Flow Cytometry and Automated Microscopy Integration for Phagocytic Analysis of Hypervirulent Klebsiella pneumoniae in Dictyostelium discoideum.

This chapter introduces protocols for culturing and maintaining Dictyostelium discoideum and methods for conducting virulence assays in this organism to study bacterial pathogenicity. It outlines advanced techniques, such as automated microscopy and flow cytometry, for detailed cellular analysis and traditional microbiological approaches. These comprehensive protocols will enable researchers to probe the virulence factors of pathogens like Klebsiella pneumoniae and to elucidate the details of host-pathogen interactions within a cost-effective and adaptable laboratory framework.

A Comprehensive Guide to Quality Assessment and Data Submission for Genomic Surveillance of Enteric Pathogens.

This document outlines the steps necessary to assemble and submit the standard data package required for contributing to the global genomic surveillance of enteric pathogens. Although targeted to GenomeTrakr laboratories and collaborators, these protocols are broadly applicable for enteric pathogens collected for different purposes. There are five protocols included in this chapter: (1) quality control (QC) assessment for the genome sequence data, (2) validation for the contextual data, (3) data submission for the standard pathogen package or Pathogen Data Object Model (DOM) to the public repository, (4) viewing and querying data at NCBI, and (5) data curation for maintaining relevance of public data. The data are available through one of the International Nucleotide Sequence Database Consortium (INSDC) members, with the National Center for Biotechnology Information (NCBI) being the primary focus of this document. NCBI Pathogen Detection is a custom dashboard at NCBI that provides easy access to pathogen data plus results for a standard suite of automated cluster and genotyping analyses important for informing public health and regulatory decision-making.

Embracing multiple infection models to tackle Q fever: A review of in vitro, in vivo, and lung ex vivo models.

Multiple animal and cell culture models are employed to study pathogenesis of Coxiella burnetii, the causative agent of acute and chronic human Q fever. C. burnetii is a lung pathogen that is aerosolized in contaminated products and inhaled by humans to cause acute disease that can disseminate to other organs and establish chronic infection. Cellular models of Q fever include a variety of tissue-derived cell lines from mice and humans such as lung alveolar ex vivo cells. These models have the advantage of being cost-effective and reproducible. Similarly, animal models including mice and guinea pigs are cost-effective, although only immunocompromised SCID mice display a severe disease phenotype in response to Nine Mile I and Nine Mile II isolates of C. burnetii while immunocompetent guinea pigs display human-like symptoms and robust immune responses. Non-human primates such as macaques and marmosets are the closest model of human disease but are costly and largely used for adaptive immune response studies. All animal models are used for vaccine development but many differences exist in the pathogen's ability to establish lung infection when considering infection routes, bacterial isolates, and host genetic background. Similarly, while cellular models are useful for characterization of host-pathogen mechanisms, future developments should include use of a lung infection platform to draw appropriate conclusions. Here, we summarize the current state of the C. burnetii lung pathogenesis field by discussing the contribution of different animal and cell culture models and include suggestions for continuing to move the field forward.

Identification, characterization, and structure of a tRNA splicing enzyme RNA 5'-OH kinase from the pathogenic fungi Mucorales.

Fungal Trl1 is an essential tRNA splicing enzyme composed of C-terminal cyclic phosphodiesterase and central polynucleotide kinase end-healing domains that convert the 2',3'-cyclic-PO4 and 5'-OH ends of tRNA exons into the 3'-OH,2'-PO4 and 5'-PO4 termini required for sealing by an N-terminal ATP-dependent ligase domain. Trifunctional Trl1 enzymes are present in most human fungal pathogens and are untapped targets for anti-fungal drug discovery. Mucorales species, deemed high priority human pathogens by WHO, elaborate a noncanonical tRNA splicing apparatus in which a stand-alone monofunctional RNA ligase enzyme joins 3'-OH,2'-PO4 and 5'-PO4 termini. Here we identify a stand-alone Mucor circinelloides polynucleotide kinase (MciKIN) and affirm its biological activity in tRNA splicing by genetic complementation in yeast. Recombinant MciKIN catalyzes magnesium-dependent phosphorylation of 5'-OH RNA and DNA ends in vitro. MciKIN displays a strong preference for GTP as the phosphate donor in the kinase reaction, a trait shared with the stand-alone RNA kinase homologs from Mucorales species Rhizopus azygosporus (RazKIN) and Lichtheimia corymbifera (LcoKIN) and with the kinase domains of fungal Trl1 enzymes. We report a 1.65 Šcrystal structure of RazKIN in complex with GDP•Mg2+ that illuminates the basis for guanosine nucleotide specificity.

