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The purine adenosine 5′-triphosphate (ATP) is not only a universal intracellular energy carrier but plays also an important role as extracellular signaling molecule. Purinergic signaling is involved in many physiological and pathological processes like coagulation, inflammation, or sepsis in mammals. ATP is well-known as a messenger for intercellular communications in multicellular organisms, but phylogenetically much older unicellular organisms like yeast or bacteria use ATP as an extracellular signaling molecule as well. However, the mechanisms of ATP secretion by bacteria and its extracellular implications still have to be elucidated. This review will provide an overview of the current knowledge about bacterial extracellular ATP (eATP) under homeostatic conditions and during growth. Possible secretion mechanisms of ATP by bacteria will be discussed and implications of bacterial ATP are shown, with a focus on bacteria–host interactions.

Sepsis causes millions of deaths per year worldwide and is a current global health priority declared by the WHO. Sepsis-related deaths are a result of dysregulated inflammatory immune responses indicating the need to develop strategies to target inflammation. An important mediator of inflammation is extracellular adenosine triphosphate (ATP) that is released by inflamed host cells and tissues, and also by bacteria in a strain-specific and growth-dependent manner. Here, we investigated the mechanisms by which bacteria release ATP. Using genetic mutant strains of Escherichia coli ( E. coli ), we demonstrate that ATP release is dependent on ATP synthase within the inner bacterial membrane. In addition, impaired integrity of the outer bacterial membrane notably contributes to ATP release and is associated with bacterial death. In a mouse model of abdominal sepsis, local effects of bacterial ATP were analyzed using a transformed E. coli bearing an arabinose-inducible periplasmic apyrase hydrolyzing ATP to be released. Abrogating bacterial ATP release shows that bacterial ATP suppresses local immune responses, resulting in reduced neutrophil counts and impaired survival. In addition, bacterial ATP has systemic effects via its transport in outer membrane vesicles (OMV). ATP-loaded OMV are quickly distributed throughout the body and upregulated expression of genes activating degranulation in neutrophils, potentially contributing to the exacerbation of sepsis severity. This study reveals mechanisms of bacterial ATP release and its local and systemic roles in sepsis pathogenesis. Sepsis is a severe condition often caused by the body’s immune system overreacting to bacterial infections. This can lead to excessive inflammation which damages organs and requires urgent medical care. With sepsis claiming millions of lives each year, new and improved ways to treat this condition are urgently needed. One potential strategy for treating sepsis is to target the underlying mechanisms controlling inflammation. Inflamed and dying cells release molecules called ATP (the energy carrier of all living cells), which strongly influence the immune system, including during sepsis. In the early stages of an infection, ATP acts as a danger signal warning the body that something is wrong. However, over time, it can worsen infections by disturbing the immune response. Similar to human cells, bacteria release their own ATP, which can have different impacts depending on the type of bacteria and where they are located in the body. However, it is not well understood how bacterial ATP influences severe infections like sepsis. To investigate this question, Spari et al analysed how ATP is released from Escherichia coli , a type of bacteria that causes severe infections . This revealed that the bacteria secrete ATP directly in to their environment and via small membrane-bound structures called vesicles. Spari et al. then probed a mouse model of abdominal sepsis which had been infected with E. coli that release either normal or low levels of ATP. They found that the ATP released from E. coli impaired the mice’s survival and lowered the number of neutrophils (immune cells which are important for defending against bacteria) at the site of the infection. The ATP secreted via vesicles also altered the role of neutrophils but in more distant regions, and it is possible that these changes may be contributing to the severity of sepsis. These findings provide a better understanding of how ATP released from bacteria impacts the immune system during sepsis. While further investigation is needed, these findings may offer new therapeutic targets for treating sepsis.

