Impact of Paracoccin Gene Silencing on Paracoccidioides brasiliensis Virulence

ABSTRACT Among the endemic deep mycoses in Latin America, paracoccidioidomycosis (PCM), caused by thermodimorphic fungi of the Paracoccidioides genus, is a major cause of morbidity. Disease development and its manifestations are associated with both host and fungal factors. Concerning the latter, several recent studies have employed the methodology of gene modulation in P. brasiliensis using antisense RNA (AsRNA) and Agrobacterium tumefaciens-mediated transformation (ATMT) to identify proteins that influence fungus virulence. Our previous observations suggested that paracoccin (PCN), a multidomain fungal protein with both lectin and enzymatic activities, may be a potential P. brasiliensis virulence factor. To explore this, we used AsRNA and ATMT methodology to obtain three independent PCN-silenced P. brasiliensis yeast strains (AsPCN1, AsPCN2, and AsPCN3) and characterized them with regard to P. brasiliensis biology and pathogenicity. AsPCN1, AsPCN2, and AsPCN3 showed relative PCN expression levels that were 60%, 40%, and 60% of that of the wild-type (WT) strain, respectively. PCN silencing led to the aggregation of fungal cells, blocked the morphological yeast-to-mycelium transition, and rendered the yeast less resistant to macrophage fungicidal activity. In addition, mice infected with AsPCN1, AsPCN2, and AsPCN3 showed a reduction in fungal burden of approximately 96% compared with those inoculated with the WT strain, which displayed a more extensive destruction of lung tissue. Finally, mice infected with the PCN-silenced yeast strains had lower mortality than those infected with the WT strain. These data demonstrate that PCN acts as a P. brasiliensis contributory virulence factor directly affecting fungal pathogenesis. IMPORTANCE The nonexistence of efficient genetic transformation systems has hampered studies in the dimorphic fungus Paracoccidioides brasiliensis, the etiological agent of the most frequent systemic mycosis in Latin America. The recent development of a method for gene expression knockdown by antisense RNA technology, associated with an Agrobacterium tumefaciens-mediated transformation system, provides new strategies for studying P. brasiliensis. Through this technology, we generated yeasts that were silenced for paracoccin (PCN), a P. brasiliensis component that has lectin and enzymatic properties. By comparing the phenotypes of PCN-silenced and wild-type strains of P. brasiliensis, we identified PCN as a virulence factor whose absence renders the yeasts unable to undergo the transition to mycelium and causes a milder pulmonary disease in mice, with a lower mortality rate. Our report highlights the importance of the technology used for P. brasiliensis transformation and demonstrates that paracoccin is a virulence factor acting on fungal biology and pathogenesis. IMPORTANCE The nonexistence of efficient genetic transformation systems has hampered studies in the dimorphic fungus Paracoccidioides brasiliensis, the etiological agent of the most frequent systemic mycosis in Latin America. The recent development of a method for gene expression knockdown by antisense RNA technology, associated with an Agrobacterium tumefaciens-mediated transformation system, provides new strategies for studying P. brasiliensis. Through this technology, we generated yeasts that were silenced for paracoccin (PCN), a P. brasiliensis component that has lectin and enzymatic properties. By comparing the phenotypes of PCN-silenced and wild-type strains of P. brasiliensis, we identified PCN as a virulence factor whose absence renders the yeasts unable to undergo the transition to mycelium and causes a milder pulmonary disease in mice, with a lower mortality rate. Our report highlights the importance of the technology used for P. brasiliensis transformation and demonstrates that paracoccin is a virulence factor acting on fungal biology and pathogenesis.

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EXPERIMENTAL PARACOCCIDIOIDOMYCOSIS IN PREGNANT RATS

Revista do Instituto de Medicina Tropical de São Paulo ◽

10.1590/s0036-46652015000600010 ◽

2015 ◽

Vol 57 (6) ◽

pp. 515-518 ◽

Cited By ~ 1

Author(s):

Eduardo Alexandre LOTH ◽

Vanessa CECATTO ◽

Samia Khalil BIAZIM ◽

José Henrique Fermino FERREIRA ◽

Caroline DANIELLI ◽

...

