Iron Acquisition Research Articles

Two ferrous iron transporter-like proteins independently participate in asexual development under iron limitation and virulence in Beauveria bassiana

Reductive assimilation pathway involves ferric reductase and ferrous iron transporter, which is integral for fungal iron acquisition. A family of ferric reductase-like proteins has been functionally characterized in the filamentous entomopathogenic fungus Beauveria bassiana. In this investigation, two ferrous iron transporter-like proteins (Ftr) were functionally annotated in B. bassiana. BbFtr1 and BbFtr2 displayed high similarity in structure and were associated with the plasma and nuclear membrane. Their losses had no negatively influence on fungal growth on various nutrients and development under the iron-replete condition. Single mutants of BbFTR1 and BbFTR2 displayed the iron-availability dependent developmental defects, and double mutant exhibited the significantly impaired developmental potential under the iron-limited conditions. In insect bioassay, the double mutant also showed the weaker virulence than either of two single disruption mutants. These results suggested that two ferrous iron transporter-like proteins function independently in fungal physiologies under the iron-deficient condition. Intriguingly, a bZIP transcription factor BbHapX was required for expression of BbFTR1 and BbFTR2 under iron-depleted conditions. This study enhances our understanding of the iron uptake system in the filamentous entomopathogenic fungi.

Iron efflux by IetA enhances β-lactam aztreonam resistance and is linked to oxidative stress through cellular respiration in Riemerella anatipestifer.

Riemerella anatipestifer encodes an iron acquisition system, but whether it encodes the iron efflux pump and its role in antibiotic resistance are largely unknown. To screen and identify an iron efflux gene in R. anatipestifer and determine whether and how the iron efflux gene is involved in antibiotic resistance. In this study, gene knockout, streptonigrin susceptibility assay and inductively coupled plasma mass spectrometry were used to screen for the iron efflux gene ietA. The MIC measurements, scanning electron microscopy and reactive oxygen species (ROS) detection were used to verify the role of IetA in aztreonam resistance and its mechanism. Mortality and colonization assay were used to investigate the role of IetA in virulence. The deletion mutant ΔietA showed heightened susceptibility to streptonigrin, and prominent intracellular iron accumulation was observed in ΔfurΔietA under excess iron conditions. Additionally, ΔietA exhibited increased sensitivity to H2O2-produced oxidative stress. Under aerobic conditions with abundant iron, ΔietA displayed increased susceptibility to the β-lactam antibiotic aztreonam due to heightened ROS production. However, the killing efficacy of aztreonam was diminished in both WT and ΔietA under anaerobic or iron restriction conditions. Further experiments demonstrated that the efficiency of aztreonam against ΔietA was dependent on respiratory complexes Ⅰ and Ⅱ. Finally, in a duckling model, ΔietA had reduced virulence compared with the WT. Iron efflux is critical to alleviate oxidative stress damage and β-lactam aztreonam killing in R. anatipestifer, which is linked by cellular respiration.

Bacterioplankton taxa compete for iron along the early spring-summer transition in the Arctic Ocean.

Microbial assemblages under the sea ice of the Dease Strait, Canadian Arctic, were sequenced for metagenomes of a small size fraction (0.2-3 μm). The community from early March was typical for this season, with Alpha- and Gammaproteobacteria as the dominant taxa, followed by Thaumarchaeota and Bacteroidetes. Toward summer, Bacteroidetes, and particularly the genus Polaribacter, became increasingly dominant, followed by the Gammaproteobacteria. Analysis of genes responsible for microbial acquisition of iron showed an abundance of ABC transporters for divalent cations and ferrous iron. The most abundant transporters, however, were the outer membrane TonB-dependent transporters of iron-siderophore complexes. The abundance of iron acquisition genes suggested this element was essential for the microbial assemblage. Interestingly, Gammaproteobacteria were responsible for most of the siderophore synthesis genes. On the contrary, Bacteroidetes did not synthesize siderophores but accounted for most of the transporters, suggesting a role as cheaters in the competition for siderophores as public goods. This cheating ability of the Bacteroidetes may have contributed to their dominance in the summer.

