Effects of iron and desferrioxamine on Rhizopus infection

To investigate the association among iron, desferrioxamine, and a Rhizopus infection, the influence of iron and/or desferrioxamine on experimental mucormycosis in mice was examined. All mice pretreated with iron, desferrioxamine, or a combination of iron and desferrioxamine died within 5 days after the inoculation of R. oryzae. In the mice fungal lesions were observed in the brain which resembled human cerebral mucormycosis. By contrast, the mortality in the control mice with R. oryzae was 20% through the 3-week experimental period. Therefore, it was demonstrated that iron as well as desferrioxamine administration markedly promotes the growth of R. oryzae. The increased susceptibility to R. oryzae was considered to be due to increased serum iron in the animals pretreated with iron only; however, pretreatment with desferrioxamine did not affect the amount of serum ion. Thus, the data suggest that desferrioxamine acts as a siderophore to R. oryzae and exerts an adverse effect on mucormycosis. This study has shown that the presence of iron and desferrioxamine enhances the virulence and pathogenicity of R. oryzae by serving as a growth factor.     

Fob1 and Fob2 Proteins Are Virulence Determinants of Rhizopus oryzae via Facilitating Iron Uptake from Ferrioxamine

Dialysis patients with chronic renal failure receiving deferoxamine for treating iron overload are uniquely predisposed for mucormycosis, which is most often caused by Rhizopus oryzae. Although the deferoxamine siderophore is not secreted by Mucorales, previous studies established that Rhizopus species utilize iron from ferrioxamine (iron-rich form of deferoxamine). Here we determined that the CBS domain proteins of Fob1 and Fob2 act as receptors on the cell surface of R. oryzae during iron uptake from ferrioxamine. Fob1 and Fob2 cell surface expression was induced in the presence of ferrioxamine and bound radiolabeled ferrioxamine. A R. oryzae strain with targeted reduced Fob1/Fob2 expression was impaired for iron uptake, germinating, and growing on medium with ferrioxamine as the sole source of iron. This strain also exhibited reduced virulence in a deferoxamine-treated, but not the diabetic ketoacidotic (DKA), mouse model of mucormycosis. The mechanism by which R. oryzae obtains iron from ferrioxamine involves the reductase/permease uptake system since the growth on ferrioxamine supplemented medium is associated with elevated reductase activity and the use of the ferrous chelator bathophenanthroline disulfonate abrogates iron uptake and growth on medium supplemented with ferrioxamine as a sole source of iron. Finally, R. oryzae mutants with reduced copies of the high affinity iron permease (FTR1) or with decreased FTR1 expression had an impaired iron uptake from ferrioxamine in vitro and reduced virulence in the deferoxamine-treated mouse model of mucormycosis. These two receptors appear to be conserved in Mucorales, and can be the subject of future novel therapy to maintain the use of deferoxamine for treating iron-overload.     

Candida albicans possess a highly versatile and dynamic high‐affinity iron transport system important for its commensal‐pathogenic lifestyle

Iron is an essential nutrient for nearly all organisms, but iron overdose is toxic. The human commensal‐pathogenic fungus Candida albicans traverses host niches with markedly different iron availability. During systemic infection, C. albicans must activate the high‐affinity iron permease Ftr1 to acquire iron sequestered by the host's iron‐withholding defense and suppresses iron uptake while residing in the iron‐rich gut to avoid toxicity. Ftr1 associates with a ferroxidase to form an iron transporter. C. albicans contains four permeases and five ferroxidase homologs, suggesting 20 possible subunit combinations. Here, we investigated the iron‐dependent expression, cellular localization and interacting partners of all permeases and ferroxidases and the significance of each subunit for gastrointestinal colonization and systemic infection in mice. We uncovered three distinct patterns of iron‐dependent expression and highly flexible ferroxidase‐permease partnerships, which underlie a dynamic iron transport system that can be deftly tuned according to iron availability. We found functional differentiation as well as redundancy among the ferroxidases and permeases during both gastrointestinal colonization and bloodstream infection. We propose that C. albicans possesses a sophisticated iron acquisition and utilization system befitting its commensal‐pathogenic lifestyle. Our findings reveal new possibilities for medical intervention of C. albicans infection.     