Keyword(s):

Latin America ◽

Wistar Rats ◽

Paracoccidioides Brasiliensis ◽

Severe Infection ◽

Pathological Examination ◽

Lower Body ◽

Systemic Mycosis ◽

Dimorphic Fungus ◽

Pregnant Rats ◽

Experimental Paracoccidioidomycosis

Paracoccidioidomycosis (PCM), caused by the dimorphic fungus Paracoccidioides brasiliensis (Pb), is the most prevalent systemic mycosis in Latin America. There are few reports in the literature about the disease damages during pregnancy and the consequences to the fetuses and breeding. This study evaluated the implications of PCM during pregnancy on offspring and mothers in Wistar rats. Groups of rats were submitted to systemic Pb infection, by intraperitoneal infusion, and mated 30 days after the infection date. Immediately after birth, rats and neonates were sacrificed to obtain organs for standard histological examination, morphometric analysis, fungi recovery by plating (CFU) and dosing of anti-Pb antibodies by ELISA. There were no stillbirths or miscarriages, however, the fetuses from infected pregnant rats had lower body and organ weight but the fertility rate was 100%. The largest number of CFU was recovered from the organ of pregnant rats, the pathological examination revealed more severe infection in the same group, further on the largest number of granulomas and fungal field. It can be concluded that the PCM was more severe in the group of pregnant rats, with implications to the weight of offspring.

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Biliary Paracoccidioidomycosis: An Unusual Infection Simulating Malignant Neoplasm

Annals of Hepatology ◽

10.5604/01.3001.0012.2235 ◽

2018 ◽

Vol 17 (5) ◽

pp. 0-10

Author(s):

Klaus Steinbrück ◽

Reinaldo Fernandes

Keyword(s):

Differential Diagnosis ◽

Latin America ◽

Lymph Nodes ◽

Paracoccidioides Brasiliensis ◽

Case Series ◽

Malignant Neoplasm ◽

Granulomatous Disease ◽

Dimorphic Fungus ◽

Abdominal Organs ◽

Endemic Areas

Paracoccidioidomycosis is a systemic granulomatous disease caused by the dimorphic fungus Paracoccidioides brasiliensis and is restricted to Latin America. It normally affects lungs, skin and lymph nodes. Abdominal organs are usually not involved. In rare cases paracoccidioidomycosis may simulate neoplasm. Herein we describe our experience with four cases of paracoccidioidomycosis mimicking cholangiocarcinoma. To the best of our knowledge, this is the largest case series on this subject produced in English. Paracoccidioidomycosis must be considered as a differential diagnosis of cholangiocarcinoma, especially in individuals who come from endemic areas.

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Farnesol secretion as a possible driving force for maintaining Candida albicans as a diploid

Access Microbiology ◽

10.1099/acmi.cc2021.po0117 ◽

2021 ◽

Vol 3 (12) ◽

Author(s):

Kenneth Nickerson ◽

Cory Boone ◽

Kory Parker

Keyword(s):