PGPg_finder: A comprehensive and user-friendly pipeline for identifying plant growth-promoting genes in genomic and metagenomic data

Identifying and comparing plant growth-promoting traits (PGPT) within whole-genome and metagenomic sequencing data can significantly advance agricultural research and promote sustainable crop production. This study introduces PGPg_finder, a comprehensive pipeline designed to annotate and compare PGPT from both whole-genome and metagenome sequencing datasets. This pipeline utilizes direct sequence annotation alongside de novo assembly methods to accurately detect PGPT. By cross-referencing sequences from the PLaBAse database, it identifies and quantifies the presence of these genes within the original datasets, facilitating an intuitive comparison of the abundance and distribution of PGPT across various samples. We evaluated the performance of PGPg_finder by analyzing genomes from five rhizobacterial strains: Paenibacillus vini, Paenibacillus polymyxa, Fictibacillus sp., Brevibacillus agri, and Bacillus cereus, and also metagenomic samples from bulk soils subjected to forest-to-pasture conversion in the Amazon rainforest. The genomic workflow revealed several genes associated with substrate utilization, abiotic stress neutralization, phosphate solubilization, and iron acquisition. It also identified genes unique to specific lineages, including those associated with colonization and plant-derived substrate usage in P. polymyxa, quorum sensing response and biofilm formation in P. vini, heavy metal detoxification and nitrogen acquisition in B. agri, and spore production and neutralizing biotic stress in B. cereus. The strain Fictibacillus sp. presented several unique genes related to surface attachment, stress response, xenobiotic degradation, phosphate solubilization, and phytohormone production. The use of PGPg_finder highlights its potential to uncover novel inoculants and strains. The metagenomic workflow distinguished plant-growth promotion gene profiles between soils from the Amazon rainforest and pasture, with the latter showing a profile more aligned with simple carbohydrate consumption, abiotic stress tolerance, motility and chemotaxis, and phosphorus mineralization. Native forests exhibited a profile associated with the degradation of complex organic matter, oxidative stress tolerance, xenobiotic degradation, bactericidal activity, iron acquisition, and volatile pathways. These findings underscore the effectiveness and sensitivity of PGPg_finder in accurately identifying and comparing PGPT genes, highlighting both commonalities and variations across samples. The application of this pipeline has the potential to significantly facilitate the identification of plant growth-promoting microbes.

Iron mobilization from intact ferritin: effect of differential redox activity of quinone derivatives with NADH/O2 and in situ-generated ROS.

Ferritins are multimeric nanocage proteins that sequester/concentrate excess of free iron and catalytically synthesize a hydrated ferric oxyhydroxide bio-mineral. Besides functioning as the primary intracellular iron storehouses, these supramolecular assemblies also oversee the controlled release of iron to meet physiologic demands. By virtue of the reducing nature of the cytosol, reductive dissolution of ferritin-iron bio-mineral by physiologic reducing agents might be a probable pathway operating in vivo. Herein, to explore this reductive iron-release pathway, a series of quinone analogs differing in size, position/nature of substituents and redox potentials were employed to relay electrons from physiologic reducing agent, NADH, to the ferritin core. Quinones are well known natural electron/proton mediators capable of facilitating both 1/2 electron transfer processes and have been implicated in iron/nutrient acquisition in plants and energy transduction. Our findings on the structure-reactivity of quinone mediators highlight that iron release from ferritin is dictated by electron-relay capability (dependent on E1/2 values) of quinones, their molecular structure (i.e., the presence of iron-chelation sites and the propensity for H-bonding) and the type/amount of reactive oxygen species (ROS) they generate in situ. Juglone/Plumbagin released maximum iron due to their intermediate E1/2 values, presence of iron chelation sites, the ability to inhibit in situ generation of H2O2 and form intramolecular H-bonding (possibly promotes semiquinone formation). This study may strengthen our understanding of the ferritin-iron-release process and their significance in bioenergetics/O2-based cellular metabolism/toxicity while providing insights on microbial/plant iron acquisition and the dynamic host-pathogen interactions.

