Differential gene expression analysis of Paracoccidioides brasiliensis during keratinocyte infection

Abstract

Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis, one of the most important systemic fungal diseases in Latin America. This initiates in lung tissue and can subsequently disseminate to other tissues. Clinical manifestations range from localized forms to disseminated disease that can progress to lethality, probably depending on the relationships among the virulence of the fungus, the immune response and the ability to interact with the surface structures and invade epithelial cells and mononuclear cells of the host. It is generally regarded as a multifocal disease, with oral lesions as the prominent feature. The aim of this study was to evaluate P. brasiliensis yeast infection in normal oral keratinocytes (NOKs). The differential expression of mRNAs and proteins was also determined when the fungus was placed in contact with the cell in order to characterize differentially expressed genes and proteins during P. brasiliensis infection. After contact with NOKs, the fungus appeared to induce alterations in the cells, which showed cellular extensions and cavitations, probably resulting from changes in the actin cytoskeleton seen at 5 and 8 h after infection. Levels of protein expression were higher after reisolation of the fungus from infected NOK culture compared with culture of the fungus in medium. The analysis identified transcripts related to 19 proteins involved in different biological processes. Transcripts were found with multiple functions including induction of cytokines, protein metabolism, alternative carbon metabolism, zinc transport and the stress response during contact with NOKs. The proteins found suggested that the yeast was in a stress situation, as indicated by the presence of RDS1. Nevertheless, the yeast seemed to be proliferating and metabolically active, as shown by the presence of a proteasome, short-chain acetylator, glucosamine-6-phosphate isomerase and ADP/ATP carrier transcripts. Additionally, metabolic pathways may have been activated in order to eliminate toxic substances from the cell as a zinc transporter was detected, which is a potential target for the development of future drugs. INTRODUCTIONParacoccidioides brasiliensis is a thermodimorphic pathogenic fungus, the aetiological agent of paracoccidioidomycosis (PCM), an endemic disease geographically limited to Latin America and one of the most prevalent human deep systemic mycoses found in Brazil, Colombia and Venezuela (San-Blas et al., 2002). The transition from mycelium at 25 u C to yeast at 37 u C is essential for P. brasiliensis to establish the disease (Franco et al., 1997). P. brasiliensis may be inhaled and, once in the lungs, the fungus initiates a process of morphological transition into the pathogenic yeast form (San-Blas et al., 2002).PCM has a multiplicity of clinical presentations, from cutaneous to systemic forms, and can attack various tissues, especially the lungs (Benard & Franco, 2007). Oral mucosal lesions are commonly multiple, with a granular, mulberry-like surface and microulcerations, and ...

Gene Expression of Putative Pathogenicity-Related Genes in Verticillium dahliae in Response to Elicitation with Potato Extracts and during Infection Using Quantitative Real-Time PCR

“…Ubiquitin-conjugating enzyme variant MMS2 transcripts contribute more in the highly aggressive isolate during the infection process. This is also similar to the findings on the human pathogen Paracoccidioides brasiliensis , where MMS2 was significantly elevated while infecting oral keratinocytes cell [ 86 ], indicating its function during infection.…”

Importance of adhesins in virulence of Paracoccidioides spp.

“…The data were analyzed using the 2 −ΔΔCT method. The cycle threshold values for the triplicate PCRs of each RNA sample were averaged, and then the 2 −ΔΔCT values were calculated using the control gene 60S ribosomal L34, which is a constitutive housekeeping gene that is widely used as a control in different studies of paracoccidioidomycosis (Felipe et al, 2005a ; Bailão et al, 2006 , 2007 ; Brito et al, 2011 ; Parente et al, 2011 ; Peres da Silva et al, 2011 ; Lima et al, 2014 ). A sample that contained all reagents except P. brasiliensis cDNA was used as a negative control.…”

Paracoccidioidomycosis: Current Perspectives from Brazil

“…The structures of the cytoskeletons of pulmonary epithelial cells and keratinocytes and their morphological features, including actin, tubulin and cytokeratin, could be affected by the interactions between the host and Paracoccidioides sp [ 127 , 128 ]. Keratinocyte parasitism may represent a possible mechanism by which fungal cells can evade local immune mechanisms [ 129 ].…”