Candida Albicans ◽

Quorum Sensing ◽

Mating Type ◽

Virulence Factor ◽

Driving Force ◽

Prior Work ◽

Wild Type ◽

Dimorphic Fungus ◽

Peptide Pheromone ◽

Quorum Sensing Molecule

Candida albicans is a pathogenic dimorphic fungus which is invariably found as a diploid in patients. C. albicans secretes the sesquiterpene farnesol both as a quorum sensing molecule which blocks the yeast to hypha conversion and as a virulence factor for pathogenicity. 20-25 μM farnesol kills other competing yeasts and fungi, often by triggering apoptosis, and yet wild type diploid C. albicans tolerates 300-500 μM farnesol. The recent availability of 10 haploid strains of C. albicans (5 mating type aand 5 mating type α) allowed us to compare their production of and sensitivity to farnesol. On average, the heterozygous diploid strains of C. albicans were 2.4 times more resistant to 20-40 μM farnesol than MTLa haploid cells and 4.6 times more resistant than MTLα haploid cells. Furthermore, the MTLa haploids produce approximately 10 times more farnesol than do the MTLα haploids. Prior work concluded that haploid strains exhibited such low fitness that C. albicans was thought to be an obligate diploid. We now suggest that increased farnesol secretion by the MTLa haploids and increased farnesol sensitivity of the MTLα haploids is a mechanism for maintaining the dominant heterozygous diploid status of C. albicans. This idea is based on the observation that the a-factor peptide pheromone is farnesylated but the α-factor pheromone is not farnesylated. Our working hypothesis is that farnesol is secreted in part via Ste6 and imported in part via Ste3, the proteins which export and import the farnesylated a-pheromone. We also examined whether farnesol was excreted in extracellular vesicles.

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Effects of an Autoregulatory Senescence-inhibitor Gene Construct on Nicotiana alata Link and Otto.

HortScience ◽

10.21273/hortsci.33.3.519d ◽

1998 ◽

Vol 33 (3) ◽

pp. 519d-519 ◽

Cited By ~ 4

Author(s):

Kenneth R. Schroeder ◽

Dennis P. Stimart

Keyword(s):

Agrobacterium Tumefaciens ◽

Plant Height ◽

Dry Weight ◽

Nicotiana Alata ◽

Wild Type ◽

Gene Encoding ◽

Delayed Senescence ◽

Gene Construct ◽

Shoot Dry Weight ◽

Inhibitor System

Nicotiana alata Link and Otto. was transformed via Agrobacterium tumefaciens encoding a senescence-specific promoter SAG12 cloned from Arabidopsis thaliana fused to a Agrobacterium tumefaciens gene encoding isopentenyl transferase (IPT) that catalyzes cytokinin synthesis. This was considered an autoregulatory senescence-inhibitor system. In 1996, we reported delayed senescence of intact flowers by 2 to 6 d and delayed leaf senescence of transgenic vs. wild-type N. alata. Further evaluations in 1997 revealed several other interesting effects of the SAG12-IPT gene construct. Measurement of chlorophyll content of mature leaves showed higher levels of both chlorophyll a and b in transgenic material under normal fertilization and truncated fertilization regimes. At 4 to 5 months of age transgenic plants expressed differences in plant height, branching, and dry weight. Plant height was reduced by 3 to 13 cm; branch counts increased 2 to 3 fold; and shoot dry weight increased up to 11 g over wild-type N. alata. These observations indicate the system is not tightly autoregulated and may prove useful to the floriculture industry for producing compact and more floriferous plants.

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Optimizing Transformation Frequency of Cryptococcus neoformans and Cryptococcus gattii Using Agrobacterium tumefaciens

Journal of Fungi ◽

10.3390/jof7070520 ◽

2021 ◽

Vol 7 (7) ◽

pp. 520

Author(s):

Jianmin Fu ◽

Nohelli E. Brockman ◽

Brian L. Wickes

Keyword(s):

Agrobacterium Tumefaciens ◽

Yeast Cell ◽

Transformation Efficiency ◽

Transformation Frequency ◽

Yeast Cells ◽

Agar Concentration ◽

Genetic Tool ◽

Number Of Factors ◽

Cryptococcus Spp ◽

Transformation Systems

The transformation of Cryptococcus spp. by Agrobacterium tumefaciens has proven to be a useful genetic tool. A number of factors affect transformation frequency. These factors include acetosyringone concentration, bacterial cell to yeast cell ratio, cell wall damage, and agar concentration. Agar concentration was found to have a significant effect on the transformant number as transformants increased with agar concentration across all four serotypes. When infection time points were tested, higher agar concentrations were found to result in an earlier transfer of the Ti-plasmid to the yeast cell, with the earliest transformant appearing two h after A. tumefaciens contact with yeast cells. These results demonstrate that A. tumefaciens transformation efficiency can be affected by a variety of factors and continued investigation of these factors can lead to improvements in specific A. tumefaciens/fungus transformation systems.

