Effect of olive oil mill wastewater on extracellular ligninolytic enzymes produced by Phanerochaete flavido-alba

Four genes, fagA, B, C and D, encoding products with 32-47% identity to proteins involved in bacterial iron uptake systems, were identified immediately downstream of the Corynebacterium pseudotuberculosis phospholipase D gene. beta-Galactosidase assays on a C. pseudotuberculosis strain carrying a fagA-lacZ fusion indicated that the putative fagABC operon was poorly expressed in iron-rich media. However, similar experiments in iron-limited media resulted in an approximately three-fold increase in beta-galactosidase activity, suggesting that this operon is regulated by iron in vitro. Although no defect in iron utilization could be determined for a C. pseudotuberculosis fagB(C) mutant in vitro, this mutant showed reduced virulence compared to wild-type in a goat model of caseous lymphadenitis. Thus, expression of the fag genes in the host appears to contribute to virulence.     

Effect of iron on the virulence of Trichomonas vaginalis

The role of iron was evaluated with respect to the virulence of Trichomonas vaginalis in mice. Iron-supplemented and iron-depleted Diamond's trypticase-yeast extract-maltose (TYM) media were prepared by adding 360 microM of ferrous sulfate and 100 microM of 2,2'-dipyridyl. Trophozoites cultivated from normal TYM and iron-supplemented TYM media produced subcutaneous abscesses; however, trichomonads grown in an iron-deficient TYM medium failed to produce any pathology. In addition to the increased virulence of trophozoites in mice, iron affects the level of adherence and the cytotoxicity of trichomonads to HeLa cells, which are significantly reduced in trophozoites grown in iron-deficient medium. In conclusion, it is suggested that under iron-depleted conditions such as that induced by 2,2'-dipyridyl the virulence of T. vaginalis is reduced.     

Intracellular iron availability modulates the requirement for Leishmania Iron Regulator 1 (LIR1) during macrophage infections

The Leishmania plasma membrane transporter Leishmania Iron Regulator 1 (LIR1) facilitates iron export and is required for parasite virulence. By modulating macrophage iron content, we investigated the host site where LIR1 regulates Leishmania amazonensis infectivity. In bone marrow-derived macrophages, LIR1 null mutants demonstrated a paradoxical increase in virulence during infections in heme-depleted media, while wild-type growth was inhibited under the same conditions. Loading the endocytic pathway of macrophages with cationized ferritin prior to infection reversed the effect of heme depletion on both strains. Thus, LIR1 contributes to Leishmania virulence by protecting the parasites from toxicity resulting from iron accumulation inside parasitophorous vacuoles.     

Ferritin mutants of Escherichia coli are iron deficient and growth impaired, and fur mutants are iron deficient

Escherichia coli contains at least two iron storage proteins, a ferritin (FtnA) and a bacterioferritin (Bfr). To investigate their specific functions, the corresponding genes (ftnA and bfr) were inactivated by replacing the chromosomal ftnA and bfr genes with disrupted derivatives containing antibiotic resistance cassettes in place of internal segments of the corresponding coding regions. Single mutants (ftnA::spc and bfr::kan) and a double mutant (ftnA::spc bfr::kan) were generated and confirmed by Western and Southern blot analyses. The iron contents of the parental strain (W3110) and the bfr mutant increased by 1.5- to 2-fold during the transition from logarithmic to stationary phase in iron-rich media, whereas the iron contents of the ftnA and ftnA bfr mutants remained unchanged. The ftnA and ftnA bfr mutants were growth impaired in iron-deficient media, but this was apparent only after the mutant and parental strains had been precultured in iron-rich media. Surprisingly, ferric iron uptake regulation (fur) mutants also had very low iron contents (2.5-fold less iron than Fur+ strains) despite constitutive expression of the iron acquisition systems. The iron deficiencies of the ftnA and fur mutants were confirmed by M?ssbauer spectroscopy, which further showed that the low iron contents of ftnA mutants are due to a lack of magnetically ordered ferric iron clusters likely to correspond to FtnA iron cores. In combination with the fur mutation, ftnA and bfr mutations produced an enhanced sensitivity to hydroperoxides, presumably due to an increase in production of "reactive ferrous iron." It is concluded that FtnA acts as an iron store accommodating up to 50% of the cellular iron during postexponential growth in iron-rich media and providing a source of iron that partially compensates for iron deficiency during iron-restricted growth. In addition to repressing the iron acquisition systems, Fur appears to regulate the demand for iron, probably by controlling the expression of iron-containing proteins. The role of Bfr remains unclear.     

