Biology subjects

Bellinzoni, M.

Publications and source records attributed to Bellinzoni, M..

7 recordsLinked to original sources

bioRxiv2026

A Seven-Protein Assembly Promotes Stability, Neutralisation and Secretion of the T7SSb LXG-effector TelE

Streptococcus gallolyticus subsp. gallolyticus (SGG) is a gut pathobiont associated with colorectal cancer. Like many Firmicutes, SGG utilizes a specialized Type VII Secretion System (T7SSb) to export WXG100 and LXG proteins, the latter involved in bacterial competition. We previously identified TelE, an LXG protein whose C-terminus mediates membrane pore formation in Escherichia coli. In SGG UCN34, TelE (Gallo_0562) is co-expressed with six proteins (Gallo_0559 to Gallo_0565), including its immunity protein TipE (Gallo_0565). Here we show that the absence of those co-expressed proteins affects TelE stability and secretion. These proteins associate with TelE to form a soluble and stable seven-protein complex. Gallo_0559 and Gallo_0560 interact with the N-terminal LXG domain of TelE, Gallo_0561 binds to its central region, while the six-transmembrane protein Gallo_0563, together with Gallo_0564 and TipE associates with its C-terminal domain. These findings describe a new modular complex that stabilizes TelE while reducing its toxicity and optimizing its T7SSb-mediated delivery.

microbiology↗

bioRxiv2024

The chemoreceptor controlling the Wsp-like transduction pathway in Halomonas titanicae KHS3 binds purine derivatives

The chemosensory pathway HtChe2 from the marine bacterium Halomonas titanicae KHS3 controls the activity of a diguanylate cyclase. Constitutive activation of the pathway results in colony morphology alterations and increased ability to form biofilm. Such characteristics resemble the behaviour of the Wsp pathway of Pseudomonas. In this work we investigate the specificity of Htc10, the only chemoreceptor coded within the HtChe2 gene cluster. Thermal shift analyses performed with the Htc10 ligand-binding domain led to the identification of purine derivatives as ligands. This ligand-binding domain was crystallized in the presence of guanine or hypoxanthine and its structure was solved by X-ray protein crystallography. The sensor domain adopts a double-cache folding, with ligands bound to the membrane-distal pocket. A high-resolution structure of the occupied guanine-binding pocket allowed the identification of the involved residues. These residues were validated by site directed mutagenesis and thermal shift or isothermal calorimetry analyses of the protein variants. The dissociation constants for guanine or hypoxanthine of the intact domain were in the low micromolar range. To our knowledge, this is the first description of binding specificity for a chemoreceptor that controls the activity of an associated diguanylate cyclase, and opens the way for dynamic studies of the signalling behaviour of this kind of sensory complex. A comparison between Htc10 and the functionally equivalent WspA receptor from Pseudomonas revealed no significant sequence similarities. In contrast, highly conserved Htc10-like receptors were found in distant bacteria carrying HtChe2-like clusters.

microbiology↗

bioRxiv2023

High resolution cryo-EM and crystallographic snapshots of the large actinobacterial 2-oxoglutarate dehydrogenase: an all-in-one fusion with unique properties

Actinobacteria possess unique ways to regulate the oxoglutarate node located in the central position of the tricarboxylic acid cycle, a crossroad between energy conservation and nitrogen metabolism. Here, we studied the decarboxylative oxidation route that leads, through the 2-oxoglutarate dehydrogenase (ODH) complex, to the generation of succinyl-CoA and reduced equivalents to feed the respiratory chain. Compared to most organisms in which the oxidative decarboxylation and reductive acylation steps are carried out by different enzymes within the ODH complex, actinobacteria rely on an all-in-one protein (OdhA) in which both activities are carried out by the same polypeptide. We describe high-resolution cryo-EM and X-ray crystallography snapshots of representative enzymes from Mycobacterium smegmatis and Corynebacterium glutamicum, showing that OdhA is an 800-kDa homohexamer that folds into a three-blade propeller shape. The obligate trimeric and dimeric states of the acyltransferase and dehydrogenase domains, respectively, are critical for maintaining the overall assembly, where both domains interact via subtle readjustments of their interfaces. Complexes obtained with substrate analogues, reaction products and allosteric regulators illustrate how these domains operate. Furthermore, we provide additional insights into the phosphorylation-dependent regulation of this enzymatic machinery by the FHA (Fork-Head Associated) signalling protein OdhI, delivering new molecular details on how this actinobacterial-specific switching mechanism operates. Overall, the quaternary organization of OdhA represents a new piece of the fascinating puzzle of the synergistic, mixed pyruvate dehydrogenase/2-oxoglutarate dehydrogenase actinobacterial supercomplex.

biochemistry↗

bioRxiv2021

A TPR scaffold couples signal detection to OdhI phosphorylation in metabolic control by the protein kinase PknG

