UpaG, a new member of the Trimeric Autotransporter family of Adhesins in Uropathogenic Escherichia coli

The ability of Escherichia coli to colonize both intestinal and extraintestinal sites is driven by the presence of specific virulence factors, among which are the autotransporter (AT) proteins. Members of the trimeric AT adhesin family are important virulence factors for several gram-negative pathogens and mediate adherence to eukaryotic cells and extracellular matrix (ECM) proteins. In this study, we characterized a new trimeric AT adhesin (UpaG) from uropathogenic E. coli (UPEC). Molecular analysis of UpaG revealed that it is translocated to the cell surface and adopts a multimeric conformation. We demonstrated that UpaG is able to promote cell aggregation and biofilm formation on abiotic surfaces in CFT073 and various UPEC strains. In addition, UpaG expression resulted in the adhesion of CFT073 to human bladder epithelial cells, with specific affinity to fibronectin and laminin. Prevalence analysis revealed that upaG is strongly associated with E. coli strains from the B2 and D phylogenetic groups, while deletion of upaG had no significant effect on the ability of CFT073 to colonize the mouse urinary tract. Thus, UpaG is a novel trimeric AT adhesin from E. coli that mediates aggregation, biofilm formation, and adhesion to various ECM proteins.

Uropathogenic Escherichia coli mediated urinary tract infection

Urinary tract infection (UTI) is among the most common infectious diseases of humans and is the most common nosocomial infection in the developed world. They cause significant morbidity and mortality, with approximately 150 million cases globally per year. It is estimated that 40-50% of women and 5% of men will develop a UTI in their lifetime, and UTI accounts for more than 1 million hospitalizations and $1.6 billion in medical expenses each year in the USA. Uropathogenic E. coli (UPEC) is the primary cause of UTI. This review presents an overview of the primary virulence factors of UPEC, the major host responses to infection of the urinary tract, the emergence of specific multidrug resistant clones of UPEC, antibiotic treatment options for UPEC-mediated UTI and the current state of vaccine strategies as well as other novel anti-adhesive and prophylactic approaches to prevent UTI. New and emerging themes in UPEC research are also discussed in the context of future outlooks.

Relevance of the deletion polymorphisms of the glutathione S-transferases GSTT1 and GSTM1 in pharmacology and toxicology

Although cytosolic glutathione S-transferase (GST) enzymes occupy a key position in biological detoxification processes, two of the most relevant human isoenzymes, GSTT1-1 and GSTM1-1, are genetically deleted (non-functional alleles GSTT1*0 and GSTM1*0) in a high percentage of the human population, with major ethnic differences. The structures of the GSTT and GSTM gene areas explain the underlying genetic processes. GSTT1-1 is highly conserved during evolution and plays a major role in phase-II biotransformation of a number of drugs and industrial chemicals, e.g. cytostatic drugs, hydrocarbons and halogenated hydrocarbons. GSTM1-1 is particularly relevant in the deactivation of carcinogenic intermediates of polycyclic aromatic hydrocarbons. Several lines of evidence suggest that hGSTT1-1 and/or hGSTM1-1 play a role in the deactivation of reactive oxygen species that are likely to be involved in cellular processes of inflammation, ageing and degenerative diseases. There is cumulating evidence that combinations of the GSTM1*0 state with other genetic traits affecting the metabolism of carcinogens (CYP1A1, GSTP1) may predispose the aero-digestive tract and lung, especially in smokers, to a higher risk of cancer. The GSTM1*0 status appears also associated with a modest increase in the risk of bladder cancer, consistent with a GSTM1 interaction with carcinogenic tobacco smoke constituents. Both human GST deletions, although largely counterbalanced by overlapping substrate affinities within the GST superfamily, have consequences when the organism comes into contact with distinct man-made chemicals. This appears relevant in industrial toxicology and in drug metabolism.

