In Campylobacter jejuni, a new type of chaperone receives heme from ferrochelatase

Funding Information: JZ is a recipient of the MSCA-IF-2019 Individual Fellowship H2020-WF-02-2019, 101003441. FS acknowledges support from the European Research Council (ERC) under the European Union’s Horizon 2020 Research and Innovation program (grant agreement 803768). This work was also financially supported by Fundação para a Ciência e Tecnologia (Portugal) through PTDC/BIA-BQM/28642/2017 grant (LS), the MOSTMICRO-ITQB R&D Unit (UIDB/04612/2020 and UIDP/04612/2020), and the LS4FUTURE Associated Laboratory (LA/P/0087/2020). Publisher Copyright: Copyright © 2023 Zamarreño Beas, Videira, Karavaeva, Lourenço, Almeida, Sousa and Saraiva.Intracellular heme formation and trafficking are fundamental processes in living organisms. Bacteria and archaea utilize three biogenesis pathways to produce iron protoporphyrin IX (heme b) that diverge after the formation of the common intermediate uroporphyrinogen III (uro’gen III). In this study, we identify and provide a detailed characterization of the enzymes involved in the transformation of uro’gen III into heme in Campylobacter jejuni, demonstrating that this bacterium utilizes the protoporphyrin-dependent (PPD) pathway. In general, limited knowledge exists regarding the mechanisms by which heme b reaches its target proteins after this final step. Specifically, the chaperones necessary for trafficking heme to prevent the cytotoxic effects associated with free heme remain largely unidentified. In C. jejuni, we identified a protein named CgdH2 that binds heme with a dissociation constant of 4.9 ± 1.0 µM, and this binding is impaired upon mutation of residues histidine 45 and 133. We demonstrate that C. jejuni CgdH2 establishes protein–protein interactions with ferrochelatase, suggesting its role in facilitating heme transfer from ferrochelatase to CgdH2. Furthermore, phylogenetic analysis reveals that C. jejuni CgdH2 is evolutionarily distinct from the currently known chaperones. Therefore, CgdH2 is the first protein identified as an acceptor of intracellularly formed heme, expanding our knowledge of the mechanisms underlying heme trafficking within bacterial cells.publishersversionpublishe

Species-specific activity of antibacterial drug combinations

International audienceThe spread of antimicrobial resistance has become a serious public health concern, making once treatable diseases deadly again and undermining breakthrough achievements of modern medicine 1,2. Drug combinations can aid in fighting multi-drug resistant (MDR) bacterial infections, yet, are largely unexplored and rarely used in clinics. To identify general principles for antibacterial drug combinations and understand their potential, we profiled ~3,000 dose-resolved combinations of antibiotics, human-targeted drugs and food additives in 6 strains from three Gram-negative pathogens, Escherichia coli, Salmonella Typhimurium and Pseudomonas aeruginosa. Despite their phylogenetic relatedness, more than 70% of the detected drug-drug interactions are species-specific and 20% display strain specificity, revealing a large potential for narrow-spectrum therapies. Overall, antagonisms are more common than synergies and occur almost exclusively between drugs targeting different cellular processes, whereas synergies are more conserved and enriched in drugs targeting the same process. We elucidate mechanisms underlying this dichotomy and further use our resource to dissect the interactions of the food additive, vanillin. Finally, we demonstrate that several synergies are effective against MDR clinical isolates in vitro and during Galleria mellonella infections with one reverting resistance to the last-resort antibiotic, colistin

Regulation of bacterial haem biosynthesis

Funding Information: This work was financially supported by Fundação para a Ciência e Tecnologia (Portugal) by grant PTDC/BIA-BQM/28642/2017 and through R&D unit LISBOA-01-0145-FEDER007660 (MostMicro) cofounded by FCT/MCTES and FEDER funds under the PT2020 Partnership Agreement. We also acknowledge funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No. 810856. JB is recipient of the MSCA-IF-2019 Individual Fellowship H2020-WF-02-2019, 101003441. Funding Information: This work was financially supported by Fundação para a Ciência e Tecnologia (Portugal) by grant PTDC/BIA-BQM/28642/2017 and through R&D unit LISBOA-01-0145-FEDER007660 (MostMicro) cofounded by FCT/MCTES and FEDER funds under the PT2020 Partnership Agreement. We also acknowledge funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 810856. JB is recipient of the MSCA-IF-2019 Individual Fellowship H2020-WF-02-2019, 101003441. Publisher Copyright: © 2021 Elsevier B.V.Haem b and sirohaem are two iron-chelated modified tetrapyrroles that serve as prosthetic groups in proteins with crucial roles in a variety of biological functions, such as gas transport, respiration, and nitrite and sulphite reduction. These tetrapyrroles are synthesised from 5-aminolaevulinic acid and share a common pathway until the formation of uroporphyrinogen III, from where the synthesis diverges. In bacteria, sirohaem is produced from uroporphyrinogen III through the activities of one, two or three separate proteins, while haem b is synthesised through three distinct pathways. The biosynthesis of haem b and sirohaem comprises intermediates and end-products that are unstable or potentially hazardous to the cell. Therefore, the cellular metabolic fluxes of tetrapyrroles need to be tightly controlled by substrate channelling and/or other regulatory processes. This review summarises the recent advances on the regulation and protein–protein interactions controlling the formation of sirohaem and haem b in bacteria.publishersversionpublishe