Ehrlichiosis is a potentially life-threatening disease caused by infection with the obligatory intracellular bacteria Ehrlichia species. Ehrlichia japonica infection of mice provides an animal model of ehrlichiosis as it recapitulates full-spectrum and lethal ehrlichiosis in humans. The E. japonica transposon mutant of EHF0962 , which encodes a previously uncharacterized hypothetical protein, is attenuated in both infection and virulence in mice. EHF0962 was hence named here as resistance-inducing protein of Ehrlichia (RipE). Using this Δ ripE mutant, we studied how RipE protein contributes to Ehrlichia pathogenesis. Ehrlichia species have an intracellular developmental cycle and a brief extracellular stage to initiate a new cycle of infection. Majority of RipE proteins were expressed on the surface of the smaller infectious dense-core stage of bacteria. Extracellular Δ ripE E. japonica contained significantly less adenosine triphosphate (ATP) and lost infectivity more rapidly in culture compared with wild-type (WT) E. japonica . Genetic complementation in the Δ ripE mutant or overexpression of ripE in WT E. japonica significantly increased bacterial ATP levels, and RipE-overexpressing E. japonica was more virulent in mice than WT E. japonica . RipE is conserved among Ehrlichia species. Immunization of mice with recombinant RipE induced an in vitro infection-neutralizing antibody, significantly prolonged survival time after a lethal dose of E. japonica challenge, and cross-protected mice from infection by Ehrlichia chaffeensis , the agent of human monocytic ehrlichiosis. Our findings shed light on the extracellular stage of Ehrlichia , highlighting the importance of RipE and ATP levels in Ehrlichia for extracellular resistance and the next cycle of infection. Thus, RipE is a critical Ehrlichia protein for infection as such can be a potential vaccine target for ehrlichiosis.

Five bacterial species that are most likely to have putative prokaryotic inward rectifier K+ (Kir) channels were selected by in silico sequence homology and membrane topology analyses with respect to the number of transmembrane domains (TMs) and the presence of K+ selectivity filter and/or ATP binding sites in reference to rabbit heart inward rectifier K+ channel (Kir6.2). A dot blot assay with genomic DNAs when probed with whole rabbit Kir6.2 cDNA further supported the in silico analysis by exhibiting a stronger hybridization in species with putative Kir's compared to one without a Kir. Among them, Chromobacterium violaceum gave rise to a putative Kir channel gene, which was PCR-cloned into the bacterial expression vector pET30b(+), and its expression was induced in Escherichia coli and confirmed by gel purification and immunoblotting. On the other hand, this putative bacterial Kir channel was functionally expressed in Xenopus oocytes and its channel activity was measured electrophysiologically by using two electrode voltage clamping (TEVC). Results revealed a K+ current with characteristics similar to those of the ATP-sensitive K+ (K-ATP) channel. Collectively, cloning and functional characterization of bacterial ion channels could be greatly facilitated by combining the in silico analysis and heterologous expression in Xenopus oocytes.

Heat shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone which is essential in eukaryotes. It is required for the activation and stabilization of a wide variety of client proteins and many of them are involved in important cellular pathways. Since Hsp90 affects numerous physiological processes such as signal transduction, intracellular transport, and protein degradation, it became an interesting target for cancer therapy. Structurally, Hsp90 is a flexible dimeric protein composed of three different domains which adopt structurally distinct conformations. ATP binding triggers directionality in these conformational changes and leads to a more compact state. To achieve its function, Hsp90 works together with a large group of cofactors, termed co-chaperones. Co-chaperones form defined binary or ternary complexes with Hsp90, which facilitate the maturation of client proteins. In addition, posttranslational modifications of Hsp90, such as phosphorylation and acetylation, provide another level of regulation. They influence the conformational cycle, co-chaperone interaction, and inter-domain communications. In this review, we discuss the recent progress made in understanding the Hsp90 machinery.

Target protection proteins confer resistance to the host organism by directly binding to the antibiotic target. One class of such proteins are the antibiotic resistance (ARE) ATP-binding cassette (ABC) proteins of the F-subtype (ARE-ABCFs), which are widely distributed throughout Gram-positive bacteria and bind the ribosome to alleviate translational inhibition from antibiotics that target the large ribosomal subunit. Here, we present single-particle cryo-EM structures of ARE-ABCF-ribosome complexes from three Gram-positive pathogens: Enterococcus faecalis LsaA, Staphylococcus haemolyticus VgaA LC and Listeria monocytogenes VgaL. Supported by extensive mutagenesis analysis, these structures enable a general model for antibiotic resistance mediated by these ARE-ABCFs to be proposed. In this model, ABCF binding to the antibiotic-stalled ribosome mediates antibiotic release via mechanistically diverse long-range conformational relays that converge on a few conserved ribosomal RNA nucleotides located at the peptidyltransferase center. These insights are important for the future development of antibiotics that overcome such target protection resistance mechanisms. Mitochondrial ribosomes (mitoribosomes) are characterized by a distinct architecture and thus biogenesis pathway. Here, cryo-EM structures of mitoribosome large subunit assembly intermediates elucidate final steps of 16 S rRNA folding, methylation and peptidyl transferase centre (PTC) completion, as well as functions of several mitoribosome assembly factors.

