Stress adaptation in a pathogenic fungus.

Candida albicans is a major fungal pathogen of humans. This yeast is carried by many individuals as a harmless commensal, but when immune defences are perturbed it causes mucosal infections (thrush). Additionally, when the immune system becomes severely compromised, C. albicans often causes life-threatening systemic infections. A battery of virulence factors and fitness attributes promote the pathogenicity of C. albicans. Fitness attributes include robust responses to local environmental stresses, the inactivation of which attenuates virulence. Stress signalling pathways in C. albicans include evolutionarily conserved modules. However, there has been rewiring of some stress regulatory circuitry such that the roles of a number of regulators in C. albicans have diverged relative to the benign model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. This reflects the specific evolution of C. albicans as an opportunistic pathogen obligately associated with warm-blooded animals, compared with other yeasts that are found across diverse environmental niches. Our understanding of C. albicans stress signalling is based primarily on the in vitro responses of glucose-grown cells to individual stresses. However, in vivo this pathogen occupies complex and dynamic host niches characterised by alternative carbon sources and simultaneous exposure to combinations of stresses (rather than individual stresses). It has become apparent that changes in carbon source strongly influence stress resistance, and that some combinatorial stresses exert non-additive effects upon C. albicans. These effects, which are relevant to fungus-host interactions during disease progression, are mediated by multiple mechanisms that include signalling and chemical crosstalk, stress pathway interference and a biological transistor.

Fungal iron availability during deep seated candidiasis is defined by a complex interplay involving systemic and local events.

Nutritional immunity--the withholding of nutrients by the host--has long been recognised as an important factor that shapes bacterial-host interactions. However, the dynamics of nutrient availability within local host niches during fungal infection are poorly defined. We have combined laser ablation-inductively coupled plasma mass spectrometry (LA-ICP MS), MALDI imaging and immunohistochemistry with microtranscriptomics to examine iron homeostasis in the host and pathogen in the murine model of systemic candidiasis. Dramatic changes in the renal iron landscape occur during disease progression. The infection perturbs global iron homeostasis in the host leading to iron accumulation in the renal medulla. Paradoxically, this is accompanied by nutritional immunity in the renal cortex as iron exclusion zones emerge locally around fungal lesions. These exclusion zones correlate with immune infiltrates and haem oxygenase 1-expressing host cells. This local nutritional immunity decreases iron availability, leading to a switch in iron acquisition mechanisms within mature fungal lesions, as revealed by laser capture microdissection and qRT-PCR analyses. Therefore, a complex interplay of systemic and local events influences iron homeostasis and pathogen-host dynamics during disease progression.

Differential adaptation of Candida albicans in vivo modulates immune recognition by dectin-1.

The ß-glucan receptor Dectin-1 is a member of the C-type lectin family and functions as an innate pattern recognition receptor in antifungal immunity. In both mouse and man, Dectin-1 has been found to play an essential role in controlling infections with Candida albicans, a normally commensal fungus in man which can cause superficial mucocutaneous infections as well as life-threatening invasive diseases. Here, using in vivo models of infection, we show that the requirement for Dectin-1 in the control of systemic Candida albicans infections is fungal strain-specific; a phenotype that only becomes apparent during infection and cannot be recapitulated in vitro. Transcript analysis revealed that this differential requirement for Dectin-1 is due to variable adaptation of C. albicans strains in vivo, and that this results in substantial differences in the composition and nature of their cell walls. In particular, we established that differences in the levels of cell-wall chitin influence the role of Dectin-1, and that these effects can be modulated by antifungal drug treatment. Our results therefore provide substantial new insights into the interaction between C. albicans and the immune system and have significant implications for our understanding of susceptibility and treatment of human infections with this pathogen.

Extreme zinc tolerance in acidophilic microorganisms from the bacterial and archaeal domains.