Markers of genetic susceptibility in human environmental hygiene and toxicology : the role of selected CYP, NAT and GST genes

Inherited genetic traits co-determine the susceptibility of an individual to a toxic chemical. Special emphasis has been put on individual responses to environmental and industrial carcinogens, but other chronic diseases are of increasing interest. Polymorphisms of relevant xenobiotic metabolising enzymes may be used as toxicological susceptibility markers. A growing number of genes encoding enzymes involved in biotransformation of toxicants and in cellular defence against toxicant-induced damage to the cells has been identified and cloned, leading to increased knowledge of allelic variants of genes and genetic defects that may result in a differential susceptibility toward environmental toxicants. "Low penetrating" polymorphisms in metabolism genes tend to be much more common in the population than allelic variants of "high penetrating" cancer genes, and are therefore of considerable importance from a public health point of view. Positive associations between cancer and CYP1A1 alleles, in particular the *2C I462V allele, were found for tissues following the aerodigestive tract. Again, in most cases, the effect of the variant CYP1A1 allele becomes apparent or clearer in connection with the GSTM1 null allele. The CYP1B1 codon 432 polymorphism (CYP1B1*3) has been identified as a susceptibility factor in smoking-related head-and-neck squameous cell cancer. The impact of this polymorphic variant of CYP1B1 on cancer risk was also reflected by an association with the frequency of somatic mutations of the p53 gene. Combined genotype analysis of CYP1B1 and the glutathione transferases GSTM1 or GSTT1 has also pointed to interactive effects. Of particular interest for the industrial and environmental field is the isozyme CYP2E1. Several genotypes of this isozyme have been characterised which seem to be associated with different levels of expression of enzyme activity. The acetylator status for NAT2 can be determined by genotyping or by phenotyping. In the pathogenesis of human bladder cancer due to occupational exposure to "classical" aromatic amines (benzidine, 4-aminodiphenyl, 1-naphthylamine) acetylation by NAT2 is regarded as a detoxication step. Interestingly, the underlying European findings of a higher susceptibility of slow acetylators towards aromatic amines are in contrast to findings in Chinese workers occupationally exposed to aromatic amines which points to different mechanisms of susceptibility between European and Chinese populations. Regarding human bladder cancer, the hypothesis has been put forward that genetic polymorphism of GSTM1 might be linked with the occurrence of this tumour type. This supports the hypothesis that exposure to PAH might causally be involved in urothelial cancers. The human polymorphic GST catalysing conjugation of halomethanes, dihalomethanes, ethylene oxide and a number of other industrial compounds could be characterised as a class theta enzyme (GSTT1) by means of molecular biology. "Conjugator" and "non-conjugator" phenotypes are coincident with the presence and absence of the GSTT1 gene. There are wide variations in the frequencies of GSTT1 deletion (GSTT1 *0/0) among different ethnicities. Human phenotyping is facilitated by the GST activity towards methyl bromide or ethylene oxide in erythrocytes which is representative of the metabolic GSTT1 competence of the entire organism. Inter-individual variations in xenobiotic metabolism capacities may be due to polymorphisms of the genes coding for the enzymes themselves or of the genes coding for the receptors or transcription factors which regulate the expression of the enzymes. Also, polymorphisms in several regions of genes may cause altered ligand affinity, transactivation activity or expression levels of the receptor subsequently influencing the expression of the downstream target genes. Studies of individual susceptibility to toxicants and gene-environment interaction are now emerging as an important component of molecular epidemiology.

Genetic susceptibility to environmental toxicants : the interface between human and experimental studies in the development of new toxicological concepts

The growing knowledge of the genetic polymorphisms of enzymes metabolising xenobiotics in humans and their connections with individual susceptibility towards toxicants has created new and important interfaces between human epidemiology and experimental toxicology. The results of molecular epidemiological studies may provide new hypotheses and concepts, which call for experimental verification, and experimental concepts may obtain further proof by molecular epidemiological studies. If applied diligently, these possibilities may be combined to lead to new strategies of human-oriented toxicological research. This overview will present some outstanding examples for such strategies taken from the practically very important field of occupational toxicology. The main focus is placed on the effects of enzyme polymorphisms of the xenobiotic metabolism in association with the induction of bladder cancer and renal cell cancer after exposure to occupational chemicals. Also, smoking and induction of head and neck squamous cell cancer are considered.

