Publications

Selected publications

2025
2024
2023
2022
2021
2020

2025

Potential Utility of Combined Urine Lipocalin-2 and Copper Test in Diagnosing Acute Pyelonephritis or Cystitis

Jan Hrbáček, Tomáš Pluháček, Jiří Novák, Andrea Palyzová, Dominika Luptáková, Jiří Houšť, David A. Stevens, Radim Dobiáš, Roman Zachoval, Vladimír Havlíček

https://doi.org/10.1021/acsomega.5c05922

The noninvasive differentiation of acute pyelonephritis (AP) and complicated urinary tract infections (cUTI), from acute cystitis (AC), represents an important diagnostic challenge in urology and nephrology. In a study of 95 adults, urine from patients with AP (n = 17), cUTI (n = 6), AC (n = 28), and controls (n = 44) was examined using a multidisciplinary approach: enzyme-linked immunosorbent assays for quantitation of acute phase proteins ceruloplasmin (Cp), pentraxin-3 (Ptx3), and lipocalin-2 (Lcn2); inductively coupled plasma mass spectrometry for determination of total copper (Cu), zinc (Zn), and iron (Fe) concentrations; and liquid chromatography coupled to electrospray ionization MS (LC–MS) for identification and quantitation of bacterial metallophores. Markedly elevated levels of human Cp, Ptx3, and Lcn2, all normalized to urine creatinine (Cr), in AC, cUTI, and AP patients compared to controls identified ongoing urinary tract infection without further differentiation between AC and AP. On the contrary, total urine copper normalized to Cr (Cu/Cr index) significantly differentiated AP from AC (p = 0.0209), as well as from the controls. Bacterial metallophores aerobactin and yersiniabactin, but not enterobactin, were indicators of AP, cUTI, and AC caused by Enterobacteriaceae. Importantly, the newly proposed combined test based on the quantitation of Lcn2 normalized to Cr (Lcn2/Cr) and Cu/Cr could noninvasively differentiate AP and cUTIs from AC with a diagnostic accuracy of 78% sensitivity and 65% specificity. The combination of characteristic elemental and molecular biomarkers may represent a future research direction with the potential to improve diagnostics of UTIs.

Regenerating liver uses ammonia to support de novo pyrimidine synthesis and cell proliferation

Berwini B. Endaya, Lukáš Kučera, Dan-Diem Thi Le, Jessica B. Spinelli, Andrea Brožková, Dominika Luptáková, Kryštof Klíma, Gabriela L. Oliveira, Petra Brisudová, Klára Boháčová, Štěpána Boukalová, Renata Zobalová, František Kolář, Karel Chalupsky, Klára Dohnalová, Arash Yarmohammadi-Barzegar, Šárka Dvořáková, Evgeniya Biryukova, Marta Kaliaeva, Vladimír Havlíček, Radislav Sedláček, Jan Procházka, Libor Vítek, Sunghyouk Park, Pavel Martásek, Zdeněk Krška, Paulo J. Oliveira, Michael V. Berridge, Jiří Neužil

https://doi.org/10.1038/s41467-025-65451-2

Liver is endowed with high regenerative activity, so that the tissue regrows in mouse after partial hepatectomy within days. We reason that this requires de novo pyrimidine synthesis to support rapid progression via the cell cycle. We find that suppression of de novo pyrimidine synthesis prevents proliferation in regenerating liver, suppressing liver regrowth. Tracing studies and spatial metabolomics reveal a metabolic shift such that ammonia, normally detoxified to urea in the periportal region under homeostasis, is redirected for generating aspartate and carbamoyl phosphate periportally, and glutamine pericentrally, and these products are utilized as precursors by the de novo pyrimidine synthesis pathway. Our research uncovers a metabolic reprogramming leading to utilization of a toxic byproduct for anabolic pathways that are essential for liver regeneration.

Detection of Siderophores as a Superior Noninvasive Diagnostic Tool in Unraveling Mixed Fungal Infections

Radim Dobiáš, Milan Navrátil, Rutuja H. Patil, Dominika Luptáková, David A. Stevens, Vladimír Havlíček

