43 results found
Ronneau S, Michaux C, Helaine S, 2023, Decline in nitrosative stress drives antibiotic persister regrowth during infection., Cell Host Microbe, Vol: 31, Pages: 993-1006.e6
Internalization of pathogenic bacteria by macrophages results in formation of antibiotic-tolerant persisters. These cells are maintained in a non-growing state for extended periods of time, and it is assumed that their growth resumption causes infection relapse after cessation of antibiotic treatment. Despite this clinical relevance, the signals and conditions that drive persister regrowth during infection are not yet understood. Here, we found that after persister formation in macrophages, host reactive nitrogen species (RNS) produced in response to Salmonella infection lock persisters in growth arrest by intoxicating their TCA cycle, lowering cellular respiration and ATP production. Intracellular persisters resume growth when macrophage RNS production subsides and functionality of their TCA cycle is regained. Persister growth resumption within macrophages is slow and heterogeneous, dramatically extending the time the persister reservoir feeds infection relapse. Using an inhibitor of RNS production, we can force recalcitrant bacteria to regrow during antibiotic treatment, thereby facilitating their eradication.
Michaux C, Ronneae S, Giorgio RT, et al., 2022, Antibiotic tolerance and persistence have distinct fitness trade-offs, PLOS PATHOGENS, Vol: 18, ISSN: 1553-7366
Paulini S, Fabiani FD, Weiss AS, et al., 2022, The Biological Significance of Pyruvate Sensing and Uptake in Salmonella enterica Serovar Typhimurium, MICROORGANISMS, Vol: 10
Paulini S, Fabiani FD, Weiß AS, et al., 2022, Identification of two pyruvate transporters in<i>Salmonella enterica</i>serovar Typhimurium and their biological relevance
<jats:title>SUMMARY</jats:title><jats:p>Pyruvate (CH<jats:sub>3</jats:sub>COCOOH) is the simplest of the alpha-keto acids and is at the interface of several metabolic pathways both in prokaryotes and eukaryotes. In an amino acid-rich environment, fast-growing bacteria excrete pyruvate instead of completely metabolizing it. The role of pyruvate uptake in pathological conditions is still unclear. In this study, we identified two pyruvate-specific transporters, BtsT and CstA, in<jats:italic>Salmonella enterica</jats:italic>serovar Typhimurium (<jats:italic>S</jats:italic>. Typhimurium). Expression of<jats:italic>btsT</jats:italic>is induced by the histidine kinase/response regulator system BtsS/BtsR upon sensing extracellular pyruvate (threshold 200 μM), whereas expression of<jats:italic>cstA</jats:italic>is maximal in the stationary phase. Both pyruvate transporters were found to be important for the uptake of this compound, but also for chemotaxis to pyruvate, survival under oxidative and nitrosative stress, and persistence of<jats:italic>S</jats:italic>. Typhimurium in response to gentamicin. Compared with the wild-type, the Δ<jats:italic>btsT</jats:italic>Δ<jats:italic>cstA</jats:italic>mutant has disadvantages in antibiotic persistence in macrophages, as well as in colonization and systemic infection in gnotobiotic mice. These data demonstrate the surprising complexity of the two pyruvate uptake systems in<jats:italic>S</jats:italic>. Typhimurium.</jats:p>
Ronneau S, Hill PWS, Helaine S, 2021, Antibiotic persistence and tolerance: not just one and the same, CURRENT OPINION IN MICROBIOLOGY, Vol: 64, Pages: 76-81, ISSN: 1369-5274
Grabe GJ, Giorgio RT, Hall AMJ, et al., 2021, Auxiliary interfaces support the evolution of specific toxin-antitoxin pairing, NATURE CHEMICAL BIOLOGY, Vol: 17, Pages: 1296-1304, ISSN: 1552-4450
Moldoveanu AL, Rycroft JA, Helaine S, 2021, Impact of bacterial persisters on their host, CURRENT OPINION IN MICROBIOLOGY, Vol: 59, Pages: 65-71, ISSN: 1369-5274
Michaux C, Ronneau S, Helaine S, 2021, Studying Antibiotic Persistence During Infection, BACTERIAL PERSISTENCE, 2 EDITION, Pages: 273-289, ISSN: 1064-3745
Michaux C, Ronneau S, Helaine S, 2021, Correction to: Studying Antibiotic Persistence During Infection.
