1.
Erttmann, Saskia F., et al.
(författare)
The gut microbiota prime systemic antiviral immunity via the cGAS-STING-IFN-I axis
2022
Ingår i: Immunity. - : Elsevier BV. - 1074-7613 .- 1097-4180. ; 55:5, s. 847-861
Tidskriftsartikel (refereegranskat) abstract
The microbiota are vital for immune homeostasis and provide a competitive barrier to bacterial and fungal pathogens. Here, we investigated how gut commensals modulate systemic immunity and response to viral infection. Antibiotic suppression of the gut microbiota reduced systemic tonic type I interferon (IFN-I) and antiviral priming. The microbiota-driven tonic IFN-I-response was dependent on cGAS-STING but not on TLR signaling or direct host-bacteria interactions. Instead, membrane vesicles (MVs) from extracellular bacteria activated the cGAS-STING-IFN-I axis by delivering bacterial DNA into distal host cells. DNA-containing MVs from the gut microbiota were found in circulation and promoted the clearance of both DNA (herpes simplex virus type 1) and RNA (vesicular stomatitis virus) viruses in a cGAS-dependent manner. In summary, this study establishes an important role for the microbiota in peripheral cGAS-STING activation, which promotes host resistance to systemic viral infections. Moreover, it uncovers an underappreciated risk of antibiotic use during viral infections.
2.
Corkery, Dale, et al.
(författare)
Vibrio cholerae cytotoxin MakA induces noncanonical autophagy resulting in the spatial inhibition of canonical autophagy
2021
Ingår i: Journal of Cell Science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 134:5
Tidskriftsartikel (refereegranskat) abstract
Autophagy plays an essential role in the defense against manymicrobial pathogens as a regulator of both innate and adaptive immunity. Some pathogens have evolved sophisticated mechanisms that promote their ability to evade or subvert host autophagy. Here, we describe a novel mechanism of autophagy modulation mediated by the recently discovered Vibrio cholerae cytotoxin, motility-associatedkilling factor A (MakA). pH-dependent endocytosis of MakA by host cells resulted in the formation of a cholesterol-rich endolysosomal membrane aggregate in the perinuclear region. Aggregate formation induced the noncanonical autophagy pathway driving unconventional LC3 (herein referring to MAP1LC3B) lipidation on endolysosomal membranes. Subsequent sequestration of the ATG12-ATG5-ATG16L1 E3-like enzyme complex, required for LC3 lipidation at the membranous aggregate, resulted in an inhibition of both canonical autophagy and autophagy-related processes, including the unconventional secretion of interleukin-1β (IL-1β). These findings identify a novel mechanismof host autophagy modulation and immune modulation employed by V. cholerae during bacterial infection.
3.
Graffeuil, Antoine, et al.
(författare)
Polar mutagenesis of polycistronic bacterial transcriptional units using Cas12a
2022
Ingår i: Microbial Cell Factories. - : BioMed Central. - 1475-2859 .- 1475-2859. ; 21:1
Tidskriftsartikel (refereegranskat) abstract
Background: Functionally related genes in bacteria are often organized and transcribed as polycistronic transcrip‑ tional units. Examples are the fim operon, which codes for biogenesis of type 1 fimbriae in Escherichia coli, and the atp operon, which codes for the FoF1 ATP synthase. We tested the hypothesis that markerless polar mutations could be efficiently engineered using CRISPR/Cas12a in these loci.Results: Cas12a‑mediated engineering of a terminator sequence inside the fimA gene occurred with efficiencies between 10 and 80% and depended on the terminator’s sequence, whilst other types of mutations, such as a 97 bp deletion, occurred with 100% efficiency. Polar mutations using a terminator sequence were also engineered in the atp locus, which induced its transcriptional shutdown and produced identical phenotypes as a deletion of the whole atp locus (ΔatpIBEFHAGDC). Measuring the expression levels in the fim and atp loci showed that many supposedly non‑ polar mutants induced a significant polar effect on downstream genes. Finally, we also showed that transcriptional shutdown or deletion of the atp locus induces elevated levels of intracellular ATP during the exponential growth phase.Conclusions: We conclude that Cas12a‑mediated mutagenesis is an efficient simple system to generate polar mutants in E. coli. Different mutations were induced with varying degrees of efficiency, and we confirmed that all these mutations abolished the functions encoded in the fim and atp loci. We also conclude that it is difficult to predict which mutagenesis strategy will induce a polar effect in genes downstream of the mutation site. Furthermore the strategies described here can be used to manipulate the metabolism of E. coli as showcased by the increase in intra‑ cellular ATP in the markerless ΔatpIBEFHAGDC mutant.