https://doi.org/10.1021/acsomega.5c01914

Advances in the early diagnosis of systemic mycoses are urgently needed, given the morbidity and mortality of such infections and the correlation between delays in treatment and poor outcomes. We demonstrated the prospective application of liquid chromatography–mass spectrometry in the diagnosis of a mixed fungal infection. In this study, we compared the performance of chest radiography, galactomannan (sGM), and beta-d-glucan (sBDG) serology with a novel diagnostic method based on creatinine-indexed microbial siderophores in urine. A woman with angioblastic T-cell lymphoma presented with neutropenia following allogeneic transplantation. sGM and sBDG remained positive throughout the 28-day intensive care unit stay. A. fumigatus DNA was detected in the induced sputum samples on sampling days 0 and 18. On day 18, a CT scan showed a typical nest sign, and R. microsporus DNA was detected in sputum. The patient was discharged from the hospital on day 28 and expired 7 days later. With our novel strategy based on mass spectrometry, A. fumigatus was consistently detected in the urine from day 0 to the end of the stay by the detection of triacetylfusarinine C (uTafC), an A. fumigatus-specific hydroxamate siderophore. An additional invasive R. microsporus infection was revealed by the detection of a mucoromycete-specific carboxylate siderophore in urine, rhizoferrin (uRhf), from day seven onward. Both creatinine-normalized siderophore indices (uTafC/Cr, uRhf/Cr) were sensitive to antifungal therapy and correlated with fast relapses of the invasive disease in time. This study illustrates how such an early and specific new approach can unravel the complexities of dual fungal infections.

Time- and temperature-dependent Pentraxin 3 stability in serum and bronchoalveolar lavage fluid samples

Radim Dobiáš, Valeria Skopelidou, Andrea Langer Sermeño, Jan Strakoš, Dominika Luptáková, Hana Tomášková, Milan Raška, Jozef Škarda, Denisa Bázsóová, Vladimír Havlíček

https://doi.org/10.1093/mmy/myaf057

Pentraxin 3 (Ptx3) is an acute-phase protein that specifically targets fungal galactosaminogalactan and has been proposed as a promising biomarker for invasive fungal infections. However, its stability in clinical samples over time has not yet been established. This study aimed to evaluate the stability of Ptx3 in serum and bronchoalveolar lavage fluid (BALF) samples during mid- and long-term storage. A total of 44 serum and 52 BALF samples were examined or re-examined for Ptx3 concentrations using enzyme immunoassay in pooled and individual sample formats. Samples were stored at −80°C, −20°C, and +37°C for periods ranging from 0 to 56 months. Statistical analyses included a paired two-sample Wilcoxon signed-rank test, analysis of variance, Bonferroni test, and linear regression analysis. Ptx3 remained highly stable in serum and BALF samples for up to 8 months at −20°C, with variations ranging from −1.8% to +2.8%. Long-term stability was observed at −80°C for 48 months, followed by a slow decline in Ptx3 levels. In contrast, storage at +37°C resulted in rapid degradation, with a 36.5%–60.7% increase or a 92.9%–97% decrease in Ptx3 levels in serum and BALF, respectively. These findings confirm that Ptx3 is a stable and reliable biomarker for invasive fungal infections when appropriate storage conditions are maintained.

Precursor direct biosynthesis of odd-chain polyunsaturated fatty acids by oomycete Pythium

Andrea Palyzová, Markéta Kulišová, Katarína Majtán, Karel Fous, Irena Jarošová Kolouchová, Tomáš Řezanka

https://doi.org/10.1016/j.foodchem.2025.144204

The growing demand for improving the nutritional value of food has driven efforts to enhance the production of polyunsaturated fatty acids (PUFAs) through microbial cultivation, mainly using low-cost substrates in closed-loop systems. One promising approach is precursor-driven biosynthesis, which enables the controlled production of odd-chain PUFAs. In this study, we cultivated two oomycetes from the genus Pythium (ATCC 38472 M1 and X42) under 20 different conditions in the presence of propionic acid and TWEEN 80 to stimulate the production of odd-chain PUFAs. This approach led to an increased yield of specific odd-chain PUFAs, including C17:2ω6, C17:3ω3, C17:4ω3, C19:2ω5, C19:3ω5, C19:4ω5, and C19:5ω2. The microorganisms were grown in two different media: potato dextrose broth (PDB) and waste brewery yeast, either alone or supplemented with propionic acid and/or TWEEN 80. The addition of propionic acid facilitated the biosynthesis of odd-chain PUFAs without the need for genetically modified strains, yielding quantities suitable for fermenter-scale production.

Elucidation of new sulfamethoxazole catabolic pathways in whole-cell catalyst of bacterium Kocuria rhizophila SA117

Tomáš Řezanka, Jiří Zahradník, Sofía G. Zavala-Meneses, Helena Marešová, Michal Řezanka, Helena Pelantová, Michal Grulich, Václav Filištein, Andrea Palyzová

https://doi.org/10.1016/j.biortech.2025.132912

Sulfamethoxazole (SMX) and its residues exhibit high environmental persistence due to their resistance to conventional degradation processes. The bacterial strain Kocuria rhizophila SA117, isolated from polluted soils, was characterized biochemically, phylogenetically, and −omically. Herein, we describe a complete degradation pathway for SMX and determine two putative pathways: cleavage of the benzene ring and the degradation of the substituted isoxazole, leading to the formation of non-toxic Krebs cycle metabolites. Based on molecular structures containing 13C6-labeled carbons and 2H3 atoms, thirty metabolites were identified by high-resolution tandem mass spectrometry. Genomic and proteomic analysis of strain SA117 revealed its ability to perform a wide range of metabolic activities under sulfamethoxazole selective pressure. These activities include energy and sulfur metabolism, adaptation to stress conditions, and catabolism of aromatic compounds. This study has greatly enhanced the understanding of microbial sulfonamide degradation and highlighted the potential of the bacterium Kocuria in remediation strategies.