Cell–cell signalling promotes ferric iron uptake in Xanthomonas oryzae pv. oryzicola that contribute to its virulence and growth inside rice

Cell–cell communication mediated by diffusible signal factor (DSF) plays an important role in virulence of several Xanthomonas group of plant pathogens. In the bacterial pathogen of rice, Xanthomonas oryzae pv. oryzicola, DSF is required for virulence and in planta growth. In order to understand the role of DSF in promoting in planta growth and virulence, we have characterized the DSF deficient mutant of X. oryzae pv. oryzicola. Mutant analysis by expression analysis, radiolabelled iron uptake studies and growth under low‐iron conditions indicated that DSF positively regulates ferric iron uptake. Further, the DSF deficient mutant of X. oryzae pv. oryzicola exhibited a reduced capacity to use ferric form of iron for growth under low‐iron conditions. Exogenous iron supplementation in the rice leaves rescued the in planta growth deficiency of the DSF deficient mutant. These data suggest that DSF promotes in planta growth of X. oryzae pv. oryzicola by positively regulating functions involved in ferric iron uptake which is important for its virulence. Our results also indicate that requirement of iron uptake strategies to utilize either Fe³⁺ or Fe²⁺ form of iron for colonization may vary substantially among closely related members of the Xanthomonas group of plant pathogens.     

Functional characterization of the ferroxidase, permease high-affinity iron transport complex from Candida albicans

Saccharomyces cerevisiae expresses two proteins that together support high‐affinity Fe‐uptake. These are a multicopper oxidase, Fet3p, with specificity towards Fe²⁺ and a ferric iron permease, Ftr1p, which supports Fe‐accumulation. Homologues of the genes encoding these two proteins are found in all fungal genomes including those for the pathogens, Candida albicans and Cryptococcus neoformans. At least one of these loci represents a virulence factor for each pathogen suggesting that this complex would be an appropriate pharmacologic target. However, the mechanism by which this protein pair supports Fe‐uptake in any fungal pathogen has not been elucidated. Taking advantage of the robust molecular genetics available in S. cerevisiae, we identify the two of five candidate ferroxidases likely involved in high‐affinity Fe‐uptake in C. albicans, Fet31 and Fet34. Both localize to the yeast plasma membrane and both support Fe‐uptake along with an Ftr1 protein, either from C. albicans or from S. cerevisiae. We express and characterize Fet34, demonstrating that it is functionally homologous to ScFet3p. Using S. cerevisiae as host for the functional expression of the C. albicans Fe‐uptake proteins, we demonstrate that they support a mechanism of Fe‐trafficking that involves channelling of the CaFet34‐generated Fe³⁺ directly to CaFtr1 for transport into the cytoplasm.     

Genomic Analysis of the Basal Lineage Fungus Rhizopus oryzae Reveals a Whole-Genome Duplication

Rhizopus oryzae is the primary cause of mucormycosis, an emerging, life-threatening infection characterized by rapid angioinvasive growth with an overall mortality rate that exceeds 50%. As a representative of the paraphyletic basal group of the fungal kingdom called “zygomycetes,” R. oryzae is also used as a model to study fungal evolution. Here we report the genome sequence of R. oryzae strain 99–880, isolated from a fatal case of mucormycosis. The highly repetitive 45.3 Mb genome assembly contains abundant transposable elements (TEs), comprising approximately 20% of the genome. We predicted 13,895 protein-coding genes not overlapping TEs, many of which are paralogous gene pairs. The order and genomic arrangement of the duplicated gene pairs and their common phylogenetic origin provide evidence for an ancestral whole-genome duplication (WGD) event. The WGD resulted in the duplication of nearly all subunits of the protein complexes associated with respiratory electron transport chains, the V-ATPase, and the ubiquitin–proteasome systems. The WGD, together with recent gene duplications, resulted in the expansion of multiple gene families related to cell growth and signal transduction, as well as secreted aspartic protease and subtilase protein families, which are known fungal virulence factors. The duplication of the ergosterol biosynthetic pathway, especially the major azole target, lanosterol 14α-demethylase (ERG11), could contribute to the variable responses of R. oryzae to different azole drugs, including voriconazole and posaconazole. Expanded families of cell-wall synthesis enzymes, essential for fungal cell integrity but absent in mammalian hosts, reveal potential targets for novel and R. oryzae-specific diagnostic and therapeutic treatments.     

Identification of disulfide bond isomerase substrates reveals bacterial virulence factors

Bacterial pathogens are exposed to toxic molecules inside the host and require efficient systems to form and maintain correct disulfide bonds for protein stability and function. The intracellular pathogen Francisella tularensis encodes a disulfide bond formation protein ortholog, DsbA, which previously was reported to be required for infection of macrophages and mice. However, the molecular mechanisms by which F. tularensis DsbA contributes to virulence are unknown. Here, we demonstrate that F. tularensis DsbA is a bifunctional protein that oxidizes and, more importantly, isomerizes complex disulfide connectivity in substrates. A single amino acid in the conserved cis‐proline loop of the DsbA thioredoxin domain was shown to modulate both isomerase activity and F. tularensis virulence. Trapping experiments in F. tularensis identified over 50 F. tularensis DsbA substrates, including outer membrane proteins, virulence factors, and many hypothetical proteins. Six of these hypothetical proteins were randomly selected and deleted, revealing two novel proteins, FTL_1548 and FTL_1709, which are required for F. tularensis virulence. We propose that the extreme virulence of F. tularensis is partially due to the bifunctional nature of DsbA, that many of the newly identified substrates are required for virulence, and that the development of future DsbA inhibitors could have broad anti‐bacterial implications.     

