Rational manipulation of mRNA folding free energy allows rheostat control of pneumolysin production by Streptococcus pneumoniae

Rational manipulation of mRNA folding free energy allows rheostat control of pneumolysin production by Streptococcus pneumoniae

Insights into bacterial colony morphology evolution and diversification during infection development in cystic fibrosis

Tese de Doutoramento em Engenharia Biomédica.

Candida bracarensis: Evaluation of virulence factors and its tolerance to Amphotericin B and Fluconazole

Candida bracarensis is an uncommon Candida species found during an epidemiological study of candidiasis performed in Braga, Portugal. Initially, it was identified as C. glabrata, but recently detailed analyses pointed out their differences. So, little information is still available about C. bracarensis virulence factors and antifungal susceptibilities. Therefore, the main goal of this work is to evaluate the ability of C. bracarensis to form biofilms, to produce hydrolytic enzymes (proteases, phospholipases and hemolysins), as well as its susceptibility to amphotericin B and fluconazole. It was shown, for the first time, that all C. bracarensis strains were able to form biofilms and display proteinase and hemolytic activities. Moreover, although planktonic cells presented antifungal susceptibility, amphotericin B and fluconazole were unable to inhibit biofilm formation and eradicate pre-formed biofilms. Due to the propensity of C. bracarensis to display antifungal resistance and virulence attributes, the control of these emerging pathogens is recommended.

Bacterial cellulose as a novel substrate for retinal pigment epithelium cells transplantation in age-related macular degeneration

Tese de Doutoramento em Engenharia Química e Biológica.

Poly-N-acetyl glucosamine expression in Staphylococcus epidermidis biofilm cells affects the immune response and promotes tissue pathology in infected hosts

Staphylococcus epidermidis is a biofilm - forming bacterium and a leading etiological agent of nosocomial infections. The ability to establish biofilms on indwelling medical devices is a key virulence factor for this bacterium. Still, the influence of poly - N - acetyl glucosamine (PNAG), the major component of the extracellular biofilm matrix, in the host immune response has been scarcely studied. Here, t h is influence was assessed in mice challenged i.p. with PNAG - p roducing (WT) and isogenic - mutant lacking PNAG (M10) bacteria grown in biofilm - inducing conditions. Faster bacterial clearance was observed in the mice infected with WT bacteria than in M10 - infected counterparts , which w as accompanied by earlier neutrophil recruitment and higher IL - 6 production. Interestingly, in the WT - infected mice, but not in those infected with M10 , elevated serum IL - 10 was detected . To further study the effe ct of PNAG in the immune response, mice were primed with WT or M10 biofilm bacteria and subsequently infected with WT biofilm - released cells. WT - primed mice presented a higher frequency of splenic IFN - γ + and IL - 17 + CD4 + T cells, and more severe liver patho logy than M10 - primed counterparts. Nevertheless, T reg cells obtained from the WT - primed mice presented a higher suppressive function than those obtained from M10 - primed mice. This effect was abrogated when IL - 10 - deficient mice were similarly primed and infected indicating that PNAG promotes the differentiati on of highly suppressive T reg cells by a mechanism dependent on IL - 10. Altogether, these results provide evidence help ing explain ing the coexistence of inflammation and bacterial persistence often observed in biofilm - originated S. epidermidis infections

The influence of biofilm formation by Gardnerella vaginalis and other anaerobes on bacterial vaginosis

Bacterial vaginosis (BV) is the worldwide leading vaginal disorder in women of reproductive age. BV is characterized by the replacement of beneficial lactobacilli and the augmentation of anaerobic bacteria. Gardnerella vaginalis is a predominant bacterial species, however, BV is also associated with other numerous anaerobes, such as Atopobium vaginae, Mobiluncus mulieris, Prevotella bivia, Fusobacterium nucleatum and Peptoniphilus sp.. Currently, the role of G. vaginalis in the etiology of BV remains a matter of controversy. It is however known that, in BV patients, a biofilm is usually formed on the vaginal epithelium and G. vaginalis is typically the predominant species. So, the current paradigm is that the establishment of a biofilm plays a key role in the pathogenesis of BV. This review provides background on the influence of biofilm formation by G. vaginalis and other anaerobes in the polymicrobial etiology of BV, through its initial adhesion until biofilm formation and discusses the commensal and synergic interactions established between them to understand the phenotypic shift of G. vaginalis' biofilms into BV establishment.

Engineering modular viral scaffolds for targeted bacterial population editing

Bacteria are central to human health and disease, but existing tools to edit microbial consortia are limited. For example, broad-spectrum antibiotics are unable to precisely manipulate bacterial communities. Bacteriophages can provide highly specific targeting of bacteria, but assembling well-defined phage cocktails solely with natural phages can be a time-, labor- and cost-intensive process. Here, we present a synthetic biology strategy to modulate phage host ranges by engineering phage genomes in Saccharomyces cerevisiae. We used this technology to redirect Escherichia coli phage scaffolds to target pathogenic Yersinia and Klebsiella bacteria, and conversely, Klebsiella phage scaffolds to target E. coli by modular swapping of phage tail components. The synthetic phages achieved efficient killing of their new target bacteria and were used to selectively remove bacteria from multi-species bacterial communities with cocktails based on common viral scaffolds. We envision this approach accelerating phage biology studies and enabling new technologies for bacterial population editing.