2024

Exploring the Effects of Cyclosporin A to Isocyclosporin A Rearrangement on Ion Mobility Separation

Hynek Mácha, Jakub Zápal, Marek Kuzma, Dominika Luptáková, Karel Lemr, Vladimír Havlíček

https://doi.org/10.1021/acs.analchem.3c05165

Cyclosporin A (CycA) is a peptide secondary metabolite derived from fungi that plays a crucial role in transplantation surgery. Cyclic traveling wave ion mobility mass spectrometry (IM-MS) revealed an N → O peptidyl shift in singly protonated CycA to isocyclosporin A (isoA), whereas no such isomerization was observed for doubly protonated and sodiated molecules. CycA and isoA were able to be separated by considering doubly protonated precursors using a specific ion fragment. In parallel, sodium ion stabilization facilitated the simultaneous separation and quantitation of singly charged cyclosporin isomers with the limit of detection and coefficient of determination of 1.3% and 0.9908 for CycA in isoA and 1.0% and 0.9830 for isoA in CycA, respectively. Finally, 1H–13C gHSQC NMR experiments permitted parallel recording of up to 11 cyclosporin conformers. The ratios were determined by integrating the volume of cross-peaks of the upfield resonating hydrogen in the diastereotopic methylene group of sarcosine-3
Hypoxic-Ischemic Insult Alters Polyamine and Neurotransmitter Abundance in the Specific Neonatal Rat Brain Subregions

Hynek Mácha, Dominika Luptáková, Ivo Juránek, Per E. Andrén, Vladimír Havlíček

https://doi.org/10.1021/acschemneuro.4c00190

Neonatal hypoxic-ischemic (HI) brain insult is a major cause of neonatal mortality and morbidity. To assess the underlying pathological mechanisms, we mapped the spatiotemporal changes in polyamine, amino acid, and neurotransmitter levels, following HI insult (by the Rice–Vannucci method) in the brains of seven-day-old rat pups. Matrix-assisted laser desorption/ionization mass spectrometry imaging of chemically modified small-molecule metabolites by 4-(anthracen-9-yl)-2-fluoro-1-methylpyridin-1-ium iodide revealed critical HI-related metabolomic changes of 22 metabolites in 14 rat brain subregions, much earlier than light microscopy detected signs of neuronal damage. For the first time, we demonstrated excessive polyamine oxidation and accumulation of 3-aminopropanal in HI neonatal brains, which was later accompanied by neuronal apoptosis enhanced by increases in glycine and norepinephrine in critically affected brain regions. Specifically, putrescine, cadaverine, and 3-aminopropanal increased significantly as early as 12 h postinsult, mainly in motor and somatosensory cortex, hippocampus, and midbrain, followed by an increase in norepinephrine 24 h postinsult, which was predominant in the caudate putamen, the region most vulnerable to HI. The decrease of γ-aminobutyric acid (GABA) and the continuous dysregulation of the GABAergic system together with low taurine levels up to 36 h sustained progressive neurodegenerative cellular processes. The molecular alterations presented here at the subregional rat brain level provided unprecedented insight into early metabolomic changes in HI-insulted neonatal brains, which may further aid in the identification of novel therapeutic targets for the treatment of neonatal HI encephalopathy.
Proteogenomic Characterization of Pseudomonas veronii SM-20 Growing on Phenanthrene as Only Carbon and Energy Source

Zavala-Meneses, S.G.; Firrincieli, A.; Chalova, P.; Pajer, P.; Checcucci, A.; Skultety, L.; Cappelletti, M.

