Molecular biology of the dimorphic fungi Paracoccidioides SPP

Paracoccidioides spp, herein commonly referred as Paracoccidioides brasiliensis, is the etiological agent of racoccidioidomycosis (PCM), the most prevalent systemic mycosis endemic in Latin America. Many aspects of the biology of P. brasiliensis remain unknown, in particular its ecology and the apparent lack of a sexual reproduction stage in its life cycle. This review will highlight the current knowledge on the genetics and genomics of P. brasiliensis, its most important putative virulence factors and the challenges for developing genetic tools in this organism. P. brasiliensis is a dimorphic ascomycete fungus belonging to the order Onygenales, family Ajellomycetaceae. The P. brasiliensis pathogenic yeast form is haracterized by a multiple-budding and multinucleate nature, with a highly polymorphic cellular shape. Successful infection and dissemination by P. brasiliensis requires initial interaction of the fungus with host cells. The fungus has to adhere to host cells after which internalization of the fungus takes place. Gp43 is a 43-kDa glycoprotein that participates in the interaction with the host at different levels. There are very few putative virulence factors described in P. brasiliensis,amongthem an extracellular phospholipase B, a 32-kDa haloacid dehalogenase PbHad32 that was shown to bind laminin, fibrinogen, and fibronectin, and to be important for initial adhesion to pulmonary epithelial cells, the pigment melanin, and the Rho-like GTPase PbCdc42. The morphological transition of P. brasiliensis from mycelium to the yeast form is a key process for the infectivity of the fungus. There are several transcriptional profiling studies addressing which genes have increased or decreased mRNA accumulation during mycelium-to-yeast transitions. Functional genomics studies in P. brasiliensis have been hampered by the absence of efficient molecular techniques that enable targeted gene inactivation in this fungus. However, an optimized Agrobacterium tumefaciens-mediated transformation method has been developed and was used to knock-down expression of the genes encoding the Rho-like GTPase PbCdc42 and the HAD-type hydrolase PbHad32. A challenge for the future is the development of mutagenesis methods that allow for the creation of targeted insertional gene mutants in Paracoccidioides spp. The complete genome sequencing of three isolates of Paracoccidioides species provides the opportunity to perform more complete evaluations of the transcriptomic and proteomic data, and to understand the biology and virulence of these important pathogenic fungi.Fundação para a Ciência e Tecnologia (FCT) - Bolsa nº PTDC/BIA-MIC/108309/2008Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento. Científico e Tecnológic

Functionality of the paracoccidioides mating α-pheromone-receptor system

Recent evidence suggests that Paracoccidioides species have the potential to undergo sexual reproduction, although no sexual cycle has been identified either in nature or under laboratory conditions. In the present work we detected low expression levels of the heterothallic MAT loci genes MAT1-1 and MAT1-2, the a-pheromone (PBa) gene, and the a- and apheromone receptor (PREB and PREA) genes in yeast and mycelia forms of several Paracoccidioides isolates. None of the genes were expressed in a mating type dependent manner. Stimulation of P. brasiliensis MAT1-2 strains with the synthetic a pheromone peptide failed to elicit transcriptional activation of MAT1-2, PREB or STE12, suggesting that the strains tested are insensitive to a-pheromone. In order to further evaluate the biological functionality of the pair a-pheromone and its receptor, we took advantage of the heterologous expression of these Paracoccidioides genes in the corresponding S. cerevisiae null mutants. We show that S. cerevisiae strains heterologously expressing PREB respond to Pba pheromone either isolated from Paracoccidioides culture supernatants or in its synthetic form, both by shmoo formation and by growth and cell cycle arrests. This allowed us to conclude that Paracoccidioides species secrete an active a-pheromone into the culture medium that is able to activate its cognate receptor. Moreover, expression of PREB or PBa in the corresponding null mutants of S. cerevisiae restored mating in these non-fertile strains. Taken together, our data demonstrate pheromone signaling activation by the Paracoccidioides a-pheromone through its receptor in this yeast model, which provides novel evidence for the existence of a functional mating signaling system in Paracoccidioides.MHJS and JFM were supported by Fundacão para a Ciência e Tecnologia (FCT) grants. This work was supported by a grant from FCT (PTDC/BIA-MIC/ 108309/2008)

