An investigation of functional diversity of endogenous cellulase and expression of other digestive enzyme genes in freshwater crayfish (Cherax) species

The project evaluated potential of soluble cellulose as a cheap feed ingredient for major farmed Australian freshwater crayfish species testing their growth performance, digestive enzyme activity and digestive enzyme gene expression patterns. Test animals showed an innate capacity to utilise a range of carbohydrate sources including complex structural polysaccharides. Results suggest that more plant-derived ingredient can be incorporated in formulated low-cost feeds for the culture industry.

Identification of genes important for growth of asymptomatic bacteriuria Escherichia coli in urine

Escherichia coli is the most important etiological agent of urinary tract infections (UTIs). Unlike uropathogenic E. coli, which causes symptomatic infections, asymptomatic bacteriuria (ABU) E. coli strains typically lack essential virulence factors and colonize the bladder in the absence of symptoms. While ABU E. coli can persist in the bladder for long periods of time, little is known about the genetic determinants required for its growth and fitness in urine. To identify such genes, we have employed a transposon mutagenesis approach using the prototypic ABU E. coli strain 83972 and the clinical ABU E. coli strain VR89. Six genes involved in the biosynthesis of various amino acids and nucleobases were identified (carB, argE, argC, purA, metE, and ilvC), and site-specific mutants were subsequently constructed in E. coli 83972 and E. coli VR89 for each of these genes. In all cases, these mutants exhibited reduced growth rates and final cell densities in human urine. The growth defects could be complemented in trans as well as by supplementation with the appropriate amino acid or nucleobase. When assessed in vivo in a mouse model, E. coli 83972carAB and 83972argC showed a significantly reduced competitive advantage in the bladder and/or kidney during coinoculation experiments with the parent strain, whereas 83972metE and 83972ilvC did not. Taken together, our data have identified several biosynthesis pathways as new important fitness factors associated with the growth of ABU E. coli in human urine.

UafB is a serine-rich repeat adhesin of Staphylococcus saprophyticus that mediates binding to fibronectin, fibrinogen and human uroepithelial cells

Staphylococcus saprophyticus is an important cause of urinary tract infection (UTI), particularly among young women, and is second only to uropathogenic Escherichia coli as the most frequent cause of UTI. The molecular mechanisms of urinary tract colonization by S. saprophyticus remain poorly understood. We have identified a novel 6.84 kb plasmid-located adhesin-encoding gene in S. saprophyticus strain MS1146 which we have termed uro-adherence factor B (uafB). UafB is a glycosylated serine-rich repeat protein that is expressed on the surface of S. saprophyticus MS1146. UafB also functions as a major cell surface hydrophobicity factor. To characterize the role of UafB we generated an isogenic uafB mutant in S. saprophyticus MS1146 by interruption with a group II intron. The uafB mutant had a significantly reduced ability to bind to fibronectin and fibrinogen. Furthermore, we show that a recombinant protein containing the putative binding domain of UafB binds specifically to fibronectin and fibrinogen. UafB was not involved in adhesion in a mouse model of UTI; however, we observed a striking UafB-mediated adhesion phenotype to human uroepithelial cells. We have also identified genes homologous to uafB in other staphylococci which, like uafB, appear to be located on transposable elements. Thus, our data indicate that UafB is a novel adhesin of S. saprophyticus that contributes to cell surface hydrophobicity, mediates adhesion to fibronectin and fibrinogen, and exhibits tropism for human uroepithelial cells.

Molecular analysis of type 3 fimbrial genes from Escherichia coli, Klebsiella and Citrobacter species

Background Catheter-associated urinary tract infection (CAUTI) is the most common nosocomial infection in the United States and is caused by a range of uropathogens. Biofilm formation by uropathogens that cause CAUTI is often mediated by cell surface structures such as fimbriae. In this study, we characterised the genes encoding type 3 fimbriae from CAUTI strains of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Citrobacter koseri and Citrobacter freundii. Results Phylogenetic analysis of the type 3 fimbrial genes (mrkABCD) from 39 strains revealed they clustered into five distinct clades (A-E) ranging from one to twenty-three members. The majority of sequences grouped in clade A, which was represented by the mrk gene cluster from the genome sequenced K. pneumoniae MGH78578. The E. coli and K. pneumoniae mrkABCD gene sequences clustered together in two distinct clades, supporting previous evidence for the occurrence of inter-genera lateral gene transfer. All of the strains examined caused type 3 fimbriae mediated agglutination of tannic acid treated human erythrocytes despite sequence variation in the mrkD-encoding adhesin gene. Type 3 fimbriae deletion mutants were constructed in 13 representative strains and were used to demonstrate a direct role for type 3 fimbriae in biofilm formation. Conclusions The expression of functional type 3 fimbriae is common to many Gram-negative pathogens that cause CAUTI and is strongly associated with biofilm growth. Our data provides additional evidence for the spread of type 3 fimbrial genes by lateral gene transfer. Further work is now required to substantiate the clade structure reported here by examining more strains as well as other bacterial genera that make type 3 fimbriae and cause CAUTI.