Zinc can occur in extremely high concentrations in acidic, heavy metal polluted environments inhabited by acidophilic prokaryotes. Although these organisms are able to thrive in such severely contaminated ecosystems their resistance mechanisms have not been well studied. Bioinformatic analysis of a range of acidophilic bacterial and archaeal genomes identified homologues of several known zinc homeostasis systems. These included primary and secondary transporters, such as the primary heavy metal exporter ZntA and Nramp super-family secondary importer MntH. Three acidophilic model microorganisms, the archaeon 'Ferroplasma acidarmanus', the Gram negative bacterium Acidithiobacillus caldus, and the Gram positive bacterium Acidimicrobium ferrooxidans, were selected for detailed analyses. Zinc speciation modeling of the growth media demonstrated that a large fraction of the free metal ion is complexed, potentially affecting its toxicity. Indeed, many of the putative zinc homeostasis genes were constitutively expressed and with the exception of 'F. acidarmanus' ZntA, they were not up-regulated in the presence of excess zinc. Proteomic analysis revealed that zinc played a role in oxidative stress in At. caldus and Am. ferrooxidans. Furthermore, 'F. acidarmanus' kept a constant level of intracellular zinc over all conditions tested whereas the intracellular levels increased with increasing zinc exposure in the remaining organisms.

Oxidative stress response in the opportunistic oral pathogen Fusobacterium nucleatum.

The anaerobic, Gram-negative bacillus Fusobacterium nucleatum plays a vital role in oral biofilm formation and the development of periodontal disease. The organism plays a central bridging role between early and late colonizers within dental plaque and plays a protective role against reactive oxygen species. Using a two-dimensional gel electrophoresis and mass spectrometry approach, we have annotated 78 proteins within the proteome of F. nucleatum subsp. nucleatum and identified those proteins whose apparent intracellular concentrations change in response to either O(2)- or H(2)O(2)-induced oxidative stress. Three major protein systems were altered in response to oxidative stress: (i) proteins of the alkyl hydroperoxide reductase/thioredoxin reductase system were increased in intracellular concentration; (ii) glycolytic enzymes were modified by oxidation (i.e. D-glyceraldehyde 3-phosphate dehydrogenase, and fructose 6-phosphate aldolase) or increased in intracellular concentration, with an accompanying decrease in ATP production; and (iii) the intracellular concentrations of molecular chaperone proteins and related proteins (i.e. ClpB, DnaK, HtpG, and HrcA) were increased.

Iron homeostasis and responses to iron limitation in extreme acidophiles from the Ferroplasma genus.

Extremely acidophilic archaea from the genus Ferroplasma inhabit iron-rich biomining environments and are important constituents of naturally occurring microbial consortia that catalyze the production of acid mine drainage. A combined bioinformatic, transcript profiling, and proteomic approach was used to elucidate iron homeostasis mechanisms in "F. acidarmanus" Fer1 and F. acidiphilum Y(T) . Bioinformatic analysis of the "F. acidarmanus" Fer1 genome sequence revealed genes encoding proteins hypothesized to be involved in iron-dependent gene regulation and siderophore biosynthesis; the Fhu and NRAMP cation acquisition systems; iron storage proteins; and the SUF machinery for the biogenesis of Fe-S clusters. A subset of homologous genes was identified on the F. acidiphilum Y(T) chromosome by direct PCR probing. In both strains, some of the genes appeared to be regulated in a ferrous/ferric iron-dependent manner, as indicated by RT-PCR. A detailed gel-based proteomics analysis of responses to iron depletion showed that a putative isochorismatase, presumably involved in siderophore biosynthesis, and the SufBCD system were upregulated under iron-limiting conditions. No evidence was obtained for iron sparing response during iron limitation. This study constitutes the first detailed investigation of iron homeostasis in extremely acidophilic archaea.

Substrate-driven gene expression in Roseburia inulinivorans: importance of inducible enzymes in the utilization of inulin and starch.

Roseburia inulinivorans is a recently identified motile representative of the Firmicutes that contributes to butyrate formation from a variety of dietary polysaccharide substrates in the human large intestine. Microarray analysis was used here to investigate substrate-driven gene-expression changes in R. inulinivorans A2-194. A cluster of fructo-oligosaccharide/inulin utilization genes induced during growth on inulin included one encoding a ß-fructofuranosidase protein that was prominent in the proteome of inulin-grown cells. This cluster also included a 6-phosphofructokinase and an ABC transport system, whereas a distinct inulin-induced 1-phosphofructokinase was linked to a fructose-specific phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS II transport enzyme). Real-time PCR analysis showed that the ß-fructofuranosidase and adjacent ABC transport protein showed greatest induction during growth on inulin, whereas the 1-phosphofructokinase enzyme and linked sugar phosphotransferase transport system were most strongly up-regulated during growth on fructose, indicating that these two clusters play distinct roles in the use of inulin. The R. inulinivorans ß-fructofuranosidase was overexpressed in Escherichia coli and shown to hydrolyze fructans ranging from inulin down to sucrose, with greatest activity on fructo-oligosaccharides. Genes induced on starch included the major extracellular α-amylase and two distinct α-glucanotransferases together with a gene encoding a flagellin protein. The latter response may be concerned with improving bacterial access to insoluble starch particles.