Macrophages largely contribute to heterologous anti‐Propionibacterium acnes antibody‐mediated protection from Actinobacillus pleuropneumoniae infection in mice

Actinobacillus pleuropneumoniae is the causative agent of acute and chronic pleuropneumonia. Propionibacterium acnes is a facultative anaerobic gram‐positive corynebacterium. We have previously found that anti‐P. acnes antibodies can prevent A. pleuropneumoniae infections in mice. To investigate the role of macrophages in this process, affinity‐purified anti‐P. acnes IgG and anti‐A. pleuropneumoniae IgG were used in opsonophagocytosis assays. Additionally, the efficacy of passive immunization with P. acnes serum against A. pleuropneumoniae was tested in macrophage‐depleted mice. It was found that anti‐P. acnes IgG had an effect similar to that of anti‐A. pleuropneumoniae IgG (P > 0.05), which significantly promotes phagocytosis of A. pleuropneumoniae by macrophages (P < 0.01). It was also demonstrated that, after passive immunization with anti‐P. acnes serum, macrophage‐replete mice had the highest survival rate (90%), whereas the survival rate of macrophage‐depleted mice was only 40% (P < 0.05). However, macrophage‐depleted mice that had been passively immunized with naïve serum had the lowest survival rate (20%), this rate being lower than that of macrophage‐replete mice that had been passively immunized with naïve serum. Overall, anti‐P. acnes antibodies did not prevent A. pleuropneumoniae infection under conditions of macrophage depletion (P > 0.05). Furthermore, in mice that had been passively immunized with anti‐P. acnes serum, macrophage depletion resulted in a greater A. pleuropneumoniae burden and more severe pathological features of pneumonia in lung tissues than occurred in macrophage‐replete mice. It was concluded that macrophages are essential for the process by which anti‐P. acnes antibody prevents A. pleuropneumoniae infection in mice.     

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.     

The phage shock protein PspA facilitates divalent metal transport and is required for virulence of Salmonella enterica sv. Typhimurium

The phage shock protein (Psp) system is induced by extracytoplasmic stress and thought to be important for the maintenance of proton motive force. We investigated the contribution of PspA to Salmonella virulence. A pspA deletion mutation significantly attenuates the virulence of Salmonella enterica serovar Typhimurium following intraperitoneal inoculation of C3H/HeN (Ity^r) mice. PspA was found to be specifically required for virulence in mice expressing the natural resistance‐associated macrophage protein 1 (Nramp1) (Slc11a1) divalent metal transporter, which restricts microbial growth by limiting the availability of essential divalent metals within the phagosome. Salmonella competes with Nramp1 by expressing multiple metal uptake systems including the Nramp‐homologue MntH, the ABC transporter SitABCD and the ZIP family transporter ZupT. PspA was found to facilitate Mn²⁺ transport by MntH and SitABCD, as well as Zn²⁺ and Mn²⁺ transport by ZupT. In vitro uptake of ⁵⁴Mn²⁺ by MntH and ZupT was reduced in the absence of PspA. Transport‐deficient mutants exhibit reduced viability in the absence of PspA when grown under metal‐limited conditions. Moreover, the ZupT transporter is required for Salmonella enterica serovar Typhimurium virulence in Nramp1‐expressing mice. We propose that PspA promotes Salmonella virulence by maintaining proton motive force, which is required for the function of multiple transporters mediating bacterial divalent metal acquisition during infection.     

Peroxisomal fission is induced during appressorium formation and is required for full virulence of the rice blast fungus

Peroxisomes are involved in various metabolic processes and are important for virulence in different pathogenic fungi. How peroxisomes rapidly emerge in the appressorium during fungal infection is poorly understood. Here, we describe a gene, PEF1, which can regulate peroxisome formation in the appressorium by controlling peroxisomal fission, and is required for plant infection in the rice blast fungus Magnaporthe oryzae. Targeted deletion of PEF1 resulted in a reduction in virulence and a delay in penetration and invasive growth in host cells. PEF1 was particularly expressed during appressorial development, and its encoding protein was co‐localized with peroxisomes during appressorial development. Compared with the massive vesicle‐shaped peroxisomes formed in the wild‐type appressorium, the Δpef1 mutant could only form stringy linked immature peroxisomes, suggesting that PEF1 was involved in peroxisomal fission during appressorium formation. We also found that the Δpef1 mutant could not utilize fatty acids efficiently, which can improve significantly the expression level of PEF1 and induce peroxisomal fission. As expected, the Δpef1 mutant showed reduced intracellular production of reactive oxygen species (ROS) during appressorium formation and induced ROS accumulation in host cells during infection. Taken together, PEF1‐mediated peroxisomal fission is important for fungal infection by controlling the number of peroxisomes in the appressorium.     