https://doi.org/10.3390/microorganisms12040753

In this study, we conducted an extensive investigation of the biodegradation capabilities and stress response of the newly isolated strain Pseudomonas veronii SM-20 in order, to assess its potential for bioremediation of sites contaminated with polycyclic aromatic hydrocarbons (PAHs). Initially, phenotype microarray technology demonstrated the strain’s proficiency in utilizing various carbon sources and its resistance to certain stressors. Genomic analysis has identified numerous genes involved in aromatic hydrocarbon metabolism. Biodegradation assay analyzed the depletion of phenanthrene (PHE) when it was added as a sole carbon and energy source. We found that P. veronii strain SM-20 degraded approximately 25% of PHE over a 30-day period, starting with an initial concentration of 600 µg/mL, while being utilized for growth. The degradation process involved PHE oxidation to an unstable arene oxide and 9,10-phenanthrenequinone, followed by ring-cleavage. Comparative proteomics provided a comprehensive understanding of how the entire proteome responded to PHE exposure, revealing the strain’s adaptation in terms of aromatic metabolism, surface properties, and defense mechanism. In conclusion, our findings shed light on the promising attributes of P. veronii SM-20 and offer valuable insights for the use of P. veronii species in environmental restoration efforts targeting PAH-impacted sites.

Nitrile Imines as Peptide and Oligonucleotide Photo-Cross-Linkers in Gas-Phase Ions

Jiahao Wan, Marianna Nytka, Haocheng Qian, Kim Vu, Karel Lemr, František Tureček

https://doi.org/10.1021/jasms.3c00379

Nitrile imines produced by photodissociation of 2,5-diaryltetrazoles undergo cross-linking reactions with amide groups in peptide-tetrazole (tet-peptide) conjugates and a tet-peptide-dinucleotide complex. Tetrazole photodissociation in gas-phase ions is efficient, achieving ca. 50% conversion with 2 laser pulses at 250 nm. The formation of cross-links was detected by CID-MS3 that showed structure-significant dissociations by loss of side-chain groups and internal peptide segments. The structure and composition of cross-linking products were established by a combination of UV–vis action spectroscopy and cyclic ion mobility mass spectrometry (c-IMS). The experimental absorption bands were found to match the bands calculated for vibronic absorption spectra of nitrile imines and cross-linked hydrazone isomers. The calculated collision cross sections (CCSth) for these ions were related to the matching experimental CCSexp from multipass c-IMS measurements. Loss of N2 from tet-peptide conjugates was calculated to be a mildly endothermic reaction with ΔH0 = 80 kJ mol–1 in the gas phase. The excess energy in the photolytically formed nitrile imine is thought to drive endothermic proton transfer, followed by exothermic cyclization to a sterically accessible peptide amide group. The exothermic nitrile imine reaction with peptide amides is promoted by proton transfer and may involve an initial [3 + 2] cycloaddition followed by cleavage of the oxadiazole intermediate. Nucleophilic groups, such as cysteine thiol, did not compete with the amide cyclization. Nitrile imine cross-linking to 2′-deoxycytidylguanosine was found to be >80% efficient and highly specific in targeting guanine. The further potential for exploring nitrile-imine cross-linking for biomolecular structure analysis is discussed.

2023

Siderophore-Based Noninvasive Differentiation of Aspergillus fumigatus Colonization and Invasion in Pulmonary Aspergillosis

Dominika Luptáková, Rutuja H. Patil, Radim Dobiáš, David A. Stevens, Tomáš Pluháček, Andrea Palyzová, Marcela Káňová, Milan Navrátil, Zbyněk Vrba, Petr Hubáček, Vladimír Havlíček

https://doi.org/10.1128/spectrum.04068-22

Germination from conidia to hyphae and hyphal propagation of Aspergillus fumigatus are the key pathogenic steps in the development of invasive pulmonary aspergillosis (IPA). By applying in vitro observations in a clinical study of 13 patients diagnosed with probable IPA, here, we show that the transition from colonization to the A. fumigatus invasive stage is accompanied by the secretion of triacetylfusarinine C (TafC), triacetylfusarinine B (TafB), and ferricrocin (Fc) siderophores into urine, with strikingly better sensitivity performance than serum sampling. The best-performing index, the TafC/creatinine index, with a median value of 17.2, provided 92.3% detection sensitivity (95% confidence interval [CI], 64.0 to 99.8%) and 100% specificity (95% CI, 84.6 to 100%), i.e., substantially better than the corresponding indications provided by galactomannan (GM) and β-d-glucan (BDG) serology. For the same patient cohort, the serum GM and BDG sensitivities were 46.2 and 76.9%, respectively, and their specificities were 86.4 and 63.6%, respectively. The time-dependent specific appearance of siderophores in the host’s urine represents an impactful clinical diagnostic advantage in the early discrimination of invasive aspergillosis from colonization. A favorable concentration of TafC in a clinical specimen distant from a deep infection site enables the noninvasive sampling of patients suffering from IPA.