The Increased Prevalence of rmpA Gene in Klebsiella pneumoniae Isolates Coharboring blaNDM and blaOXA-48-like Genes.

The emergence of carbapenemase-producing Klebsiella pneumoniae poses a substantial risk to public health. It is essential to comprehend the influence of carbapenemase on the virulence characteristics of K. pneumoniae in order to devise successful strategies for combating these infections. In this study, we explored the distribution disparity of virulence determinants between carbapenemase-producing (CP-Kp, n = 52) and carbapenemase-nonproducing (CN-Kp, n = 43) isolates. The presence of carbapenemases was detected via the modified carbapenem inactivation method and confirmed by PCR. The New Delhi metallo-β-lactamase (blaNDM) and Oxacillinase-48-like (blaOXA-48-like) genes were the most prevalent (94.23% and 76.92%, respectively) in CP-Kp isolates. Coexistence of blaNDM and blaOXA-48-like was observed in 71.15% of isolates, whereas 5.77% coharbored blaNDM and blaKPC. PCR analysis revealed the presence of several virulence genes, including adhesins (fimH, 92.63%, mrkD, 97.89%), capsule-associated virulence (uge, 90.53%), the K2 capsule serotype (k2, 6.32%), the iron acquisition system (kfu, 23.16%), and the regulator of mucoid phenotype (rmpA, 28.42%). A significantly higher prevalence of rmpA was detected in the CP-Kp compared with the CN-Kp (24/52 vs. 3/43, p < 0.0001), indicating a potential association between rmpA and carbapenemase acquisition. In addition, the majority of rmpA (22/24) positive isolates in the CP-Kp isolates coharbored blaNDM and either blaOXA-48-like or blaKPC.

Comparative genomics of Vibrio toranzoniae strains.

Vibrio toranzoniae is a marine bacterium belonging to the Splendidus clade, originally isolated from healthy clams in Galicia (NW Spain). Its isolation from different hosts and seawater indicated two lifestyles and wide geographical distribution. The aim of the present study was to determine the differences at genome level among strains, as well as to determine their phylogeny. For this purpose, whole genomes were sequenced by different technologies and the resulting sequences corrected. Genomes were annotated and compared with different online tools. Furthermore, the study of core and pan genome was examined, and the phylogeny was inferred. The content of the core genome ranged from 2,953 to 2,766 genes and that of the pangenome from 6,278 to 6,132, depending on the tool used. The comparison revealed that although the strains shared certain homology, with DDH values ranging from 77.10 to 82.30 and values of OrthoANI higher than 97%,notable differences were found related to motility, capsule synthesis, iron acquisition system or mobile genetic elements. The phylogenetic analysis of the core genome did not reveal a differentiation of the strains according to their lifestyle, but that of the pangenome pointed out certain geographical isolation in the same growing area. The study led to a reclassification of some isolates formerly described as V. toranzoniae and manifested the importance of cured deposited sequences to proper phylogenetic assignment.