Biofilm development in the extremely acidophilic archaeon 'Ferroplasma acidarmanus' Fer1.

'Ferroplasma acidarmanus' Fer1 is an iron-oxidizing extreme acidophile isolated from the Iron Mountain mine, California, USA. This archaeon is predominantly found in biofilm-associated structures in the environment, and produces two distinct biofilm morphologies. Bioinformatic analysis of the 'F. acidarmanus' Fer1 genome identified genes annotated as involved in attachment and biofilm formation. No putative quorum sensing signaling genes were identified and no N-acyl homoserine lactone-like compounds were found in 'F. acidarmanus' Fer1 biofilm supernatant. Scanning confocal microscopy analysis of biofilm development on the surface of pyrite demonstrated the temporal and spatial development of biofilm growth. Furthermore, two-dimensional polyacrylamide gel electrophoresis was used to examine differential protein expression patterns between biofilm and planktonic populations. Ten up-regulated proteins were identified that included six enzymes associated with anaerobic growth, suggesting that the dominating phenotype in the mature biofilm was associated with anaerobic modes of growth. This report increases our knowledge of the genetic and proteomic basis of biofilm formation in an extreme acidophilic archaeon.

Kinetic characterization and quaternary structure of glutamate racemase from the periodontal anaerobe Fusobacterium nucleatum.

Cofactor-independent glutamate racemases (GRs) that supply the d-glutamate required for biosynthesis of the peptidoglycan that encapsulates bacterial cells are attractive targets for the development of antibacterial drugs. Recombinant GR from Fusobacterium nucleatum (FnGR), a Gram-negative anaerobe involved in periodontal disease, was overproduced, purified, and characterized. Unlike most other GRs, FnGR is a pseudosymmetric enzyme, catalyzing the racemization of glutamate enantiomers with similar kinetic parameters (k(cat)(L-->D)=17.4+/-0.8s(-1), K(m)(L-->D)=1.04+/-0.07mM, k(cat)(D-->L)=26+/-1s(-1), and K(m)(D-->L)=1.7+/-0.1mM; [corrected] pH optimum approximately 8.5). Mutational analysis of residue 151 (A151V) located at the entryway to the active site revealed that FnGR is very sensitive to increased steric bulk at this position. Blue native-polyacrylamide gel electrophoresis, Ferguson plot analyses, and cross-linking studies, indicated that FnGR existed predominately as dimers. Unlike Bacillus subtilis GR, the presence of glutamate did not significantly alter the position of the monomer-dimer equilibrium of FnGR.

Proteomic investigation of amino acid catabolism in the indigenous gut anaerobe Fusobacterium varium.

The butyrate-producing anaerobe Fusobacterium varium is an integral constituent of human gut microflora. Unlike many gut microorganisms, F. varium is capable of fermenting both amino acids and glucose. Although F. varium has been implicated in beneficial as well as pathological bacterium-host interactions, its genome has not been sequenced. To obtain a better understanding of the metabolic processes associated with amino acid fermentation by F. varium, we used a gel-based proteomic approach to examine the changes in the soluble proteome accompanying the utilization of eight different growth substrates: glucose, L- and D-glutamate, L-histidine, L- and D-lysine, and L- and D-serine. Using LC-MS/MS to analyze approximately 25% of the detected protein spots, we were able to identify 47 distinct proteins. While the intracellular concentrations of enzymes characteristic of a catabolic pathway for a specific amino acid were selectively increased in response to the presence of an excess of that amino acid in the growth medium, the concentrations of the core acetate-butyrate pathway enzymes remained relatively constant. Our analysis revealed (i) high intracellular concentrations of glutamate mutase and beta-methylaspartate ammonia-lyase under all growth conditions, underscoring the importance of the methylaspartate pathway of glutamate catabolism in F. varium (ii) the presence of two enzymes of the hydroxyglutarate pathway of glutamate degradation in the proteome of F. varium ((R)-2-hydroxyglutaryl-CoA dehydratase and NAD-specific glutamate dehydrogenase) specifically when L-glutamate was the main energy source (iii) the presence of genes in the genome of F. varium encoding each of the enzymes of the hydroxyglutarate pathway (iv) the presence of both L- and D-serine ammonia-lyases (dehydratases) which permit F. varium to thrive on either L- or D-serine, respectively, and (v) the presence of aspartate-semialdehyde dehydrogenase and dihydrodipicolinate synthase, consistent with the ability of F. varium to synthesize meso-2,6-diaminopimelic acid as a component of its peptidoglycan. Proteins involved in other cellular processes, including oxidation-reduction reactions, protein synthesis and turnover, and transport were also identified.

