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Pore-forming toxins (PFTs) are recognized as major virulence factors produced by both Gram-positive and Gram-negative bacteria. While the effects of PFTs have been extensively investigated using mammalian cells as a model system, their interactions with the environmental host, Acanthamoeba castellanii remains less understood. This study employed high-throughput image screening (HTI), advanced microscopy, western blot analysis, and cytotoxicity assays to evaluate the impact of PFT-producing bacterial species on their virulence against A. castellanii. Our unbiased HTI data analysis reveals that the cyst induction of A. castellanii in response to various bacterial species does not correlate with the presence of PFT-producing bacteria. Moreover, A. castellanii demonstrates resistance to PFT-mediated cytotoxicity, in contrast to mammalian macrophages. Notably, Vibrio anguillarum and Ralstonia eutropha triggered a high frequency of cyst formation and cytotoxicity in infected A. castellanii. In summary, our findings reveal that A. castellanii exhibits a unique resistance to PFTs, unlike mammalian cells, suggesting its potential ecological role as a reservoir for diverse pathogenic species and its influence on their persistence and proliferation in the environment. (Figure presented.)

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.

Acinetobacter baumannii has emerged as one of the most common extensive drug-resistant nosocomial bacterial pathogens. Not only can the bacteria survive in hospital settings for long periods, but they are also able to resist adverse conditions. However, underlying regulatory mechanisms that allow A. baumannii to cope with these conditions and mediate its virulence are poorly understood. Here, we show that bi-stable expression of the Csu pili, along with the production of poly-N-acetyl glucosamine, regulates the formation of Mountain-like biofilm-patches on glass surfaces to protect bacteria from the bactericidal effect of colistin. Csu pilus assembly is found to be an essential component of mature biofilms formed on glass surfaces and of pellicles. By using several microscopic techniques, we show that clinical isolates of A. baumannii carrying abundant Csu pili mediate adherence to epithelial cells. In addition, Csu pili suppressed surface-associated motility but enhanced colonization of bacteria into the lungs, spleen, and liver in a mouse model of systemic infection. The screening of c-di-GMP metabolizing protein mutants of A. baumannii 17978 for the capability to adhere to epithelial cells led us to identify GGDEF/EAL protein AIS_2337, here denoted PdeB, as a major regulator of Csu pili-mediated virulence and biofilm formation. Moreover, PdeB was found to be involved in the type IV pili-regulated robustness of surface-associated motility. Our findings suggest that the Csu pilus is not only a functional component of mature A. baumannii biofilms but also a major virulence factor promoting the initiation of disease progression by mediating bacterial adherence to epithelial cells.

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.

Acinetobacter baumannii is a Gram-negative bacterium increasingly implicated in hospital-acquired infections and outbreaks. Effective prevention and control of such infections are commonly challenged by the frequent emergence of multidrug-resistant strains. Here we introduce Ab-web (https://www.acinetobacterbaumannii.no), the first online platform for sharing expertise on A. baumannii. Abweb is a species-centric knowledge hub, initially with 10 articles organized into two main sections, 'Overview' and 'Topics', and three themes, 'epidemiology', 'antibiotic resistance', and 'virulence'. The 'workspace' section provides a spot for colleagues to collaborate, build, and manage joint projects. Ab-web is a community-driven initiative amenable to constructive feedback and new ideas.

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.

Recently, we demonstrated that a novel bacterial cytotoxin, the protein MakA which is released by Vibrio cholerae, is a virulence factor, causing killing of Caenorhabditis elegans when the worms are grazing on the bacteria. Studies with mammalian cell cultures in vitro indicated that MakA could affect eukaryotic cell signalling pathways involved in lipid biosynthesis. MakA treatment of colon cancer cells in vitro caused inhibition of growth and loss of cell viability. These findings prompted us to investigate possible signalling pathways that could be targets of the MakA-mediated inhibition of tumour cell proliferation. Initial in vivo studies with MakA producing V. cholerae and C. elegans suggested that the MakA protein might target the PIP5K1α phospholipid-signalling pathway in the worms. Intriguingly, MakA was then found to inhibit the PIP5K1α lipid-signalling pathway in cancer cells, resulting in a decrease in PIP5K1α and pAkt expression. Further analyses revealed that MakA inhibited cyclin-dependent kinase 1 (CDK1) and induced p27 expression, resulting in G2/M cell cycle arrest. Moreover, MakA induced downregulation of Ki67 and cyclin D1, which led to inhibition of cell proliferation. This is the first report about a bacterial protein that may target signalling involving the cancer cell lipid modulator PIP5K1α in colon cancer cells, implying an anti-cancer effect.

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.

Escherichia coli arbitrarily encompasses facultative anaerobic, rod-shaped bacteria with defined respiratory and fermentative types of metabolism. The species diversification has been further advanced by atypical strains whose features deviate from the essential species-specific morphological and metabolic cutoff. The morphological cutoff is exemplified by bacterial filamentation. E. coli filamentation has been studied from two different perspectives: the first considers filamentation as a result of adaptive strategies and response to stress, while the second is based on findings from the cell division of E. coli’s conditional mutants. Another cutoff is represented by E. coli’s inability to use citrate as a sole carbon and energy source. In this study, we compared two atypical E. coli strains that belong to the same neuroinvasive ecovar but exhibit either of the two phenotypes that deviate from the species’ features. While E. coli RS218 exists in the form of filaments incapable of growth on citrate, strain IHE3034 is represented as normal-sized bacteria able to ferment citrate under oxic conditions in the presence of glucose; in this paper, we show that these two phenotypes result from a bona fide trade-off. With the help of comparative proteomics and metabolomics, we discovered the proteome required for the upkeep of these phenotypes. The metabolic profiles of both strains reveal that under aerobic conditions, RS218 undergoes oxidative metabolism, while IHE3034 undergoes anaerobic respiration. Finally, we show that the use of citrate and filament formation are both linked in a trade-off occurring via a c-di-GMP-dependent phase variation event. IMPORTANCE Aerobic use of citrate and filamentous growth are arbitrary cutoffs for the Escherichia coli species. The strains that exhibit them as stable phenotypes are called atypical. In this study, we compare two atypical neuroinvasive E. coli strains, which alternatively display either of these phenotypes. We present the proteome and metabolome required for the maintenance of filamentous growth and show that anaerobic nitrate respiration is the main requirement for the use of citrate. The fact that the two phenotypes are differentially expressed by each strain prompted us to check if they are part of a trade-off. Indeed, these atypical characters are reversible and result from a c-di-GMP phase variation event. Thus, we revealed hidden links between stable morphological and metabolic phenotypes and provided information about alternative evolutionary pathways for the survival of E. coli strains in various host niches.

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.