Virginia Carvalhais

Dormancy within Staphylococcus epidermidis biofilms: a transcriptomic analysis by RNA-seq

The proportion of dormant bacteria within Staphylococcus epidermidis biofilms may determine its inflammatory profile. Previously, we have shown that S. epidermidis biofilms with higher proportions of dormant bacteria have reduced activation of murine macrophages. RNA-sequencing was used to identify the major transcriptomic differences between S. epidermidis biofilms with different proportions of dormant bacteria. To accomplish this goal, we used an in vitro model where magnesium allowed modulation of the proportion of dormant bacteria within S. epidermidis biofilms. Significant differences were found in the expression of 147 genes. A detailed analysis of the results was performed based on direct and functional gene interactions. Biological processes among the differentially expressed genes were mainly related to oxidation-reduction processes and acetyl-CoA metabolic processes. Gene set enrichment revealed that the translation process is related to the proportion of dormant bacteria. Transcription of mRNAs involved in oxidation-reduction processes was associated with higher proportions of dormant bacteria within S. epidermidis biofilm. Moreover, the pH of the culture medium did not change after the addition of magnesium, and genes related to magnesium transport did not seem to impact entrance of bacterial cells into dormancy.

Dormant bacteria within Staphylococcus epidermidis biofilms have low inflammatory properties and maintain tolerance to vancomycin and penicillin after entering planktonic growth

Staphylococcus epidermidis is the most commonly isolated aetiological agent of nosocomial infections, mainly due to its ability to establish biofilms on indwelling medical devices. Detachment of bacteria from S. epidermidis biofilms and subsequent growth in the planktonic form is a hallmark of the pathogenesis of these infections leading to dissemination. Here we showed that S. epidermidis cells collected from biofilms cultured in conditions that promote cell viability present marked changes in their physiological status upon initiating a planktonic mode of growth. When compared to cells growing in biofilms, they displayed an increased SYBR green I staining intensity, increased transcription of the rpiA gene, decreased transcription of the icaA gene, as well as higher susceptibility to vancomycin and penicillin. When bacteria collected from biofilms with high proportions of dormant cells were subsequently cultured in the planktonic mode, a large proportion of cells maintained a low SYBR green I staining intensity and increased resistance to vancomycin and penicillin, a profile typical of dormant cells. This phenotype further associated with a decreased ability of these biofilm-derived cells to induce the production of pro-inflammatory cytokines by bone marrow-derived dendritic cells in vitro. These results demonstrated that cells detached from the biofilm maintain a dormant cell-like phenotype, having a low pro-inflammatory effect and decreased susceptibility to antibiotics, suggesting these cells may contribute to the recalcitrant nature of biofilm infections.

Alterations in the Staphylococcus epidermidis biofilm transcriptome following interaction with whole human blood

Staphylococcus epidermidis biofilm formation on the surface of intravenous catheters is responsible for 22% of the cases of bloodstream infections, in patients in intensive care units in the USA. The ability of S. epidermidis to withstand the high bactericidal activity of human blood is therefore crucial for systemic dissemination. To identify the genes involved in the bacteriums survival, the transcriptome of S. epidermidis biofilms, upon contact with human blood, was assessed using an ex vivo model. Our results showed an increased transcription of genes involved in biosynthesis and metabolism of amino acids, small molecules, carboxylic and organic acids, and cellular ketones. One of the striking changes observed 4 h of S. epidermidis exposure to human blood was an increased expression of genes involved in iron utilization. This finding suggests that iron acquisition is an important event for S. epidermidis survival in human blood.