Proteomic investigation of glucose metabolism in the butyrate-producing gut anaerobe Fusobacterium varium.

A proteome survey and MS analysis were conducted to investigate glucose metabolism in Fusobacterium varium, a butyrate-producing constituent of the indigenous human gut microflora. The bacterium was capable of catabolizing glucose as the main energy source via the Embden-Meyerhof-Parnas pathway. 2-DE analyses revealed that the apparent concentrations of the six identified glycolytic enzymes (pyruvate kinase, enolase, glucose-6-phosphate isomerase, phosphoglycerate kinase, triosephosphate isomerase, and glyceraldehyde-3-phosphate dehydrogenase) were specifically increased in response to the presence of glucose in the chemically defined minimal growth medium, and did not diminish when the medium was additionally supplemented with L-glutamate, an amino acid readily fermented by members of the Fusobacterium genus. A substrate pool depletion study revealed that the sugar, and not the amino acid, is the more efficient growth substrate. Both proteomics and substrate pool depletion studies revealed that F. varium can simultaneously utilize both glucose and L-glutamate as energy sources. Enzymes involved in L-glutamate metabolism were also identified, including an NAD-dependent glutamate dehydrogenase and two enzymes of the methylaspartate pathway of L-glutamate catabolism (glutamate mutase and methylaspartate ammonia-lyase). Their apparent intracellular concentrations were elevated when the bacterium was cultured in media supplemented with excess L-glutamate. Our observation that the apparent concentrations of specific proteins were elevated in response to a particular growth substrate supplied as an energy source provides the first evidence for the presence of a nutrient-responsive mechanism governing intracellular protein concentration in F. varium.

The ypdI gene codes for a putative lipoprotein involved in the synthesis of colanic acid in Escherichia coli.

In Escherichia coli, the synthesis of colanic acid, an extracellular polysaccharide imminent in biofilm development, is a complicated process involving numerous genes and not yet wholly elucidated. Using a plasmid-borne E. coli K-12 gene library, we have identified a clone whose presence conferred mucoid colony phenotype onto E. coli CM2555 strain. Our results indicate that overexpression of a gene previously catalogued as ypdI, which encodes a putative lipoprotein, is responsible for this phenotype. We show that the mucoidy of ypdI -overexpressing bacteria is due to increased production of colanic acid. This phenotype depends on the function of the rcsA gene, but not on that of rcsF. These results suggest that the ypdI gene product might be an additional factor playing a role in colanic acid synthesis, indicating that this process can be even more complicated than supposed to date. However, no obvious phenotype was observed in the DeltaypdI::kan mutant cultivated under standard laboratory conditions.

The acrAB locus is involved in modulating intracellular acetyl coenzyme A levels in a strain of Escherichia coli CM2555 expressing the chloramphenicol acetyltransferase (cat) gene.

Recently, an Escherichia coli CM2555 strain was described as sensitive to chloramphenicol when expressing the chloramphenicol resistance gene (cat) from a multicopy plasmid. This sensitivity was linked to dysfunction of the acrA gene, which encodes a component of the AcrAB-TolC multidrug efflux pump. Preliminary data indicate that the sensitivity phenotype might be due to a decline in intracellular acetyl coenzyme A concentration accompanying the reaction catalyzed by chloramphenicol acetyltransferase, the cat-encoded resistance protein. Here, we demonstrate that the acrA dysfunction is the factor impairing the intracellular acetyl coenzyme A levels in the cat-expressing CM2555 strain. This effect might be alleviated by the interplay of proteins constituting two homologous efflux systems: AcrAB-TolC and AcrEF-TolC. However, our results show also that this is a genetic background-specific phenomenon, as the decrease in acetyl coenzyme A level is not evident in a cat-bearing DeltaacrAB derivative of the commonly used strain C600.