Signal transduction is essential for bacteria to adapt to changing environmental conditions. Among many forms of post-translational modifications, reversible protein phosphorylation has evolved as a ubiquitous molecular mechanism of protein regulation in response to specific stimuli. The Ser/Thr protein kinase PknG modulates the fate of intracellular glutamate by controlling the phosphorylation status of the 2-oxoglutarate dehydrogenase regulator OdhI, a function that is conserved among diverse actinobacteria. PknG has a modular organization characterized by the presence of regulatory domains surrounding the catalytic domain. Here we present an investigation through in vivo experiments as well as biochemical and structural methods of the molecular bases of the regulation of PknG from C. glutamicum (CgPknG), in the light of previous knowledge available for the kinase from M. tuberculosis (MtbPknG). We found that OdhI phosphorylation by CgPknG is regulated by a conserved mechanism that depends on a C-terminal domain composed of tetratricopeptide repeats (TPR) essential for metabolic homeostasis. Furthermore, we identified a conserved structural motif that physically connects the TPR domain and a flexible N-terminal extension of the kinase that is involved in docking interactions with OdhI. Based on our results and previous reports, we propose a model in which the TPR domain of PknG couples signal detection to the specific phosphorylation of OdhI. Overall, the available data indicate that conserved PknG domains in distant actinobacteria retain their roles in kinase regulation in response to nutrient availability. IMPORTANCEBacteria control the metabolic processes by which they obtain nutrients and energy in order to adapt to the environment. In this way, the metabolic characteristics of a microorganism determine its ecological role and its usefulness in industrial processes. Here, we use genetic, biochemical, and structural approaches to study a key component in a system that regulates glutamate production in C. glutamicum, a species that is used for the industrial production of amino acids. We elucidated molecular mechanisms involved in metabolic control in C. glutamicum, which are conserved in related pathogenic bacteria. The findings have broader significance for diverse actinobacteria, including microorganisms that cause disease as well as environmental species used to produce billions of dollars of amino acids and antibiotics every year.

biochemistry↗

bioRxiv2021

Proteome remodeling in the Mycobacterium tuberculosis PknG knockout: molecular evidence for the role of this kinase in cell envelope biogenesis and hypoxia response.

Mycobacterium tuberculosis, the etiological agent of tuberculosis, is among the deadliest human pathogens. One of M. tuberculosiss pathogenic hallmarks is its ability to persist in a dormant state in the host for long periods, reinitiating the infectious cycle when favorable environmental conditions are found. Thus, it is not surprising that this pathogen has developed different mechanisms to withstand the stressful conditions found in the host. In particular, the Ser/Thr protein kinase PknG has gained special relevance since it regulates nitrogen metabolism and facilitates bacterial survival inside macrophages. Nevertheless, the molecular mechanisms underlying these effects are far from being elucidated. To further investigate these issues, we performed quantitative proteomics analyses of protein extracts from M. tuberculosis H37Rv and a mutant derivative lacking pknG. Our results showed that in the absence of PknG the mycobacterial proteome was remodeled since 5.7% of the proteins encoded by M. tuberculosis presented significant changes in its relative abundance when compared to the wild-type strain. The main biological processes affected by pknG deletion were the biosynthesis of cell envelope components and the response to hypoxic conditions. As many as 13 DosR-regulated proteins were underrepresented in the pknG deletion mutant, including the distinctive Hrp-1, which was found to be 12-fold decreased according to Parallel Reaction Monitoring experiments. Altogether, the results presented here allow us to postulate that PknG regulation of bacterial adaptation to stress conditions might be an important mechanism underlying its reported effect on intracellular bacterial survival.

biochemistry↗

bioRxiv2020

Actinobacteria challenge the paradigm: a unique protein architecture for a well-known central metabolic complex

-ketoacid dehydrogenase complexes are large, tripartite enzymatic machineries carrying out key reactions in central metabolism. Extremely conserved across the tree of life, they have so far all considered to be structured around a high molecular weight hollow core, consisting of up to 60 subunits of the acyltransferase component. We provide here evidence that Actinobacteria break the rule by possessing an acetyltranferase component reduced to its minimally active, trimeric unit, characterized by a unique C-terminal helix that affects the oligomerization and the full 3D architecture of the complex. We show that this unique feature is characterized by an insertion, which together with OdhA is found spread over Actinobacteria. This phylum includes organisms or great interest for agriculture, industrial bio-production and many human pathogens as Mycobacterium tuberculosis. Moreover, components of this complex are key for M. tuberculosis survival in the human host, and its unique core and protein-protein interactions represent potentially "druggable" targets.

microbiology↗

bioRxiv2020

Central role and structure of the membrane pseudokinase YukC in the antibacterial Bacillus subtilis Type VIIb Secretion System.

Type VIIb Secretion System (T7SSb) has been recently identified in Firmicutes resembling the mycobacterial T7SSa. Despite limited sequence homology, T7SSa and T7SSb have substrates with striking structural similarities, the WXG100 proteins. Recent advances in Staphylococcus spp. proposed that T7SSb is involved in intra-species competition. However, the architecture and mechanism of action of this secretion complex remain largely obscure. Here, we investigate the T7SSb of Bacillus subtilis as a model system. We report the first evidence of B. subtilis ability to mediate intra- and inter-species antibacterial activity in a T7SSb-dependent manner. Then, we present the first systematic investigation of the T7SSb protein-protein network, revealing novel interactions and highlighting the central role of the pseudokinase subunit YukC in the assembly of the system. Its direct interaction with a T7SSb-secreted toxin supports its role in recruiting substrates to the secretion machinery. Finally, we solved the crystal structure of full-length transmembrane YukC defining novel structural motifs and suggesting that intrinsic flexibility modulates the orientation of the pseudokinase domains and YukC function. Overall, our results provide a better understanding on the role and molecular organisation of the T7SSb, opening new perspectives for the comprehension of this poorly characterized molecular machine.

microbiology↗