The response of Paracoccidioides spp. to nitrosative stress

Paracoccidioidomycosis (PCM) is an endemic disease in Latin America caused by species belonging to the genus Paracoccidioides. During infection, immune cells present a variety of defense mechanisms against pathogens. One of these defensive strategies is the production and release of nitric oxide (NO) and S-nitroso thiols (e.g., S-nitrosoglutathione, GSNO), which produce reactive nitrogen species (RNS). This results in damage to DNA and membranes, inhibition of respiration and inactivation of cellular enzymes. In response to nitrosative stress, human pathogenic fungi possess defense mechanisms to prevent the adverse effects of NO, which helps them survive during initial contact with the host immune system. To understand how Paracoccidioides spp. respond to nitrosative stress, we conducted this study to identify genes and proteins that might contribute to this response. The results of proteomic analysis demonstrated that nitrosative stress induced a reduction in the expression of proteins related to the mitochondrial electron transport chain. This hypothesis was supported by the reduced mitochondrial activity observed in the presence of GSNO. Additionally, lipids and branched chain amino acid metabolism enzymes were altered. The role played by enzymes acting in oxidative stress in the RNS response was remarkable. This interface among enzymes acting in both stress responses was confirmed by using a RNA approach to silence the ccp gene in Paracoccidioides. It was observed that mutants with low expression of the ccp gene were more sensitive to nitrosative stress.     

Host Response to Meloidodera spp. (Heteroderidae)

Host responses to Meloidodera floridensis Chitwood et al., 1956, M. charis Hooper, 1960, and M. belli Wouts, 1973 were examined on loblolly pine, peony, and sage, respectively, with light, scanning, and transmission electron microscopy. In each case the nematodes induce a single uninucleate giant cell. The giant cell is initiated in the pericycle and expands as it matures. The mature giant cell induced by M. floridensis is surrounded by vascular parenchyma, whereas that caused by M. charts and M. belli coutacts xylem and phloem. The cell wall of giant cells induced by all three Meloidodera spp. is generally thicker than that of surrounding cells, with the thickest part adjacent to the lip region of the nematode. The thinner portion of the wall includes numerous pit fields with plasmodesmata, but wall ingrowths were not detected in a thorough examination of the entire wall. The nucleus of a giant cell induced by M. goridensis is highly irregular in shape with deep invaginations, whereas those caused by M. charis and M. belli include a cluster of apparently interconnected nuclear units. Organelles, including mitochondria, endoplasmic reticulum, and plastids of giant cells caused by Meloidodera, are typical of those reported in host responses of other Heteroderidae. The formation of a single uninucleate giant cell by Meloidodera, Cryphodera, Hylonerna, and Sarisodera, but a syncytium by Atalodera and Heterodera sensu lato, might be considered in conjunction with additional characters to determine the most parsimonious pattern of phylogeny of Heteroderidae.     

Response of Rhizobium spp. and pea seedlings to glycine

Summary Inhibition of growth of Rhizobium spp. by glycine and by the D-form of an unnatural amino acid, norvaline, was established. Rhizobium trifolii, strain Coryn, was completely inhibited at a glycine concentration of 10 g/ml while in the case of Rhizobium leguminosarum, strain S 310a, at the same glycine concentration only bacteroid-like forms were found.The amino-acid composition of pea and maize seedlings was altered by introducing amino acids into the cotyledons. It was found that the amino compounds were transported to the roots. In the case of peas, glycine was completely converted to serine while in maize this was not the case. L-serine, sarcosine and D-norvaline were found unchanged during the experimental period. re]19750515     

Identification and Analysis of the Role of Superoxide Dismutases Isoforms in the Pathogenesis of Paracoccidioides spp.

The ability of Paracoccidioides to defend itself against reactive oxygen species (ROS) produced by host effector cells is a prerequisite to survive. To counteract these radicals, Paracoccidioides expresses, among different antioxidant enzymes, superoxide dismutases (SODs). In this study, we identified six SODs isoforms encoded by the Paracoccidioides genome. We determined gene expression levels of representative isolates of the phylogenetic lineages of Paracoccidioides spp. (S1, PS2, PS3 and Pb01-like) using quantitative RT-PCR. Assays were carried out to analyze SOD gene expression of yeast cells, mycelia cells, the mycelia-to-yeast transition and the yeast-to-mycelia germination, as well as under treatment with oxidative agents and during interaction with phagocytic cells. We observed an increased expression of PbSOD1 and PbSOD3 during the transition process, exposure to oxidative agents and interaction with phagocytic cells, suggesting that these proteins could assist in combating the superoxide radicals generated during the host-pathogen interaction. Using PbSOD1 and PbSOD3 knockdown strains we showed these genes are involved in the response of the fungus against host effector cells, particularly the oxidative stress response, and in a mouse model of infection. Protein sequence analysis together with functional analysis of knockdown strains seem to suggest that PbSOD3 expression is linked with a pronounced extracellular activity while PbSOD1 seems more related to intracellular requirements of the fungus. Altogether, our data suggests that P. brasiliensis actively responds to the radicals generated endogenously during metabolism and counteracts the oxidative burst of immune cells by inducing the expression of SOD isoforms.     

Transcriptome Analysis of Poplar during Leaf Spot Infection with Sphaerulina spp.