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MetR-Regulated Vibrio cholerae Metabolism Is Required for Virulence

mBio ◽

10.1128/mbio.00236-12 ◽

2012 ◽

Vol 3 (5) ◽

Cited By ~ 28

Author(s):

Ryan W. Bogard ◽

Bryan W. Davies ◽

John J. Mekalanos

Keyword(s):

Amino Acid ◽

Vibrio Cholerae ◽

Virulence Factor ◽

Current Knowledge ◽

Intestinal Colonization ◽

Wild Type ◽

Pathogenic Potential ◽

Content Type ◽

Mouse Intestine

ABSTRACTLysR-type transcriptional regulators (LTTRs) are the largest, most diverse family of prokaryotic transcription factors, with regulatory roles spanning metabolism, cell growth and division, and pathogenesis. Using a sequence-defined transposon mutant library, we screened a panel ofV. choleraeEl Tor mutants to identify LTTRs required for host intestinal colonization. Surprisingly, out of 38 LTTRs, only one severely affected intestinal colonization in the suckling mouse model of cholera: the methionine metabolism regulator, MetR. Genetic analysis of genes influenced by MetR revealed thatglyA1andmetJwere also required for intestinal colonization. Chromatin immunoprecipitation of MetR and quantitative reverse transcription-PCR (qRT-PCR) confirmed interaction with and regulation ofglyA1, indicating that misregulation ofglyA1is likely responsible for the colonization defect observed in themetRmutant. TheglyA1mutant was auxotrophic for glycine but exhibited wild-type trimethoprim sensitivity, making folate deficiency an unlikely cause of its colonization defect. MetJ regulatory mutants are not auxotrophic but are likely altered in the regulation of amino acid-biosynthetic pathways, including those for methionine, glycine, and serine, and this misregulation likely explains its colonization defect. However, mutants defective in methionine, serine, and cysteine biosynthesis exhibited wild-type virulence, suggesting that these amino acids can be scavenged in vivo. Taken together, our results suggest that glycine biosynthesis may be required to alleviate an in vivo nutritional restriction in the mouse intestine; however, additional roles for glycine may exist. Irrespective of the precise nature of this requirement, this study illustrates the importance of pathogen metabolism, and the regulation thereof, as a virulence factor.IMPORTANCEVibrio choleraecontinues to be a severe cause of morbidity and mortality in developing countries. Identification ofV. choleraefactors critical to disease progression offers the potential to develop or improve upon therapeutics and prevention strategies. To increase the efficiency of virulence factor discovery, we employed a regulator-centric approach to multiplex our in vivo screening capabilities and allow whole regulons inV. choleraeto be interrogated for pathogenic potential. We identified MetR as a new virulence regulator and serine hydroxymethyltransferase GlyA1 as a new MetR-regulated virulence factor, both required byV. choleraeto colonize the infant mouse intestine. Bacterial metabolism is a prerequisite to virulence, and current knowledge of in vivo metabolism of pathogens is limited. Here, we expand the known role of amino acid metabolism and regulation in virulence and offer new insights into the in vivo metabolic requirements ofV. choleraewithin the mouse intestine.

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Paracoccidioides brasiliensis Isolated from Nine-Banded Armadillos (Dasypus novemcinctus) Reveal Population Structure and Admixture in the Amazon Basin

Journal of Fungi ◽

10.3390/jof7010054 ◽

2021 ◽

Vol 7 (1) ◽

pp. 54

Author(s):

Eduardo Bagagli ◽

Daniel Ricardo Matute ◽

Hans Garcia Garces ◽

Bernardo Guerra Tenório ◽

Adalberto Garcia Garces ◽

...