Effect of Lactobacillus salivarius Bacteriocin Abp118 on the Mouse and Pig Intestinal Microbiota

Lactobacilli are Gram-positive bacteria that are a subdominant element in the human gastrointestinal microbiota, and which are commonly used in the food industry. Some lactobacilli are considered probiotic, and have been associated with health benefits. However, there is very little culture-independent information on how consumed probiotic microorganisms might affect the entire intestinal microbiota. We therefore studied the impact of the administration of Lactobacillus salivarius UCC118, a microorganism well characterized for its probiotic properties, on the composition of the intestinal microbiota in two model animals. UCC118 has anti-infective activity due to production of the bacteriocin Abp118, a broad-spectrum class IIb bacteriocin, which we hypothesized could impact the microbiota. Mice and pigs were administered wild-type (WT) L. salivarius UCC118 cells, or a mutant lacking bacteriocin production. The microbiota composition was determined by pyrosequencing of 16S rRNA gene amplicons from faeces. The data show that L. salivarius UCC118 administration had no significant effect on proportions of major phyla comprising the mouse microbiota, whether the strain was producing bacteriocin or not. However, L. salivarius UCC118 WT administration led to a significant decrease in Spirochaetes levels, the third major phylum in the untreated pig microbiota. In both pigs and mice, L. salivarius UCC118 administration had an effect on Firmicutes genus members. This effect was not observed when the mutant strain was administered, and was thus associated with bacteriocin production. Surprisingly, in both models, L. salivarius UCC118 administration and production of Abp118 had an effect on Gram-negative microorganisms, even though Abp118 is normally not active in vitro against this group of microorganisms. Thus L. salivarius UCC118 administration has a significant but subtle impact on mouse and pig microbiota, by a mechanism that seems at least partially bacteriocin-dependent

P. brasiliensis virulence is affected by SconC, the negative regulator of inorganic sulfur assimilation

Conidia/mycelium-to-yeast transition of Paracoccidioidesbrasiliensis is a critical step for the establishment of paracoccidioidomycosis, a systemic mycosis endemic in Latin America. Thus, knowledge of the factors that mediate this transition is of major importance for the design of intervention strategies. So far, the only known pre-requisites for the accomplishment of the morphological transition are the temperature shift to 37°C and the availability of organic sulfur compounds. In this study, we investigated the auxotrophic nature to organic sulfur of the yeast phase of Paracoccidioides, with special attention to P. brasiliensis species. For this, we addressed the role of SconCp, the negative regulator of the inorganic sulfur assimilation pathway, in the dimorphism and virulence of this pathogen. We show that down-regulation of SCONC allows initial steps of mycelium-to-yeast transition in the absence of organic sulfur compounds, contrarily to the wild-type fungus that cannot undergo mycelium-to-yeast transition under such conditions. However, SCONC down-regulated transformants were unable to sustain yeast growth using inorganic sulfur compounds only. Moreover, pulses with inorganic sulfur in SCONC down-regulated transformants triggered an increase of the inorganic sulfur metabolism, which culminated in a drastic reduction of the ATP and NADPH cellular levels and in higher oxidative stress. Importantly, the down-regulation of SCONC resulted in a decreased virulence of P. brasiliensis, as validated in an in vivo model of infection. Overall, our findings shed light on the inability of P. brasiliensis yeast to rely on inorganic sulfur compounds, correlating its metabolism with cellular energy and redox imbalances. Furthermore, the data herein presented reveal SconCp as a novel virulence determinant of P. brasiliensis.J.F.M. and J.G.R. were supported by a PhD grant from Fundacao para a Ciencia e Tecnologia (FCT). This work was supported by a grant from FCT (PTDC/BIA-MIC/108309/2008). M. Sturme. and M. Saraiva are Ciencia 2008 fellows. The authors would also like to thank FAPESP (Fundacao para Amparo a Pesquisa do Estado de Sao Paulo) and CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico) for financial support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