Diseases of poplar caused by the native fungal pathogen Sphaerulina musiva and related species are of growing concern, particularly with the increasing interest in intensive poplar plantations to meet growing energy demands. Sphaerulina musiva is able to cause infection on leaves, resulting in defoliation and canker formation on stems. To gain a greater understanding of the different responses of poplar species to infection caused by the naturally co-evolved Sphaerulina species, RNA-seq was conducted on leaves of Populus deltoides, P. balsamifera and P. tremuloides infected with S. musiva, S. populicola and a new undescribed species, Ston1, respectively. The experiment was designed to contain the pathogen in a laboratory environment, while replicating disease development in commercial plantations. Following inoculation, trees were monitored for disease symptoms, pathogen growth and host responses. Genes involved in phenylpropanoid, terpenoid and flavonoid biosynthesis were generally upregulated in P. balsamifera and P. tremuloides, while cell wall modification appears to play an important role in the defense of P. deltoides. Poplar defensive genes were expressed early in P. balsamifera and P. tremuloides, but their expression was delayed in P. deltoides, which correlated with the rate of disease symptoms development. Also, severe infection in P. balsamifera led to leaf abscission. This data gives an insight into the large differences in timing and expression of genes between poplar species being attacked by their associated Sphaerulina pathogen.     

Transcriptome analysis and molecular studies on sulfur metabolism in the human pathogenic fungus Paracoccidioides brasiliensis

The dimorphic pathogenic fungus Paracoccidioides brasiliensis can grow as a prototroph for organic sulfur as a mycelial (non-pathogenic) form, but it is unable to assimilate inorganic sulfur as a yeast (pathogenic) form. Temperature and the inability to assimilate inorganic sulfur are the single conditions known to affect P. brasiliensis mycelium-to-yeast (M-Y) dimorphic transition. For a comprehensive evaluation of genes that have their expression modulated during the M-Y transition in different culture media, we performed a large-scale analysis of gene expression using a microarray hybridization approach. The results of the present work demonstrate the use of microarray hybridization analysis to examine gene expression during the M-Y transition in minimal medium and compare these results with the M-Y transition in complete medium. Our results showed that about 95% of the genes in our microarray are mainly responding to the temperature trigger, independently of the media where the M-Y transition took place. As a preliminary step to understand the inorganic sulfur inability in P. brasiliensis yeast form, we decided to characterize the mRNA accumulation of several genes involved in different aspects of both organic and inorganic sulfur assimilation. Our results suggest that although P. brasiliensis cannot use inorganic sulfur as a single sulfur source to initiate both M-Y transition and Y growth, the fungus can somehow use both organic and inorganic pathways during these growth processes.     

Response of Paracoccidioides brasiliensis Pb01 to stressor agents and cell wall osmoregulators

In its attempt to survive, the fungal cell can change the cell wall composition and/or structure in response to environmental stress. The molecules involved in these compensatory mechanisms are a possible target for the development of effective antifungal agents. In the thermodimorphic fungus Paracoccidioides brasiliensis Pb01, the main polymers that compose the cell wall are chitin and glucans. These polymers form a primary barrier that is responsible for the structural integrity and formation of the cell wall. In this study the behaviour of P. brasiliensis was evaluated under incubation with cell wall stressor agents such as Calcofluor White (CFW), Congo Red (CR), Sodium Dodecyl Sulphate (SDS), NaCl, KCl, and Sorbitol. Use of concentrations at which the fungus is visually sensitive to those agents helped to explain some of the adaptive mechanisms used by P. brasiliensis in response to cell wall stress. Our results show that 1,3-β-D-glucan synthase (PbFKS1), glucosamine-6-phosphate synthase (PbGFA1) and β-1,3-glucanosyltransferase (PbGEL3)as well as 1,3-β-D-glucan and N-acetylglucosamine (GlcNAc) residues in the cell wall are involved in compensatory mechanisms against cell wall damage.     

RNA-seq Transcriptome Profiling of the Halophyte Salicornia persica in Response to Salinity

Journal of Plant Growth Regulation - Salinity adversely affects growth and yield of crop plants. Plants respond to salinity using a series of physiological and biochemical mechanisms through an...     

Response of Salvinia spp. to different nitrogen sources: the acid-base regulation approach

This paper shows the importance of acid-base analyses and ¹³C measurements in the evaluation of the responses of Salvinia species' responses to different N sources. It also highlights the importance of these methodologies as potential tools in the study of differences between habitats and nutrient acquisition, particularly N. This study used three different species of Salvinia cultured in the absence of combined N or in the presence of either NO _inf3 ^sup– or NH _inf4 ^sup+ as N sources. The interaction between NO _inf3 ^sup– or NH _inf4 ^sup+ as N source and organic acid metabolism, and the information on diazotrophy from the organic acid measurements, were also examined. Nevertheless, the results presented may not be used per se to assign diazotrophy. Carboxylate (C-A) levels in the different Salvinia species are much lower than the norm for bryophytes and tracheophytes, consistent with previously published work on Azolla. This might be related to the aquatic life form of these plants, since they seem to have no potential to increase the availability of Fe or P by the acidification of their rooting medium (water) that a larger net synthesis of organic acids, with cation-H⁺ exchange, could achieve.