First Pangenome of Corynebacterium rouxii, a Potentially Toxigenic Species of Corynebacterium diphtheriae Complex

Corynebacterium rouxii is one of the recently described species of the Corynebacterium diphtheriae complex. As this species can potentially infect different hosts and harbor the tox gene, producing diphtheria toxin, we present its first pangenomic analysis in this work. A total of fifteen genomes deposited in online databases were included. After confirming the taxonomic position of the isolates by genomic taxonomy, the genomes were submitted to genomic plasticity, gene synteny, and pangenome prediction analyses. In addition, virulence and antimicrobial resistance genes were investigated. Finally, epidemiological data were obtained through molecular typing, clustering, and phylogenetic analysis. Our data demonstrated genetic diversity within the species with low synteny. However, the gene content is extensively conserved, and the pangenome is composed of 2606 gene families, of which 1916 are in the core genome and 80 are related to unique genes. Prophages, insertion sequences, and genomic islands were found. A type I-E CRISPR-Cas system was also detected. Besides the tox gene, determinants involved in adhesion and iron acquisition and two putative antimicrobial resistance genes were predicted. These findings provide valuable insight about this species’ pathogenicity, evolution, and diversity. In the future, our data can contribute to different areas, including vaccinology and epidemiology.

New insight into protective effect against oxidative stress and biosynthesis of exopolysaccharides produced by Lacticaseibacillus paracasei NC4 from fermented eggplant.

Fermented eggplant is a traditional fermented food, however lactic acid bacteria capable of producing exopolysaccharide (EPS) have not yet been exploited. The present study focused on the production and protective effects against oxidative stress of an EPS produced by Lacticaseibacillus paracasei NC4 (NC4-EPS), in addition to deciphering its genomic features and EPS biosynthesis pathway. Among 54 isolates tested, strain NC4 showed the highest EPS yield and antioxidant activity. The maximum EPS production (2.04 ± 0.11g/L) was achieved by culturing in MRS medium containing 60g/L sucrose at 37°C for 48h. Under 2mM H2O2 stress, the survival of a yeast model Saccharomyces cerevisiae treated with 0.4mg/mL NC4-EPS was 2.4-fold better than non-treated cells, which was in agreement with the catalase and superoxide dismutase activities measured from cell lysates. The complete genome of NC4 composed of a circular chromosome of 2,888,896bp and 3 circular plasmids. The NC4 genome comprises more genes with annotated function in nitrogen metabolism, phosphorus metabolism, cell division and cell cycle, and iron acquisition and metabolism as compared to other reported L. paracasei. Of note, the eps gene cluster is not conserved across L. paracasei. Pathways of sugar metabolism for EPS biosynthesis were proposed for the first time, in which gdp pathway only present in few plant-derived bacteria was identified. These findings shed new light on the cell-protective activity and biosynthesis of EPS produced by L. paracasei, paving the way for future efforts to enhance yield and tailor-made EPS production for food and pharmaceutical industries.

Bicarbonate affects the expression of iron acquisition and translocation genes involved in chlorosis in Medicago lupulina

Bicarbonate affects the expression of iron acquisition and translocation genes involved in chlorosis in Medicago lupulina

The Transcription Factors AcuK and AcuM Influence Siderophore Biosynthesis of Aspergillus fumigatus.

The mold Aspergillus fumigatus employs two high-affinity uptake systems, reductive iron assimilation (RIA) and siderophore-mediated iron acquisition (SIA), for the acquisition of the essential trace element iron. SIA has previously been shown to be crucial for virulence in mammalian hosts. Here, we show that a lack of AcuK or AcuM, transcription factors required for the activation of gluconeogenesis, decreases the production of both extra- and intracellular siderophores in A. fumigatus. The lack of AcuM or AcuK did not affect the expression of genes involved in RIA and SIA, suggesting that these regulators do not directly regulate iron homeostasis genes, but indirectly affect siderophore production through their influence on metabolism. Consistent with this, acetate supplementation reversed the intracellular siderophore production defect of ΔacuM and ΔacuK. Moreover, ΔacuM and ΔacuK displayed a similar growth defect under iron limitation and iron sufficiency, which suggests they have a general role in carbon metabolism apart from gluconeogenesis. In agreement with a potential role of the glyoxylate cycle in adaptation to iron starvation, transcript levels of the malate synthase-encoding acuE were found to be upregulated by iron limitation that is partially dependent on AcuK and AcuM. Together, these data demonstrate the influence of iron availability on carbon metabolism.