Pham THM, Brewer SM, Thurston T, et al., 2020, Salmonella-driven polarization of granuloma macrophages antagonizes TNF-mediated pathogen restriction during persistent infection, Cell Host and Microbe, Vol: 27, Pages: 54-67.E5, ISSN: 1931-3128
Many intracellular bacteria can establish chronic infection and persist in tissues within granulomas composed of macrophages. Granuloma macrophages exhibit heterogeneous polarization states, or phenotypes, that may be functionally distinct. Here, we elucidate a host-pathogen interaction that controls granuloma macrophage polarization and long-term pathogen persistence during Salmonella Typhimurium ( STm) infection. We show that STm persists within splenic granulomas that are densely populated by CD11b +CD11c +Ly6C + macrophages. STm preferentially persists in granuloma macrophages reprogrammed to an M2 state, in part through the activity of the effector SteE, which contributes to the establishment of persistent infection. We demonstrate that tumor necrosis factor (TNF) signaling limits M2 granuloma macrophage polarization, thereby restricting STm persistence. TNF neutralization shifts granuloma macrophages toward an M2 state and increases bacterial persistence, and these effects are partially dependent on SteE activity. Thus, manipulating granuloma macrophage polarization represents a strategy for intracellular bacteria to overcome host restriction during persistent infection.
Panagi I, Jennings E, Zeng J, et al., 2020, Salmonella effector SteE converts the mammalian serine/threonine kinase GSK3 into a tyrosine kinase to direct macrophage polarization., Cell Host and Microbe, Vol: 27, Pages: 41-53.e6, ISSN: 1931-3128
Many Gram-negative bacterial pathogens antagonize anti-bacterial immunity through translocated effector proteins that inhibit pro-inflammatory signaling. In addition, the intracellular pathogen Salmonella enterica serovar Typhimurium initiates an anti-inflammatory transcriptional response in macrophages through its effector protein SteE. However, the target(s) and molecular mechanism of SteE remain unknown. Here, we demonstrate that SteE converts both the amino acid and substrate specificity of the host pleiotropic serine/threonine kinase GSK3. SteE itself is a substrate of GSK3, and phosphorylation of SteE is required for its activity. Remarkably, phosphorylated SteE then forces GSK3 to phosphorylate the non-canonical substrate signal transducer and activator of transcription 3 (STAT3) on tyrosine-705. This results in STAT3 activation, which along with GSK3 is required for SteE-mediated upregulation of the anti-inflammatory M2 macrophage marker interleukin-4Rα (IL-4Rα). Overall, the conversion of GSK3 to a tyrosine-directed kinase represents a tightly regulated event that enables a bacterial virulence protein to reprogram innate immune signaling and establish an anti-inflammatory environment.
Hill P, Helaine S, 2019, Antibiotic Persisters and Relapsing Salmonella enterica Infections., Persister Cells and Infectious Disease, Editors: Lewis, Publisher: Springer Nature, ISBN: 9783030252410
Antibiotic persistence is defined as the ability of a subpopulation of bacteria within a clonal antibiotic-susceptible population to survive antibiotic treatment. Studies on antibiotic persistence have traditionally been carried out on bacteria cultured in laboratory media. However, over recent years, there has been a push to study antibiotic persisters in more physiologically relevant systems. Thus, the concept of antibiotic persistence during infection, which refers to the ability of a subpopulation of bacteria to survive combined host and antibiotic challenges, has emerged as a major new frontier of research. Here, we discuss the relevance and principles of this concept using relapsing Salmonella enterica infections as an example. We critically evaluate the clinical and experimental evidence for the existence and importance of antibiotic persisters in relapsing Salmonella infections; we outline our current understanding of the molecular mechanisms that enable successful antibiotic persistence during infection; and, finally, we discuss the challenges for this nascent field going forward.