Role of a VPS41 homologue in starvation response, intracellular survival and virulence of Cryptococcus neoformans

Previous studies have demonstrated an important role for the vacuole in the virulence of the fungus Cryptococcus and studies in yeast have implicated the vacuolar protein Vps41 in copper loading of proteins such as iron transporters. However, our studies found that a cryptococcal vps41Delta strain displayed wild-type growth on media containing iron and copper chelators and normal activity of the copper-containing virulence factor laccase as well as almost normal growth at 37 degrees C and wild-type production of the virulence factor capsule. Despite these attributes, the vps41Delta mutant strain showed a dramatic attenuation of virulence in mice and co-incubation of mutant cells with the macrophage cell line, J774.16, resulted in a dramatic loss in viability of the vps41Delta mutant strain at 10 h compared with wild-type and complemented strains. Closer examination revealed that the vps41Delta mutant displayed a dramatic loss in viability after nutrient starvation which was traced to a failure to undergo G2 arrest, but there was no defect in the formation of autophagic or proteolytic vesicles. Our results indicate that VPS41 plays a key role in regulating starvation response in this pathogenic organism and that defects in cell cycle arrest are associated with attenuated pathogenic fitness in mammalian hosts.     

A putative P-type ATPase required for virulence and resistance to haem toxicity in Listeria monocytogenes

Regulation of iron homeostasis in many pathogens is principally mediated by the ferric uptake regulator, Fur. Since acquisition of iron from the host is essential for the intracellular pathogen Listeria monocytogenes, we predicted the existence of Fur-regulated systems that support infection. We examined the contribution of nine Fur-regulated loci to the pathogenicity of L. monocytogenes in a murine model of infection. While mutating the majority of the genes failed to affect virulence, three mutants exhibited a significantly compromised virulence potential. Most striking was the role of the membrane protein we designate FrvA (Fur regulated virulence factor A; encoded by frvA [lmo0641]), which is absolutely required for the systemic phase of infection in mice and also for virulence in an alternative infection model, the Wax Moth Galleria mellonella. Further analysis of the ΔfrvA mutant revealed poor growth in iron deficient media and inhibition of growth by micromolar concentrations of haem or haemoglobin, a phenotype which may contribute to the attenuated growth of this mutant during infection. Uptake studies indicated that the ΔfrvA mutant is unaffected in the uptake of ferric citrate but demonstrates a significant increase in uptake of haem and haemin. The data suggest a potential role for FrvA as a haem exporter that functions, at least in part, to protect the cell against the potential toxicity of free haem.     

Effects of host iron transport compounds on growth kinetics and outer-membrane protein expression of Bilophila wadsworthia

Since the environmental iron concentration has emerged as an important attribute in the expression of bacterial virulence, the purpose of this study was to determine the effects of transferrin, lactoferrin, heme compounds, and inorganic iron sources (ferric and ferrous sulfate) on the growth of Bilophila wadsworthia and to study its outer membrane composition when grown under these different simulated in vivo conditions. Lactoferrin, transferrin, hemin and hemoglobin supported full growth of the bacteria in media lacking other iron sources. Bilophila wadsworthia was also capable of growing in the presence of ferrous and ferric sulfate. Profiles obtained by SDS-PAGE showed two iron-regulated outer membrane proteins (IROMPs) of 190 kDa and 88 kDa. The 190 kDa was susceptible to proteinase K cleavage in whole cells, indicating its exposure at the cell surface. These two major IROMPs were expressed in iron-restricted media supplemented with iron-bound organic sources and repressed by the addition of inorganic iron sources.     

Strain-dependent effects of environmental signals on the production of extracellular phospholipase by Cryptococcus neoformans

Extracellular phospholipase (PL) activities comprising phospholipase B, lysophospholipase and lysophospholipase transacylase have been identified in culture supernatants of Cryptococcus neoformans and contribute to virulence. We found that PL production was optimal after fungal growth at 30 degrees C and secretion at 37 degrees C for all six C. neoformans isolates studied (four C. neoformans var. neoformans and two C. neoformans var. gattii). No increase in PL activity was found in one strain, NU-2, in low iron or tissue culture media, conditions where upregulation of other virulence factors has been reported. The most virulent strains in an intravenous mouse model of infection were best able to produce PL at growth and secretion temperatures of 37 degrees C, in tissue culture media and under assay conditions of pH 7.0.     

