c-di-AMP, a likely master regulator of bacterial K + homeostasis machinery, activates a K + exporter

bis-(3',5')-cyclic diadenosine monophosphate (c-di-AMP) is a second messenger with roles in virulence, cell wall and biofilm formation, and surveillance of DNA integrity in many bacterial species, including pathogens. Strikingly, it has also been proposed to coordinate the activity of the components of K+ homeostasis machinery, inhibiting K+ import, and activating K+ export. However, there is a lack of quantitative evidence supporting the direct functional impact of c-di-AMP on K+ transporters. To gain a detailed understanding of the role of c-di-AMP on the activity of a component of the K+ homeostasis machinery in B. subtilis, we have characterized the impact of c-di-AMP on the functional, biochemical, and physiological properties of KhtTU, a K+/H+ antiporter composed of the membrane protein KhtU and the cytosolic protein KhtT. We have confirmed c-di-AMP binding to KhtT and determined the crystal structure of this complex. We have characterized in vitro the functional properties of KhtTU and KhtU alone and quantified the impact of c-di-AMP and of pH on their activity, demonstrating that c-di-AMP activates KhtTU and that pH increases its sensitivity to this nucleotide. Based on our functional and structural data, we were able to propose a mechanism for the activation of KhtTU by c-di-AMP. In addition, we have analyzed the impact of KhtTU in its native bacterium, providing a physiological context for the regulatory function of c-di-AMP and pH. Overall, we provide unique information that supports the proposal that c-di-AMP is a master regulator of K+ homeostasis machinery.We acknowledge the SOLEIL and ALBA synchrotrons for access and thank their staff for help with data collection. Support of the Biochemical and Biophysical Technologies, Cell Culture and Genotyping and X-ray Crystallography scientific platforms of i3S (Porto, Portugal) is also acknowledged. Mass spectrometry analysis was performed by Hugo Osório at the i3S Proteomics Scientific Platform. This work had support from the Portuguese Mass Spectrometry Network, integrated in the National Roadmap of Research Infrastructures of Strategic Relevance (ROTEIRO/0028/2013; LISBOA-01-0145-FEDER-022125). Work was supported by FEDER - Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020-Operational Programme for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through FCT - Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the projects POCI-01–0145-FEDER-029863(PTDC/BIABQM/29863/2017) and by Fundação Luso-Americana para o Desenvolvimento through the FLAD Life Science 2020 award ‘Bacterial K+ transporters are potential antimicrobial targets: mechanisms of transport and regulation’

