Biology subjects

Sgro, G. G.

Publications and source records attributed to Sgro, G. G..

6 recordsLinked to original sources

bioRxiv2026

Bordetella pertussis BctCBA Mediates Citrate-Dependent Zn2+ and Ni2+ binding

Bordetella pertussis, the causative agent of whooping cough, is a reemerging public health threat. While the Tripartite Tricarboxylate Transporter (TTT) system BctCBA was previously implicated solely in citrate uptake, we demonstrate that the solute-binding protein BctC specifically binds citrate chelated with Zn{superscript 2} and Ni{superscript 2}. To elucidate the molecular mechanism of this interaction, we determined the crystal structures of BctC in three states: apo, open, and closed (citrate-zinc-bound), defining the structural determinants for metal-citrate recognition. Comparative analyses suggest that citrate-mediated divalent cation binding is a widespread feature among bacterial TTT homologs. Finally, in silico modeling of the full BctCBA complex predicts an elevator-type transport mechanism. Together, these findings redefine the functional scope of BctCBA, revealing a sophisticated strategy by which B. pertussis exploits organic chelators to acquire essential trace metals during infection.

biochemistry↗

bioRxiv2026

Cryo-EM structure analysis of phage {Phi}Xacm4-11 that infects the phytopathogen Xanthomonas citri

Very few bacteriophages that infect Xanthomonas species have been characterized genetically and only one 3D structure, the capsid of a siphovirus that infects the phytopathogen Xanthomonas citri, has been determined at high resolution. This study presents the annotated DNA sequence and detailed structural analysis of {Phi}Xacm4-11, a podovirus that infects Xanthomonas citri, shedding light on its unique architecture and functional attributes, providing insights into the molecular mechanisms underlying host recognition and infection. Annotation of the genome revealed conserved features among related phages, but also distinct genetic elements that may contribute to {Phi}Xacm4-11s specificity toward X. citri. Genes associated with host recognition and infection were identified, including the genes potentially coding for the receptor-binding proteins (RBPs) at the tail fibre tip, offering insights into their role in bacterial attachment. Using high-resolution cryo-electron microscopy, we resolved the architecture of the mature, pre-released virion, revealing a T7-like head-tail assembly with a well-defined portal-tail complex embedded at a unique fivefold vertex. Our findings provide a detailed view of the structural and functional components of {Phi}Xacm4-11, furthering our understanding of its molecular interactions with X. citri and its potential application in phage therapy against phytopathogens. SIGNIFICANCE STATEMENTBacteriophages are increasingly recognized as powerful tools to control bacterial pathogens in medicine and agriculture, yet the structural basis of host recognition and genome delivery remains poorly understood for most phages. Here, we present a comprehensive structural and functional analysis of {Phi}Xacm4-11, a podovirus that infects the plant pathogen Xanthomonas citri. By combining genome annotation, proteomics, and high-resolution cryo-electron microscopy, we reveal the complete architecture of the mature virion and its specialized portal-tail machinery. Our results show how this short-tailed phage deploys an internal injection device to penetrate the bacterial cell envelope and highlight structural features linked to type IV pilus-dependent infection. These findings provide insights into phage entry mechanisms and establish {Phi}Xacm4-11 as a model for engineering biocontrol strategies.

biochemistry↗

bioRxiv2025

Global identification of Chromobacterium violaceum T6SS effectors reveals an Rhs antibacterial toxin featuring FIX and ADP-ribosyltransferase domains

Bacteria coexist in polymicrobial communities where they engage in complex interactions, including interbacterial antagonism. We recently reported that the environmental bacterial pathogen Chromobacterium violaceum has an active type VI secretion system (T6SS), which plays a crucial role in interbacterial competition. However, the arsenal of toxic effectors delivered by this T6SS remains unknown. Here, we identify the repertoire of C. violaceum T6SS effectors and characterize a novel antibacterial Rhs-family effector, RhsF (Rhs with a FIX domain), and its cognate immunity protein, RhsFi. Using mass spectrometry analyses of secreted proteins and proteins co-immunoprecipitated with VgrG3, we identified six novel effector candidates, namely four phospholipases, a protein of unknown function, and the previously-uncharacterized Rhs protein, RhsF (CV_1431). RhsF contains an N-terminal FIX domain and was shown to intoxicate susceptible bacteria in a T6SS-dependent manner. The action of the C-terminal toxin domain of RhsF (RhsF-CT) is prevented in the presence of RhsFi (CV_1430), confirming that RhsF-RhsFi comprises an effector-immunity pair. The structure of the RhsF-CT/RhsFi complex determined by X-ray crystallography (1.85 [A] resolution) revealed that RhsF-CT shares structural similarity with ADP-ribosyltransferase toxins and that RhsFi inhibits toxicity via direct occlusion of the RhsF-CT catalytic site. Functional assays confirmed that RhsF toxicity requires a catalytic triad composed of R1403, Y1456, and E1497 residues. Overall, our findings reveal the effectors secreted by the T6SS of C. violaceum, establish RhsF as a potent antibacterial toxin, and confirm T6SS-dependent delivery of a FIX-containing Rhs protein, expanding the known repertoire of bacterial arms involved in microbial competition.