Season variability of iron effects on periodic culture of microalgae Dunaliella viridis Teod.

Effects of different iron concentrations (final concentrations of iron in Artari's medium: 3.7, 37.0, 74.0, and 185.0mmol·L-1) on growth rate and contents of protein, triacylglycerides, and β-carotene in Dunaliella viridis cells at cultivation in different months were investigated. It was shown that the dose-dependent effects of iron were notable for season variability. In the 1stexperimental series (October, 2007), iron at researched concentrations did not affect growth rate of culture and protein, triacylglyceride, and [3-carotene contents in cells. In experimental series conducted respectively in Novem-ber 2007, December 2007, and February 2008, the dose-dependent stimulation of microalgae growth was observed. For each of these experimental series, there were particular dose dependences of protein, triacylglyceride, and β-carotene contents in microalgae cells at cultivation on media with iron at different concentrations. Meanwhile, for all of the four experimental series conducted in different months, variability of growth rate and analyzed parameters of microalgae Dunaliella viridis as control (cultivation without iron) was shown. It is suggested that these functional differences of control cultures of microalgae in different months caused variability in the dose-dependent effects of iron in a Dunaliella viridis culture. The possibility of iron usage for increasing microalgae biomass and for enriching it by β-carotene in Dunaliella viridis culture with initial low productivity and low β-carotene content is considered.     

Gene expression patterns during swarming in Salmonella typhimurium: genes specific to surface growth and putative new motility and pathogenicity genes

Swarming is a specialized form of surface motility displayed by several flagellated bacterial genera, which shares features with other surface phenomenon such as biofilm formation and host invasion. Swarmer cells are generally more flagellated and longer than vegetative cells of the same species propagated in liquid media, and move within an encasement of polysaccharide 'slime'. Signals and signalling pathways controlling swarm cell differentiation are largely unknown. In order to test whether there is a genetic programme specific to swarming, we have determined global gene expression profiles of Salmonella typhimurium over an 8 h time course during swarming, and compared the microarray data with a similar time course of growth in liquid media as well as on harder agar where the bacteria do not swarm. Our data show that bacteria growing on the surface of agar have a markedly different physiology from those in broth, as judged by differential regulation of nearly one-third of the functional genome. The large number of genes showing surface-specific upregulation included those for lipopolysaccharide synthesis, iron metabolism and type III secretion. Although swarming-specific induction of flagellar gene expression was not generally apparent, genes for iron metabolism were strongly induced specifically on swarm agar. Surface-dependent regulation of many virulence genes suggests that growth on an agar surface could serve as a model for gene expression during the initial stages of host infection. Based on cluster analysis of distinctive expression patterns, we report here the identification of putative new genes involved in motility and virulence.     

Cloning and functional characterization of the Rhizopus oryzae high affinity iron permease (rFTR1) gene

Rhizopus oryzae is the most common etiologic agent of mucormycosis. Clinical and animal model data clearly demonstrate that the presence of elevated available serum iron predisposes the host to develop mucormycosis. Therefore, the high affinity iron permease (rFTR1) which encodes a protein required to scavenge iron from the environment, is highly likely to be a critical determinant of virulence for R. oryzae. We have cloned rFTR1 by using a PCR approach relying on degenerate primers designed from the conserved regions of Saccharomyces cerevisiae high affinity iron permease. Sequence analysis of a 2.0 kb EcoRI genomic clone revealed a single open reading frame of 1107 bp that lacked introns. The putative rFtr1p had significant homology to known fungal high affinity iron permeases from Candida albicans (46% identity) and S. cerevisiae (44% identity). In R. oryzae, rFTR1 was expressed in iron-depleted and not in iron-rich media. Finally, rFTR1 restored the ability of an ftr1 null mutant of S. cerevisiae to grow on iron-limited medium and to take up radiolabeled iron, whereas S. cerevisiae transformed with the empty vector did not. These data demonstrate that we have cloned the gene encoding a R. oryzae high affinity iron permease and the putative rFtr1p is involved in assimilation of iron from iron-depleted environments.     