Keyword(s):

Population Structure ◽

Latin America ◽

Endemic Area ◽

Amazon Basin ◽

Paracoccidioides Brasiliensis ◽

Pathogenic Fungi ◽

Sensu Stricto ◽

Species Level ◽

Dasypus Novemcinctus ◽

Show Evidence

Paracoccidioidomycosis is an endemic fungal disease to Latin America caused by at least five species-level genotypes of Paracoccidioides, named P. lutzii, P. brasiliensis (S1a and S1b populations), P. americana, P. restrepiensis, and P. venezuelensis. In this manuscript, we report on Paracoccidioides sp. sampling efforts in armadillos from two different areas in Brazil. We sequenced the genomes of seven Paracoccidioides isolates and used phylogenomics and populations genetics for genotyping. We found that P. brasiliensis and P. lutzii are both present in the Amazon region. Additionally, we identified two Paracoccidioides isolates that seem to be the result of admixture between divergent populations within P. brasiliensis sensu stricto. Both of these isolates were recovered from armadillos in a P. lutzii endemic area in Midwestern Brazil. Additionally, two isolates from human patients also show evidence of resulting from admixture. Our results suggest that the populations of P. brasiliensis sensu stricto exchange genes in nature. More generally, they suggest that population structure and admixture within species is an important source of variation for pathogenic fungi.

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Quorum Sensing: a Transcriptional Regulatory System Involved in the Pathogenicity of Burkholderia mallei

Infection and Immunity ◽

10.1128/iai.72.11.6589-6596.2004 ◽

2004 ◽

Vol 72 (11) ◽

pp. 6589-6596 ◽

Cited By ~ 57

Author(s):

Ricky L. Ulrich ◽

David DeShazer ◽

Harry B. Hines ◽

Jeffrey A. Jeddeloh

Keyword(s):

Quorum Sensing ◽

Virulence Factor ◽

Regulatory System ◽

In Silico Analysis ◽

Homoserine Lactone ◽

Burkholderia Mallei ◽

Wild Type ◽

Transcriptional Regulatory ◽

A Cell

ABSTRACT Numerous gram-negative bacterial pathogens regulate virulence factor expression by using a cell density mechanism termed quorum sensing (QS). An in silico analysis of the Burkholderia mallei ATCC 23344 genome revealed that it encodes at least two luxI and four luxR homologues. Using mass spectrometry, we showed that wild-type B. mallei produces the signaling molecules N-octanoyl-homoserine lactone and N-decanoyl-homoserine lactone. To determine if QS is involved in the virulence of B. mallei, we generated mutations in each putative luxIR homologue and tested the pathogenicities of the derivative strains in aerosol BALB/c mouse and intraperitoneal hamster models. Disruption of the B. mallei QS alleles, especially in RJ16 (bmaII) and RJ17 (bmaI3), which are luxI mutants, significantly reduced virulence, as indicated by the survival of mice who were aerosolized with 104 CFU (10 50% lethal doses [LD50s]). For the B. mallei transcriptional regulator mutants (luxR homologues), mutation of the bmaR5 allele resulted in the most pronounced decrease in virulence, with 100% of the challenged animals surviving a dose of 10 LD50s. Using a Syrian hamster intraperitoneal model of infection, we determined the LD50s for wild-type B. mallei and each QS mutant. An increase in the relative LD50 was found for RJ16 (bmaI1) (>967 CFU), RJ17 (bmaI3) (115 CFU), and RJ20 (bmaR5) (151 CFU) compared to wild-type B. mallei (<13 CFU). These findings demonstrate that B. mallei carries multiple luxIR homologues that either directly or indirectly regulate the biosynthesis of an essential virulence factor(s) that contributes to the pathogenicity of B. mallei in vivo.

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