biochemistry↗

bioRxiv2024

Velamins: the first green-light emitting class of wild-type Ca2+-regulated photoproteins isolated from the ctenophore Velamen parallelum

Ca2+-regulated photoproteins (CaPhs) consist of single-chain globular proteins to which coelenterazine, a widely distributed marine luminogenic substrate (the luciferin), binds along with molecular oxygen, producing a stable peroxide. Upon Ca2+ addition, CaPhs undergo conformational changes leading to the cyclization of the peroxide and the formation of a high-energy intermediate. Subsequently, its decomposition yields coelenteramide in an excited state and results in the emission of a flash of light. To date, all CaPhs reported produce blue light ({lambda}max 465-495 nm). Here, we report the cloning and functional characterization of a novel class of wild-type CaPhs capable of emitting green light: velamins, isolated from the bioluminescent ctenophore Velamen parallelum. Ten unique photoprotein-like sequences were recovered and grouped in three main clusters. Representative sequences were cloned, expressed, purified, and regenerated into the active His-tagged -, {beta}-, and {gamma}-velamins. Upon injection of a calcium-containing buffer into the velamin, a flash of green light ({lambda}max 500-508 nm) was observed across pH values ranging from 7 to 9. Whilst -velamin isoforms exhibited the highest light emission activity, {beta}- and {gamma}-velamins were found to be more thermostable at higher temperatures. Velamins are the only known wild-type Ca2+-regulated photoproteins that exhibit the longest wavelength in light emission, making them a promising model for studying spectral modulation. As a result, velamins hold potential for enhancing the sensitivity of signal detection in analytical systems, particularly when dealing with complex biological matrices.

biochemistry↗

bioRxiv2023

The protective function of an immunity protein against the cis-toxic effects of a Xanthomonas Type IV Secretion System Effector

Many bacterial species use specialized secretion systems to translocate proteinaceous toxic effectors into target bacterial cells. In most cases, effectors are encoded in bicistronic operons with their cognate immunity proteins. The current model is that immunity proteins could, in principle, provide protection in two different ways: i) by avoiding self-intoxication (suicide or cis-intoxication) or ii) by inhibiting intoxication due to "friendly-fire" translocation from neighboring sister cells (fratricide or trans-intoxication). Here, we set out to distinguish between these two protection mechanisms in the case of the bactericidal Xanthomonas citri Type IV Secretion System (X-T4SS), where killing is due to the action of a cocktail of secreted effectors (X-Tfes) that are inhibited by their cognate immunity proteins (X-Tfis). We use a set of X. citri mutants lacking multiple X-Tfe/X-Tfi pairs to show that X-Tfis are not absolutely required to protect against trans-intoxication. Our investigation then focused on the in vivo function of the lysozyme-like effector X-TfeXAC2609 and its cognate immunity protein X-TfiXAC2610. We observe the accumulation of damage in the X. citri cell envelope and inhibition of biofilm formation due to the action of X-TfeXAC2609 in the absence of X-TfiXAC2610. We show that X-TfeXAC2609 toxicity is independent of an active X-T4SS and that X-TfiXAC2610 protects the cell colony against X-TfeXAC2609-induced cis-intoxication via autolysis. In vitro assays employing X-TfiXAC2610 mutants were used to test and validate an AlphaFold2-derived model of the X-TfeXAC2609-X-TfiXAC2610 complex which presents topological similarities with the distantly related Tse1/Tsi1 complex from P. aeruginosa and the the i-type lysozyme from Meretrix lusoria (MI-iLys) in complex with PliI-Ah from Aeromonas hydrophila. While immunity proteins in other systems have been shown to protect against attacks by sister cells (trans-intoxication), this is the first description of an antibacterial secretion system in which the immunity proteins are dedicated to protecting cells against cis-intoxication.

biochemistry↗

bioRxiv2020

Zebrafish studies on the vaccine candidate to COVID-19, the Spike protein: Production of antibody and adverse reaction

Establishing new experimental animal models to assess the safety and immune response to the antigen used in the development of COVID-19 vaccine is an imperative issue. Based on the advantages of using zebrafish as a model in research, herein we suggest doing this to test the safety of the putative vaccine candidates and to study immune response against the virus. We produced a recombinant N-terminal fraction of the Spike SARS-CoV-2 protein and injected it into adult female zebrafish. The specimens generated humoral immunity and passed the antibodies to the eggs. However, they presented adverse reactions and inflammatory responses similar to severe cases of human COVID-19. The analysis of the structure and function of zebrafish and human Angiotensin-converting enzyme 2, the main human receptor for virus infection, presented remarkable sequence similarities. Moreover, bioinformatic analysis predicted protein-protein interaction of the Spike SARS-CoV-2 fragment and the Toll-like receptor pathway. It might help in the choice of future therapeutic pharmaceutical drugs to be studied. Based on the in vivo and in silico results presented here, we propose the zebrafish as a model for translational research into the safety of the vaccine and the immune response of the vertebrate organism to the SARS-CoV-2 virus.

immunology↗