Ronneau S, Helaine S, 2019, Clarifying the Link between Toxin-Antitoxin Modules and Bacterial Persistence, JOURNAL OF MOLECULAR BIOLOGY, Vol: 431, Pages: 3462-3471, ISSN: 0022-2836
Panagi I, Jennings E, Zeng J, et al., 2019, The Salmonella Effector SteE Converts the Mammalian Serine/Threonine Kinase GSK3 into a Tyrosine Kinase, Publisher: Elsevier BV
Balaban NQ, Helaine S, Lewis K, et al., 2019, Definitions and guidelines for research on antibiotic persistence (vol 17, pg 441, 2019), NATURE REVIEWS MICROBIOLOGY, Vol: 17, Pages: 460-460, ISSN: 1740-1526
Balaban NQ, Helaine S, Lewis K, et al., 2019, Definitions and guidelines for research on antibiotic persistence, Nature Reviews Microbiology, Vol: 17, Pages: 441-448, ISSN: 1740-1526
Increasing concerns about the rising rates of antibiotic therapy failure and advances in single-cell analyses have inspired a surge of research into antibiotic persistence. Bacterial persister cells represent a subpopulation of cells that can survive intensive antibiotic treatment without being resistant. Several approaches have emerged to define and measure persistence, and it is now time to agree on the basic definition of persistence and its relation to the other mechanisms by which bacteria survive exposure to bactericidal antibiotic treatments, such as antibiotic resistance, heteroresistance or tolerance. In this Consensus Statement, we provide definitions of persistence phenomena, distinguish between triggered and spontaneous persistence and provide a guide to measuring persistence. Antibiotic persistence is not only an interesting example of non-genetic single-cell heterogeneity, it may also have a role in the failure of antibiotic treatments. Therefore, it is our hope that the guidelines outlined in this article will pave the way for better characterization of antibiotic persistence and for understanding its relevance to clinical outcomes.
Gollan B, Grabe G, Michaux C, et al., 2019, Bacterial Persisters and Infection: Past, Present, and Progressing, ANNUAL REVIEW OF MICROBIOLOGY, VOL 73, Vol: 73, Pages: 359-385, ISSN: 0066-4227
Stapels DAC, Hill PWS, Westermann AJ, et al., 2018, Salmonella persisters undermine host immune defenses during antibiotic treatment, Science, Vol: 362, Pages: 1156-1160, ISSN: 0036-8075
Many bacterial infections are hard to treat and tend to relapse, possibly due to the presence of antibiotic-tolerant persisters. In vitro, persister cells appear to be dormant. After uptake of Salmonella species by macrophages, nongrowing persisters also occur, but their physiological state is poorly understood. In this work, we show that Salmonella persisters arising during macrophage infection maintain a metabolically active state. Persisters reprogram macrophages by means of effectors secreted by the Salmonella pathogenicity island 2 type 3 secretion system. These effectors dampened proinflammatory innate immune responses and induced anti-inflammatory macrophage polarization. Such reprogramming allowed nongrowing Salmonella cells to survive for extended periods in their host. Persisters undermining host immune defenses might confer an advantage to the pathogen during relapse once antibiotic pressure is relieved.
