Since January 2020, the world is facing the COVID-19 pandemic caused by SARS-CoV-2. In a big effort to cope with this outbreak, two Uruguayan institutions, Institut Pasteur de Montevideo and Universidad de la República, have developed and implemented a diagnosis pipeline based on qRT-PCR using entirely local resources. In this context, we performed comparative quantitative proteomic analysis from oro- and naso-pharyngeal swabs used for diagnosis. Tryptic peptides obtained from five positive and five negative samples were analysed by nano-LC-MS/MS using a Q-Exactive Plus mass spectrometer. Data analysis was performed using PatternLab for Proteomics software. From all SARS-CoV-2 positive swabs we were able to detect peptides of the SARS-CoV-2 nucleoprotein that encapsulates and protect the RNA genome. Additionally, we detected an average of 1100 human proteins from each sample. The most abundant proteins exclusively detected in positive swabs were “Guanylate-binding protein 1”, “Tapasin” and “HLA class II histocompatibility antigen DR beta chain”. The biological processes overrepresented in infected host cells were “SRP-dependent cotranslational protein targeting to membrane”, “nuclear-transcribed mRNA catabolic process, nonsense-mediated decay”, “viral transcription” and “translational initiation”. Data is available via ProteomeXchange with identifier PXD020394. We expect that this data can contribute to the future development of mass spectrometry based approaches for COVID-19 diagnosis. Also, we share this preliminary proteomic characterization concerning the host response to infection for its reuse in basic investigation.

Background: South America has become the new epicenter of the COVID-19 pandemic with more than 1.1M reported cases and >50,000 deaths (June 2020). Conversely, Uruguay stands out as an outlier managing this health crisis with remarkable success. Methods: We developed a molecular diagnostic test to detect SARS-CoV-2. This methodology was transferred to research institutes, public hospitals and academic laboratories all around the country, creating a COVID-19 diagnostic lab network. Uruguay also implemented active epidemiological surveillance following the Test, Trace and Isolate (TETRIS) strategy coupled to real-time genomic epidemiology. Results: Three months after the first cases were detected, the number of positive individuals reached 826 (23 deaths, 112 active cases and 691 recovered). The Uruguayan strategy was based in a close synergy established between the national health authorities and the scientific community. In turn, academia rapidly responded to develop national RT-qPCR tests. Consequently, Uruguay was able to perform ~1,000 molecular tests per day in a matter of weeks. The COVID-19 diagnostic lab network performed more than 54% of the molecular tests in the country. This, together with real-time genomics, were instrumental to implement the TETRIS strategy, helping to contain domestic transmission of the main outbreaks registered so far. Conclusions: Uruguay has successfully navigated the first trimester of the COVID-19 health crisis in South America. A rapid response by the scientific community to increase testing capacity, together with national health authorities seeking out the support from the academia were fundamental to successfully contain, until now, the COVID-19 outbreak in the country.

PknG from Mycobacterium tuberculosis is a multidomain Serine/Threonine protein kinase that regulates bacterial metabolism as well as the pathogen's ability to survive inside the host by still uncertain mechanisms. To uncover PknG interactome we developed an affinity purification-mass spectrometry strategy to stepwise recover PknG substrates and interactors; and to identify those involving PknG autophosphorylated docking sites. We report a confident list of 7 new putative substrates and 66 direct or indirect partners indicating that PknG regulates many physiological processes, such as nitrogen and energy metabolism, cell wall synthesis and protein translation. GarA and the 50S ribosomal protein L13, two previously reported substrates of PknG, were recovered in our interactome. Comparative proteome analyses of wild type and pknG null mutant M. tuberculosis strains provided evidence that two kinase interactors, the FHA-domain containing protein GarA and the enzyme glutamine synthetase, are indeed endogenous substrates of PknG, stressing the role of this kinase in the regulation of nitrogen metabolism. Interestingly, a second FHA protein was identified as a PknG substrate. Our results show that PknG phosphorylates specific residues in both glutamine synthetase and FhaA in vitro, and suggest that these proteins are phosphorylated by PknG in living mycobacteria.