Proteomic profile of dormancy within Staphylococcus epidermidis biofilms using iTRAQ and label-free strategies

Staphylococcus epidermidis is an important nosocomial bacterium among carriers of indwelling medical devices, since it has a strong ability to form biofilms. The presence of dormant bacteria within a biofilm is one of the factors that contribute to biofilm antibiotic tolerance and immune evasion. Here, we provide a detailed characterization of the quantitative proteomic profile of S. epidermidis biofilms with different proportions of dormant bacteria. A total of 427 and 409 proteins were identified by label-free and label-based quantitative methodologies, respectively. From these, 29 proteins were found to be differentially expressed between S. epidermidis biofilms with prevented and induced dormancy. Proteins overexpressed in S. epidermidis with prevented dormancy were associated with ribosome synthesis pathway, which reflects the metabolic state of dormant bacteria. In the opposite, underexpressed proteins were related to catalytic activity and ion binding, with involvement in purine, arginine, and proline metabolism. Additionally, GTPase activity seems to be enhanced in S. epidermidis biofilm with induced dormancy. The role of magnesium in dormancy modulation was further investigated with bioinformatics tool based in predicted interactions. The main molecular function of proteins, which strongly interact with magnesium, was nucleic acid binding. Different proteomic strategies allowed to obtain similar results and evidenced that prevented dormancy led to an expression of a markedly different repertoire of proteins in comparison to the one of dormant biofilms.

Anti-tumoral activity of human salivary peptides

Chemotherapy continues to be the standard treatment for advanced or metastasized cancer. However, commonly used chemotherapeutic agents may induce damage in healthy cells and tissues. Thus, in recent years, there has been an increased focus on the development of new, efficient anticancer drugs exhibiting low toxicity and that are not affected by mechanisms of chemoresistance. In the present work, we tested synthetic and naturally obtained human salivary peptides against breast, prostate, colon, osteosarcoma and bladder cancer cell lines (T47-D, PC-3, HT-29, MG63, T-24, respectively). Results have showed that there is a reduced cell population increase that is peptide-, cell- and possibly pathway-specific, with the most potent effect observed in observed in T-47D breast cancer cells. Protein expression and microscopy results further indicate that, in this cell line, the peptide with the sequence GPPPQGGRPQG (GG peptide) interferes with the ability of cell adhesion proteins to stabilize adherens junctions, such as E-cadherin, leading to apoptosis. These promising results encourage future works aimed at disclosing the vast potential of salivary peptides as new therapeutic agents.

An immunoproteomic approach for characterization of dormancy within Staphylococcus epidermidis biofilms.

Virulence of Staphylococcus epidermidis is mainly attributed to surface colonization and biofilm formation in indwelling medical devices. Physiological heterogeneity of biofilms may influence host immune response and sensitivity to antibiotics. Dormant cells, among others, contribute to biofilm heterogeneity. The aim of this study was to identify immunogenic proteins of S. epidermidis biofilms associated with dormancy mechanism, by using two-dimensional electrophoresis (2-DE) immunoblotting and mass spectrometry (MS). A total of 19 bacterial proteins, recognized by human serum samples, were identified. These proteins were mainly involved in small molecule metabolic biological processes. Catalytic activity and ion binding were the most representative molecular functions. CodY and GpmA proteins were more reactive to sera when biofilm dormancy was induced, while FtnA and ClpP were more reactive when dormancy was prevented. This is the first work that identifies differences in immunoreactive proteins within bacterial biofilms with induced or prevented dormancy. Considering the importance of dormancy within biofilms, further evaluation of these proteins can provide insights into the mechanisms related to dormancy and help to improve current understanding on how dormancy affects the host immune response.

Proteome signatures—how are they obtained and what do they teach us?

The dawn of a new Proteomics era, just over a decade ago, allowed for large-scale protein profiling studies that have been applied in the identification of distinctive molecular cell signatures. Proteomics provides a powerful approach for identifying and studying these multiple molecular markers in a vast array of biological systems, whether focusing on basic biological research, diagnosis, therapeutics, or systems biology. This is a continuously expanding field that relies on the combination of different methodologies and current advances, both technological and analytical, which have led to an explosion of protein signatures and biomarker candidates. But how are these biological markers obtained? And, most importantly, what can we learn from them? Herein, we briefly overview the currently available approaches for obtaining relevant information at the proteome level, while noting the current and future roles of both traditional and modern proteomics. Moreover, we provide some considerations on how the development of powerful and robust bioinformatics tools will greatly benefit high-throughput proteomics. Such strategies are of the utmost importance in the rapidly emerging field of immunoproteomics, which may play a key role in the identification of antigens with diagnostic and/or therapeutic potential and in the development of new vaccines. Finally, we consider the present limitations in the discovery of new signatures and biomarkers and speculate on how such hurdles may be overcome, while also offering a prospect for the next few years in what could be one of the most significant strategies in translational medicine research.