4.
Pakharukova, Natalia, et al.
(författare)
Archaic chaperone-usher pili self-secrete into superelastic zigzag springs
2022
Ingår i: Nature. - : Nature Publishing Group. - 0028-0836 .- 1476-4687. ; 609:7926, s. 335-340
Tidskriftsartikel (refereegranskat) abstract
Adhesive pili assembled via the chaperone-usher pathway (CUP) are hair-like appendages that mediate host tissue colonization and biofilm formation of Gram-negative bacteria 1-3. Archaic CUP pili, the most diverse and widespread CUP adhesins, are promising vaccine and drug targets due to their prevalence in the most troublesome multidrug-resistant (MDR) pathogens 1,4,5. However, their architecture and assembly-secretion process remain unknown. Here, we present the 3.4 Å resolution cryo-electron microscopy structure of the prototypical archaic Csu pilus that mediates biofilm formation of Acinetobacter baumannii, a notorious MDR nosocomial pathogen. In contrast to the thick helical tubes of the classical type 1 and P pili, archaic pili assemble into a conceptually novel ultrathin zigzag architecture secured by an elegant clinch mechanism. The molecular clinch provides the pilus with high mechanical stability as well as superelasticity, a property observed now for the first time in biomolecules, while enabling a more economical and faster pilus production. Furthermore, we demonstrate that clinch formation at the cell surface drives pilus secretion through the outer membrane. These findings suggest that clinch-formation inhibitors might represent a new strategy to fight MDR bacterial infections.
5.
Zlatkov, Nikola, 1985-, et al.
(författare)
Unconventional Cyclic di-GMP Signaling in Escherichia coli
2020. - 1
Ingår i: Microbial Cyclic Di-Nucleotide Signaling. - Chambridge : Springer International Publishing. - 9783030333072 - 9783030333089 ; , s. 487-517
Bokkapitel (övrigt vetenskapligt/konstnärligt) abstract
The species Escherichia coli represents an unfathomable variety of commensal, pathogenic, and environmental strains. The conventional cyclic di-GMP signaling in E. coli controls sessility-motility changes linked to commensalism and/or pathogenicity. Extraintestinal Pathogenic E. coli (ExPEC) are “commensals” that can cause an array of infections outside the gastrointestinal tract. To accommodate their pathogenic lifestyle with the commensal one, ExPEC biology is shaped not only by the presence of specific virulence genes and pathoadaptive mutations but also by regulatory adaptations. Bioinformatic and genetic studies indicate that the cyclic di-GMP signaling network is included in the adaptation process. For example, some neuroinvasive ExPEC were found to maintain reduced cyclic di-GMP levels due to RpoS deactivation, resulting in loss of appearance of the rugose morphotype. Moreover, ExPEC has a diversified repertoire of cyclic di-GMP degrading enzymes obtained by acquisition of novel genes often associated with fimbrial biogenesis gene clusters (e.g., sfaY/papY/focY) and by modification or deletion of specific core genome genes. For example, the majority of ExPEC contains a shortened allelic variant of the ycgG gene and some ExPEC strains do not even carry the genetic locus. New combinations of regulators offer a new cyclic di-GMP platform for S-fimbrial biogenesis and for new metabolic capabilities leading to citrate utilization and ferric citrate uptake. In this review, we outline the prerequisites for the unconventional signaling network, the rationale behind its existence in ExPEC, and future perspectives in studies of ExPEC.
6.
Ahsan, Umaira, et al.
(författare)
Emergence of high colistin resistance in carbapenem resistant Acinetobacter baumannii in Pakistan and its potential management through immunomodulatory effect of an extract from Saussurea lappa
2022
Ingår i: Frontiers in Pharmacology. - : Frontiers Media S.A.. - 1663-9812. ; 13
Tidskriftsartikel (refereegranskat) abstract
Carbapenem resistant Acinetobacter baumannii has emerged as one of the most difficult to treat nosocomial bacterial infections in recent years. It was one of the major causes of secondary infections in Covid-19 patients in developing countries. The polycationic polypeptide antibiotic colistin is used as a last resort drug to treat carbapenem resistant A. baumannii infections. Therefore, resistance to colistin is considered as a serious medical threat. The purpose of this study was to assess the current status of colistin resistance in Pakistan, a country where carbapenem resistant A. bumannii infections are endemic, to understand the impact of colistin resistance on virulence in mice and to assess alternative strategies to treat such infections. Out of 150 isolates collected from five hospitals in Pakistan during 2019–20, 84% were carbapenem resistant and 7.3% were additionally resistant to colistin. There were two isolates resistant to all tested antibiotics and 83% of colistin resistant isolates were susceptible to only tetracycline family drugs doxycycline and minocycline. Doxycycline exhibited a synergetic bactericidal effect with colistin even in colistin resistant isolates. Exposure of A. baumannii 17978 to sub inhibitory concentrations of colistin identified novel point mutations associated with colistin resistance. Colistin tolerance acquired independent of mutations in lpxA, lpxB, lpxC, lpxD, and pmrAB supressed the proinflammatory immune response in epithelial cells and the virulence in a mouse infection model. Moreover, the oral administration of water extract of Saussuria lappa, although not showing antimicrobial activity against A. baumannii in vitro, lowered the number of colonizing bacteria in liver, spleen and lung of the mouse model and also lowered the levels of neutrophils and interleukin 8 in mice. Our findings suggest that the S. lappa extract exhibits an immunomodulatory effect with potential to reduce and cure systemic infections by both opaque and translucent colony variants of A. baumannii.