TLR9 activation dampens the early inflammatory response to paracoccidioides brasiliensis, Impacting host survival

Background: Paracoccidioides brasiliensis causes paracoccidioidomycosis, one of the most prevalent systemic mycosis in Latin America. Thus, understanding the characteristics of the protective immune response to P. brasiliensis is of interest, as it may reveal targets for disease control. The initiation of the immune response relies on the activation of pattern recognition receptors, among which are TLRs. Both TLR2 and TLR4 have been implicated in the recognition of P. brasiliensis and regulation of the immune response. However, the role of TLR9 during the infection by this fungus remains unclear.J.F. Menino was supported by a grant from Fundacao para a Ciencia e Tecnologia (FCT), Portugal (SFRH/BD/33446/2008). This work was supported by a grant from FCT (PTDC/BIA-MIC/108309/2008). M. Saraiva is a Ciencia 2007 fellow and M. Sturme is a Ciencia 2008 fellow. We would also like to thank FAPESP (Fundacao para Amparo a Pesquisa do Estado de Sao Paulo) and CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico) for financial support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Stable transformation of the green algae Acutodesmus obliquus and Neochloris oleoabundans based on E. coli conjugation

Microalgae are an ideal platform for the production of high-value chemicals, nutritional products and biofuels. Genetic engineering could speed up the development of microalgae derived products and reduce the overall production costs. Genetic methods such as particle bombardment, electroporation, Agrobacterium tumefaciens mediated transformation (ATMT), and agitation with glass beads and silicon carbide whiskers have been developed for the genetic transformation of microalgae. However, the transformation efficiency is species dependent, so a variety of transformation methods are required to engineer a wide range of microalgae species. The oleaginous microalgae Acutodesmus obliquus and Neochloris oleoabundans have a great potential as production platforms due to their ability to produce large amounts of triacylglycerol (TAG). Genetic modification techniques however are required to increase TAG levels further or to modify the fatty acid composition. Recently, a conjugation-based method for the delivery of episomes from bacteria to diatom microalgae has been reported. In this study, we have achieved the successful transformation of green oleaginous microalgal strains by transferring an expression vector via conjugation from E. coli. Since delivery of exogenous DNA into the microalgae cells is only the first step in obtaining transgenic microalgae, we further analyzed transformation efficiencies by PCR and expression of the Clover fluorescent protein in the targeted species

Stable transformation of the green algae Acutodesmus obliquus and Neochloris oleoabundans based on E. coli conjugation

Microalgae are an ideal platform for the production of high-value chemicals, nutritional products and biofuels. Genetic engineering could speed up the development of microalgae derived products and reduce the overall production costs. Genetic methods such as particle bombardment, electroporation, Agrobacterium tumefaciens mediated transformation (ATMT), and agitation with glass beads and silicon carbide whiskers have been developed for the genetic transformation of microalgae. However, the transformation efficiency is species dependent, so a variety of transformation methods are required to engineer a wide range of microalgae species. The oleaginous microalgae Acutodesmus obliquus and Neochloris oleoabundans have a great potential as production platforms due to their ability to produce large amounts of triacylglycerol (TAG). Genetic modification techniques however are required to increase TAG levels further or to modify the fatty acid composition. Recently, a conjugation-based method for the delivery of episomes from bacteria to diatom microalgae has been reported. In this study, we have achieved the successful transformation of green oleaginous microalgal strains by transferring an expression vector via conjugation from E. coli. Since delivery of exogenous DNA into the microalgae cells is only the first step in obtaining transgenic microalgae, we further analyzed transformation efficiencies by PCR and expression of the Clover fluorescent protein in the targeted species.</p

Large Intergenic Cruciform-Like Supermotifs in the Lactobacillus plantarum Genome▿ †

Twenty-four Lactobacillus plantarum supermotifs (LPSMs) with lengths from ∼800 to 1,000 nucleotides were identified in the L. plantarum genome. LPSMs were conserved in other L. plantarum strains but not in other species. Secondary structure analysis predicted that LPSMs may fold into cruciform-like structures. Preliminary experiments indicate that the LPSMs are transcribed