Whole-genome multiparametric screening to identify modulators of epithelial-to-mesenchymal transition

Metastasis accounts for the poor prognosis of the majority of solid tumors. The phenotypic transition of nonmotile epithelial tumor cells to migratory and invasive “mesenchymal” cells (epithelial-to-mesenchymal transition [EMT]) enables the transit of cancer cells from the primary tumor to distant sites. There is no single marker of EMT; rather, multiple measures are required to define cell state. Thus, the multiparametric capability of high-content screening is ideally suited for the comprehensive analysis of EMT regulators. The aim of this study was to generate a platform to systematically identify functional modulators of tumor cell plasticity using the bladder cancer cell line TSU-Pr1-B1 as a model system. A platform enabling the quantification of key EMT characteristics, cell morphology and mesenchymal intermediate filament vimentin, was developed using the fluorescent whole-cell-tracking reagent CMFDA and a fluorescent promoter reporter construct, respectively. The functional effect of genome-wide modulation of protein-coding genes and miRNAs coupled with those of a collection of small-molecule kinase inhibitors on EMT was assessed using the Target Activation Bioapplication integrated in the Cellomics ArrayScan platform. Data from each of the three screens were integrated to identify a cohort of targets that were subsequently examined in a validation assay using siRNA duplexes. Identification of established regulators of EMT supports the utility of this screening approach and indicated capacity to identify novel regulators of this plasticity program. Pathway analysis coupled with interrogation of cancer-related expression profile databases and other EMT-related screens provided key evidence to prioritize further experimental investigation into the molecular regulators of EMT in cancer cells.

Ecionines A and B, two new cytotoxic pyridoacridine alkaloids from the Australian marine sponge, Ecionemia geodides

Chemical investigations of the Australian marine sponge Ecionemia geodides resulted in the isolation of two new pyridoacridine alkaloids, ecionines A (1) and B (2), along with the previously isolated marine natural products, biemnadin (3) and meridine (4). Compounds 1 and 2 both contain an imine moiety, which is rare for the pyridoacridine structure class. The chemical structures of 1 and 2 were determined by extensive 1D and 2D NMR and MS data analyses. All compounds were tested against a panel of human bladder cancer cell lines, the increasingly metastatic TSU-Pr1 series (TSU-Pr1, TSU-Pr1-B1 and TSU-Pr1- B2) and the superficial bladder cancer cell line 5637. Ecionine A (1) displayed cytotoxicity against all cell lines, with IC50 values ranging from 3 to 7 mM. This is the first report of chemistry from the sponge genus Ecionemia.

The Cressbrook Creek alluvial aquifer system, Southeast Queensland : hydrochemistry and isotopes to determine hydrological processes and response to floods

This study developed an understanding of hydrological processes within the Cressbrook Creek catchment of the upper Brisbane River, in particular for the alluvial aquifers. Those aquifers within the lower catchment are used for intensive irrigation, and have been impacted by long-term drought followed by flooding. The study utilised water chemistry, isotopic characters and hydraulic measurements to determine factors such as recharge, links between creeks and groundwater, and variations in water quality. The catchment-wide study will enable improved management of the local water resources.

The co-transcriptome of uropathogenic Escherichia coli-infected mouse macrophages reveals new insights into host-pathogen interactions

Urinary tract infections (UTI) are among the most common infections in humans. Uropathogenic Escherichia coli (UPEC) can invade and replicate within bladder epithelial cells, and some UPEC strains can also survive within macrophages. To understand the UPEC transcriptional program associated with intramacrophage survival, we performed host–pathogen co-transcriptome analyses using RNA sequencing. Mouse bone marrow-derived macrophages (BMMs) were challenged over a 24 h time course with two UPEC reference strains that possess contrasting intramacrophage phenotypes: UTI89, which survives in BMMs, and 83972, which is killed by BMMs. Neither of these strains caused significant BMM cell death at the low multiplicity of infection that was used in this study. We developed an effective computational framework that simultaneously separated, annotated, and quantified the mammalian and bacterial transcriptomes. BMMs responded to the two UPEC strains with a broadly similar gene expression program. In contrast, the transcriptional responses of the UPEC strains diverged markedly from each other. We identified UTI89 genes upregulated at 24 h post-infection, and hypothesized that some may contribute to intramacrophage survival. Indeed, we showed that deletion of one such gene (pspA) significantly reduced UTI89 survival within BMMs. Our study provides a technological framework for simultaneously capturing global changes at the transcriptional level in co-cultures, and has generated new insights into the mechanisms that UPEC use to persist within the intramacrophage environment.