Characterization of an in vitro fed-batch model to obtain cells released from S. epidermidis biofilms

Both dynamic and fed-batch systems have been used for the study of biofilms. Dynamic systems, whose hallmark is the presence of continuous flow, have been considered the most appropriate for the study of the last stage of the biofilm lifecycle: biofilm disassembly. However, fed-batch is still the most used system in the biofilm research field. Hence, we have used a fed-batch system to collect cells released from Staphylococcus epidermidis biofilms, one of the most important etiological agents of medical device-associated biofilm infections. Herein, we showed that using this model it was possible to collect cells released from biofilms formed by 12 different S. epidermidis clinical and commensal isolates. In addition, our data indicated that biofilm disassembly occurred by both passive and active mechanisms, although the last occurred to a lesser extent. Moreover, it was observed that S. epidermidis biofilm-released cells presented higher tolerance to vancomycin and tetracycline, as well as a particular gene expression phenotype when compared with either biofilm or planktonic cells. Using this model, biofilm-released cells phenotype and their interaction with the host immune system could be studied in more detail, which could help providing significant insights into the pathophysiology of biofilm-related infections.

Comparative proteomic and transcriptomic profile of Staphylococcus epidermidis biofilms grown in glucose-enriched medium

Staphylococcus epidermidis is an important nosocomial agent among carriers of indwelling medical devices, due to its strong ability to form biofilms on inert surfaces. Contrary to some advances made in the transcriptomic field, proteome characterization of S. epidermidis biofilms is less developed. To highlight the relation between transcripts and proteins of S. epidermidis biofilms, we analyzed the proteomic profile obtained by two mechanical lysis methods (sonication and bead beating), associated with two distinct detergent extraction buffers, namely SDS and CHAPS. Based on gel electrophoresis-LC-MS/MS, we identified a total of 453 proteins. While lysis with glass beads provided greater amounts of protein, CHAPS extraction buffer allowed identification of a higher number of proteins compared to SDS. Our data shows the impact of different protein isolation methods in the characterization of the S. epidermidis biofilm proteome. Furthermore, the correlation between proteomic and transcriptomic profiles was evaluated. The results confirmed that proteomic and transcriptomic data should be analyzed simultaneously in order to have a comprehensive understanding of a specific microbiological condition.

Controlled RNA contamination and degradation and its impact on qPCR gene expression in S. epidermidis biofilms

RNA quality is of utmost importance to perform gene expression quantification by qPCR. The classical methods used to determine RNA quality are based on electrophoresis and spectrophotometer assessment, namely A(260)/A(280) and A(260)/A(230) ratios. It was previously shown that due to the complex nature of Staphylococcus epidermidis biofilms, RNA extraction procedures could impact mRNA quality and thus accurate quantification. Herein, we contaminated and degraded RNA extracted from S. epidermidis biofilms, and assessed the effect on gene expression by qPCR. As expected, thermal degradation of RNA had a significant impact on gene expression on two out of the three tested genes. On the other hand, the contamination of the extracted RNA yielded an interesting result: while most contaminants did not changed the purity indicators or the integrity of RNA, significant changes on gene expression levels were found. This work confirms that poor RNA extraction has an important impact in qPCR quantification, emphasizing the consequences of carry-over contaminants on gene expression studies. Additionally, our results show that the parameters commonly used to assess the quality of extracted RNA from bacterial cultures seem to be insufficient to ensure reliable gene expression determination.