Wilcox B, Osterman I, Serebryakova M, et al., 2018, Escherichia coli ItaT is a type II toxin that inhibits translation by acetylating isoleucyl-tRNA(Ile), Nucleic Acids Research, Vol: 46, Pages: 7873-7885, ISSN: 0305-1048
Prokaryotic toxin–antitoxin (TA) modules are highly abundant and are involved in stress response and drug tolerance. The most common type II TA modules consist of two interacting proteins. The type II toxins are diverse enzymes targeting various essential intracellular targets. The antitoxin binds to cognate toxin and inhibits its function. Recently, TA modules whose toxins are GNAT-family acetyltransferases were described. For two such systems, the target of acetylation was shown to be aminoacyl-tRNA: the TacT toxin targets aminoacylated elongator tRNAs, while AtaT targets the amino acid moiety of initiating tRNAMet. We show that the itaRT gene pair from Escherichia coli encodes a TA module with acetyltransferase toxin ItaT that specifically and exclusively acetylates Ile-tRNAIle thereby blocking translation and inhibiting cell growth. ItaT forms a tight complex with the ItaR antitoxin, which represses the transcription of itaRT operon. A comprehensive bioinformatics survey of GNAT acetyltransferases reveals that enzymes encoded by validated or putative TA modules are common and form a distinct branch of the GNAT family tree. We speculate that further functional analysis of such TA modules will result in identification of enzymes capable of specifically targeting many, perhaps all, aminoacyl tRNAs.
Rycroft J, Gollan B, Grabe G, et al., 2018, Activity of acetyltransferase toxins involved in Salmonella persister formation during macrophage infection, Nature Communications, Vol: 9, ISSN: 2041-1723
Non-typhoidal Salmonella strains are responsible for invasive infections associated withhigh mortality and recurrence in sub-Saharan Africa and there is strong evidence for clonalrelapse following antibiotic treatment. Persisters are non-growing bacteria that are thought tobe responsible for the recalcitrance of many infections to antibiotics. Toxin-antitoxin systemsare stress-responsive elements that are important for Salmonella persister formation,specifically during infection. Here we report analysis of persister formation of clinical invasive strains of S. Typhimurium and Enteritidis in human primary macrophages. We show that allthe invasive clinical isolates of both serovars that we tested produce high levels of persisters following internalization by human macrophages. Our genome comparison reveals that S.Enteritidis and S. Typhimurium strains contain three acetyltransferase toxins that we characterize structurally and functionally. We show that all induce the persister state byinhibiting translation through acetylation of aminoacyl-tRNAs. However, they differ in theirpotency and target partially different subsets of aminoacyl-tRNAs, potentially accounting fortheir non-redundant effect.
Fisher RA, Gollan B, Helaine S, 2017, Persistent bacterial infections and persister cells, Nature Reviews Microbiology, Vol: 15, Pages: 453-464, ISSN: 1740-1526
Many bacteria can infect and persist inside their hosts for long periods of time. This can be due to immunosuppression of the host, immune evasion by the pathogen and/or ineffective killing by antibiotics. Bacteria can survive antibiotic treatment if they are resistant or tolerant to a drug. Persisters are a subpopulation of transiently antibiotic-tolerant bacterial cells that are often slow-growing or growth-arrested, and are able to resume growth after a lethal stress. The formation of persister cells establishes phenotypic heterogeneity within a bacterial population and has been hypothesized to be important for increasing the chances of successfully adapting to environmental change. The presence of persister cells can result in the recalcitrance and relapse of persistent bacterial infections, and it has been linked to an increase in the risk of the emergence of antibiotic resistance during treatment. If the mechanisms of the formation and regrowth of these antibiotic-tolerant cells were better understood, it could lead to the development of new approaches for the eradication of persistent bacterial infections. In this Review, we discuss recent developments in our understanding of bacterial persisters and their potential implications for the treatment of persistent infections.
Hall A, Gollan B, Helaine S, 2017, Toxin-antitoxin systems: reversible toxicity, Current Opinion in Microbiology, Vol: 36, Pages: 102-110, ISSN: 1879-0364
Toxin–antitoxin (TA) systems encoded on the plasmids and chromosomes of bacteria are emerging as key players in stress adaptation. In particular, they have been implicated in the induction of persisters non-growing cells that can evade antibiotic exposure. TA toxins operate by a diverse range of mechanisms, either destructive or conservative, leading to the reversible growth arrest of bacterial cells. Whilst the molecular mechanisms of intoxication are now well understood, we still have very little information on how corrupted cells reawaken. Alongside the phenomenon of conditional cooperativity, new evidence suggests that the effects of some TA toxins can be reversed, allowing non-growing cells to be detoxified and growth to resume.