The interplay between electron transport chain function and iron regulatory factors influences melanin formation in Cryptococcus neoformans.

Mitochondrial functions are critical for the ability of the fungal pathogen Cryptococcus neoformans to cause disease. However, mechanistic connections between key functions such as the mitochondrial electron transport chain (ETC) and virulence factor elaboration have yet to be thoroughly characterized. Here, we observed that inhibition of ETC complex III suppressed melanin formation, a major virulence factor. This inhibition was partially overcome by defects in Cir1 or HapX, two transcription factors that regulate iron acquisition and use. In this regard, loss of Cir1 derepresses the expression of laccase genes as a potential mechanism to restore melanin, while HapX may condition melanin formation by controlling oxidative stress. We hypothesize that ETC dysfunction alters redox homeostasis to influence melanin formation. Consistent with this idea, inhibition of growth by hydrogen peroxide was exacerbated in the presence of the melanin substrate L-DOPA. In addition, loss of the mitochondrial chaperone Mrj1, which influences the activity of ETC complex III and reduces ROS accumulation, also partially overcame antimycin A inhibition of melanin. The phenotypic impact of mitochondrial dysfunction was consistent with RNA-Seq analyses of WT cells treated with antimycin A or L-DOPA, or cells lacking Cir1 that revealed influences on transcripts encoding mitochondrial functions (e.g., ETC components and proteins for Fe-S cluster assembly). Overall, these findings reveal mitochondria-nuclear communication via ROS and iron regulators to control virulence factor production in C. neoformans.IMPORTANCEThere is a growing appreciation of the importance of mitochondrial functions and iron homeostasis in the ability of fungal pathogens to sense the vertebrate host environment and cause disease. Many mitochondrial functions such as heme and iron-sulfur cluster biosynthesis, and the electron transport chain (ETC), are dependent on iron. Connections between factors that regulate iron homeostasis and mitochondrial activities are known in model yeasts and are emerging for fungal pathogens. In this study, we identified connections between iron regulatory transcription factors (e.g., Cir1 and HapX) and the activity of complex III of the ETC that influence the formation of melanin, a key virulence factor in the pathogenic fungus Cryptococcus neoformans. This fungus causes meningoencephalitis in immunocompromised people and is a major threat to the HIV/AIDS population. Thus, understanding how mitochondrial functions influence virulence may support new therapeutic approaches to combat diseases caused by C. neoformans and other fungi.

Transcriptional regulation of the yersiniabactin receptor fyuA gene by the ferric uptake regulator in Klebsiella pneumoniae NTUH-K2044.

The ferric uptake regulator (Fur) is a global regulator that influences the expression of virulence genes in Klebsiella pneumoniae. Bioinformatics analysis suggests Fur may involve in iron acquisition via the identified regulatory box upstream of the yersiniabactin receptor gene fyuA. To observe the impact of the gene fyuA on the virulence of K. pneumoniae, the gene fyuA knockout strain and complementation strain were constructed and then conducted a series of phenotypic experiments including chrome azurol S(CAS) detection, crystal violet staining, and wax moth virulence experiment. To examine the regulatory relationship between Fur and the gene fyuA, green fluorescent protein (GFP) reporter gene fusion assay, real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR), gel migration assay (EMSA), and DNase I footprinting assay were used to clarify the regulatory mechanism of Fur on fyuA. CAS detection revealed that the gene fyuA could affect the generation of iron carriers in K. pneumoniae. Crystal violet staining experiment showed that fyuA could positively influence biofilm formation. Wax moth virulence experiment indicated that the deletion of the fyuA could weaken bacterial virulence. GFP reporter gene fusion experiment and RT-qPCRanalysis revealed that Fur negatively regulated the expression of fyuA in iron-sufficient environment. EMSA experiment demonstrated that Fur could directly bind to the promoter region of fyuA, and DNase I footprinting assay further identified the specific binding site sequences. The study showed that Fur negatively regulated the transcriptional expression of fyuA by binding to upstream of the gene promoter region, and then affected the virulence of K. pneumoniae.

