Microbiology-Sgm最新文献

Fluorescent pseudomonads catabolize purines via uric acid and allantoin, a pathway whose end-product is glyoxylate. In this work, we show that in Pseudomonas aeruginosa strain PAO1, the ORFs PA1498-PA1502 encode a pathway that converts the resulting glyoxylate into pyruvate. The expression of this cluster of ORFs was stimulated in the presence of allantoin, and mutants containing transposon insertions in the cluster were unable to grow on allantoin as a sole carbon source. The likely operonic structure of the cluster is elucidated. We also show that the purified proteins encoded by PA1502 and PA1500 have glyoxylate carboligase (Gcl) and tartronate semialdehyde (TSA) reductase (GlxR) activity, respectively, in vitro. Gcl condenses two molecules of glyoxylate to yield TSA, which is then reduced by GlxR to yield d-glycerate. GlxR displayed much greater specificity (k _cat/K_M) for Gcl-derived TSA than it did for the TSA tautomer, hydroxypyruvate. This is relevant because TSA can potentially spontaneously tautomerize to yield hydroxypyruvate at neutral pH. However, kinetic and [¹H]-NMR evidence indicate that PA1501 (which encodes a putative hydroxypyruvate isomerase, Hyi) increases the rate of the Gcl-catalysed reaction, possibly by minimizing the impact of this unwanted tautomerization. Finally, we use X-ray crystallography to show that apo-GlxR is a configurationally flexible enzyme that can adopt two distinct tetrameric assemblies in vitro.

Acinetobacter baumannii is associated with severe hospital-acquired, multi-drug-resistant infections worldwide, causing significant mortality and morbidity in intensive care patients or those under prolonged hospitalization. Multiple studies have recently shown that a proportion of circulating clinical isolates establish a transient multiplication niche inside phagocytic and non-phagocytic eukaryotic cells. We have previously demonstrated that the A. baumannii ABC141 strain invades human endothelial and epithelial cells, where it efficiently multiplies without induction of cytotoxicity. Here, we show that ABC141 adhesion, invasion and intracellular multiplication depend on the growth stage, being most efficient in the exponential growth phase. To define the gene expression signature most favourable to an intracellular lifestyle, a transcriptomic comparison was carried out between exponentially grown ABC141 and cultures in the stationary phase. Although most of the pathways identified reflected growth-related metabolic changes, we observed an up-regulation of the twin-arginine translocation (Tat) export system. Analysis of a mutant strain lacking the tatABC operon revealed that this export system is required only for adhesion to host cells, but not for invasion or intracellular multiplication. These data highlight a new role for the Tat export pathway in A. baumannii pathogenesis.

The human body hosts a complex and dynamic microbial community that is crucial for maintaining health. While bacteria dominate this system, fungal communities, collectively called the mycobiome, are increasingly recognized as vital contributors. However, fungi remain understudied due to challenges in culturing many species, limiting our understanding of their roles, interactions and effects on human biology. Advances in next-generation sequencing have transformed mycobiome research, revealing fungal diversity and its impact on health and disease. This review examines the mycobiome's composition and function across major body sites, including the gut, mouth, lungs, reproductive tract and skin. It also explores connections between fungal imbalances (dysbiosis) and diseases such as neurological disorders, cancer and post-COVID-19 complications. Despite progress, challenges persist, including the need for better culture-independent diagnostic tools and standardized research methods. Combining culturomics and metagenomics could help overcome these limitations and identify new treatment targets. By summarizing current knowledge and highlighting research gaps, this review aims to guide future studies on the mycobiome's role in human health.

The Staphylococcus aureus type VIIb secretion system (T7SSb) is a multiprotein secretion system that secretes toxins with antibacterial activity, but which is also required for full virulence in animal models of infection. S. aureus strains carry one of four T7SSb locus types, named essC1 to essC4, each of which encodes a characteristic LXG-family substrate at the T7SS locus. In essC2 strains, this LXG-domain protein is EsxX, which has a glycine zipper sequence in its C-terminus and has potent antibacterial, membrane-depolarizing activity. In this work, we recognize conserved features of the essC2 and essC3 systems, identifying the LXG protein SAR0287 as structurally and functionally similar to EsxX. Using a zebrafish larval hindbrain ventricle infection model, we demonstrate that the T7SSb of essC2 and essC3 representative strains contributes to bacterial replication and zebrafish mortality. However, there is no significant loss of virulence in the model system if EsxX or SAR0287 is absent. These findings indicate that there is no discernible role for either toxin in this virulence model.

The CRISPR-associated protein 9 (Cas9) produced by disease-associated strains of Campylobacter jejuni contributes to full virulence, including immune evasion and bacterial survival inside eukaryotic cells. In this work, we explored the role of C. jejuni Cas9 (CjeCas9) in cell envelope integrity, antibiotic resistance, intracellular survival inside Caco-2 intestinal epithelial cells and Toll-like receptor 2 (TLR-2) activation. We show that CjeCas9 modulates the permeability of the C. jejuni cell envelope, sialylated lipooligosaccharide expression and susceptibility to ciprofloxacin, the most commonly prescribed antibiotic to treat C. jejuni infections. Moreover, we reveal that WT production of CjeCas9 increased intracellular survival of C. jejuni inside Caco-2 intestinal epithelial cells by a factor of 550 compared to the respective cas9 gene deletion mutant and that intracellular survival was associated with the activation of TLR-2. In conclusion, we established that CjeCas9 modulates C. jejuni (intracellular) virulence traits, including intracellular survival.