The combined use of chemical and biochemical markers to assess water quality in two low-stream rivers (NE Spain).

Carps (Cyprinus carpio) and red swamp crayfish (Procambarus clarkii) were sampled from two lowstream Mediterranean rivers (Anoia and Cardener) receiving extensive urban and industrial waste water discharges. Tissue residues of selected pollutants (organochlorinated compounds, polycyclic aromatic hydrocarbons (PAHs)) were determined in conjunction with different biochemical responses (cytochrome P450, phase II enzymes) with the aim of investigating whether resident organisms were responsive to changes in water quality. Biota inhabiting those rivers were highly exposed to complex mixtures of polychlorobiphenyls and dichlorodiphenyltrichloroethanes (up to 19 ng/g w.w.) and PAHs (up to 6097 ng/g of hydroxylated PAHs in bile), the highest residues being observed in carps from Cardener. This has a reflection on 7-ethoxyresorufin O-deethylase (EROD) activity; that in carps from Cardener ranged between 350 and 550 pmol/min/mg protein, whereas in carps from Anoia ranged between was 90 and 250 pmol/min/mg protein. The highest activity recorded was downstream of the sewage treatment plants in both rivers. Phase II enzymes were less sensitive to polutant exposure than EROD; nonetheless, both glutathione S-transferase and UDP-glucuronyl transferase were higher in carps from Cardener. The results support the usefulness of the combined use of chemical and biochemical responses to assess and diagnose pollution in highly stressed ecosystems.

Inactivation of the acrA gene is partially responsible for chloramphenicol sensitivity of Escherichia coli CM2555 strain expressing the chloramphenicol acetyltransferase gene.

An Escherichia coli CM2555 strain, sensitive to chloramphenicol when expressing the cat gene and producing active chloramphenicol acetyltransferase (CAT), was described recently. It was proposed that this sensitivity is due to decreased levels of acetyl coenzyme A (Acetyl CoA) in cat-expressing CM2555 cells in the presence of chloramphenicol. CAT catalyzes transfer of the acetyl moiety from Acetyl CoA to a chloramphenicol molecule. Thus, a very efficient acetylation of chloramphenicol may cause deprivation of Acetyl CoA and cell death. A specific mutation causing the chloramphenicol sensitivity phenotype of CM2555 was not reported to date. Therefore, we aimed to identify a genetic defect causing this phenotype. Here, we found that overexpression of the acrEF genes, encoding a transmembrane pump, or the acrE gene alone, results in restoration of chloramphenicol-resistance of cat-expressing CM2555 strain. Although no mutation exists in the CM2555 acrE locus, a nonsense mutation in the 67th codon of the acrA gene, which encodes a component of another transmembrane pump, has been found. Although introduction of the deltaacrAB allele into CM732, a parental strain of CM2555, and into some other commonly used E. coli strains led to their chloramphenicol sensitivity in the presence of CAT, the same genetic manipulation did not result in such a phenotype in other genetic backgrounds, including "wild-type" E. coli MG1655. These results suggest that the acrA dysfunction is one of more mutations responsible for chloramphenicol sensitivity of cat-expressing CM2555 strain.

Assessment of organotin pollution along the Polish coast (Baltic Sea) by using mussels and fish as sentinel organisms.

Levels of tributyltin (TBT) and its degradation products, mono- (MBT) and dibutyltin (DBT), as well as triphenyltin (TPT), were monitored in 10 stations along the Polish coast (Baltic Sea). Mussel-Mytilus edulis-and fish-Platichthys flesus-were used as sentinel organisms. The bioaccumulation patterns of butyltin and phenyltin compounds varied substantially. Butyltins were detected in mussel tissue from all the sampled stations. Among them, organisms from the Gulf of Gdansk showed the highest residues (68 ng/g w.w. as Sn) in conjunction with elevated TBT/DBT ratios, which suggest recent inputs of TBT in the area. Additionally, flatfish were sampled in the Gulf of Gdansk, and different tissues (liver, digestive tube and gills) were analyzed separately. TPT, although undetected in mussels, was always present in fish. The highest organotin concentration was observed in the liver (369 ng/g w.w. as Sn) of fish caught near Gdansk port. Relatively high concentrations were observed in digestive tube, which points out the ingestion of organotin contaminated food as an important uptake route of those compounds in P. flesus.