Bacterial vaginosis biofilms: challenges to current therapies and emerging solutions

Bacterial vaginosis (BV) is the most common genital tract infection in women during their reproductive years and it has been associated with serious health complications, such as preterm delivery and acquisition or transmission of several sexually transmitted agents. BV is characterized by a reduction of beneficial lactobacilli and a significant increase in number of anaerobic bacteria, including Gardnerella vaginalis, Atopobium vaginae, Mobiluncus spp., Bacteroides spp. and Prevotella spp.. Being polymicrobial in nature, BV etiology remains unclear. However, it is certain that BV involves the presence of a thick vaginal multi-species biofilm, where G. vaginalis is the predominant species. Similar to what happens in many other biofilm-related infections, standard antibiotics, like metronidazole, are unable to fully eradicate the vaginal biofilm, which can explain the high recurrence rates of BV. Furthermore, antibiotic therapy can also cause a negative impact on the healthy vaginal microflora. These issues sparked the interest in developing alternative therapeutic strategies. This review provides a quick synopsis of the currently approved and available antibiotics for BV treatment while presenting an overview of novel strategies that are being explored for the treatment of this disorder, with special focus on natural compounds that are able to overcome biofilm-associated antibiotic resistance.

A novel small-caliber bacterial cellulose vascular prosthesis: production, characterization, and preliminary in vivo testing

Vascular grafts are used to bypass damaged or diseased blood vessels. Bacterial cellulose (BC) has been studied for use as an off-the-shelf graft. Herein, we present a novel, cost-effective, method for the production of small caliber BC grafts with minimal processing or requirements. The morphology of the graft wall produced a tensile strength above that of native vessels, performing similarly to the current commercial alternatives. As a result of the production method, the luminal surface of the graft presents similar topography to that of native vessels. We have also studied the in vivo behavior of these BC graft in order to further demonstrate their viability. In these preliminary studies, 1 month patency was achieved, with the presence of neo-vessels and endothelial cells on the luminal surface of the graft.

The impact of carbon source in Candida albicans virulence: participation of RLM1 in pathogen host interaction

Dissertação de mestrado em Genética Molecular

The relevance of bacterial interactions on oral health

Dissertação de mestrado integrado em Engenharia Biomédica (área de especialização em Engenharia Clínica)

Acetylated bacterial cellulose coated with urinary bladder matrix as a substrate for retinal pigment epithelium

This work evaluated the effect of acetylated bacterial cellulose (ABC) substrates coated with urinary bladder matrix (UBM) on the behavior of Retinal Pigment Epithelium (RPE), as assessed by cell adhesion, proliferation and development of cell polarity exhibiting transepithelial resistance and polygonal shaped-cells with microvilli. Acetylation of bacterial cellulose (BC) generated a moderate hydrophobic surface (around 65°) while the adsorption of UBM onto these acetylated substrates did not affect significantly the surface hydrophobicity. The ABS substrates coated with UBM enabled the development of a cell phenotype closer to that of native RPE cells. These cells were able to express proteins essential for their cytoskeletal organization and metabolic function (ZO-1 and RPE65), while showing a polygonal shaped morphology with microvilli and a monolayer configuration. The coated ABC substrates were also characterized, exhibiting low swelling effect (between 1.52.0 swelling/mm3), high mechanical strength (2048 MPa) and non-pyrogenicity (2.12 EU/L). Therefore, the ABC substrates coated with UBM exhibit interesting features as potential cell carriers in RPE transplantation that ought to be further explored.

A spin-offs journey into achieving marketable products from bacterial cellulose

[Excerpt] Academic spin-offs, technological ventures born inside Universities, have increasingly strengthen the connections between the scholarship and the economy, by fostering the role of technology transfer and knowledge commercialization. This presentation will outline the major steps in taking an idea or a technology to market, growing the venture and aiming at securing a successful exit. Also, it will present BCTechnologies (Bacterial Cellulose Technologies), a spin-off from the University of Minho (Portugal). (...)

Structural and enzymatic characterization of ABgp46, a novel phage endolysin with broad anti-Gram-negative bacterial activity

The present study demonstrates the antibacterial potential of a phage endolysin against Gram-negative pathogens, particularly against multidrug resistant strains of Acinetobacter baumannii. We have cloned, heterologously expressed and characterized a novel endolysin (ABgp46) from Acinetobacter phage vb_AbaP_CEB1 and tested its antibacterial activity against several multidrug-resistant A. baumannii strains. LC-MS revealed that ABgp46 is an N-acetylmuramidase, that is also active over a broad pH range (4.0-10.0) and temperatures up to 50°C. Interestingly, ABgp46 has intrinsic and specific anti-A. baumannii activity, reducing multidrug resistant strains by up to 2 logs within 2 hours. By combining ABgp46 with several organic acids that act as outer membrane permeabilizing agents, it is possible to increase and broaden antibacterial activity to include other Gram-negative bacterial pathogens. In the presence of citric and malic acid, ABgp46 reduces A. baumannii below the detection limit (> 5 log) and more than 4 logs P. aeruginosa and Salmonella Typhimurium strains. Overall, this globular endolysin exhibits a broad and high activity against Gram-negative pathogens, that can be enhanced in presence of citric and malic acid, and be used in human and veterinary medicine.