Bioenergetic State of Escherichia coli Controls Aminoglycoside Susceptibility

International audienceAminoglycosides (AG) have been used against Gram-negative bacteria for decades. Yet, how bacterial metabolism and environmental conditions modify AG toxicity is poorly understood. Here, we show that the level of AG susceptibility varies depending on the nature of the respiratory chain that Escherichia coli uses for growth, i.e., oxygen, nitrate, or fumarate. We show that all components of the fumarate respiratory chain, namely, hydrogenases 2 and 3, the formate hydrogenlyase complex, menaquinone, and fumarate reductase are required for AG-mediated killing under fumarate respiratory conditions. In addition, we show that the AAA+ ATPase RavA and its Von Wildebrand domain-containing partner, ViaA, are essential for AG to act under fumarate respiratory conditions. This effect was true for all AG that were tested but not for antibiotics from other classes. In addition, we show that the sensitizing effect of RavA-ViaA is due to increased gentamicin uptake in a proton motive force-dependent manner. Interestingly, the sensitizing effect of RavA-ViaA was prominent in poor energy conservation conditions, i.e., with fumarate, but dispensable under high energy conservation conditions, i.e., in the presence of nitrate or oxygen. We propose that RavA-ViaA can facilitate uptake of AG across the membrane in low-energy cellular states. IMPORTANCE Antibiotic resistance is a major public health, social, and economic problem. Aminoglycosides (AG) are known to be highly effective against Gram-negative bacteria, but their use is limited to life-threatening infections because of their nephrotoxicity and ototoxicity at therapeutic dose. Elucidation of AG-sensitization mechanisms in bacteria would allow reduced effective doses of AG. Here, we have identified the molecular components involved in anaerobic fumarate respiration that are required for AG to kill. In addition to oxidoreductases and menaquinone, this includes new molecular players, RavA, an AAA+ ATPase, and ViaA, its partner that has the VWA motif. Remarkably, the influence of RavA-ViaA on AG susceptibility varies according to the type of bioenergetic metabolism used by E. coli. This is a significant advance because anaerobiosis is well known to reduce the antibacterial activity of AG. This study highlights the critical importance of the relationship between culture conditions, metabolism, and antibiotic susceptibility

Oxidative stress antagonizes fluoroquinolone drug sensitivity via the SoxR-SUF Fe-S cluster homeostatic axis

International audienceThe level of antibiotic resistance exhibited by bacteria can vary as a function of environmental conditions. Here, we report that phenazine-methosulfate (PMS), a redox-cycling compound (RCC) enhances resistance to fluoroquinolone (FQ) norfloxacin. Genetic analysis showed that E. coli adapts to PMS stress by making Fe-S clusters with the SUF machinery instead of the ISC one. Based upon phenotypic analysis of soxR, acrA, and micF mutants, we showed that PMS antagonizes fluoroquinolone toxicity by SoxR-mediated up-regulation of the AcrAB drug efflux pump. Subsequently, we showed that despite the fact that SoxR could receive its cluster from either ISC or SUF, only SUF is able to sustain efficient SoxR maturation under exposure to prolonged PMS period or high PMS concentrations. This study furthers the idea that Fe-S cluster homeostasis acts as a sensor of environmental conditions, and because its broad influence on cell metabolism, modifies the antibiotic resistance profile of E. coli

The gp27-like Hub of VgrG Serves as Adaptor to Promote Hcp Tube Assembly

International audienceContractile injection systems are multiprotein complexes that use a spring-like mechanism to deliver effectors into target cells. In addition to using a conserved mechanism, these complexes share a common core known as the tail. The tail comprises an inner tube tipped by a spike, wrapped by a contractile sheath, and assembled onto a baseplate. Here, using the type VI secretion system (T6SS) as a model of contractile injection systems, we provide molecular details on the interaction between the inner tube and the spike. Reconstitution into the Escherichia coli heterologous host in the absence of other T6SS components and in vitro experiments demonstrated that the Hcp tube component and the VgrG spike interact directly. VgrG deletion studies coupled to functional assays showed that the N-terminal domain of VgrG is sufficient to interact with Hcp, to initiate proper Hcp tube polymerization, and to promote sheath dynamics and Hcp release. The interaction interface between Hcp and VgrG was then mapped using docking simulations, mutagenesis, and cysteine-mediated cross-links. Based on these results, we propose a model in which the VgrG base serves as adaptor to recruit the first Hcp hexamer and initiates inner tube polymerization