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’

Using an in-vitro biofilm model to assess the virulence potential of Bacterial Vaginosis or non-Bacterial Vaginosis Gardnerella vaginalis isolates

Gardnerella vaginalis is the most common species found in bacterial vaginosis (BV). However, it is also present in a significant proportion of healthy women and G. vaginalis vaginal colonization does not always lead to BV. In an effort to better understand the differences between G. vaginalis isolated from women with a positive (BV) versus a negative (non-BV) diagnosis of BV, we compared the virulence potential of 7 BV and 7 non-BV G. vaginalis isolates and assessed the virulence factors related to biofilm formation, namely: initial adhesion and cytotoxic effect, biofilm accumulation, susceptibility to antibiotics, and transcript levels of the known vaginolysin, and sialidase genes. Furthermore, we also determined the ability of G. vaginalis to displace lactobacilli previously adhered to HeLa cells. Our results showed that non-BV strains were less virulent than BV strains, as suggested by the lower cytotoxicity and initial adhesion to Hela cells. Significant differences in expression of known virulence genes were also detected, further suggesting a higher virulence potential of the BV associated G. vaginalis. Importantly, we demonstrated that BV associated G. vaginalis were able to displace pre-coated vaginal protective lactobacilli and we hypothesize this to be a trigger for BV development.European Union funds (FEDER/COMPETE) and by national funds (FCT) under the project with reference FCOMP-01-0124-FEDER-008991 (PTDC/BIA-MIC/098228/2008). FCT Strategic Project of UID/BIO/04469/2013 unit the project NORTE-07-0124-FEDER-000027, co-funded by the Programa Operacional Regional do Norte(ON.2 – O Novo Norte), QREN, FEDER, and the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462). FCT individual fellowship SFRH/BD/93963/2013

Functional analysis of virulence potential from Gardnerella vaginalis and other anaerobes commonly associated with Bacterial vaginosis

In the past half century, bacterial vaginosis (BV) has been a controversial topic in medical microbiology, and despite the wealth of information on this topic, the etiological agent has not yet been definitively identified Taking these data into consideration, we hypothesized that strains of G. vaginalis that were able to form biofilms could be the causative agent of BV. To test our hypothesis, we isolated more than 30 bacterial species from BV patients and also several strains of G. vaginalis from healthy women, and tested biofilm forming ability, initial adhesion to human vaginal cells, cytotoxicity activity, antimicrobial resistance and gene expression of know virulent genes. Our results revealed that G. vaginalis outcompeted all the other bacterial species in the initial adhesion to the epithelial cells. Furthermore, when comparing BV-associated G. vaginalis strains to strains isolated from healthy women, we found that all 7strains from BV were more virulent than the 7 strains colonizing healthy women, as measured by the higher cytotoxicity and the higher initial adhesion to epithelial cells. No significant differences were found in antimicrobial resistance profiles. Interestingly, no significant differences in expression of known virulence genes were detected, suggesting that the higher virulence of the BV-associated G. vaginalis was due to a yet unknown virulence determinant. We then tested virulent G. vaginalis against other known BV-associated anaerobe pathogens, namely Mobiluncus mulieris, Atopobium vaginae, Prevotella bivia and Fusobacteria nucleatum in mixed biofilm formation quantification. Interestingly, while the other tested anaerobes did not reveal a higher initial adhesion, they did enhance biofilm formation by G. vaginalis. Overall, our data suggests that virulent variants of G. vaginalis have the potential to be the etiological agent of BV, while acknowledging that other anaerobes do enhance G. vaginalis virulence