Caracterização da inibição da tripsina pela boofilina

Mestrado em Biologia Molecular e CelularNo decurso da evolução, enquanto os vertebrados desenvolviam um complexo sistema para prevenir as perdas de sangue, os hematófagos criavam mecanismos anticoagulantes capazes de contra-atacar a reposta hemostática do hospedeiro, por forma a facilitar a extração, o armazenamento e a digestão do sangue. Muitos destes mecanismos assentam na inibição específica da trombina, proteína que tem um papel central na hemostasia. Apesar do elevado número de inibidores naturais de trombina, apenas uma pequena parte foi caracterizada bioquimicamente e, dentro desta, só alguns inibidores foram estudados de um ponto de vista estrutural. De todos aqueles com estrutura conhecida, a boofilina é o único que possui dois domínios canónicos do tipo Kunitz. No entanto, apesar de possuir domínios canónicos, a boofilina inibe a trombina através de um mecanismo não-canónico, no qual o seu N-terminal é inserido no centro catalítico da trombina enquanto que o domínio C-terminal interage com o exosite I da proteinase. Além disso, ao contrário de todos os outros inibidores caracterizados, a boofilina, em complexo com a trombina, é capaz de interagir com uma proteinase serínica adicional do tipo tripsina. Esta segunda interação envolve unicamente o domínio N-terminal, presumivelmente através do loop reactivo. In vivo, é possível que para além de inibir a trombina, a boofilina seja também capaz de inibir outra proteinase serínica da cascata de coagulação como o fXa, resultando não só na inibição da trombina mas também na diminuição da sua produção. Este aspecto faz da boofilina um bom modelo para a desenvolvimento de novos anticoagulantes terapêuticos. Pretende-se, por isso, clarificar este segundo mecanismo de inibição através do estudo da interação entre a boofilina e a tripsina. A boofilina (D1D2) e o seu domínio N-terminal isolado (D1) foram expressos em Pichia pastoris. Ambas as proteínas foram purificadas por cromatografia de afinidade numa coluna de tripsina seguida, no caso da D1D2, por cromatografia de troca iónica. Uma vez purificadas, a constante de inibição de cada uma das proteínas foi determinada e a D1 foi cristalizada por difusão vapor. Os cristais obtidos eram ortorrômbicos (grupo espacial P212121), tinham uma percentagem invulgarmente baixa de solvente (23%) e difrataram com uma resolução mínima de 1.80 Ǻ. A estrutura cristalográfica da D1 releva uma conformação similar à observada no complexo trombina-boofilina. O complexo tripsina-D1 foi também preparado in vitro e a sua cristalização está em curso.During evolution, while vertebrates developed a complex system to prevent blood loss, blood-feeding animals evolved anticoagulant mechanisms to counteract the haemostatic response of their hosts in order to facilitate blood drawing, storage and digestion. Many of these anticoagulant mechanisms rely on the specific inhibition of thrombin, a central enzyme in haemostasis. Although a number of a natural thrombin inhibitors from haematophagous animals have been described only a few have been fully characterised biochemically, and even fewer have been studied from a structural viewpoint. From those structurally characterised to date, only boophilin displays two tandem canonical Kunitz fold domains. However, despite its regular BPTI-like domains, boophilin inhibits thrombin by a non-canonical mechanism, in which its N-terminus inserts into the active site cleft of thrombin, while the C-terminus Kunitz domain interacts with the exosite I of the proteinase. Moreover, in contrast to all the other natural thrombin inhibitors characterised, when in complex with thrombin boophilin retains the capability to interact with an additional (non-thrombin) trypsin-like serine proteinase molecule. In this second interaction only the N-terminal domain is involved, presumably through its conserved reactive-loop. In vivo, it is likely that besides inhibiting thrombin, boophilin can target another serine proteinase of the coagulation cascade, such as fXa. This would result not only in thrombin inhibition, but also in the impairment of thrombin generation, making boophilin a good model for the design of new therapeutic anticoagulants. For this reason, this work aimed at clarifying this second inhibition mechanism by studying the trypsin-boophilin complex. Boophilin (D1D2) and its N-terminal domain (D1) were expressed in Pichia pastoris. Both proteins were purified by immobilized-trypsin affinity chromatography followed, in the case of D1D2, by ion-exchange chromatography. Once purified, the inhibition constant of both proteins was determined and D1 was crystallized by vapour diffusion. The crystals obtained were orthorhombic (space group P212121), had an unusually low solvent content of 23% and diffracted X-rays to beyond 1.80 Ǻ. The crystallographic structure of the first domain of boophilin reveals a conformation of the reactive site loop similar to that observed in the thrombin-boophilin complex. The D1-trypsin complex was also prepared in vitro and its crystallization is underway

Lactobacilli and its metabolites as potential probiotics against Gardnerella vaginalis