Chromosomal and plasmid-encoded virulence and multidrug resistance of Escherichia coli ST58/24 infecting a 2-year-old sickle cell patient with sepsis in Kampala Uganda, East Africa

Sepsis and drug resistance represent a complex of the most common global causes of mortality in intensive care units (ICUs) especially among patients with comorbidities. Extraintestinal pathogenic Escherichia coli (ExPEC) strains are highly implicated in systemic infections, with multidrug resistance exacerbating the risk of chronic conditions and patient mortality. The diversity of virulence and evolution of multidrug resistance are yet to be fully deciphered. In this work, we aimed at unveiling the pathogens and their genomic determinants of virulence and drug resistance relevant to increased sepsis in a sickle cell child admitted to ICU. From a rectal swab, we isolated a strain of E. coli from the patient and phenotypically tested it against a panel of selected beta lactams, fluoroquinolones, macrolides, aminoglycosides and colistin. We then sequenced the entire genome and integrated multiple bioinformatic pipelines to divulge the virulence and multidrug resistance profiles of the isolate. Our results revealed that the isolate belongs to the sequence type (ST) 58/24, which (ST58), is a known ExPEC. With the use of PathogenFinder, we were able to confirm that this isolate is a human pathogen (p = 0.936). The assembled chromosome and two plasmids encode virulence factors related to capsule (antiphagocytosis), serum survival and resistance, type 6 secretion system (T6SS), multiple siderophores (iron acquisition), and biosynthetic gene clusters for polyketides and nonribosomal peptides exhibiting host cell damaging activity in silico. The genome also harbors multidrug resistance genotypes including extended spectrum beta lactamase (ESBL) genes such as blaTEM-1A/B, sulfonamide resistance genes sul1/2, fluoroquinolone resistance genes dfrA5 and nonsynonymous mutations of the gene pmrB, conferring intrinsic colistin resistance. Conclusively, this pathogen holds the potential to cause systemic infection and might exacerbate sickle cell anemia in the patient. The virulence and multidrug resistance profiles are encoded by both the chromosome and plasmids. Genomic surveillance of pathogens with multidrug resistance among patients with comorbidities is crucial for effective disease management.

Multi-Omics of Campylobacter jejuni Growth in Chicken Exudate Reveals Molecular Remodelling Associated with Altered Virulence and Survival Phenotypes.

Campylobacter jejuni is the leading cause of foodborne human gastroenteritis in the developed world. Infections are largely acquired from poultry produced for human consumption and poor food handling is thus a major risk factor. Chicken exudate (CE) is a liquid produced from defrosted commercial chicken products that facilitates C. jejuni growth. We examined the response of C. jejuni to growth in CE using a multi-omics approach. Changes in the C. jejuni proteome were assessed by label-based liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). We quantified 1328 and 1304 proteins, respectively, in experiments comparing 5% CE in Mueller-Hinton (MH) medium and 100% CE with MH-only controls. These proteins represent 81.8% and 80.3% of the predicted C. jejuni NCTC11168 proteome. Growth in CE induced profound remodelling of the proteome. These changes were typically conserved between 5% and 100% CE, with a greater magnitude of change observed in 100% CE. We confirmed that CE induced C. jejuni biofilm formation, as well as increasing motility and resistance against oxidative stress, consistent with changes to proteins representing those functions. Assessment of the C. jejuni metabolome showed CE also led to increased intracellular abundances of serine, proline, and lactate that were correlated with the elevated abundances of their respective transporters. Analysis of carbon source uptake showed prolonged culture supernatant retention of proline and succinate in CE-supplemented medium. Metabolomics data provided preliminary evidence for the uptake of chicken-meat-associated dipeptides. C. jejuni exposed to CE showed increased resistance to several antibiotics, including polymyxin B, consistent with changes to tripartite efflux system proteins and those involved in the synthesis of lipid A. The C. jejuni CE proteome was also characterised by very large increases in proteins associated with iron acquisition, while a decrease in proteins containing iron-sulphur clusters was also observed. Our data suggest CE is both oxygen- and iron-limiting and provide evidence of factors required for phenotypic remodelling to enable C. jejuni survival on poultry products.