Infections arising from multidrug-resistant pathogenic bacteria are spreading rapidly throughout the world and threaten to become untreatable. The origins of resistance are numerous and complex, but one underlying factor is the capacity of bacteria to rapidly export drugs through the intrinsic activity of efflux pumps. In this Review, we describe recent advances that have increased our understanding of the structures and molecular mechanisms of multidrug efflux pumps in bacteria. Clinical and laboratory data indicate that efflux pumps function not only in the drug extrusion process but also in virulence and the adaptive responses that contribute to antimicrobial resistance during infection. The emerging picture of the structure, function and regulation of efflux pumps suggests opportunities for countering their activities.

Wall teichoic acid (WTA) is a polyol phosphate polymer that covalently decorates peptidoglycan of gram-positive bacteria, including Staphylococcus aureus . Central to WTA biosynthesis is flipping of lipid-linked precursors across the cell membrane by TarGH, a type V ABC transporter. Here, we present cryo-EM structures of S. aureus TarGH in the presence of targocil-II, a promising small-molecule lead with β-lactam antibiotic synergistic action. Targocil-II binds to the extracellular dimerisation interface of TarG, we suggest mimicking flipped but not yet released substrate. In absence of targocil-II and in complex with ATP analogue ATPγS, determined at 2.3 Å resolution, the ATPase active site is allosterically inhibited. This is due to a so far undescribed D-loop conformation, potentially minimizing spurious ATP hydrolysis in the absence of substrate. Targocil-II binding comparatively causes local and remote conformational changes through to the TarH active site, with the D-loop now optimal for ATP hydrolysis. These structures suggest an ability to modulate ATP hydrolysis in a WTA substrate dependent manner and a jammed ATPase cycle as the basis of the observed inhibition by targocil-II. The molecular insights provide an unprecedented basis for development of TarGH targeted therapeutics for treatment of multidrug-resistant S. aureus and other gram-positive bacterial infections. Cryo-EM illuminates atomic binding details and far-ranging conformational effects of the inhibitor targocil-II on wall teichoic acid ABC transporter TarGH, a virulence target from the antibiotic resistant, often hospital acquired pathogen S. aureus .

A number of species of Gram-negative bacteria can use insoluble minerals of Fe(III) and Mn(IV) as extracellular respiratory electron acceptors. In some species of Shewanella, deca-heme electron transfer proteins lie at the extracellular face of the outer membrane (OM), where they can interact with insoluble substrates. To reduce extracellular substrates, these redox proteins must be charged by the inner membrane/periplasmic electron transfer system. Here, we present a spectro-potentiometric characterization of a trans-OM icosa-heme complex, MtrCAB, and demonstrate its capacity to move electrons across a lipid bilayer after incorporation into proteoliposomes. We also show that a stable MtrAB subcomplex can assemble in the absence of MtrC; an MtrBC subcomplex is not assembled in the absence of MtrA; and MtrA is only associated to the membrane in cells when MtrB is present. We propose a model for the modular organization of the MtrCAB complex in which MtrC is an extracellular element that mediates electron transfer to extracellular substrates and MtrB is a trans-OM spanning β-barrel protein that serves as a sheath, within which MtrA and MtrC exchange electrons. We have identified the MtrAB module in a range of bacterial phyla, suggesting that it is widely used in electron exchange with the extracellular environment.

New drugs are needed to treat gram-negative bacterial infections. These bacteria are protected by an outer membrane which prevents many antibiotics from reaching their cellular targets. The outer leaflet of the outer membrane contains LPS, which is responsible for creating this permeability barrier. Interfering with LPS biogenesis affects bacterial viability. We developed a cell-based screen that identifies inhibitors of LPS biosynthesis and transport by exploiting the nonessentiality of this pathway in Acinetobacter. We used this screen to find an inhibitor of MsbA, an ATP-dependent flippase that translocates LPS across the inner membrane. Treatment with the inhibitor caused mislocalization of LPS to the cell interior. The discovery of an MsbA inhibitor, which is universally conserved in all gram-negative bacteria, validates MsbA as an antibacterial target. Because our cell-based screen reports on the function of the entire LPS biogenesis pathway, it could be used to identify compounds that inhibit other targets in the pathway, which can provide insights into vulnerabilities of the gram-negative cell envelope.