Infection metallomics for critical care in the post-COVID era

Rutuja H. Patil, Dominika Luptáková, Vladimír Havlíček

https://doi.org/10.1002/mas.21755

Infection metallomics is a mass spectrometry (MS) platform we established based on the central concept that microbial metallophores are specific, sensitive, noninvasive, and promising biomarkers of invasive infectious diseases. Here we review the in vitro, in vivo, and clinical applications of metallophores from historical and functional perspectives, and identify under-studied and emerging application areas with high diagnostic potential for the post-COVID era. MS with isotope data filtering is fundamental to infection metallomics; it has been used to study the interplay between “frenemies” in hosts and to monitor the dynamic response of the microbiome to antibiotic and antimycotic therapies. During infection in critically ill patients, the hostile environment of the host’s body activates secondary bacterial, mycobacterial, and fungal metabolism, leading to the production of metallophores that increase the pathogen’s chance of survival in the host. MS can reveal the structures, stability, and threshold concentrations of these metal-containing microbial biomarkers of infection in humans and model organisms, and can discriminate invasive disease from benign colonization based on well-defined thresholds distinguishing proliferation from the colonization steady state.
Metabolomic Study of Aging in fa/fa Rats: Multiplatform Urine and Serum Analysis

Helena Pelantová , Petra Tomášová, Blanka Šedivá, Barbora Neprašová, Lucia Mráziková, Jaroslav Kuneš, Blanka Železná, Lenka Maletínská and Marek Kuzma

https://www.mdpi.com/2218-1989/13/4/552

Zucker fatty (fa/fa) rats represent a well-established and widely used model of genetic obesity. Because previous metabolomic studies have only been published for young fa/fa rats up to 20 weeks of age, which can be considered early maturity in male fa/fa rats, the aim of our work was to extend the metabolomic characterization to significantly older animals. Therefore, the urinary profiles of obese fa/fa rats and their lean controls were monitored using untargeted NMR metabolomics between 12 and 40 weeks of age. At the end of the experiment, the rats were also characterized by NMR and LC-MS serum analysis, which was supplemented by a targeted LC-MS analysis of serum bile acids and neurotransmitters. The urine analysis showed that most of the characteristic differences detected in young obese fa/fa rats persisted throughout the experiment, primarily through a decrease in microbial co-metabolite levels, the upregulation of the citrate cycle, and changes in nicotinamide metabolism compared with the age-related controls. The serum of 40-week-old obese rats showed a reduction in several bile acid conjugates and an increase in serotonin. Our study demonstrated that the fa/fa model of genetic obesity is stable up to 40 weeks of age and is therefore suitable for long-term experiments.
The Deciphering of Growth-Dependent Strategies for Quorum-Sensing Networks in Pseudomonas aeruginosa

Tereza Juříková, Hynek Mácha, Vanda Lupjanová, Tomáš Pluháček , Helena Marešová, Barbora Papoušková, Dominika Luptáková, Rutuja H. Patil, Oldřich Benada, Michal Grulich and Andrea Palyzová

https://doi.org/10.3390/microorganisms11092329

Pseudomonas aeruginosa is recognized as a significant cause of morbidity and mortality among nosocomial pathogens. In respiratory infections, P. aeruginosa acts not only as a single player but also collaborates with the opportunistic fungal pathogen Aspergillus fumigatus. This study introduced a QS molecule portfolio as a potential new biomarker that affects the secretion of virulence factors and biofilm formation. The quantitative levels of QS molecules, including 3-o-C12-HSL, 3-o-C8- HSL, C4-HSL, C6-HSL, HHQ, PQS, and PYO, measured using mass spectrometry in a monoculture, indicated metabolic changes during the transition from planktonic to sessile cells. In the co-cultures with A. fumigatus, the profile of abundant QS molecules was reduced to 3-o-C12-HSL, C4-HSL, PQS, and PYO. A decrease in C4-HSL by 50% to 170.6 ± 11.8 ng/mL and an increase 3-o-C12-HSL by 30% up to 784.4 ± 0.6 ng/mL were detected at the stage of the coverage of the hyphae with bacteria. Using scanning electron microscopy, we showed the morphological stages of the P. aeruginosa biofilm, such as cell aggregates, maturated biofilm, and cell dispersion. qPCR quantification of the genome equivalents of both microorganisms suggested that they exhibited an interplay strategy rather than antagonism. This is the first study demonstrating the quantitative growth-dependent appearance of QS molecule secretion in a monoculture of P. aeruginosa and a co-culture with A. fumigatus.