TCA cycle activity in Staphylococcus aureus is essential for iron‐regulated synthesis of staphyloferrin A,but not staphyloferrin B: the benefit of a second citrate synthase

Staphylococcus aureus elaborates two citrate‐containing siderophores, staphyloferrin A (SA) and staphyloferrin B (SB), that enhance growth under iron‐restriction, yet, paradoxically, expression of the TCA cycle citrate synthase, CitZ, is downregulated during iron starvation. Iron starvation does, however, result in expression of SbnG, recently identified as a novel citrate synthase that is encoded from within the iron‐regulated SB biosynthetic locus, suggesting an important role for SbnG in staphyloferrin production. We demonstrate that during growth of S. aureus in iron‐restricted media containing glucose, SB is produced but, in contrast, SA production is severely repressed; accordingly, SB‐deficient mutants grow poorly in these media. Hypothesizing that reduced TCA cycle activity hinders SA production, we show that a citZ mutant is capable of SB synthesis, but not SA synthesis, providing evidence that SbnG does not generate citrate for incorporation into SA. A citZ sbnG mutant synthesizes neither staphyloferrin, is severely compromised for growth in iron‐restricted media, and is significantly more impaired for virulence than either of the single‐deletion mutants. We propose that SB is the more important of the two siderophores for S. aureus insofar as it is synthesized, and supports iron‐restricted growth, without need of TCA cycle activity.     

Differences in virulence for mice between disease and carrier strains of Neisseria meningitidis

The virulence of 11 prototype strains of Neisseria meningitidis, which had been used in the development of the serotyping scheme for serogroup B meningococci, were examined in mice treated with iron dextran. These strains, together with those previously examined, allowed for a good assessment of the virulence differences between carrier and disease strains. All of a total of 17 disease strains displayed virulence for mice (60% with high virulence), whereas only approximately 50% of 13 carrier strains possessed virulence (only 15% with high virulence). Because the ability to initiate infection in mice is independent of exogenous iron, this model system for infection appears particularly suited to studies of the physiological bases for virulence in N. meningitidis.     

Genomics of the 35-kb pvd locus and analysis of novel pvdIJK genes implicated in pyoverdine biosynthesis in Pseudomonas aeruginosa

Novel putative pyoverdine synthetase pvdIJK genes were found upstream of pvdD in the 6.2-Mb chromosome of Pseudomonas aerugilosa strain PAO1. These genes formed a locus implicated in pyoverdine biosynthesis. Sequence analysis showed that the product of these genes shared 43%, 60% and 57% identity with PvdD. PvdIJK are thought to be implicated in synthesis of pyoverdine, a siderophore chelating Fe3+. A pvdI mutant was obtained by gene disruption mutagenesis and confirmed by Southern hybridization. The pvdl mutant produced gave no significant growth on solid media supplemented with the iron chelator 2,2-dipyridyl; while the PvdI- phenotype abolished pyoverdine fluorescence. The role of PvdI in pathogenicity was tested by measuring the in vivo growth of P. aeruginosa wild-type and mutant strains in a chronic lung infection rat model, and by measuring the competitive infectivity index into a neutropenic mice model. The data obtained confirmed the importance of PvdI in virulence and iron uptake.     

Iron and fungal pathogenesis: a case study with Cryptococcus neoformans

The acquisition of iron from mammalian hosts is an important aspect of infection because microbes must compete with the host for this nutrient and iron perception often regulates virulence factor expression. For example, iron levels are known to influence the elaboration of two major virulence factors, the polysaccharide capsule and melanin, in the pathogenic fungus Cryptococcus neoformans. This pathogen, which causes meningoencephalitis in immunocompromised people, acquires iron through the use of secreted reductants, cell surface reductases, a permease/ferroxidase uptake system and siderophore transporters. In addition, a master regulator, Cir1, integrates iron sensing with the expression of virulence factors, with growth at 37°C and with signalling pathways that also influence virulence. The challenge ahead is to develop mechanistic views of the iron acquisition functions and regulatory schemes that operate when C. neoformans is in host tissue. Achieving these goals may contribute to an understanding of the notable predilection of the fungus for the mammalian central nervous system.