7.
Bala, Anju, et al.
(författare)
Insights into the genetic contexts of sulfonamide resistance among early clinical isolates of Acinetobacter baumannii
2023
Ingår i: Infection, Genetics and Evolution. - : Elsevier. - 1567-1348 .- 1567-7257. ; 112
Tidskriftsartikel (refereegranskat) abstract
Since the late 1930s, resistance to sulfonamides has been accumulating across bacterial species including Acinetobacter baumannii, an opportunistic pathogen increasingly implicated the spread of antimicrobial resistance worldwide. Our study aimed to explore events involved in the acquisition of sulfonamide resistance genes, particularly sul2, among the earliest available isolates of A. baumannii. The study utilized the genomic data of 19 strains of A. baumannii isolated before 1985. The whole genomes of 5 clinical isolates obtained from the Culture Collection University of Göteborg (CCUG), Sweden, were sequenced using the Illumina MiSeq system. Acquired resistance genes, insertion sequence elements and plasmids were detected using ResFinder, ISfinder and Plasmidseeker, respectively, while sequence types (STs) were assigned using the PubMLST Pasteur scheme. BLASTn was used to verify the occurrence of sul genes and to map their genetic surroundings. The sul1 and sul2 genes were detected in 4 and 9 isolates, respectively. Interestingly, sul2 appeared thirty years earlier than sul1. The sul2 gene was first located in the genomic island GIsul2 located on a plasmid, hereafter called NCTC7364p. With the emergence of international clone 1, the genetic context of sul2 evolved toward transposon Tn6172, which was also plasmid-mediated. Sulfonamide resistance in A. baumannii was efficiently acquired and transferred vertically, e.g., among the ST52 and ST1 isolates, as well as horizontally among non-related strains by means of a few efficient transposons and plasmids. Timely acquisition of the sul genes has probably contributed to the survival skill of A. baumannii under the high antimicrobial stress of hospital settings.
8.
Mushtaq, Fizza, et al.
(författare)
Colony phase variation switch modulates antimicrobial tolerance and biofilm formation in Acinetobacter baumannii
2024
Ingår i: Microbiology Spectrum. - : American Society for Microbiology. - 2165-0497. ; 12:2
Tidskriftsartikel (refereegranskat) abstract
Carbapenem-resistant Acinetobacter baumannii causes one of the most difficult-to-treat nosocomial infections. Polycationic drugs like polymyxin B or colistin and tetracycline drugs such as doxycycline or minocycline are commonly used to treat infections caused by carbapenem-resistant A. baumannii. Here, we show that a subpopulation of cells associated with the opaque/translucent colony phase variation by A. baumannii AB5075 displays differential tolerance to subinhibitory concentrations of colistin and tetracycline. Using a variety of microscopic techniques, we demonstrate that extracellular polysaccharide moieties mediate colistin tolerance to opaque A. baumannii at single-cell level and that mushroom-shaped biofilm structures protect opaque bacteria at the community level. The colony switch phenotype is found to alter several traits of A. baumannii, including long-term survival under desiccation, tolerance to ethanol, competition with Escherichia coli, and intracellular survival in the environmental model host Acanthamoeba castellanii. Additionally, our findings suggest that extracellular DNA associated with membrane vesicles can promote colony switching in a DNA recombinase-dependent manner.Importance: As a WHO top-priority drug-resistant microbe, Acinetobacter baumannii significantly contributes to hospital-associated infections worldwide. One particularly intriguing aspect is its ability to reversibly switch its colony morphotype on agar plates, which has been remarkably underexplored. In this study, we employed various microscopic techniques and phenotypic assays to investigate the colony phase variation switch under different clinically and environmentally relevant conditions. Our findings reveal that the presence of a poly N-acetylglucosamine-positive extracellular matrix layer contributes to the protection of bacteria from the bactericidal effects of colistin. Furthermore, we provide intriguing insights into the multicellular lifestyle of A. baumannii, specifically in the context of colony switch variation within its predatory host, Acanthamoeba castellanii.