Clinical evaluation of a multiplex PCR-based test for joint infection: a prospective diagnostic accuracy study of forty-nine patients.

PURPOSE: The purpose of this study was to evaluate the diagnostic accuracy (sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV)) of the PCR-based BioFire® Joint Infection Panel (BJI Panel) against microbiological culture growth for patients suspected of having a native or prosthetic joint infection. METHODS: Synovial fluid and tissue biopsies were prospectively collected from patients from June 2022 to June 2023. The results of the BJI Panel were compared with those of culture growth. RESULTS: 51 samples were included. Including all pathogens, the sensitivity was 69%, the specificity 89%, the PPV 73% and the NPV 86%. Including only pathogens in the BJI Panel, the sensitivity was 100%, the specificity 90%, the PPV 73% and the NPV 100%. CONCLUSION: The BJI Panel has a high accuracy for detecting the pathogens in its panel, but the absence of important common pathogens from the panel reduces its sensitivity and NPV. With a short turnaround time and precise pathogen detection, the BJI Panel has the potential to add value as a complementary diagnostic method.

First Report of Fusarium compactum Causing Fusarium Head Blight of Wheat in China.

Fusarium head blight (FHB), a devastating wheat disease caused by several species of Fusarium, threatens global wheat yield and quality (Erenstein et al. 2022). In August 2023, wheat spikes exhibiting clear FHB symptoms were observed in fields in Yunnan, China (24°16'46″ N, 102°29'46″ E), with an incidence rate of approximately 10%. Diseased wheat spikes exhibited a bleached, wilted appearance, with abundant orange sporodochia on the glumes, similar to previous reports (Osborne et al. 2007). Twenty-four symptomatic spikes were collected from a single field, and sporodochia were washed with sterile water to prepare a spore suspension of 1 × 10³ spores/ml, which was inoculated onto potato dextrose agar (PDA) to obtain monosporic cultures. Four reference strains (KUNCC 3418 to KUNCC 3420, and KUNCC 3431) were deposited at the Kunming Institute of Botany Culture Collection, Chinese Academy of Sciences (KUNCC). For species identification, four strains were cultured on PDA and carnation leaf agar (CLA) at 25°C, with incubation under a 12-hour near-UV light/dark cycle on CLA and in complete darkness for 24 hours on PDA. Colonies on PDA grew rapidly, appearing white and loosely flocculent. Abundant pale orange, translucent sporodochia formed on CLA. Sporodochial conidiogenous cells were monophialidic or polyphialidic, subulate to subcylindrical, 9.5-12 µm × 3-3.5 µm. Sporodochial macroconidia were naviculate to fusiform, with an elongate, tapering apical cell and a foot-shaped basal cell, 3-6-septate, 33-67.5 µm × 3.5-5.5 µm. The ITS, tef1-α, rpb1, rpb2, and cam regions were amplified and sequenced using primers ITS1/ITS4, EF-1/EF-2, rpb1-F7/G2R, rpb2-5F2/11aR, and CL1/CL2A, respectively (White et al. 1990; O'Donnell et al. 2000; O'Donnell et al. 2010; Reeb et al. 2004; O'Donnell et al. 1998). These sequences were deposited in GenBank for cam (PP951603 to PP951606), ITS (PP946846 to PP946849), tef-1α (PP719217, PP731572 to PP731574), rpb1 (PP719219, PP737839 to PP737841), and rpb2 (PP719218, PP951607 to PP951609). BLASTn analyses of these sequences showed an identity range of 99.7% to 100% with the epitype strain NRRL 36323 of F. compactum (GenBank: cam = GQ505560, ITS = MH855177, tef-1α = GQ505648, and rpb2 = GQ505826), with base pair matches of 663/665 bp for cam, 488/488 bp for ITS, 641/641 bp for tef-1α, and 892/892 bp for rpb2. Both morphological and BLASTn analyses confirmed these isolates as F. compactum (Leslie & Summerell 2006; Han et al. 2023). Pathogenicity tests were performed by spraying 1 ml of spore suspension (1 × 108 spores/ml) of F. compactum strains onto spikes of the wheat cultivar Yunmai 126 at the flowering stage (n = 9). Controls (n = 9) were treated only with sterile water. Following treatment, the wheat spikes were covered with plastic bags and incubated at 25°C for 10 days. After 14 days, the inoculated spikes turned bleached and dry, showing FHB symptoms, while the wheat spikes in the control treatment remained asymptomatic. The pathogenic fungus re-isolated from all diseased samples was confirmed as F. compactum. It has been frequently reported in association with crown and root rot of wheat, particularly in regions such as Turkey and Iran (Tunali et al. 2008; Besharati et al. 2017). To our knowledge, this is the first report of F. compactum on diseased wheat spikes in China. This finding provides valuable insights into the spread of F. compactum.