Molecular analysis of asymptomatic bacteriuria escherichia coli strain VR50 reveals adaptation to the urinary tract by gene acquisition

Urinary tract infections (UTIs) are among the most common infectious diseases of humans, with Escherichia coli responsible for >80% of all cases. One extreme of UTI is asymptomatic bacteriuria (ABU), which occurs as an asymptomatic carrier state that resembles commensalism. To understand the evolution and molecular mechanisms that underpin ABU, the genome of the ABU E. coli strain VR50 was sequenced. Analysis of the complete genome indicated that it most resembles E. coli K-12, with the addition of a 94-kb genomic island (GI-VR50-pheV), eight prophages, and multiple plasmids. GI-VR50-pheV has a mosaic structure and contains genes encoding a number of UTI-associated virulence factors, namely, Afa (afimbrial adhesin), two autotransporter proteins (Ag43 and Sat), and aerobactin. We demonstrated that the presence of this island in VR50 confers its ability to colonize the murine bladder, as a VR50 mutant with GI-VR50-pheV deleted was attenuated in a mouse model of UTI in vivo. We established that Afa is the island-encoded factor responsible for this phenotype using two independent deletion (Afa operon and AfaE adhesin) mutants. E. coli VR50afa and VR50afaE displayed significantly decreased ability to adhere to human bladder epithelial cells. In the mouse model of UTI, VR50afa and VR50afaE displayed reduced bladder colonization compared to wild-type VR50, similar to the colonization level of the GI-VR50-pheV mutant. Our study suggests that E. coli VR50 is a commensal-like strain that has acquired fitness factors that facilitate colonization of the human bladder.

Are we getting accurate measurements of Ksat for sodic clay soils?

In this paper we discuss the use of a series of column experiments to improve understanding of the effect irrigation water chemistry (saline solutions) has on measurements of saturated hydraulic conductivity (Ksat) of a sodic clay soil. We highlight in particular the use of extended leaching periods to determine whether the duration of leaching affects the results. In the experiments, mixed cation solutions of two different salinity levels, 50 meq/L and 100 meq/L, were applied under constant head to columns of a repacked sodic clay soil using three replicates for each treatment. The maximum Ksat measured during leaching with the 100 meq/L solution was approximately double the maximum Ksat measured during leaching with the 50 meq/L solution. Measured flow rates were found to increase rapidly after flow commenced then decrease gradually until flow rates became stable. The final, stable flow rate was roughly 80% less than the maximum flow rate measured. Reasons for these changes in saturated hydraulic conductivity are discussed. The key finding from these experiments is that long term leaching, involving significantly more pore volumes than is commonly reported in the literature, is required to obtain a ‘stable’ Ksat. We recommend that further studies be carried out to (1) determine whether similar behaviour in Ksat occurs in a wide range of sodic clay soils and (2) to help build a better understanding of the causes and implications of the observed behaviour in Ksat.

Applying HYDRUS to flow in a sodic clay soil with solution composition–dependent hydraulic conductivity

Management of sodic soils under irrigation often requires application of chemical ameliorants to improve permeability combined with leaching of excess salts. Modeling irrigation, soil treatments, and leaching in these sodic soils requires a model that can adequately represent the physical and chemical changes in the soil associated with the amelioration process. While there are a number of models that simulate reactive solute transport, UNSATCHEM and HYDRUS-1D are currently the only models that also include an ability to simulate the impacts of soil chemistry on hydraulic conductivity. Previous researchers have successfully applied these models to simulate amelioration experiments on a sodic loam soil. To further gauge their applicability, we extended the previous work by comparing HYDRUS simulations of sodic soil amelioration with the results from recently published laboratory experiments on a more reactive, repacked sodic clay soil. The general trends observed in the laboratory experiments were able to be simulated using HYDRUS. Differences between measured and simulated results were attributed to the limited flexibility of the function that represents chemistry-dependent hydraulic conductivity in HYDRUS. While improvements in the function could be made, the present work indicates that HYDRUS-UNSATCHEM captures the key changes in soil hydraulic properties that occur during sodic clay soil amelioration and thus extends the findings of previous researchers studying sodic loams.