2022

NMR- and MS-Based Untargeted Metabolomic Study of Stool and Serum Samples from Patients with Anorexia Nervosa

Petra Tomášová, Petra Procházková, Radka Roubalová, Jiří Dvořák, Helena Tlaskalová-Hogenová, Martina Čermáková, Helena Pelantová, Blanka Šedivá, Marek Vecka, Hana Papežová, Marek Kuzma

https://doi.org/10.1021/acs.jproteome.1c00537

Anorexia nervosa (AN), a pathological restriction of food intake, leads to metabolic dysregulation. We conducted a metabolomics study to reveal changes caused by AN and the effect of hospital realimentation on metabolism. Both stool and serum from patients with AN and healthy controls were analyzed by NMR and MS. Statistical analysis revealed several altered biochemical and anthropometric parameters and 50 changed metabolites, including phospholipids, acylcarnitines, amino acids, derivatives of nicotinic acid, nucleotides, and energy metabolism intermediates. Biochemical and anthropometric parameters were correlated with metabolomic data. Metabolic changes in patients with AN described in our study imply serious system disruption defects, such as the development of inflammation and oxidative stress, changed free thyroxine (fT4) and thyroid-stimulating hormone (TSH) levels, a deficit of vitamins, muscle mass breakdown, and a decrease in ketone bodies as an important source of energy for the brain and heart. Furthermore, our data indicate only a very slight improvement after treatment. However, correlations of metabolomic results with body weight, interleukin 6, tumor necrosis factor α, fT4, and TSH might entail better prognoses and treatment effectiveness in patients with better system parameter status.

2021

Noninvasive Combined Diagnosis and Monitoring of Aspergillus and Pseudomonas Infections: Proof of Concept

Radim Dobiáš , Anton Škríba , Tomáš Pluháček, Miloš Petřík, Andrea Palyzová, Marcela Káňová, Eva Čubová, Jiří Houšt’ , Jiří Novák, David A. Stevens, Goran Mitulovič, Eva Krejčí, Petr Hubáček and Vladimír Havlíček

https://doi.org/10.3390/jof7090730

In acutely ill patients, particularly in intensive care units or in mixed infections, time to a microbe-specific diagnosis is critical to a successful outcome of therapy. We report the application of evolving technologies involving mass spectrometry to diagnose and monitor a patient’s course. As proof of this concept, we studied five patients and used two rat models of mono-infection and coinfection. We report the noninvasive combined monitoring of Aspergillus fumigatus and Pseudomonas aeruginosa infection. The invasive coinfection was detected by monitoring the fungal triacetylfusarinine C and ferricrocin siderophore levels and the bacterial metabolites pyoverdin E, pyochelin, and 2-heptyl-4-quinolone, studied in the urine, endotracheal aspirate, or breath condensate. The coinfection was monitored by mass spectrometry followed by isotopic data filtering. In the rat infection model, detection indicated 100-fold more siderophores in urine compared to sera, indicating the diagnostic potential of urine sampling. The tools utilized in our studies can now be examined in large clinical series, where we could expect the accuracy and speed of diagnosis to be competitive with conventional methods and provide advantages in unraveling the complexities of mixed infections.
Killing Effect of Bacillus velezensis FZB42 on a Xanthomonas campestris pv. Campestris (Xcc) Strain Newly Isolated from Cabbage Brassica oleracea Convar. Capitata (L.): A Metabolomic Study

Hynek Mácha, Helena Marešová, Tereza Juříková, Magdaléna Švecová, Oldřich Benada, Anton Škríba , Miroslav Baránek, Čeněk Novotný and Andrea Palyzová

https://doi.org/10.3390/microorganisms9071410

The potential use of Bacillus velezensis FZB42 for biological control of various phytopathogens has been documented over the past few years, but its antagonistic interactions with xanthomon- ads has not been studied in detail. Novel aspects in this study consist of close observation of the death of Xanthomonas campestris pv. campestris cells in a co-culture with B. velezensis FZB42, and quantification of lipopeptides and a siderophore, bacillibactin, involved in the killing process. A new robust Xcc-SU isolate tolerating high concentrations of ferric ions was used. In a co-culture with the antagonist, the population of Xcc-SU was entirely destroyed within 24–48 h, depending on the number of antagonist cells used for inoculation. No inhibitory effect of Xcc-SU on B. velezensis was observed. Bacillibactin and lipopeptides (surfactin, fengycin, and bacillomycin) were present in the co-culture and the monoculture of B. velezensis. Except for bacillibactin, the maximum contents of lipopeptides were higher in the antagonist monoculture compared with the co-culture. Scanning electron microscopy showed that the death of Xcc-SU bacteria in co-culture was caused by cell lysis, leading to an enhanced occurrence of distorted cells and cell ghosts. Analysis by mass spectrometry showed four significant compounds, bacillibactin, surfactin, fengycin, and bacillomycin D amongst a total of 24 different forms detected in the co-culture supernatant: Different forms of surfactin and fengycin with variations in their side-chain length were also detected. These results demonstrate the ability of B. velezensis FZB42 to act as a potent antagonistic strain against Xcc.
MassSpecBlocks: a web-based tool to create building blocks and sequences of nonribosomal peptides and polyketides for tandem mass spectra analysis