First Report of Leaf Rot on Catalpa bungei caused by Fusarium verticillioides in China.

Catalpa bungei originates from China. Because of its well-developed root system and strong resistance to wind and soil, it is one of the top ten recommended species of ecological management (Jian et al., 2022). In September 2023, a severe leaf rot of C. bungei was observed at Lanlake farm (500 acres) in Nanyang (33°3'23" N, 112°28'50" E), China. The incidence rate of leaf rot reached 45% (n = 100). The pale-yellow spots initially appeared on the adaxial surface of leaf margins, subsequently enlarging to form irregular black rot lesions, with the yellow halos around the necrotic area of the lesion, ultimately causing the entire leaves to wither. Diseased leaves (20) were collected, cut into pieces, sterilized, and then placed on potato dextrose agar (PDA). A total of 25 purified fungal strains were isolated, and three strains (QS2-1, QS2-2, QS2-3) from distinct areas were selected for further analysis. Each strain produced abundant aerial mycelium, initially white, which later developed purple pigments. The aerial conidiophores were sparsely branched, ending with verticillate phialides. The strains generally produced many more microconidia than macroconidia on PDA media. Microconidia were clavate and measured 3.9 to 6.6 × 1.1 to 2.4 µm (n = 50). To produce macroconidia, we used YPG liquid medium (0.3% yeast extract, 1% peptone, and 2% glucose) with shaking (200 r.p.m.) for 5 days. Macroconidia were slender, straight, and measured 19.5 to 27.1 × 1.9 to 3.5 µm, with 3 to 5 septa (n = 50). The morphological characteristics matched the species description of Fusarium verticillioides (Sacc.) Nirenberg 1976 (Leslie and Summerell, 2006). The rDNA internal transcribed spacer (ITS), ß-tubulin gene (tub2), translation elongation factor 1-alpha gene (tef1), calmodulin (cmdA), RNA polymerase II largest subunit (rpb1), and RNA polymerase II second largest subunit (rpb2) were amplified for molecular identification (O'Donnell et al., 2022). The sequences were submitted to GenBank with accession numbers OR741762, OR741763, OR741765 (ITS), OR762222, OR762223, OR939807 (tub2), OR939799, OR939800, PQ035927 (tef1), OR778611, OR939808, OR939809 (cmdA), PQ035921, PQ035922, PQ035923 (rpb1), and PQ035924, PQ035925, PQ035926 (rpb2). BLASTn analysis of QS2-1 sequences exhibited 99% similarity with F. verticillioides sequences (strains CBS 576.78) MT010888 of cmdA (711/713, 99%), MT010956 of rpb1 (1790/1791, 99%), and MT010972 of rpb2 (868/870, 99%). A phylogenetic tree was constructed using concatenated sequences along with the sequences of the type strains employing the neighbor-joining method, showing the three strains formed a clade with the type strain CBS 576.78. Pathogenicity was tested on 10 healthy potted seedlings by spraying them with a conidial suspension (106 conidia ml-1), while 5 seedlings were sprayed with sterilized water as a control. The plants were placed in climate incubators. Ten days after inoculation, typical lesions were observed on the treated plants, but not on the control group. The reisolated strains were identified as F. verticillioides through morphological characterization, thus fulfilling Koch's postulates. F. verticillioides is known to cause Fusarium ear rot on maize, and other plants including Brassica rapa (Akram et al., 2020) and Schizonepeta tenuifolia (Li et al., 2024). This is the first documented instance of F. verticillioides causing leaf rot on C. bungei globally. Identifying the pathogen is critical to implementing effective disease management strategies, especially in choosing proper pesticide agents and screening disease-resistant varieties.

