidate.

Mutants of Salmonella enterica serovar Typhimurium that lack the transcriptional regulator RfaH are efficient as live oral vaccines against salmonellosis in mice. We show that the attenuation of the vaccine candidate strain is associated with reduced net growth in epithelial and macrophage cells. In order to identify the relevant RfaH-dependent genes, the RfaH regulon was determined with S. enterica serovars Enteritidis and Typhimurium using whole-genome Salmonella microarrays. As well as impacting the expression of genes involved in lipopolysaccharide (LPS) core and O-antigen synthesis, the loss of RfaH results in a marked down-regulation of SPI-4 genes, the flagellum/chemotaxis system, and type III secretion system 1. However, a proportion of these effects could have been the indirect consequence of the altered expression of genes required for LPS biosynthesis. Direct and indirect effects of the rfaH mutation were dissociated by genome-wide transcriptional profiling of a structural deep-rough LPS mutant (waaG). We show that truncation of LPS itself is responsible for the decreased intracellular yield observed for DeltarfaH strains. LPS mutants do not differ in replication ability; rather, they show increased susceptibility to antimicrobial peptides in the intracellular milieu. On the other hand, evidence that deletion of rfaH, as well as some other genes involved in LPS biosynthesis, results in enhanced invasion of various mammalian cells is shown. Exposure of common minor antigens in the absence of serovar-specific antigens might be responsible for the observed cross-reactive nature of the elicited immune response upon vaccination. Increased invasiveness of the Salmonella rfaH mutant into antigen-presenting cells, combined with increased intracellular killing and the potential for raising a cross-protective immune response, renders the rfaH mutant an ideal vaccine candidate.

Escherichia coli induces DNA double-strand breaks in eukaryotic cells.

Transient infection of eukaryotic cells with commensal and extraintestinal pathogenic Escherichia coli of phylogenetic group B2 blocks mitosis and induces megalocytosis. This trait is linked to a widely spread genomic island that encodes giant modular nonribosomal peptide and polyketide synthases. Contact with E. coli expressing this gene cluster causes DNA double-strand breaks and activation of the DNA damage checkpoint pathway, leading to cell cycle arrest and eventually to cell death. Discovery of hybrid peptide-polyketide genotoxins in E. coli will change our view on pathogenesis and commensalism and open new biotechnological applications.

A Salmonella fim homologue in Citrobacter freundii mediates invasion in vitro and crossing of the blood-brain barrier in the rat pup model.

From the invasive Citrobacter freundii strain 3009, an invasion determinant was cloned, sequenced, and expressed. Sequence analysis of the determinant showed high homology with the fim determinant from Salmonella enterica serovar Typhimurium. The genes of the invasion determinant directed invasion of recombinant Escherichia coli K-12 strains into human epithelial cell lines of the bladder and gut as well as mannose-sensitive yeast agglutination and were termed fim(Cf) genes. Expression of the Fim(Cf) proteins was shown by (35)S labeling and/or Western blotting. In the infant rat model of experimental hematogenous meningitis, C. freundii strain 3009 and the in vitro invasive recombinant E. coli K-12 strain harboring the fim(Cf) determinant reached the cerebrospinal fluid, in contrast to the case for the control strain. The fim determinant was also necessary for efficient in vitro invasion by C. freundii, because a deletion mutant was strongly reduced in its invasion efficiency. The mutation could be complemented in trans by the corresponding genes. Invasion by C. freundii could be blocked only by d-mannose, GlcNAc, and chitin hydrolysate and not by other carbohydrates tested. In contrast, yeast agglutination was not affected by GlcNAc or chitin hydrolysate. This finding indicated mannose residues to be essential for both yeast agglutination and invasion, whereas GlcNAc (oligomer) residues of host cells are involved exclusively in invasion. These results showed the fim determinant of C. freundii to be responsible for d-mannose- and GlcNAc-dependent in vitro invasion without being assembled into pili and for crossing of the blood-brain barrier in the infant rat model.

The PPIase active site of Legionella pneumophila Mip protein is involved in the infection of eukaryotic host cells.

We analysed eight monoclonal antibodies (mAbs) directed against the Mip (macrophage infectivity potentiator) protein, a virulence factor of the intracellular pathogen Legionella pneumophila. Mip belongs to the FK506-binding proteins (FKBPs) and exhibits peptidyl prolyl cis/trans isomerase (PPIase) activity. Five of the mAbs recognised epitopes in the C-terminal, FKBP-homologous domain of Mip, which is highly conserved among all Legionella species. Upon immunological binding to Mip, all but one of these mAbs caused inhibition of the PPIase activity in vitro. mAb binding to the N-terminal domain of Mip did not influence its enzymatic activity. All but one of the PPIase inhibiting mAbs were able to significantly inhibit the early establishment and initiation of an intracellular infection of the bacteria in Acanthamoeba castellanii, the natural host, and in the human phagocytic cell line U937. These data demonstrate for the first time that for the virulence-enhancing property of the L. pneumophila Mip protein, an intact active site of the enzyme is an essential requirement.

Functional analysis of a vacuolar ABC transporter in wild-type Candida albicans reveals its involvement in virulence.

ATP-driven transport proteins belonging to the ATP-binding cassette (ABC) superfamily perform important functions in cell metabolism and detoxification. Compounds can be actively transported across membranes, including the plasma membrane or organellar membranes. The vacuole is an important organelle in fungal cells required for compartmentalization of metabolites as well as toxic substances. Sequestration into the vacuole is often energy-dependent. We present the first isolation and molecular analysis of a vacuolar ABC transporter gene in the opportunistic fungal pathogen Candida albicans. The protein encoded by the MLT1 gene is highly similar to Multiple Drug Resistance-associated Protein (MRP)-like transporters of yeast and higher organisms that form the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)/MRP subfamily of ABC transporters, a class of proteins so far not characterized in C. albicans. MLT1 expression is extensively growth phase-regulated, and gene transcripts are inducible by metabolic poisons. Gene replacement mutants generated in wild-type C. albicans with the dominant selection marker MPAR showed a profound reduction in virulence in a mouse peritonitis model that was reversed by complementation with an intact MLT1 gene. Hence, this report provides primary evidence for the involvement of vacuolar ABC transporters in fungal virulence.