Jan Přívratský, Jiří Novák

https://doi.org/10.1186/s13321-021-00530-2

Nonribosomal peptides and polyketides are natural products commonly synthesized by microorganisms. They are widely used in medicine, agriculture, environmental protection, and other fields. The structures of natural products are often analyzed by high-resolution tandem mass spectrometry, which becomes more popular with its increasing availability. However, the characterization of nonribosomal peptides and polyketides from tandem mass spectra is a nontrivial task because they are composed of many uncommon building blocks in addition to proteinogenic amino acids. Moreover, many of them have cyclic and branch-cyclic structures. Here, we introduce MassSpecBlocks – an open-source and web-based tool that converts the input chemical structures in SMILES format into sequences of building blocks. The structures can be searched in public databases PubChem, ChemSpider, ChEBI, NP Atlas, COCONUT, and Norine and edited in a user-friendly graphical interface. Although MassSpecBlocks can serve as a stand-alone database, our primary goal was to enable easy construction of custom sequence and building block databases, which can be used to annotate mass spectra in CycloBranch software. CycloBranch is an open-source, cross-platform, and stand-alone tool that we recently released for annotating spectra of linear, cyclic, branched, and branch-cyclic nonribosomal peptides and polyketide siderophores. The sequences and building blocks created in MassSpecBlocks can be easily exported into a plain text format used by CycloBranch. MassSpecBlocks is available online or can be installed entirely offline. It offers a REST API to cooperate with other tools.

2020

Rhizoferrin Glycosylation in Rhizopus microsporus

Anton Škríba, Rutuja Hiraji Patil, Petr Hubáček, Radim Dobiáš, Andrea Palyzová, Helena Marešová, Tomáš Pluháček and Vladimír Havlíček

https://doi.org/10.3390/jof6020089

Rhizopus spp. are the most common etiological agents of mucormycosis, causing over 90% mortality in disseminated infections. The diagnosis relies on histopathology, culture, and/or polymerase chain reaction. For the first time, the glycosylation of rhizoferrin (RHF) was described in a Rhizopus microsporus clinical isolate by liquid chromatography and accurate tandem mass spectrometry. The fermentation broth lyophilizate contained 345.3 ± 13.5, 1.2 ± 0.03, and 0.03 ± 0.002 mg/g of RHF, imido-RHF, and bis-imido-RHF, respectively. Despite a considerable RHF secretion rate, we did not obtain conclusive RHF detection from a patient with disseminated mucormycosis caused by the same R. microsporus strain. We hypothesize that parallel antimycotic therapy, RHF biotransformation, and metabolism compromised the analysis. On the other hand, the full profile of posaconazole metabolites was retrieved by our in house software CycloBranch.
CycloBranch 2: Molecular Formula Annotations Applied to imzML Data Sets in Bimodal Fusion and LC-MS Data Files

Jiří Novák, Anton Škríba, Vladimír Havlíček

https://doi.org/10.1021/acs.analchem.0c00170

Natural product chemistry, microbiology, and food, human, and plant metabolomics represent a few sources of complex metabolomics data generated by mass spectrometry. Among the medley of software tools used to handle these data sets, no universal tool can qualitatively, quantitatively, or statistically address major biological questions or tasks. CycloBranch 2, an open and platform-free software, at least now provides the de novo generation of molecular formulas of unknown compounds in both liquid chromatography/mass spectrometry and mass spectrometry imaging datafiles. For imaging files, this database-free approach was documented in the bimodal image fusion and characterization of three small molecules, including metallophores. The fine isotope ratio data filtering step distinguished 34S/13C2 and 41K/13C2 features. The standalone software package is implemented in C++ and can be downloaded from https://ms.biomed.cas.cz/cyclobranch/ and used under GNU General Public License.
Antifungal Drugs

Jiří Houšť, Jaroslav Spížek, Vladimír Havlíček

https://doi.org/10.3390/metabo10030106

We reviewed the licensed antifungal drugs and summarized their mechanisms of action, pharmacological profiles, and susceptibility to specific fungi. Approved antimycotics inhibit 1,3-β-d-glucan synthase, lanosterol 14-α-demethylase, protein, and deoxyribonucleic acid biosynthesis, or sequestrate ergosterol. Their most severe side effects are hepatotoxicity, nephrotoxicity, and myelotoxicity. Whereas triazoles exhibit the most significant drug–drug interactions, echinocandins exhibit almost none. The antifungal resistance may be developed across most pathogens and includes drug target overexpression, efflux pump activation, and amino acid substitution. The experimental antifungal drugs in clinical trials are also reviewed. Siderophores in the Trojan horse approach or the application of siderophore biosynthesis enzyme inhibitors represent the most promising emerging antifungal therapies.
Bringing SEM and MSI Closer Than Ever Before: Visualizing Aspergillus and Pseudomonas Infection in the Rat Lungs