You can bring plankton to fecal indicator organisms, but you cannot make the plankton graze: particle contribution to E. coli and MS2 inactivation in surface waters.

Organisms that are associated with feces ("fecal indicator organisms") are monitored to assess the potential for fecal contamination of surface water bodies in the United States. However, the effect of the complex mixtures of chemicals and the natural microbial community within surface water ("particles") on fecal indicator organism persistence is not well characterized. We aimed to better understand how particles, including biological (e.g., potential grazers) and inert (e.g., minerals) types, affect the fecal indicator organisms Escherichia coli K-12 ("E. coli") and bacteriophage MS2 in surface waters. A gradient of particles captured by a 0.2-µm-pore-size filter ("large particles") was generated, and the additional particles and dissolved constituents that passed through the filter were deemed "small particles." We measured the ratio of MS2 and E. coli that survived over a 24-h incubation period for each condition (0%-1,000% large-particle concentration in raw water) and completed a linear regression that included large- and small-particle coefficients. Particles were characterized by quantifying plankton, total bacterial cells, and total solids. E. coli and MS2 persistence was not significantly affected by large particles, but small particles had an effect in most waters. Small particles in higher-salinity waters had the largest, negative effect on E. coli and MS2 survival ratios: Significant small-particle coefficients ranged from -1.7 to -5.5 day-1 in the marine waters and -0.89 to -3.2 day-1 in the fresh and estuarine waters. This work will inform remediation efforts for impaired surface water bodies.IMPORTANCEMany surface water bodies in the United States have organisms associated with fecal contamination that exceed regulatory standards and prevent safe recreation. The process to remediate impaired water bodies is complicated because these fecal indicator organisms are affected by the local environmental conditions. For example, the effect of particles in surface water on fecal indicator concentrations are difficult to quantify in a way that is comparable between studies and water bodies. We applied a method that overcomes this limitation to assess the effects of large particles, including natural plankton that could consume the seeded fecal indicator organisms. Even in environmental water samples with diverse communities of plankton present, no effect of large particles on fecal indicator concentrations was observed. These findings have implications for the interpretation and design of future studies, including that particle characterization of surface water may be necessary to assess the fate of fecal indicators.

Comparing the diagnostic value of targeted with metagenomic next-generation sequencing in immunocompromised patients with lower respiratory tract infection.

BACKGROUND: Accurate identification of the etiology of lower respiratory tract infections (LRTI) is crucial, particularly for immunocompromised patients with more complex etiologies. The advent of next-generation sequencing (NGS) has enhanced the effectiveness of pathogen detection. However, assessments of the clinical diagnostic value of targeted NGS (tNGS) in immunocompromised patients with LRTI are limited. METHODS: To evaluate the diagnostic value of tNGS in immunocompromised patients with LRTI, a total of 88 patients, of whom 54 were immunocompromised, were enrolled. These patients underwent tNGS testing of bronchoalveolar lavage fluid (BALF). Results from both metagenomic next-generation sequencing (mNGS) and conventional microbiological tests (CMT) were also available for all participants. The performance of tNGS was assessed by comparing its findings against mNGS, CMT, and the clinical composite diagnosis. RESULTS: In the cohort of 88 patients, tNGS showed comparable diagnostic value to mNGS and was significantly superior to CMT. Compared to CMT and composite reference standard, tNGS showed sensitivity of 94.55% and 90.48%, respectively. In immunocompromised patients, despite a more diverse pathogen variety, tNGS maintained similar sensitivity to mNGS and outperformed CMT. tNGS positively influenced etiologic diagnosis and antibiotic decision-making in 72.72% of cases, leading to a change in antibiotic regimen in 17.05% of cases. We also compared the detection of microbial nucleic acids by tNGS with mNGS and found that tNGS could identify 87.99% of the microbial nucleic acids identified by mNGS. CONCLUSION: In summary, our study demonstrated that tNGS offers promising clinical diagnostic accuracy in immunocompromised patients, as evidenced by its favorable comparison with CMT, the composite reference standard, and mNGS.