Saliba A-E, Li L, Westermann AJ, et al., 2016, Single-cell RNA-seq ties macrophage polarization to growth rate of intracellular Salmonella, Nature Microbiology, Vol: 2, Pages: 1-8, ISSN: 2058-5276
Intracellular bacterial pathogens can exhibit large heterogeneity in growth rate inside host cells, with major consequences for the infection outcome. If and how the host responds to this heterogeneity remains poorly understood. Here, we combined a fluorescent reporter of bacterial cell division with single-cell RNA-sequencing analysis to study the macrophage response to different intracellular states of the model pathogen Salmonella enterica serovar Typhimurium. The transcriptomes of individual infected macrophages revealed a spectrum of functional host response states to growing and non-growing bacteria. Intriguingly, macrophages harbouring non-growing Salmonella display hallmarks of the proinflammatory M1 polarization state and differ little from bystander cells, suggesting that non-growing bacteria evade recognition by intracellular immune receptors. By contrast, macrophages containing growing bacteria have turned into an anti-inflammatory, M2-like state, as if fast-growing intracellular Salmonella overcome host defence by reprogramming macrophage polarization. Additionally, our clustering approach reveals intermediate host functional states between these extremes. Altogether, our data suggest that gene expression variability in infected host cells shapes different cellular environments, some of which may favour a growth arrest of Salmonella facilitating immune evasion and the establishment of a long-term niche, while others allow Salmonella to escape intracellular antimicrobial activity and proliferate.
Cheverton AM, Gollan B, Przydacz M, et al., 2016, A Salmonella Toxin Promotes Persister Formation through Acetylation of tRNA., Molecular cell, Vol: 63, Pages: 86-96, ISSN: 1097-2765
The recalcitrance of many bacterial infections to antibiotic treatment is thought to be due to the presence of persisters that are non-growing, antibiotic-insensitive cells. Eventually, persisters resume growth, accounting for relapses of infection. Salmonella is an important pathogen that causes disease through its ability to survive inside macrophages. After macrophage phagocytosis, a significant proportion of the Salmonella population forms non-growing persisters through the action of toxin-antitoxin modules. Here we reveal that one such toxin, TacT, is an acetyltransferase that blocks the primary amine group of amino acids on charged tRNA molecules, thereby inhibiting translation and promoting persister formation. Furthermore, we report the crystal structure of TacT and note unique structural features, including two positively charged surface patches that are essential for toxicity. Finally, we identify a detoxifying mechanism in Salmonella wherein peptidyl-tRNA hydrolase counteracts TacT-dependent growth arrest, explaining how bacterial persisters can resume growth.
Santos AJM, Durkin C, Helaine S, et al., 2016, Clustered intracellular Salmonella Typhimurium Blocks Host Cell Cytokinesis, Infection and Immunity, Vol: 84, Pages: 2149-2158, ISSN: 1098-5522
Several bacterial pathogens and viruses interfere with the cell cycle of their host cells to enhance virulence. This is especially apparent in bacteria that colonise the gut epithelium, where inhibition of the cell cycle of infected cells enhances the intestinal colonisation. We found that intracellular Salmonella enterica Typhimurium induced the binucleation of a large proportion of epithelial cells by 14 hours post invasion, which was dependent on an intact Salmonella pathogenicity island-2 (SPI-2) type 3 secretion system. The SPI-2 effectors SseF and SseG were required to induce binucleation. SseF and SseG are known to maintain microcolonies of Salmonella-containing vacuoles close to the microtubule organising centre of infected epithelial cells. During host cell division these clustered microcolonies prevented the correct localisation of members of the chromosomal passenger complex and mitotic kinesin-like protein 1, and consequently prevented cytokinesis. Tetraploidy, arising from a cytokinesis defect, is known to have a deleterious effect on subsequent cell divisions, either resulting in chromosomal instabilities or cell cycle arrest. In infected mice, proliferation of small intestinal epithelial cells was compromised in an SseF/SseG-dependent manner, suggesting that cytokinesis failure caused by S. Typhimurium delays epithelial cell turnover in the intestine.