Tereza Juříková, Dominika Luptáková , Olga Kofroňová , Anton Škríba, Jiří Novák, Helena Marešová , Andrea Palyzová, Miloš Petřík, Vladimír Havlíček and Oldřich Benada

https://doi.org/10.3390/jof6040257

A procedure for processing frozen rat lung tissue sections for scanning electron microscopy (SEM) from deeply frozen samples initially collected and stored for matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was developed. The procedure employed slow thawing of the frozen sections while floating on the surface and melting in a fixative solution. After the float-washing step, the sections were dehydrated in a graded ethanol series and dried in a critical point dryer. The SEM generated images with well-preserved structures, allowing for monitoring of bacterial cells and fungal hyphae in the infected tissue. Importantly, the consecutive nonfixed frozen sections were fully compatible with MALDI-MSI, providing molecular biomarker maps of Pseudomonas aeruginosa. The protocol enables bimodal image fusion in the in-house software CycloBranch, as demonstrated by SEM and MALDI-MSI.
Bacterial nanotubes as a manifestation of cell death

Jiří Pospíšil, Dragana Vítkovská, Olga Kofroňová, Katarína Muchová, Hana Šanderová, Martin Hubálek, Michaela Šiková, Martin Modrák, Oldřich Benada, Imrich Barák, Libor Krásný

https://doi.org/10.1038/s41467-020-18800-2

Bacterial nanotubes are membranous structures that have been reported to function as conduits between cells to exchange DNA, proteins, and nutrients. Here, we investigate the morphology and formation of bacterial nanotubes using Bacillus subtilis. We show that nanotube formation is associated with stress conditions, and is highly sensitive to the cells’ genetic background, growth phase, and sample preparation methods. Remarkably, nanotubes appear to be extruded exclusively from dying cells, likely as a result of biophysical forces. Their emergence is extremely fast, occurring within seconds by cannibalizing the cell membrane. Subsequent experiments reveal that cell-to-cell transfer of non-conjugative plasmids depends strictly on the competence system of the cell, and not on nanotube formation. Our study thus supports the notion that bacterial nanotubes are a post mortem phenomenon involved in cell disintegration, and are unlikely to be involved in cytoplasmic content exchange between live cells.
Lipid Profiling in Epicardial and Subcutaneous Adipose Tissue of Patients with Coronary Artery Disease

Petra Tomášová, Martina Čermáková, Helena Pelantová, Marek Vecka, Helena Kratochvílová, Michal Lipš, Jaroslav Lindner, Peter Ivák, Ivan Netuka, Blanka Šedivá, Martin Haluzík, Marek Kuzma

https://doi.org/10.1021/acs.jproteome.0c00269

Coronary artery disease is one of the most frequent causes of morbidity and mortality worldwide. It is even more prevalent in patients with type 2 diabetes mellitus who suffer from obesity and increased accumulation of epicardial fat with a possible contributing role in the development of coronary artery disease. We performed an MS-based lipidomic analysis of subcutaneous and epicardial adipose tissue in 23 patients with coronary artery disease stratified for the presence/absence of type 2 diabetes mellitus and a control group of 13 subjects aiming at identification of factors from epicardial fat contributing to the development of coronary artery disease. The samples of adipose tissues were obtained during elective cardiac surgery. They were extracted and analyzed with and without previous triacylglycerols separation by high-pressure liquid chromatography–mass spectrometry (HPLC-MS). Multivariate and univariate analyses were performed. Lipidomics data were correlated with biochemical parameters. We identified multiple changes in monoacylglycerols, diacylglycerols, triacylglycerols, glycerophosphatidylserines, glycerophosphatidylethanolamines, glycerophosphatidylcholines, ceramides, sphingomyelins, and derivatives of cholesterol. Observed changes included molecules with fatty acids with odd (15:0, 15:1, 17:0, 17:1) and even (10:0, 12:0, 14:0, 16:0, 16:1, 18:0, 18:1, 18:2, 20:4, 20:1, 22:0) fatty acids in both types of adipose tissue. More pronounced changes were detected in epicardial adipose tissue compared to subcutaneous adipose tissue of patients with coronary artery disease and type 2 diabetes. Lipidomic analysis of subcutaneous and epicardial adipose tissue revealed different profiles for patients with coronary artery disease and type 2 diabetes, which might be related to coronary artery disease and the presence of type 2 diabetes.
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