Advances in nucleic acid aptamer-based detection of respiratory virus and bacteria: a mini review.

Respiratory pathogens infecting the human respiratory system are characterized by their diversity, high infectivity, rapid transmission, and acute onset. Traditional detection methods are time-consuming, have low sensitivity, and lack specificity, failing to meet the needs of rapid clinical diagnosis. Nucleic acid aptamers, as an emerging and innovative detection technology, offer novel solutions with high specificity, affinity, and broad target applicability, making them particularly promising for respiratory pathogen detection. This review highlights the progress in the research and application of nucleic acid aptamers for detecting respiratory pathogens, discussing their selection, application, potential in clinical diagnosis, and future development. Notably, these aptamers can significantly enhance the sensitivity and specificity of detection when combined with detection techniques such as fluorescence, colorimetry and electrochemistry. This review offers new insights into how aptamers can address the limitations of traditional diagnostic methods and advance clinical diagnostics. It also highlights key challenges and future research directions for the clinical application of nucleic acid aptamers.

The Science of Cholesteatoma.

Cholesteatoma is a potential end-stage outcome of chronic ear infections that can result in the destruction of temporal bone structures with potential resultant hearing loss, vertigo, and intracranial infectious complications. There is currently no treatment apart from surgery for this condition, and despite years of study, the histopathogenesis of this disease remains poorly understood. This review is intended to summarize our accumulated knowledge of the mechanisms of cholesteatoma development and the underlying molecular biology. Attention will be directed particularly to recent developments, covering many potential pharmacologic targets that could be used to treat this disease in the future.

Nectriaceae species associated to root rot of nursery and young Eucalyptus smithii trees in Uruguay with Ilyonectria charruensis as novel species.

Forestry constitutes an important agronomical activity in Uruguay, involving the cultivation of exotic trees mainly for cellulose pulp production with Eucalyptus species. Over the last decade, E. smithii emerged as a species of interest for cellulose pulping. However, its rapid expansion has coincided with high mortality rates among young trees ranging from 5 to 85%, especially during the first and second summer after plantation. Disease surveys conducted on nine E. smithii commercial fields and three nurseries in southern and eastern Uruguay, yielded a collection of 25 isolates from E. smithii root rot belonging to the Nectriaceae family. In this study, we aimed to identify and characterize these isolates employing phenotypical and molecular studies and to assess their pathogenicity on E. smithii seedlings. Based on morphological features, the Nectriaceae isolates were subdivided into two groups, one resembling Calonectria (n=15) and another Cylindrocarpon-like (n=10). DNA sequences of the partial histone H3 (his3), actine, calmodulin, RNA polymerase II second largest subunit, translation elongation factor 1-alpha (tef1) and ß-tubulin (tub2) genes were amplified for Calonectria, as well as partial his3, tef1, tub2 and internal-transcribed spacer and intervening 5.8S (ITS) for the Cylindrocarpon-like group. Based on phylogenetic analysis and phenotypical features three species were identified and characterized; Calonectria pauciramosa (n=15), Dactylonectria novozelandica (n=2), and a novel taxon which we describe here as Ilyonectria charruensis sp. nov. (n=8). The pathogenicity trials revealed that isolates from the three species significantly reduced both shoot and root dry weights of inoculated E. smithii seedlings compared to control plants.

First Report of Botryosphaeria dothidea Causing Leaf Blight on Parrotia subaequalis in China.