High-risk Escherichia coli clones that cause neonatal meningitis and association with recrudescent infection

Neonatal meningitis is a devastating disease associated with high mortality and neurological sequelae. Escherichia coli is the second most common cause of neonatal meningitis in full-term infants (herein NMEC) and the most common cause of meningitis in preterm neonates. Here, we investigated the genomic relatedness of a collection of 58 NMEC isolates spanning 1974–2020 and isolated from seven different geographic regions. We show NMEC are comprised of diverse sequence types (STs), with ST95 (34.5%) and ST1193 (15.5%) the most common. No single virulence gene profile was conserved in all isolates; however, genes encoding fimbrial adhesins, iron acquisition systems, the K1 capsule, and O antigen types O18, O75, and O2 were most prevalent. Antibiotic resistance genes occurred infrequently in our collection. We also monitored the infection dynamics in three patients that suffered recrudescent invasive infection caused by the original infecting isolate despite appropriate antibiotic treatment based on antibiogram profile and resistance genotype. These patients exhibited severe gut dysbiosis. In one patient, the causative NMEC isolate was also detected in the fecal flora at the time of the second infection episode and after treatment. Thus, although antibiotics are the standard of care for NMEC treatment, our data suggest that failure to eliminate the causative NMEC that resides intestinally can lead to the existence of a refractory reservoir that may seed recrudescent infection.

In Silico Analysis of the Ga3+/Fe3+ Competition for Binding the Iron-Scavenging Siderophores of P. aeruginosa-Implementation of Three Gallium-Based Complexes in the "Trojan Horse" Antibacterial Strategy.

The emergence of multidrug-resistant (MDR) microorganisms combined with the ever-draining antibiotic pipeline poses a disturbing and immensely growing public health challenge that requires a multidisciplinary approach and the application of novel therapies aimed at unconventional targets and/or applying innovative drug formulations. Hence, bacterial iron acquisition systems and bacterial Fe2+/3+-containing enzymes have been identified as a plausible target of great potential. The intriguing "Trojan horse" approach deprives microorganisms from the essential iron. Recently, gallium's potential in medicine as an iron mimicry species has attracted vast attention. Different Ga3+ formulations exhibit diverse effects upon entering the cell and thus supposedly have multiple targets. The aim of the current study is to specifically distinguish characteristics of great significance in regard to the initial gallium-based complex, allowing the alien cation to effectively compete with the native ferric ion for binding the siderophores pyochelin and pyoverdine secreted by the bacterium P. aeruginosa. Therefore, three gallium-based formulations were taken into consideration: the first-generation gallium nitrate, Ga(NO3)3, metabolized to Ga3+-hydrated forms, the second-generation gallium maltolate (tris(3-hydroxy-2-methyl-4-pyronato)gallium), and the experimentally proven Ga carrier in the bloodstream-the protein transferrin. We employed a reliable in silico approach based on DFT computations in order to understand the underlying biochemical processes that govern the Ga3+/Fe3+ rivalry for binding the two bacterial siderophores.

High-risk Escherichia coli clones that cause neonatal meningitis and association with recrudescent infection.