Characterization of two homologous disulfide bond systems involved in virulence factor Biogenesis in Uropathogenic Escherichia coli CFT073

Disulfide bond (DSB) formation is catalyzed by disulfide bond proteins and is critical for the proper folding and functioning of secreted and membrane-associated bacterial proteins. Uropathogenic Escherichia coli (UPEC) strains possess two paralogous disulfide bond systems: the well-characterized DsbAB system and the recently described DsbLI system. In the DsbAB system, the highly oxidizing DsbA protein introduces disulfide bonds into unfolded polypeptides by donating its redox-active disulfide and is in turn reoxidized by DsbB. DsbA has broad substrate specificity and reacts readily with reduced unfolded proteins entering the periplasm. The DsbLI system also comprises a functional redox pair; however, DsbL catalyzes the specific oxidative folding of the large periplasmic enzyme arylsulfate sulfotransferase (ASST). In this study, we characterized the DsbLI system of the prototypic UPEC strain CFT073 and examined the contributions of the DsbAB and DsbLI systems to the production of functional flagella as well as type 1 and P fimbriae. The DsbLI system was able to catalyze disulfide bond formation in several well-defined DsbA targets when provided in trans on a multicopy plasmid. In a mouse urinary tract infection model, the isogenic dsbAB deletion mutant of CFT073 was severely attenuated, while deletion of dsbLI or assT did not affect colonization.

Phage library screening for the rapid identification and in vivo testing of candidate genes for a DNA vaccine against Mycoplasma mycoides subsp. mycoides small colony biotype

A new strategy for rapidly selecting and testing genetic vaccines has been developed, in which a whole genome library is cloned into a bacteriophage λ ZAP Express vector which contains both prokaryotic (Plac) and eukaryotic (PCMV) promoters upstream of the insertion site. The phage library is plated on Escherichia coli cells, immunoblotted, and probed with hyperimmune and/or convalescent-phase antiserum to rapidly identify vaccine candidates. These are then plaque purified and grown as liquid lysates, and whole bacteriophage particles are then used directly to immunize the host, following which PCMV-driven expression of the candidate vaccine gene occurs. In the example given here, a semirandom genome library of the bovine pathogen Mycoplasma mycoides subsp. mycoides small colony (SC) biotype was cloned into λ ZAP Express, and two strongly immunodominant clones, λ-A8 and λ-B1, were identified and subsequently tested for vaccine potential against M. mycoides subsp. mycoides SC biotype-induced mycoplasmemia. Sequencing and immunoblotting indicated that clone λ-A8 expressed an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible M. mycoides subsp. mycoides SC biotype protein with a 28-kDa apparent molecular mass, identified as a previously uncharacterized putative lipoprotein (MSC_0397). Clone λ-B1 contained several full-length genes from the M. mycoides subsp. mycoides SC biotype pyruvate dehydrogenase region, and two IPTG-independent polypeptides, of 29 kDa and 57 kDa, were identified on immunoblots. Following vaccination, significant anti-M. mycoides subsp. mycoides SC biotype responses were observed in mice vaccinated with clones λ-A8 and λ-B1. A significant stimulation index was observed following incubation of splenocytes from mice vaccinated with clone λ-A8 with whole live M. mycoides subsp. mycoides SC biotype cells, indicating cellular proliferation. After challenge, mice vaccinated with clone λ-A8 also exhibited a reduced level of mycoplasmemia compared to controls, suggesting that the MSC_0397 lipoprotein has a protective effect in the mouse model when delivered as a bacteriophage DNA vaccine. Bacteriophage-mediated immunoscreening using an appropriate vector system offers a rapid and simple technique for the identification and immediate testing of putative candidate vaccines from a variety of pathogens.

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.

Progesterone regulation of implantation-related genes: new insights into the role of oestrogen

Genomic profiling was performed on explants of late proliferative phase human endometrium after 24-h treatment with progesterone (P) or oestradiol and progesterone (17 beta-E-2+P) and on explants of menstrual phase endometrium treated with 17 beta-E-2+P. Gene expression was validated with real-time PCR in the samples used for the arrays, in endometrium collected from early and mid-secretory phase endometrium, and in additional experiments performed on new samples collected in the menstrual and late proliferative phase. The results show that late proliferative phase human endometrium is more responsive to progestins than menstrual phase endometrium, that the expression of several genes associated with embryo implantation (i.e. thrombomodulin, monoamine oxidase A, SPARC-like 1) can be induced by P in vitro, and that genes that are fully dependent on the continuous presence of 17 beta-E-2 during P exposure can be distinguished from those that are P-dependent to a lesser extent. Therefore, 17 beta-E-2 selectively primes implantation-related genes for the effects of P.