Parrotia subaequalis is of great ornamental value due to its unique bark, featuring interesting textures and colors, and its large, striking galls. These characteristics make it a popular choice for bonsai cultivation. (Yan et al. 2022) . In July 2023, an outbreak of leaf blight was observed on 40, six-month-old P. subaequalis seedlings in Anqing, Anhui, China, with an incidence rate of 80%. In the early stages of infection, small brown spots appear on the leaf surface, which gradually become round or irregular and darken to a deep brown color. As the disease progresses, the affected areas expand from the leaf margins towards the center, causing the leaf surface to become concave, wilt, and necrotize. This resulted in restricted plant growth, and in severe cases, partial or complete plant death. For isolation, 30 tissue slices (5 × 5 mm) were taken from the leaves of 10 symptomatic seedlings and surface sterilized with 75% ethanol for 5 seconds, followed by five rinses with sterilized distilled water. After two days of dark incubation at 28°C, hyphal tips of fungi were transferred onto new potato dextrose agar (PDA) plates and incubated until conidia production. Six unidentified isolates with similar morphological characteristics were obtained. The colonies, initially white, darken to black after 7 to 10 days of incubation. They produced colorless, aseptate conidia that were oblong or fusiform, measuring 18-26 µm in length and 5-8 µm in width (n=50). The morphological characteristics of the isolates resembled those of Botryosphaeria (Udayanga et al. 2015) . Isolate IS2116-1 was further confirmed through molecular methods. The rDNA internal transcribed spacer (ITS) region, translation elongation factor 1-α (TEF1-α), and beta-tubulin (TUB2) genes were amplified and sequenced using the primers ITS1/ITS4 (White et al., 1990), EF1-728F/EF1-986R, and Bt2a/Bt2b (Ferreira et al., 2021; Carbone et al., 1999), respectively. BLAST analysis revealed that the ITS (OR958722) sequence was 100% similar to the B. dothidea isolate HZ5(MH329650.1), TEF1-a (PP214058) sequence was 100% similar to the B. dothidea strain JZB310220(ON890458.1), and strain TUB2 (PP214057) sequence was 99.78% similar to the B. dothidea strain L14 (KR260833.1). Maximum likelihood analyses were performed for the combined ITS、TUB2、TEF datasets using PhyloSuite v1.2.2, the resulting phylogenetic tree indicated that isolate IS2116-1 clustered together with Botryosphaeria dothidea in a clade with 97% bootstrap support(Zheng et al. 2020) . Pathogenicity tests were conducted on 3-6 month-old P. subaequalis seedlings (n = 5) grown in a greenhouse. A conidial suspension (106 spores/ml) collected from the isolates was sprayed onto P. subaequalis seedlings, while the control was treated with distilled water. All plants were maintained in a growth chamber at 28°C with a 12-h photoperiod. The experiment was conducted twice independently . After 20 days of inoculation, brownish lesions similar to those observed in the field appeared on the treated plants, while the noninoculated control plants remained symptomless. The pathogen was reisolated from the leaves of the obviously diseased seedlings and confirmed as B. dothidea through morphological and sequence analysis. No isolates were obtained from uninoculated control plants, thus fulfilling Koch's hypothesis. This report marks the first record of B. dothidea causing leaf blight in P. subaequalis. In light of the rarity of natural P. subaequalis populations, it is imperative to assess both the extent of disease spread and its economic impact. These insights are crucial for devising strategies to protect this endangered species from disease threats and to preserve its ecological significance.

Two draft genomes of fungal leaf endophytes from tropical gymnosperms.

Two ascomycetes, Neofusicoccum sp. and Xylaria sp., were isolated from healthy leaves of the tropical gymnosperms Zamia pseudoparasitica (Z2) and Zamia nana (Z50) from Panama. The two draft genomes possess a broad predicted repertoire of carbohydrate-degrading CAZymes, peptidases, and secondary metabolites, with more secondary metabolite clusters in the Xylaria isolate.

Antibiofilm activity of Morganella morganii JB8F and Pseudomonas fluorescens JB3B compound to control single and multi-species of aquaculture pathogens.