R-pyocins are phage tail-like protein complexes produced by Pseudomonas aeruginosa that deliver a single, lethal hit by depolarizing the target cell membrane. Unlike phages, R-pyocins lack capsids and DNA, and their killing is highly specific, being determined by tail fibre proteins that recognize subtype-specific LPS receptors on susceptible strains. Five known subtypes (R1-R5) vary in host range, with R5 displaying the broadest activity. R-pyocin expression is tightly regulated by the SOS response, linking their release to environmental stress. Their non-replicative mechanism and metabolic independence make them especially promising for targeting multidrug-resistant and biofilm-associated P. aeruginosa infections, such as those seen in cystic fibrosis and chronic wounds. Preclinical studies support their therapeutic potential, and bioengineering approaches have extended their target range. With their high specificity, rapid action and adaptability, R-pyocins are strong candidates for next-generation precision antimicrobials.

Klebsiella pneumoniae is a prominent opportunistic pathogen increasingly associated with multidrug resistance and virulence. One of the main mechanisms of antimicrobial resistance in K. pneumoniae is active efflux, primarily mediated by the resistance-nodulation-division (RND) family of pumps. AcrAB-TolC is the key RND efflux pump in K. pneumoniae, regulated by the transcriptional activator RamA and its repressor RamR. Although overexpression of AcrAB-TolC has been linked to drug resistance in various clinical strains, its physiological roles in K. pneumoniae remain insufficiently studied. In this study, we generated isogenic deletions of acrB and ramR in both the genetically tractable K. pneumoniae Ecl8 and the virulent ATCC 43816 strains. We examined the phenotype of the ΔacrB and ΔramR mutants by assessing antimicrobial susceptibility, biofilm formation, growth under infection-related conditions and both in vitro and in vivo infection models. Loss of acrB increased susceptibility to drugs, decreased biofilm formation and reduced in vitro virulence in both Ecl8 and ATCC 43816. However, only in Ecl8 was the loss of AcrB found to diminish growth under infection-like conditions and decrease in vivo virulence in the Galleria mellonella infection model. In contrast, in ATCC 43816, it had no effect. Our findings suggest that AcrAB-TolC exhibits strain-specific physiological functions, highlighting its dual role in antimicrobial resistance and pathogenicity, and thereby broadening our understanding of efflux-mediated adaptations in K. pneumoniae. Exploring the broader functions of RND efflux pumps in K. pneumoniae can provide insights into the potential effects of targeting them with inhibitor molecules.

Deep-sea ecosystems remain poorly understood due to exploration challenges. Despite the advancements metagenomics have brought to the understanding of the ocean microbiome, the diversity of marine viruses, particularly in the deep sea, is still not well characterized. In this study, we analysed the impact of depth on the composition and diversity of marine viruses in deep-sea waters at a global scale. Raw reads from deep-sea shotgun DNA sequences were retrieved from the Tara and Malaspina expeditions, encompassing depths from 270 to 4,005 m. A total of 80 samples containing viral reads were identified and analysed through a comprehensive bioinformatics pipeline, including quality assessment, taxonomic classification and metabolic annotation. The analysis reveals that microbial viral diversity significantly decreases with depth, with shallower waters exhibiting higher species richness. We determined that a substantial proportion of deep-sea viral sequences remains unclassified - up to 31.9% at depths of 270-1,000 m and 9.6% at 2,400-4,005 m. Additionally, a higher abundance of auxiliary metabolic genes was observed at shallower depths, indicating potential roles in host metabolism and adaptation. Our findings reveal the deep ocean as a vast, largely unexplored source of microbial viral diversity. This research emphasizes how depth influences viral diversity and community makeup in deep-sea environments, underscoring the need for further exploration to fully grasp their complexity and ecological roles.

Tularemia is a zoonotic disease caused by Francisella tularensis. Most human cases are caused by F. tularensis ssp. tularensis (type A) or F. tularensis ssp. holarctica (type B), with the former considered more virulent. For this reason, type A isolates are often the benchmark for the testing of new vaccines or antibiotics. However, both subspecies cause considerable disease and can differ in their responsiveness to medical countermeasures. Accordingly, there is a need to identify and characterize representative type B isolates that are available to qualified research institutions to ensure the development of future vaccines or antibiotics is efficacious against both subspecies. The type B isolate OR96-0246 was identified as a strain that can address this need and was subsequently characterized. For in vitro characterization, the OR96-0246 strain was examined for growth in media and for its ability to form biofilm. As the LPS is an essential virulence factor, the O-antigen was characterized through western analysis. For future medical countermeasure testing for biodefence concerns, pneumonic challenges with animal modelling would be required. Therefore, using the OR96-0246 strain, we implemented animal models that encompassed BALB/c mice, Fischer 344 rats and cynomolgus macaques. Mice were challenged via intranasal instillation with varying doses of OR96-0246, and the LD₅₀ was determined to be 1 c.f.u. We progressed to Fischer 344 rats, which are a better-suited rodent model to gauge vaccine efficacy. When challenging the rats by whole body aerosolization with various doses of OR96-0246, the LD₅₀ was determined to be 138 c.f.u. Finally, a staircase challenge design was applied to three cynomolgus macaques, each receiving a different aerosolized dose of OR96-0246 to determine an estimated LD₅₀ for non-human primates (NHPs). Two out of the three NHPs succumbed to the challenge. The animal that received the lowest dose (2.1×10⁴ c.f.u.) survived but did demonstrate clinical signs of infection. Samples from the challenged rats and NHPs were collected for histopathology characterization. Generally, the pathological changes observed in both models were similar, consisting primarily of multifocal bronchopneumonia in the lung and necrotic lesions in the spleen. This animal model development with type B strains of F. tularensis will be essential to properly evaluate new antimicrobials and vaccines to protect against tularemia.