The plasticity of NBS resistance genes in sorghum is driven by multiple evolutionary processes

Background Increased disease resistance is a key target of cereal breeding programs, with disease outbreaks continuing to threaten global food production, particularly in Africa. Of the disease resistance gene families, the nucleotide-binding site plus leucine-rich repeat (NBS-LRR) family is the most prevalent and ancient and is also one of the largest gene families known in plants. The sequence diversity in NBS-encoding genes was explored in sorghum, a critical food staple in Africa, with comparisons to rice and maize and with comparisons to fungal pathogen resistance QTL. Results In sorghum, NBS-encoding genes had significantly higher diversity in comparison to non NBS-encoding genes and were significantly enriched in regions of the genome under purifying and balancing selection, both through domestication and improvement. Ancestral genes, pre-dating species divergence, were more abundant in regions with signatures of selection than in regions not under selection. Sorghum NBS-encoding genes were also significantly enriched in the regions of the genome containing fungal pathogen disease resistance QTL; with the diversity of the NBS-encoding genes influenced by the type of co-locating biotic stress resistance QTL. Conclusions NBS-encoding genes are under strong selection pressure in sorghum, through the contrasting evolutionary processes of purifying and balancing selection. Such contrasting evolutionary processes have impacted ancestral genes more than species-specific genes. Fungal disease resistance hot-spots in the genome, with resistance against multiple pathogens, provides further insight into the mechanisms that cereals use in the “arms race” with rapidly evolving pathogens in addition to providing plant breeders with selection targets for fast-tracking the development of high performing varieties with more durable pathogen resistance.

Signaling pathway genes for blood pressure, folate and cholesterol levels among hypertensives : an epistasis analysis

Irregular atrial pressure, defective folate and cholesterol metabolism contribute to the pathogenesis of hypertension. However, little is known about the combined roles of the methylenetetrahydrofolate reductase (MTHFR), apolipoprotein-E (ApoE) and angiotensin-converting enzyme (ACE) genes, which are involved in metabolism and homeostasis. The objective of this study is to investigate the association of the MTHFR 677 C>T and 1298A>C, ACE insertion–deletion (I/D) and ApoE genetic polymorphisms with hypertension and to further explore the epistasis interactions that are involved in these mechanisms. A total of 594 subjects, including 348 normotensive and 246 hypertensive ischemic stroke subjects were recruited. The MTHFR 677 C>T and 1298A>C, ACE I/D and ApoEpolymorphisms were genotyped and the epistasis interaction were analyzed. The MTHFR 677 C>T and ApoE polymorphisms demonstrated significant associations with susceptibility to hypertension in multiple logistic regression models, multifactor dimensionality reduction and a classification and regression tree. In addition, the logistic regression model demonstrated that significant interactions between the ApoE E3E3, E2E4, E2E2 and MTHFR 677 C>T polymorphisms existed. In conclusion, the results of this epistasis study indicated significant association between the ApoE and MTHFR polymorphisms and hypertension.

An analysis of DNA methylation in human adipose tissue reveals differential modification of obesity genes before and after gastric bypass and weight loss

Background Environmental factors can influence obesity by epigenetic mechanisms. Adipose tissue plays a key role in obesity-related metabolic dysfunction, and gastric bypass provides a model to investigate obesity and weight loss in humans. Results Here, we investigate DNA methylation in adipose tissue from obese women before and after gastric bypass and significant weight loss. In total, 485,577 CpG sites were profiled in matched, before and after weight loss, subcutaneous and omental adipose tissue. A paired analysis revealed significant differential methylation in omental and subcutaneous adipose tissue. A greater proportion of CpGs are hypermethylated before weight loss and increased methylation is observed in the 3′ untranslated region and gene bodies relative to promoter regions. Differential methylation is found within genes associated with obesity, epigenetic regulation and development, such as CETP, FOXP2, HDAC4, DNMT3B, KCNQ1 and HOX clusters. We identify robust correlations between changes in methylation and clinical trait, including associations between fasting glucose and HDAC4, SLC37A3 and DENND1C in subcutaneous adipose. Genes investigated with differential promoter methylation all show significantly different levels of mRNA before and after gastric bypass. Conclusions This is the first study reporting global DNA methylation profiling of adipose tissue before and after gastric bypass and associated weight loss. It provides a strong basis for future work and offers additional evidence for the role of DNA methylation of adipose tissue in obesity.