Helaine S, holden DW, sampson SL, et al., 2016, Elucidating population-wide mycobacterial replication dynamics at the single-cell level, Microbiology, Vol: 162, Pages: 966-978, ISSN: 1350-0872
Mycobacterium tuberculosis infections result in a spectrum of clinical outcomes, and frequently the infection persists in a latent, clinically asymptomatic state. The within-host bacterial population is likely to be heterogeneous, and it is thought that persistent mycobacteria arise from a small population of viable, but non-replicating (VBNR) cells. These are likely to be antibiotic tolerant and necessitate prolonged treatment. Little is known about these persistent mycobacteria, since they are very difficult to isolate. To address this, we have successfully developed a replication reporter system for use in M. tuberculosis. This approach, termed fluorescence dilution, exploits 2 fluorescent reporters; a constitutive reporter allows the tracking of bacteria, while an inducible reporter enables the measurement of bacterial replication. The application of fluorescent single-cell analysis to characterise intracellular M. tuberculosis identified a distinct subpopulation of non-growing mycobacteria in murine macrophages. The presence of VBNR and actively replicating mycobacteria was observed within the same macrophage after 48 hours of infection. Furthermore, our results suggest that macrophage uptake resulted in enrichment of non- or slowly replicating bacteria (as revealed by DCS treatment); this population is likely to be highly enriched for persisters, based on its drug tolerant phenotype. These results demonstrate the successful application of the novel dual fluorescent reporter system both in vitro and in macrophage infection models to provide a window into mycobacterial population heterogeneity.
Fisher RA, Cheverton AM, Helaine S, 2016, Analysis of Macrophage-Induced Salmonella Persisters., Methods in Molecular Biology, Vol: 1333, Pages: 177-187, ISSN: 1940-6029
A small subpopulation of non-replicating, multidrug-tolerant bacteria is present within clonal populations of many bacterial species. Known as persisters, these bacteria are probably the cause of relapsing infections such as typhoid fever. Formation of non-growing Salmonella persisters is stimulated by macrophage phagocytosis. This chapter outlines methods to identify and study persisters resulting from interactions between bacterial pathogens and their hosts. We use their antibiotic tolerance for isolation and enumeration and developed a method to study the heterogeneity of growth within clonal populations through single-cell analysis.
Figueira R, Brown DR, Ferreira D, et al., 2015, Adaptation to sustained nitrogen starvation by Escherichia coli requires the eukaryote-like serine/ threonine kinase YeaG, Scientific Reports, Vol: 5, ISSN: 2045-2322
Caly DL, Coulthurst SJ, An S-Q, et al., 2014, Communication, cooperation, and social interactions: a report from the Third Young Microbiologists Symposium on Microbe Signalling, Organisation, and Pathogenesis, Journal of Bacteriology, Vol: 196, Pages: 3527-3533, ISSN: 1098-5530
The third Young Microbiologists Symposium took place on the vibrant campus of the University of Dundee, Scotland, from the 2nd to 3rd of June 2014. The symposium attracted over 150 microbiologists from 17 different countries. The significant characteristic of this meeting was that it was specifically aimed at providing a forum for junior scientists to present their work. The meeting was supported by the Society for General Microbiology and the American Society for Microbiology, with further sponsorship from the European Molecular Biology Organization, the Federation of European Microbiological Societies, and The Royal Society of Edinburgh. In this report, we highlight some themes that emerged from the many exciting talks and poster presentations given by the young and talented microbiologists in the area of microbial gene expression, regulation, biogenesis, pathogenicity, and host interaction.
Helaine S, Kugelberg E, 2014, Bacterial persisters: formation, eradication, and experimental systems, TRENDS IN MICROBIOLOGY, Vol: 22, Pages: 417-424, ISSN: 0966-842X
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