BACKGROUND: Indonesia is a country that uses half or more aquatic foods as protein intake. The increased production in aquaculture industries might cause several problems, such as bacterial disease resulting in mass mortality and economic losses. Antibiotics are no longer effective because aquaculture pathogens can form biofilm. Biofilm is a microbial community that aggregates and firmly attaches to living or non-living surfaces. Biofilm formation can be caused by environmental stress, the presence of antibiotics, and limited nutrients. Therefore, it is important to explore antibiofilm to inhibit biofilm formation and/or eradicate mature biofilm. Phyllosphere bacteria can produce bioactive compounds for antimicrobial, antibiofilm, and anti-quorum sensing. Three aquaculture pathogens were used in this study, such as Aeromonas hydrophila, Streptococcus agalactiae, and Vibrio harveyi. RESULTS: Pseudomonas fluorescens JB3B and Morganella morganii JB8F extracts could disrupt single and multi-species biofilms. Both extracts could inhibit single biofilm formation from one to seven days of incubation time. We confirmed the destruction activity on multi-species biofilm using light microscope and scanning electron microscope. Using GC-MS analysis, indole was the most active fraction of the P. fluorescens JB3B extracts and octacosane from the M. morganii JB8F extract. We also conducted a toxicity test using brine shrimp lethality assay on P. fluorescens JB3B and M. morganii JB8F extracts. P. fluorescens JB3B, M. morganii JB8F, and a mixture of both extracts were confirmed non-toxic according to the LC50 value of the brine shrimp lethality test. CONCLUSIONS: P. fluorescens JB3B and M. morganii JB8F phyllosphere extracts had antibiofilm activity to inhibit single biofilm and disrupt single and multi-species biofilm of aquaculture pathogens. Both extracts could inhibit single species biofilm until seven days of incubation. Bioactive compounds that might contribute to antibiofilm properties were found in both extracts, such as indole and phenol. P. fluorescens JB3B, M. morganii JB8F extracts, and mixture of both extracts were non-toxic against Artemia salina.

A fungal endophyte induces local cell wall-mediated resistance in wheat roots against take-all disease.

Take-all disease, caused by the Ascomycete fungus Gaeumannomyces tritici, is one of the most important root diseases of wheat worldwide. The fungus invades the roots and destroys the vascular tissue, hindering the uptake of water and nutrients. Closely related non-pathogenic species in the Magnaporthaceae family, such as Gaeumannomyces hyphopodioides, occur naturally in arable and grassland soils and have previously been reported to reduce take-all disease in field studies. However, the mechanism of take-all protection has remained unknown. Here, we demonstrate that take-all control is achieved via local but not systemic host changes in response to prior G. hyphopodioides root colonisation. A time-course wheat RNA sequencing analysis revealed extensive transcriptional reprogramming in G. hyphopodioides-colonised tissues, characterised by a striking downregulation of key cell wall-related genes, including genes encoding cellulose synthases (CESA), and xyloglucan endotransglucosylase/hydrolases (XTH). In addition, we characterise the root infection biologies of G. tritici and G. hyphopodioides in wheat. We investigate the ultrastructure of previously described "subepidermal vesicles" (SEVs), dark swollen fungal cells produced in wheat roots by non-pathogenic G. hyphopodioides, but not by pathogenic G. tritici. We show that G. hyphopodioides SEVs share key characteristics of fungal resting structures, containing a greater number of putative lipid bodies and a significantly thickened cell wall compared to infection hyphae. We hypothesise that SEVs are fungal resting structures formed due to halted hyphal growth in the root cortex, perhaps as a stress response to locally induced wheat defence responses. In the absence of take-all resistant wheat cultivars or non-virulent G. tritici strains, studying closely related non-pathogenic G. hyphopodioides provides a much needed avenue to elucidate take-all resistance mechanisms in wheat.

Adaptor protein Abelson interactor 1 in homeostasis and disease.

Dysregulation of Abelson interactor 1 (ABI1) is associated with various states of disease including developmental defects, pathogen infections, and cancer. ABI1 is an adaptor protein predominantly known to regulate actin cytoskeleton organization processes such as those involved in cell adhesion, migration, and shape determination. Linked to cytoskeleton via vasodilator-stimulated phosphoprotein (VASP), Wiskott-Aldrich syndrome protein family (WAVE), and neural-Wiskott-Aldrich syndrome protein (N-WASP)-associated protein complexes, ABI1 coordinates regulation of various cytoplasmic protein signaling complexes dysregulated in disease states. The roles of ABI1 beyond actin cytoskeleton regulation are much less understood. This comprehensive, protein-centric review describes molecular roles of ABI1 as an adaptor molecule in the context of its dysregulation and associated disease outcomes to better understand disease state-specific protein signaling and affected interconnected biological processes.

Helicobacter pylori PldA modulates TNFR1-mediated p38 signaling pathways to regulate macrophage responses for its survival.