Heterologous expression of a gene of Magnaporthe oryzae chrysovirus 1 strain A disrupts growth of the human pathogenic fungus Cryptococcus neoformans

Magnaporthe oryzae chrysovirus 1 strain A (MoCV1‐A) is the causal agent of growth repression and attenuated virulence (hypovirulence) of the rice blast fungus, M. oryzae. We have previously reported that heterologous expression of MoCV1‐A ORF4 in Saccharomyces cerevisiae results in growth defects, a large central vacuole and other cytological changes. In this study, the effects of open reading frame (ORF) 4 expression in Cryptococcus neoformans, a human pathogenic fungus responsible for severe opportunistic infection, were investigated. Cells expressing the ORF4 gene in C. neoformans showed remarkably enlarged vacuoles, nuclear diffusion and a reduced growth rate. In addition, expression of ORF4 apparently suppressed formation of the capsule that surrounds the entire cell wall, which is one of the most important components of expression of virulence. After 5‐fluoroorotic acid treatment of ORF4‐expressing cells to remove the plasmid carrying the ORF4 gene, the resultant plasmid‐free cells recovered normal morphology and growth, indicating that heterologous expression of the MoCV1‐A ORF4 gene induces negative effects in C. neoformans. These data suggest that the ORF4 product is a candidate for a pharmaceutical protein to control disease caused by C. neoformans.     

Involvement of Gluconeogenic Pathway in Virulence of Xanthomonas oryzae pv. oryzae

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, one of the most widespread and destructive bacterial diseases of rice. A phosphoenolpyruvate synthase (ppsA)‐disrupted mutant OSPAM was generated by homologous suicide plasmid integration. The mutant was unable to grow in medium with pyruvate or C₄‐dicarboxylates as the sole carbon source, compared with the wild‐type, indicating a disruption in ppsA function. The mutant showed a reduction in virulence on rice but still induced a hypersensitive response in tobacco. When the mutant was complemented, the response was recovered to wild‐type. These results suggested that X. oryzae pv. oryzae possesses only PPSA route in gluconeogenesis, which is necessary for virulence.     

Salmonella acquires ferrous iron from haemophagocytic macrophages

Bacteria harbour both ferrous and ferric iron transporters. We now report that infection of macrophages and mice with a Salmonella enterica Typhimurium strain containing an inactivated feoB‐encoded ferrous iron transporter results in increased bacterial replication, compared to infection with wild type. Inactivation of other cation transporters, SitABCD or MntH, did not increase bacterial replication. The feoB mutant strain does not have an intrinsically faster growth rate. Instead, increased replication correlated with increased expression in macrophages of the fepB‐encoded bacterial ferric iron transporter and also required siderophores, which capture ferric iron. Co‐infection of mice with wild type and a feoB mutant strain yielded a different outcome: FeoB is clearly required for tissue colonization. In co‐infected primary mouse macrophages, FeoB is required for S. Typhimurium replication if the macrophages were IFNγ treated and contain phagocytosed erythrocytes, a model for haemophagocytosis. Haemophagocytes are macrophages that have engulfed erythrocytes and/or leucocytes and can harbour Salmonella in mice. These observations suggest that Salmonella acquires ferrous iron from haemophagocytic macrophages.     

VapA of Rhodococcus equi binds phosphatidic acid

Rhodococcus equi is a multihost, facultative intracellular bacterial pathogen that primarily causes pneumonia in foals less than six months in age and immunocompromised people. Previous studies determined that the major virulence determinant of R. equi is the surface bound virulence associated protein A (VapA). The presence of VapA inhibits the maturation of R. equi‐containing phagosomes and promotes intracellular bacterial survival, as determined by the inability of vapA deletion mutants to replicate in host macrophages. While the mechanism of action of VapA remains elusive, we show that soluble recombinant VapA^32‐189 both rescues the intramacrophage replication defect of a wild type R. equi strain lacking the vapA gene and enhances the persistence of nonpathogenic Escherichia coli in macrophages. During macrophage infection, VapA was observed at both the bacterial surface and at the membrane of the host‐derived R. equi containing vacuole, thus providing an opportunity for VapA to interact with host constituents and promote alterations in phagolysosomal function. In support of the observed host membrane binding activity of VapA, we also found that rVapA^32‐189 interacted specifically with liposomes containing phosphatidic acid in vitro. Collectively, these data demonstrate a lipid binding property of VapA, which may be required for its function during intracellular infection.