Bacterial vaginosis (BV) is one of the most common gynecological disorder affecting women in reproductive age. This condition poses a significant health risk because it predisposes women to abnormal pregnancy, pelvic inflammatory disease and an increased risk of sexual transmitted infections, including HIV [1]. Although BV etiology remains unknown, it is characterized by a decrease in vaginal lactobacilli and a proliferation of anaerobes [1, 2]. Despite of the richness and diversity found into BV anaerobes, Gardnerella vaginalis is present in over 90% of the pathologic cases and several studies report its potential as the main etiological candidate [3, 4, 5]. Current BV treatment is strictly based in antibiotic therapy resulting in an increased resistance of BV anaerobes, along with severe reduction of the healthy lactobacilli strains in the vaginal epithelium. Therefore, a more appropriate treatment is required, aiming to decrease G. vaginalis and also to promote the lactobacilli re-colonization in BV patients [6]. Previous studies showed the potential of lactobacilli in preventing vaginal colonization by pathogens, and thus the development of infections, by different mechanisms including auto-aggregation, co-aggregation with pathogenic microorganisms, and adhesion to epithelial cells and/or by producing some metabolites (such as lactic acid, hydrogen peroxide, bacteriocins and biosurfactants) that may act as growth inhibitors or anti-adhesive agents [7]. These probiotic properties have inspired new treatment strategies for vaginal infection. One alternative therapy for BV is the re-colonisation of vagina with lactobacilli species [6]. However, the major gap in this option resides in the choice of the most suitable lactobacillus species. Our goal was to evaluate the probiotic potential of intra and extracellular metabolites from a broad range of lactobacilli strains against several G. vaginalis strains. To accomplish our goal, we isolated 60 vaginal lactobacilli strains from healthy women and performed a screening by an agar spot test against 9 G. vaginalis strains (3 from culture collection and 6 clinical isolates from women with BV) in order to selected the most interesting probiotic candidates. We also included more than 30 culture collection lactobacilli strains. For the best candidates we determined the minimum inhibitory concentration (MIC) and identified the lactobacilli metabolites responsible for G. vaginalis growth inhibition. Our results showed that certain latobacilli metabolites were able to inhibit G. vaginalis growth. In spite of the detection of intracellular biosurfactants in some strains, they were unable to reduce G. vaginalis proliferation. On the other hand, extracellular products of some lactobacilli showed a significant effect on G. vaginalis growth. Overall, from the 90 lactobacilli strains tested, only 4 culture collection and 3 clinical isolate lactobacilli strains exhibited a broad probiotic activity against all the G. vaginalis strains tested. However, only culture collection strains were able to reduce G. vaginalis strains growth to 20-30% in MIC assays, illustrating an efficient probiotic activity by itself. Interestingly, none of these lactobacilli strains belong to the vaginal microflora, revealing a more pronounced probiotic activity than any of those vaginal isolate lactobacilli tested. Therefore, our data suggests the existence of non-vaginal lactobacilli strains possessing probiotic activity against numerous G. vaginalis strains, which may contribute for a new and more effective BV treatment than the currently used therapies

Influence of anaerobic conditions on vaginal microbiota recovery from bacterial vaginosis patients

Bacterial vaginosis (BV) is one of the most common infections in women of reproductive age. Clinical studies have shown an association among BV and abnormal pregnancy, pelvic inflammatory disease and increased risk of sexually transmitted infections, including HIV. This disorder was first described in 1914 by Curtis as a “white discharge” syndrome and despite the decades of research we have only limited, and clearly not conclusive, evidence of microbial cause of BV, mechanism of disease and effective treatment. The development of molecular techniques such as Denaturing Gradient Gel Electrophoresis (DGGE) and DNA sequencing produced a clearer picture of the complexities of the vaginal microbiota. It has also become more apparent that none of the microorganisms already isolated from the vagina are likely to be the sole pathogen responsible for BV. Thus, improved knowledge of the relationship between different species of bacteria and their host is critical to a better understanding of both women’s health and illness. In order to comprehend the dynamic interaction between bacteria–bacteria and bacteria–host it becomes necessary to isolate bacteria from vaginal samples and to use them in in vitro and in vivo assays. The human vagina has a very specific environment regarding pH, nutrient availability and oxygen tension, being populated by a large range of bacteria from facultative to obligated anaerobic species. To isolate bacteria from such a complex niche, rich media should be used in order to promote the growth of different species. By using Columbia Agar (CBA) with 5% horse blood and by incubating at 37°C in the presence of 5%, 10% CO or in anaerobic condition generated by AnaeroGenTM (Oxoid), we were able to recover different bacteria species, from each of the anaerobic conditions tested, from BV vaginal samples, including some species never reported, as far as we know, such as Brevibacterium ravenspurgense, Corynebacterium tuscaniense, Klebsiella variicola, Nosocomiicoccus ampullae, Staphylococcus warneri and Bacillus firmus. Despite the development of molecular methods and their unquestionable advantages in characterising the vaginal microbiota, classical bacteriology will nevertheless be required to elucidate the etiology of BV since the isolation of the pathogenic agent will be always required. Our findings revealed that slight differences in anaerobic conditions were crucial for the isolation of novel BV-associated organisms. Further in vitro studies of all the isolated bacteria will promote a better understanding of the dynamics of their interaction and will potentially reveal how BV develops and influences other sexual transmitted diseases.(undefined

Isolation of Gardnerella vaginalis from BV patients and healthy women : analysis of virulence through adherence, biofilm formation and cytotoxicity assays