9.
Tadala, Lalitha, 1991-
(författare)
Role of extracellular ATP in immune mechanisms against infections
2022
Doktorsavhandling (övrigt vetenskapligt/konstnärligt) abstract
Inflammation is driven either by infection with pathogens or sterile stimuli, such as tissue damage or autoimmune diseases. Upon tissue damage, ATP is released passively from the dead or compromised cells. During stress, ATP can be secreted from the cells. Extracellular ATP (eATP) acts as an endogenous danger signal. An increase in eATP is sensed by cell surface purinergic receptors and regulates the onset and resolution of inflammation. Extracellular ATP is an important inflammatory mediator during sterile inflammation. On the other hand, the role of eATP is poorly studied during infection, both bacterial and viral. In this thesis, I present the molecular mechanisms underlying ATP secretion during bacterial infections and the role of eATP in human hantaviral infections.During infection with certain enteropathogenic Gram-negative bacteria, intestinal epithelial cells secrete ATP via connexin hemichannels as an alert signal to activate the immune system, which triggers acute inflammation in the gut. However, neither what triggers ATP secretion nor the molecular mechanisms of ATP secretion were known. Pharmacological, genetic, and microscopy-based evidence shows that during invasive bacterial infections, the plasma membrane ruffles act as mechanical immune stimuli and activate the inherently mechanosensitive plasma membrane channel PIEZO1. Mechanically activated PIEZO1 leads to the influx of Ca2+ ions and concurrent ATP secretion. In addition, PIEZO1 also activates protective transcriptional responses. Thus, PIEZO1 acts as a sensor for invasive infection using mechanical stimuli, unlike the so-far-described immune sensors of infection, which all recognize microbial components by chemical interaction.During human hantavirus infection, the humoral immune responses are poorly studied. Our collaborators found that atypical B cells, which do not have the surface marker CD27, show increased frequency in a cohort of hantavirus-infected patients. CD27 shedding in murine lymphocytes had been previously linked to eATP-dependent activation of a purinergic receptor7. To test whether ATP levels in the circulation of hantavirus-infected patients are elevated, an approach to perform same-day eATP quantifications in human plasma was developed. This assay was used to establish the normal eATP concentration in plasma in a cohort of healthy volunteers and to show that eATP levels are elevated in the acute and convalescent stages of hantavirus infection. Further, the addition of ATP to isolated human B cells recapitulated the observed CD27 shedding via a metallomatrix proteinase-8-dependent (MMP8) mechanism. Together, these projects provide evidence for the importance of eATP in bacterial and viral infectious diseases.
10.
Ahmad, Irfan, et al.
(författare)
A Cyclic-di-GMP signalling network regulates biofilm formation and surface associated motility of Acinetobacter baumannii 17978
2020
Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 10:1
Tidskriftsartikel (refereegranskat) abstract
Acinetobacter baumannii has emerged as an increasing multidrug-resistant threat in hospitals and a common opportunistic nosocomial pathogen worldwide. However, molecular details of the pathogenesis and physiology of this bacterium largely remain to be elucidated. Here we identify and characterize the c-di-GMP signalling network and assess its role in biofilm formation and surface associated motility. Bioinformatic analysis revealed eleven candidate genes for c-di-GMP metabolizing proteins (GGDEF/EAL domain proteins) in the genome of A. baumannii strain 17978. Enzymatic activity of the encoded proteins was assessed by molecular cloning and expression in the model organisms Salmonella typhimurium and Vibrio cholerae. Ten of the eleven GGDEF/EAL proteins altered the rdar morphotype of S. typhimurium and the rugose morphotype of V. cholerae. The over expression of three GGDEF proteins exerted a pronounced effect on colony formation of A. baumannii on Congo Red agar plates. Distinct panels of GGDEF/EAL proteins were found to alter biofilm formation and surface associated motility of A. baumannii upon over expression. The GGDEF protein A1S_3296 appeared as a major diguanylate cyclase regulating macro-colony formation, biofilm formation and the surface associated motility. AIS_3296 promotes Csu pili mediated biofilm formation. We conclude that a functional c-di-GMP signalling network in A. baumannii regulates biofilm formation and surface associated motility of this increasingly important opportunistic bacterial pathogen.