Neonatal meningitis is a devastating disease associated with high mortality and neurological sequelae. Escherichia coli is the second most common cause of neonatal meningitis in full-term infants (herein NMEC) and the most common cause of meningitis in preterm neonates. Here, we investigated the genomic relatedness of a collection of 58 NMEC isolates spanning 1974-2020 and isolated from seven different geographic regions. We show NMEC are comprised of diverse sequence types (STs), with ST95 (34.5%) and ST1193 (15.5%) the most common. No single virulence gene profile was conserved in all isolates; however, genes encoding fimbrial adhesins, iron acquisition systems, the K1 capsule, and O antigen types O18, O75, and O2 were most prevalent. Antibiotic resistance genes occurred infrequently in our collection. We also monitored the infection dynamics in three patients that suffered recrudescent invasive infection caused by the original infecting isolate despite appropriate antibiotic treatment based on antibiogram profile and resistance genotype. These patients exhibited severe gut dysbiosis. In one patient, the causative NMEC isolate was also detected in the fecal flora at the time of the second infection episode and after treatment. Thus, although antibiotics are the standard of care for NMEC treatment, our data suggest that failure to eliminate the causative NMEC that resides intestinally can lead to the existence of a refractory reservoir that may seed recrudescent infection.

Genome mining of Mycobacterium tuberculosis: targeting SufD as a novel drug candidate through in silico characterization and inhibitor screening.

Tuberculosis (TB) stands as the second most fatal infectious disease globally, causing 1.3 million deaths in 2022. The resurgence of TB and the alarming rise of antibiotic resistance demand urgent call to develop novel antituberculosis drugs. Despite concerted efforts to control TB, the disease persists and spreads rapidly on a global scale. Targeting stress response pathways in Mycobacterium tuberculosis (Mtb) has become imperative to achieve complete eradication. This study employs subtractive genomics to identify and prioritize potential drug targets among the hypothetical proteins of Mtb, focusing on indispensable pathways. Amongst 177 essential hypothetical proteins, 152 were nonhomologous to human. These proteins participated in 34 pathways, and a 20-fold enrichment of SUF pathway genes led to its selection as a target pathway. Fe-S clusters are fundamental, widely distributed protein cofactors involved in vital cellular processes. The survival of Mtb in a hypoxic environment relies on the iron-sulfur (Fe-S) cluster biogenesis pathway for the repair of damaged Fe-S clusters. It also protects pathogen against drugs, ensuring controlled iron utilization and contributing to drug resistance. In Mtb, six proteins of Fe-S cluster assembly pathway are encoded by the suf operon. The present study was focused on SufD because of its role in iron acquisition and prevention of Fenton reaction. The research further delves into the in silico characterization of SufD, utilizing bioinformatics tools for sequence and structure based analysis. The protein's structural features, including the identification of conserved regions, motifs, and 3D structure prediction enhanced functional annotation. Target based virtual screening of compounds from the ChEMBL database resulted in 12 inhibitors with best binding affinities. Drug likeness and ADMET profiling of potential inhibitors identified promising compounds with favorable drug-like properties. The study also involved cloning in SUMO-pRSF-Duet1 expression vector, overexpression, and purification of recombinant SufD from E. coli BL21 (DE3) cells. Optimization of expression conditions resulted in soluble production, and subsequent purification highlighting the efficacy of the SUMO fusion system for challenging Mtb proteins in E. coli. These findings provide valuable insights into pharmacological targets for future experimental studies, holding promise for the development of targeted therapy against Mtb.

Siderophore-mediated iron acquisition by Klebsiella pneumoniae.

Both commensal and pathogenic bacteria produce small organic chelators, called siderophores, that avidly bind iron and increase its bioavailability. Klebsiella pneumoniae variably produces four siderophores that antagonize host iron sequestration: enterobactin, glucosylated enterobactin (also termed salmochelin), aerobactin, and yersiniabactin, which promote colonization of different host tissues. Abundant evidence links bacterial iron acquisition to virulence and infectious diseases. The data we report explain the recognition and transport of ferric catecholates and other siderophores, which are crucial to iron acquisition by K. pneumoniae.