Helicobacter pylori, a dominant member of the gastric microbiota was associated with various gastrointestinal diseases and presents a significant challenge due to increasing antibiotic resistance. This study identifies H. pylori's phospholipase A (PldA) as a critical factor in modulating host macrophage responses, facilitating H. pylori 's evasion of the immune system and persistence. PldA alters membrane lipids through reversible acylation and deacylation, affecting their structure and function. We found that PldA incorporates lysophosphatidylethanolamine into macrophage membranes, disrupting their bilayer structure and impairing TNFR1-mediated p38-MK2 signaling. This disruption results in reduced macrophage autophagy and elevated RIP1-dependent apoptosis, thereby enhancing H. pylori survival, a mechanism also observed in multidrug-resistant strains. Pharmacological inhibition of PldA significantly decreases H. pylori viability and increases macrophage survival. In vivo studies corroborate PldA's essential role in H. pylori persistence and immune cell recruitment. Our findings position PldA as a pivotal element in H. pylori pathogenesis through TNFR1-mediated membrane modulation, offering a promising therapeutic target to counteract bacterial resistance.

Recognition of phylogenetically diverse pathogens through enzymatically amplified recruitment of RNF213.

Innate immunity senses microbial ligands known as pathogen-associated molecular patterns (PAMPs). Except for nucleic acids, PAMPs are exceedingly taxa-specific, thus enabling pattern recognition receptors to detect cognate pathogens while ignoring others. How the E3 ubiquitin ligase RNF213 can respond to phylogenetically distant pathogens, including Gram-negative Salmonella, Gram-positive Listeria, and eukaryotic Toxoplasma, remains unknown. Here we report that the evolutionary history of RNF213 is indicative of repeated adaptation to diverse pathogen target structures, especially in and around its newly identified CBM20 carbohydrate-binding domain, which we have resolved by cryo-EM. We find that RNF213 forms coats on phylogenetically distant pathogens. ATP hydrolysis by RNF213's dynein-like domain is essential for coat formation on all three pathogens studied as is RZ finger-mediated E3 ligase activity for bacteria. Coat formation is not diffusion-limited but instead relies on rate-limiting initiation events and subsequent cooperative incorporation of further RNF213 molecules. We conclude that RNF213 responds to evolutionarily distant pathogens through enzymatically amplified cooperative recruitment.

Unveiling intraspecific diversity and evolutionary dynamics of the foodborne pathogen Bacillus paranthracis through high-quality pan-genome analysis.

Understanding the evolutionary dynamics of foodborne pathogens throughout host-associated habitats is of utmost importance. Bacterial pan-genomes, as dynamic entities, are strongly influenced by ecological lifestyles. As a phenotypically diverse species in the Bacillus cereus group, Bacillus paranthracis is recognized as an emerging foodborne pathogen and a probiotic simultaneously. This poorly understood species is a suitable study model for adaptive pan-genome evolution. In this study, we determined the biogeographic distribution, abundance, genetic diversity, and genotypic profiles of key genetic elements of B. paranthracis. Metagenomic read recruitment analyses demonstrated that B. paranthracis members are globally distributed and abundant in host-associated habitats. A high-quality pan-genome of B. paranthracis was subsequently constructed to analyze the evolutionary dynamics involved in ecological adaptation comprehensively. The open pan-genome indicated a flexible gene repertoire with extensive genetic diversity. Significant divergences in the phylogenetic relationships, functional enrichment, and degree of selective pressure between the different components demonstrated different evolutionary dynamics between the core and accessory genomes driven by ecological forces. Purifying selection and gene loss are the main signatures of evolutionary dynamics in B. paranthracis pan-genome. The plasticity of the accessory genome is characterized by horizontal gene transfer (HGT), massive gene losses, and weak purifying or positive selection, which might contribute to niche-specific adaptation. In contrast, although the core genome dominantly undergoes purifying selection, its association with HGT and positively selected mutations indicates its potential role in ecological diversification. Furthermore, host fitness-related dynamics are characterized by the loss of secondary metabolite biosynthesis gene clusters (BGCs) and CAZyme-encoding genes and the acquisition of antimicrobial resistance (AMR) and virulence genes via HGT. This study offers a case study of pan-genome evolution to investigate the ecological adaptations reflected by biogeographical characteristics, thereby advancing the understanding of intraspecific diversity and evolutionary dynamics of foodborne pathogens.