Mass Spectrometric Analysis of Urine from COVID-19 Patients for Detection of SARS-CoV-2 Viral Antigen and to Study Host Response.

SARS-CoV-2 infection has become a major public health burden and affects many organs including lungs, kidneys, the liver, and the brain. Although the virus is readily detected and diagnosed using nasopharyngeal swabs by reverse transcriptase polymerase chain reaction (RT-PCR), detection of its presence in body fluids is fraught with difficulties. A number of published studies have failed to detect viral RNA by RT-PCR methods in urine. Although microbial identification in clinical microbiology using mass spectrometry is undertaken after culture, here we undertook a mass spectrometry-based approach that employed an enrichment step to capture and detect SARS-CoV-2 nucleocapsid protein directly from urine of COVID-19 patients without any culture. We detected SARS-CoV-2 nucleocapsid protein-derived peptides from 13 out of 39 urine samples. Further, a subset of COVID-19 positive and COVID-19 negative urine samples validated by mass spectrometry were used for the quantitative proteomics analysis. Proteins with increased abundance in urine of SARS-CoV-2 positive individuals were enriched in the acute phase response, regulation of complement system, and immune response. Notably, a number of renal proteins such as podocin (NPHS2), an amino acid transporter (SLC36A2), and sodium/glucose cotransporter 5 (SLC5A10), which are intimately involved in normal kidney function, were decreased in the urine of COVID-19 patients. Overall, the detection of viral antigens in urine using mass spectrometry and alterations of the urinary proteome could provide insights into understanding the pathogenesis of COVID-19.

Multiple mechanisms contribute to lateral transfer of an organophosphate degradation (opd) island in Sphingobium fuliginis ATCC 27551.

The complete sequence of pPDL2 (37,317 bp), an indigenous plasmid of Sphingobium fuliginis ATCC 27551 that encodes genes for organophosphate degradation (opd), revealed the existence of a site-specific integrase (int) gene with an attachment site attP, typically seen in integrative mobilizable elements (IME). In agreement with this sequence information, site-specific recombination was observed between pPDL2 and an artificial plasmid having a temperature-sensitive replicon and a cloned attB site at the 3' end of the seryl tRNA gene of Sphingobium japonicum. The opd gene cluster on pPDL2 was found to be part of an active catabolic transposon with mobile elements y4qE and Tn3 at its flanking ends. Besides the previously reported opd cluster, this transposon contains genes coding for protocatechuate dioxygenase and for two transport proteins from the major facilitator family that are predicted to be involved in transport and metabolism of aromatic compounds. A pPDL2 derivative, pPDL2-K, was horizontally transferred into Escherichia coli and Acinetobacter strains, suggesting that the oriT identified in pPDL2 is functional. A well-defined replicative origin (oriV), repA was identified along with a plasmid addiction module relB/relE that would support stable maintenance of pPDL2 in Sphingobium fuliginis ATCC 27551. However, if pPDL2 is laterally transferred into hosts that do not support its replication, the opd cluster appears to integrate into the host chromosome, either through transposition or through site-specific integration. The data presented in this study help to explain the existence of identical opd genes among soil bacteria.