Biology subjects

Mateus, A.

Publications and source records attributed to Mateus, A..

24 records · Page 2Linked to original sources

bioRxiv2020

Phage proteins block and trigger retron toxin/antitoxin systems

Bacteria carry dozens of Toxin/Antitoxin systems (TAs) in their chromosomes. Upon growth, the antitoxin is co-expressed and neutralizes the toxin. TAs can be activated and inhibit growth, but when and how this occurs has largely remained enigmatic, hindering our understanding of their physiological roles. We developed TIC/TAC (Toxin Inhibition/Activation Conjugation), a high-throughput reverse genetics approach, to systematically identify molecular blockers and triggers of TAs. By applying TIC/TAC to a tripartite TA, the retron-Sen2 of Salmonella Typhimurium, we have identified multiple blockers and triggers of phage origin. We demonstrate that diverse phage functionalities are sensed by the DNA-part of the antitoxin and ultimately activate the retron toxin. Phage-origin proteins can circumvent activation by directly blocking the toxin. Some identified triggers and blockers also act on an E. coli retron-TA, Eco9. We propose that retron-TAs act as abortive-infection anti-phage defense systems, and delineate mechanistic principles by which phages trigger or block them.

microbiology↗

bioRxiv2020

Computational analysis of ligand dose range thermal proteome profiles

Detecting ligand-protein interactions in living cells is a fundamental challenge in molecular biology and drug research. Proteome-wide profiling of thermal stability as a function of ligand concentration promises to tackle this challenge. We present a statistical analysis method with reliable control of the false discovery rate and apply it to several datasets probing epigenetic drugs. We detect off-target drug engagement in unrelated protein families.

bioinformatics↗

bioRxiv2020

A dual-mechanism antibiotic targets Gram-negative bacteria and avoids drug resistance

The rise of antibiotic resistance and declining discovery of new antibiotics have created a global health crisis. Of particular concern, no new antibiotic classes have been approved for treating Gram-negative pathogens in decades. Here, we characterize a compound, SCH-79797, that kills both Gram-negative and Gram-positive bacteria through a unique dual-targeting mechanism of action (MoA) with undetectably low resistance frequencies. In an animal host model, SCH-79797 reduces pathogenesis of Acinetobacter baumannii, a drug-resistant Gram-negative pathogen. To characterize the MoA of SCH-79797 we combined quantitative imaging, proteomic, genetic, metabolomic, and cell-based assays. This pipeline shows that SCH-79797 has two independent cellular targets, folate metabolism and bacterial membrane integrity, and outperforms combination treatments with other antifolates and membrane disruptors in killing MRSA persisters. Thus, SCH-79797 represents a promising lead antibiotic and suggests that combining multiple MoAs onto a single chemical scaffold may be an underappreciated approach to target challenging bacterial pathogens.

microbiology↗

bioRxiv2020

Impact of phosphorylation on thermal stability of proteins

Reversible protein phosphorylation regulates virtually every cellular process and is arguably the most well-studied post-translational modification. Still, less than 3% of the phosphorylation sites identified in humans have annotated functions. Functionally-relevant phosphorylation sites are known to trigger conformational changes to proteins and/or to regulate their interactions with other proteins, nucleic acids and small molecules - all of which can be reflected in the thermal stability of a protein. Thus, combining thermal proteome profiling (TPP) with phosphoproteomics (phospho-TPP) provides a way to assess the functional relevance of identified phosphorylation sites on a proteome-wide scale by comparing the melting behavior of a protein and its phosphorylated form(s). We performed phospho-TPP experiments in HeLa cells with an optimized protocol, and conclude that phosphorylation does affect protein thermal stability, but to a much lesser extent than previously reported.

systems biology↗

bioRxiv2019

Towards a systematic map of the functional role of protein phosphorylation

Phosphorylation is a critical post-translational modification involved in the regulation of almost all cellular processes. However, less than 5% of thousands of recently discovered phosphorylation sites have a known function. Here, we devised a chemical genetic approach to study the functional relevance of phosphorylation in S. cerevisiae. We generated 474 phospho-deficient mutants that, along with the gene deletion library, were screened for fitness in 102 conditions. Of these, 42% exhibited growth phenotypes, suggesting these phosphosites are likely functional. We inferred their function based on the similarity of their growth profiles with that of gene deletions, and validated a subset by thermal proteome profiling and lipidomics. While some phosphomutants showed loss-of-function phenotypes, a higher fraction exhibited phenotypes not seen in the corresponding gene deletion suggestive of a gain-of-function effect. For phosphosites conserved in humans, the severity of the yeast phenotypes is indicative of their human functional relevance. This study provides a roadmap for functionally characterizing phosphorylation in a systematic manner.

systems biology↗

bioRxiv2019

The outer membrane lipoprotein NlpI nucleates hydrolases within peptidoglycan multi-enzyme complexes in Escherichia coli

The peptidoglycan (PG) sacculus provides bacteria with the mechanical strength to maintain cell shape and resist osmotic stress. Enlargement of the mesh-like sacculus requires the combined activity of PG synthases and hydrolases. In Escherichia coli, the activity of the two bifunctional PG synthases is driven by lipoproteins anchored in the outer membrane. However, the regulation of PG hydrolases is less well understood, with only regulators for PG amidases having been described. Here, we identify the lipoprotein NlpI as a general adaptor protein for PG hydrolases. NlpI binds to different classes of hydrolases and can specifically form multimeric complexes with various PG endopeptidases. In addition, NlpI seems to contribute both to PG elongation and cell division biosynthetic complexes based on its localization and genetic interactions. In line with such a role, we reconstitute PG multi-enzyme complexes containing NlpI, the PG synthesis regulator LpoA, its cognate bifunctional synthase, PBP1A, and different endopeptidases. Our results indicate that PG regulators and adaptors are part of PG biosynthetic multi-enzyme complexes, regulating and potentially coordinating the spatiotemporal action of PG synthases and hydrolases.\n\nSignificanceThe activity of PG hydrolases may cause lysis of the bacterial cell if left unregulated. Hence, the cell must have ways of regulating and coordinating their activities. Our current understanding of how this occurs is incomplete. In this work, we present the outer membrane (OM) anchored lipoprotein, NlpI, as a scaffold of peptidoglycan hydrolases. We propose that NlpI facilitates the formation of multi-enzyme complexes and that, along with other regulators, it coordinates a safe enlargement and separation of the PG layer in E. coli.

microbiology↗