Hydraulic conductivity increases in a sodic clay soil in response to gypsum applications impacts of bulk density and cation exchange

Amelioration of sodic soils is commonly achieved by applying gypsum, which increases soil hydraulic conductivity by altering soil chemistry. The magnitude of hydraulic conductivity increases expected in response to gypsum applications depends on soil properties including clay content, clay mineralogy, and bulk density. The soil analyzed in this study was a kaolinite rich sodic clay soil from an irrigated area of the Lower Burdekin coastal floodplain in tropical North Queensland, Australia. The impact of gypsum amelioration was investigated by continuously leaching soil columns with a saturated gypsum solution, until the hydraulic conductivity and leachate chemistry stabilized. Extended leaching enabled the full impacts of electrolyte effects and cation exchange to be determined. For the columns packed to 1.4 g/cm3, exchangeable sodium concentrations were reduced from 5.0 ± 0.5 mEq/100 g to 0.41 ± 0.06 mEq/100 g, exchangeable magnesium concentrations were reduced from 13.9 ± 0.3 mEq/100 g to 4.3 ± 2.12 mEq/100 g, and hydraulic conductivity increased to 0.15 ± 0.04 cm/d. For the columns packed to 1.3 g/cm3, exchangeable sodium concentrations were reduced from 5.0 ± 0.5 mEq/100 g to 0.51 ± 0.03 mEq/100 g, exchangeable magnesium concentrations were reduced from 13.9 ± 0.3 mEq/100 g to 0.55 ± 0.36 mEq/100 g, and hydraulic conductivity increased to 0.96 ± 0.53 cm/d. The results of this study highlight that both sodium and magnesium need to be taken into account when determining the suitability of water quality for irrigation of sodic soils and that soil bulk density plays a major role in controlling the extent of reclamation that can be achieved using gypsum applications.

Impacts of sodic soil amelioration on hydraulic conductivity and deep drainage in the Lower Burdekin

An understanding of the influence of soil chemistry on soil hydraulic properties is of critical importance for the management of sodic soils under irrigation. The hydraulic conductivity of sodic soils has been shown to be affected by properties of the applied solution including pH (Suarez et al. 1984), sodicity and salt concentration (McNeal and Coleman 1966). The changes in soil hydraulic conductivity are the result of changes in the spacing between clay layers in response to changes in soil solution chemistry. While the importance o f soil chemistry in controlling hydraulic conductivity is known, the exact impacts of sodic soil amelioration on hydraulic conductivity and deep drainage at a given location are difficult to predict. This is because the relationships between soil chemical factors and hydraulic conductivity are soil specific and because local site specific factors also need to be considered to determine the actual impacts on deep drainage rates.

Effect of water management practice on pesticide behavior in paddy water

The fate and transport of three herbicides commonly used in rice production in Japan were compared using two water management practices. The herbicides were simetryn, thiobencarb and mefenacet. The first management practice was an intermittent irrigation scheme using an automatic irrigation system (AI) with a high drainage gate and the second one was a continuous irrigation and overflow drainage scheme (CI) in experimental paddy fields. Dissipation of the herbicides appeared to follow first order kinetics with the half-lives (DT₅₀) of 1.6-3.4 days and the DT₉₀ (90% dissipation) of 7.4-9.8 days. The AI scheme had little drainage even during large rainfall events thus resulting in losses of less than 4% of each applied herbicide through runoff. Meanwhile the CI scheme resulted in losses of about 37%, 12% and 35% of the applied masses of simetryn, thiobencarb and mefenacet, respectively. The intermittent irrigation scheme using an automatic irrigation system with a high drainage gate saved irrigation water and prevented herbicide runoff whereas the continuous irrigation and overflow scheme resulted in significant losses of water as well as the herbicides. Maintaining the excess water storage is important for preventing paddy water runoff during significant rainfall events. The organic carbon partition coefficient K_oc seems to be a strong indicator of the aquatic fate of the herbicide as compared to the water solubility (S_W). However, further investigations are required to understand the relation between K_oc and the agricultural practices upon the pesticide fate and transport. An extension of the water holding period up to 10 days after herbicide application based on the DT₉₀ from the currently specified period of 3-4 days in Japan is recommended to be a good agricultural practice for controlling the herbicide runoff from paddy fields. Also, the best water management practice, which can be recommended for use during the water holding period, is the intermittent irrigation scheme using an automatic irrigation system with a high drainage gate. © 2006 Elsevier B.V. All rights reserved.