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

An agr-Like Two-Component Regulatory System in Lactobacillus plantarum Is Involved in Production of a Novel Cyclic Peptide and Regulation of Adherence

We have analyzed a locus on the annotated Lactobacillus plantarum WCFS1 genome that showed homology to the staphylococcal agr quorum-sensing system and designated it lam for Lactobacillus agr-like module. Production of the lamBDCA transcript was shown to be growth phase dependent. Analysis of a response regulator-defective mutant (ΔlamA) in an adherence assay showed that lam regulates adherence of L. plantarum to a glass surface. Global transcription analysis of the wild-type and ΔlamA strains in early, mid-, and late log phase of growth was performed using a clone-based microarray. Remarkably, only a small set of genes showed significant differences in transcription profiles between the wild-type and lamA mutant strains. The microarray analysis confirmed that lamBDCA is autoregulatory and showed that lamA is involved in regulation of expression of genes encoding surface polysaccharides, cell membrane proteins, and sugar utilization proteins. The lamBD genes encoding the putative autoinducing peptide precursor (LamD) and its processing protein (LamB) were overexpressed using the nisin-controlled expression system, and culture supernatants were analyzed by liquid chromatography/mass spectrometry (LC/MS) to identify overproduced LamD-derived peptides. In this way, a cyclic thiolactone pentapeptide that possesses a ring structure similar to those of autoinducing peptides of the staphylococcal agr system was identified. The peptide was designated LamD558, and its sequence (CVGIW) matched the annotated precursor peptide sequence. Time course analysis of wild-type culture supernatants by LC/MS indicated that LamD558 production was increased markedly from mid-log to late log growth phase. This is the first example of an agr-like system in nonpathogenic bacteria that encodes a cyclic thiolactone autoinducing peptide and is involved in regulation of adherence

Morphological heterogeneity of Paracoccidioides brasiliensis: relevance of the Rho-like GTPase PbCDC42

We thank Rosana Puccia for providing isolate Pb02.Paracoccidioides brasiliensis budding pattern and polymorphic growth were previously shown to be closely linked to the expression of PbCDC42 and to infl uence the pathogenesis of the fungus. In this work we conducted a detailed morphogenetic evaluation of the yeast-forms of 11 different clinical and environmental P. brasiliensis isolates comprising four phylogenetic lineages (S1, PS2, PS3 and Pb01-like), as well as a PbCDC42 knockdown strain. High variations in the shape and size of mother and bud cells of each isolate were observed but we did not fi nd a characteristic morphologic profi le for any of the phylogenetic groups. In all isolates studied, the bud size and shape were demonstrated to be highly dependent on the mother cell. Importantly, we found strong correlations between PbCDC42 expression and both the shape of mother and bud cells and the size of the buds in all isolates and the knock-down strain. Our results suggested that PbCDC42 expression can explain approximately 80% of mother and bud cell shape and 19% of bud cell size. This data support PbCDC42 expression level as being a relevant predictor of P. brasiliensis morphology. Altogether, these fi ndings quantitatively describe the polymorphic nature of the P. brasiliensis yeast form and provide additional support for the key role of PbCDC42 expression on yeast cell morphology.J.F.M. was supported by Fundação para a Ciência e Tecnologia (FCT), Portugal (SFRH/BD/33446/2008).This work was supported by a grant from FCT (PTDC/BIA-MIC/108309/2008)

Strains used in this study.

a<p>–Plasmids listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047033#pone-0047033-t002" target="_blank">Table 2</a>.</p>b<p>–Phylogenetic species as defined by Matute et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047033#pone.0047033-Matute1" target="_blank">[17]</a> and Teixeira et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047033#pone.0047033-Teixeira1" target="_blank">[18]</a>.</p

α-pheromone activates PreB receptor, inducing growth and cell cycle arrest in the <i>S. cerevisiae</i> model.

<p>Halo formation in <i>S. cerevisiae</i> AGLPreB stimulated with Pbα pheromone extracted from <i>P. lutzii</i> Pb01 monoculture (A); stimulated with 0.8, 4.0 and 40 µg synthetic Pbα pheromone (B–D respectively) or Pbα pheromone extracted from <i>S. cerevisiae</i> AGMPbα (E). <i>S. cerevisiae</i> BY4741 stimulated with 10 µg synthetic MFα (F) and with 20 µg synthetic Pbα (G). Quantification of growth inhibition zones (cm²) upon exposure to α-pheromones. Halos were recorded after 24 hrs of incubation and standard deviations indicated by error bars (H). Cell cycle arrest analysis for <i>S. cerevisiae</i> BY4741 upon addition of synthetic MFα (20 µg/ml) (I) and <i>S. cerevisiae</i> AGLPreB upon addition of synthetic Pbα pheromone (40 µg/ml) (J). (K–N) FCM profiles of α-pheromone-induced cell cycle arrest in haploid cells showing n and 2 n nuclear DNA content: BY4741 induced with 20 µg synthetic MFα (K–L) and <i>S. cerevisiae</i> AGLPreB induced with 40 µg synthetic Pbα pheromone (M–N). Growth rate evaluation of either <i>S. cerevisiae</i> BY4741 (O) or AGLPreB (P) upon addition of the cognate synthetic α-pheromone. Error bars are indicated, and asterisks show significant differences (<i>*</i>p<0.05, <i>**</i>p<0.01).</p

The genome of <i>Paracoccidioides</i> encodes an α-pheromone.

<p>(A) Mating pheromone signaling pathway described in <i>S. cerevisiae </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047033#pone.0047033-Butler1" target="_blank">[21]</a>. (B) <i>S. cerevisiae</i> mating pheromone signaling pathway components and the homologous genes annotated in <i>Paracoccidioides</i> and <i>A. fumigatus</i> databases. Genes not annotated in databases are denoted N.A. (C) Alignment of α-pheromone precursor peptide sequences from <i>Paracoccidioides</i> strains and <i>H. capsulatum</i>. The Kex2 peptidase recognition site (KR) and the mature pheromone are indicated by blue and red boxes, respectively.</p

<i>Paracoccidioides</i> expresses the α-pheromone and the a- and α-receptor genes independently of the mating type.

<p>Mating gene expression of <i>Paracoccidioides MAT1-1</i> strains (Pb01 and T8B1) and <i>MAT1-2</i> strains (ATCC60855 and Pb03) in yeast (Y) and mycelial (M) form. Expression levels of <i>MAT1-1</i> (A) and <i>MAT1-2</i> (B), <i>PBα</i> (B, C), <i>PREB</i> (E, F) and <i>PREA</i> (G, H) genes were measured. Error bars are indicated, and asterisks show significant differences (<i>*</i>p<0.05 or <i>**</i>p<0.01).</p