Bacterial vaginosis (BV) is one of the most common gynaecological disorder affecting women in the reproductive age. Microbiological analysis of BV has shown Gardnerella vaginalis to be the most frequent organism in BV. However, G. vaginalis colonization do not always lead to BV. This raised the question whether there are pathogenic and commensal lineages within this species. In an effort to understand the differences between G. vaginalis strains, we performed in vitro assays to compare virulence properties of recently isolated 14 G. vaginalis strains from Portuguese women with and without BV. G. vaginalis strains were characterised for their initial adhesion ability to a monolayer of HeLa cells by incubating the bacteria with this monolayer and quantifying the adhesion by staining with DAPI and fluorescence microscopy. These assays revealed that the BV isolates of G. vaginalis had a stronger initial adhesion capability than non-BV isolates. The biofilm-forming capacity was then assessed by allowing each of the strains to form biofilms under anaerobic conditions for 48 hours and using different growth media. It was possible to observe that BV isolates tend to growth preferentially as biofilms while non-BV isolates had a lower intrinsic tendency towards biofilm formation. In addition, BV isolates of G. vaginalis displayed robust cytotoxicity in the epithelial cells after 3 hours in the contact with a monolayer of HeLa cells. Thus, this study outlines two distinct variants of G. vaginalis, one apparently commensal and one pathogenic, and presents evidence for disparate virulence potentials

Gardnerella vaginalis virulence potential outcompetes with 30 other microorganisms isolated from BV patients

Bacterial vaginosis (BV) is the most common vaginal disorder affecting millions of women every year, and is usually associated with several adverse health outcomes, including preterm birth and acquisition of sexually transmitted diseases. However, the etiology of BV is still under debate. Recently, new fastidious anaerobic bacteria have been associated with BV, but there are very few studies that comprehensively evaluate the virulence potential of these microorganisms, mainly due to difficult growth conditions. However, in order to find answers to some of the questions related to BV, isolation and characterization of such bacteria will be required. In this work, samples of vaginal exudate from BV women were collected and 31 different microorganisms were isolated, including 6 novel species never described in BV before. Each microorganism was characterized for their ability to adhere to HeLa epithelial cells and cause cytopathogenic changes; their intrinsic biofilm-forming capability; and finally their antimicrobial susceptibility tests for antibiotics commonly used in the treatment of BV. Despite the strong evidence that G. vaginalis outcompeted the other species in the defined virulence assays, our results also demonstrate that other bacteria showed significant biofilm-forming capability, but not initial adhesion, such as Actinomyces turicensis and Corynebacterium tuscaniense. This work supports the evidence that G. vaginalis is the main colonizer in multi-species related BV and further describes novel microorganisms with enhanced virulence potential. Finally, this is the first characterization of Portuguese microbiota associated with BV

Gardnerella vaginalis outcompetes 29 other bacterial species isolated from BV patients in an in vitro biofilm formation model

Despite the worldwide prevalence of bacterial vaginosis (BV), its etiology is still unknown. Although BV has been associated with the presence of biofilm, the ability of BV-associated bacteria to form biofilms is still largely unknown. Here, we isolated 30 BV-associated species and characterized their virulence, using an in vitro biofilm formation model. Our data suggests that Gardnerella vaginalis had the highest virulence potential, as defined by higher initial adhesion and cytotoxicity of epithelial cells, as well as the greater propensity to form a biofilm. Interestingly, we also demonstrated that most of the BV-associated bacteria had a tendency to grow as biofilms.This work was supported by the European Union (FEDER/COMPETE funds) and the Fundacao para a Ciencia e a Tecnologia (reference FCOMP-01-0124-FEDER-008991 [PTDC/BIA-MIC/098228/2008] and RECI/EBB-EBI/0179/2012 [FCOMP-01-0124-FEDER-027462])

In silico vs in vitro analysis of primer specificity for the detection of Gardnerella vaginalis, atopobium vaginae and lactobacillus spp

Bacterial vaginosis (BV) is a common pathology of women in reproductive age that can lead to serious health complications, and is associated with shifts in the normal microflora from predominance of Lactobacillus spp. to a proliferation of other anaerobes such as G. vaginalis and A vaginae, which can be detected by PCR. The optimal PCR pathogen detection assay relies mainly on the specificity and sensitivity of the primers used. Findings Here we demonstrate that in silico analytical testing of primer specificity is not a synonym to in vitro analytical specificity by testing a range of published and newly designed primers with both techniques for the detection of BV-associated microorganisms. Conclusions By testing primer in vitro specificity with a sufficient range of bacterial strains, we were able to design primers with higher specificity and sensitivity. Also by comparing the results obtained for the newly designed primers with other previously published primers, we confirmed that in silico analysis is not sufficient to predict in vitro specificity. As such care must be taken when choosing the primers for a detection assay.(undefined