International Journal of Molecular Sciences

2023

Jump to: 2022, 2021

24 pages, 4013 KiB

Open AccessArticle

Mitochondrial Metabolism in the Spotlight: Maintaining Balanced RNAP III Activity Ensures Cellular Homeostasis

by Roza Szatkowska, Emil Furmanek, Andrzej M. Kierzek, Christian Ludwig and Malgorzata Adamczyk

Int. J. Mol. Sci. 2023, 24(19), 14763; https://doi.org/10.3390/ijms241914763 - 29 Sep 2023

Viewed by 1505

Abstract

RNA polymerase III (RNAP III) holoenzyme activity and the processing of its products have been linked to several metabolic dysfunctions in lower and higher eukaryotes. Alterations in the activity of RNAP III-driven synthesis of non-coding RNA cause extensive changes in glucose metabolism. Increased [...] Read more.

RNA polymerase III (RNAP III) holoenzyme activity and the processing of its products have been linked to several metabolic dysfunctions in lower and higher eukaryotes. Alterations in the activity of RNAP III-driven synthesis of non-coding RNA cause extensive changes in glucose metabolism. Increased RNAP III activity in the S. cerevisiae maf1Δ strain is lethal when grown on a non-fermentable carbon source. This lethal phenotype is suppressed by reducing tRNA synthesis. Neither the cause of the lack of growth nor the underlying molecular mechanism have been deciphered, and this area has been awaiting scientific explanation for a decade. Our previous proteomics data suggested mitochondrial dysfunction in the strain. Using model mutant strains maf1Δ (with increased tRNA abundance) and rpc128-1007 (with reduced tRNA abundance), we collected data showing major changes in the TCA cycle metabolism of the mutants that explain the phenotypic observations. Based on ¹³C flux data and analysis of TCA enzyme activities, the present study identifies the flux constraints in the mitochondrial metabolic network. The lack of growth is associated with a decrease in TCA cycle activity and downregulation of the flux towards glutamate, aspartate and phosphoenolpyruvate (PEP), the metabolic intermediate feeding the gluconeogenic pathway. rpc128-1007, the strain that is unable to increase tRNA synthesis due to a mutation in the C128 subunit, has increased TCA cycle activity under non-fermentable conditions. To summarize, cells with non-optimal activity of RNAP III undergo substantial adaptation to a new metabolic state, which makes them vulnerable under specific growth conditions. Our results strongly suggest that balanced, non-coding RNA synthesis that is coupled to glucose signaling is a fundamental requirement to sustain a cell’s intracellular homeostasis and flexibility under changing growth conditions. The presented results provide insight into the possible role of RNAP III in the mitochondrial metabolism of other cell types. Full article

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15 pages, 5433 KiB

Open AccessArticle

Exploring Extracellular Vesicles of Probiotic Yeast as Carriers of Biologically Active Molecules Transferred to Human Intestinal Cells

by Jolanta Mierzejewska, Patrycja Kowalska, Klaudia Marlicka, Sara Dworakowska, Ewa Sitkiewicz, Maciej Trzaskowski, Agata Głuchowska, Grażyna Mosieniak and Małgorzata Milner-Krawczyk

Int. J. Mol. Sci. 2023, 24(14), 11340; https://doi.org/10.3390/ijms241411340 - 12 Jul 2023

Cited by 7 | Viewed by 3318

Abstract

Extracellular vesicles (EVs) are nanoparticles containing various bioactive cargos—e.g., proteins, RNAs, and lipids—that are released into the environment by all cell types. They are involved in, amongst other functions, intercellular communication. This article presents studies on EVs produced by the probiotic yeast Saccharomyces [...] Read more.

Extracellular vesicles (EVs) are nanoparticles containing various bioactive cargos—e.g., proteins, RNAs, and lipids—that are released into the environment by all cell types. They are involved in, amongst other functions, intercellular communication. This article presents studies on EVs produced by the probiotic yeast Saccharomyces boulardii CNCM I-745. The size distribution and concentration of EVs in the liquid culture of yeast were estimated. Moreover, the vesicles of S. boulardii were tested for their cytotoxicity against three model human intestinal cell lines. This study did not show any significant negative effect of yeast EVs on these cells under tested conditions. In addition, EVs of S. boulardii were verified for their ability to internalize in vitro with human cells and transfer their cargo. The yeast vesicles were loaded with doxorubicin, an anticancer agent, and added to the cellular cultures. Subsequently, microscopic observations revealed that these EVs transferred the compound to human intestinal cell lines. A cytotoxicity test confirmed the activity of the transferred doxorubicin. Detailed information about the proteins present in EVs might be important in terms of exploring yeast EVs as carriers of active molecules. Thus, proteomic analysis of the EV content was also conducted within the present study, and it allowed the identification of 541 proteins after matching them to the Saccharomyces Genome Database (SGD). Altogether, this study provides strong evidence that the EVs of the probiotic CNCM I-745 strain could be considered a drug delivery system. Full article

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30 pages, 6561 KiB

Open AccessArticle

Interplay between the RNA Chaperone Hfq, Small RNAs and Transcriptional Regulator OmpR Modulates Iron Homeostasis in the Enteropathogen Yersinia enterocolitica

by Karolina Jaworska, Julia Konarska, Patrycja Gomza, Paula Rożen, Marta Nieckarz, Agata Krawczyk-Balska, Katarzyna Brzostek and Adrianna Raczkowska

Int. J. Mol. Sci. 2023, 24(13), 11157; https://doi.org/10.3390/ijms241311157 - 6 Jul 2023

Cited by 1 | Viewed by 1772

Abstract

Iron is both essential for and potentially toxic to bacteria, so the precise maintenance of iron homeostasis is necessary for their survival. Our previous study indicated that in the human enteropathogen Yersinia enterocolitica, the regulator OmpR directly controls the transcription of the [...] Read more.

Iron is both essential for and potentially toxic to bacteria, so the precise maintenance of iron homeostasis is necessary for their survival. Our previous study indicated that in the human enteropathogen Yersinia enterocolitica, the regulator OmpR directly controls the transcription of the fur, fecA and fepA genes, encoding the ferric uptake repressor and two transporters of ferric siderophores, respectively. This study was undertaken to determine the significance of the RNA chaperone Hfq and the small RNAs OmrA and RyhB1 in the post-transcriptional control of the expression of these OmpR targets. We show that Hfq silences fur, fecA and fepA expression post-transcriptionally and negatively affects the production of FLAG-tagged Fur, FecA and FepA proteins. In addition, we found that the fur gene is under the negative control of the sRNA RyhB1, while fecA and fepA are negatively regulated by the sRNA OmrA. Finally, our data revealed that the role of OmrA results from a complex interplay of transcriptional and post-transcriptional effects in the feedback circuit between the regulator OmpR and the sRNA OmrA. Thus, the expression of fur, fecA and fepA is subject to complex transcriptional and post-transcriptional regulation in order to maintain iron homeostasis in Y. enterocolitica. Full article

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20 pages, 21404 KiB

Open AccessArticle

Biofilm Formation and Genetic Diversity of Microbial Communities in Anaerobic Batch Reactor with Polylactide (PLA) Addition

by Agnieszka A. Pilarska, Anna Marzec-Grządziel, Emil Paluch, Krzysztof Pilarski, Agnieszka Wolna-Maruwka, Adrianna Kubiak, Tomasz Kałuża and Tomasz Kulupa

Int. J. Mol. Sci. 2023, 24(12), 10042; https://doi.org/10.3390/ijms241210042 - 12 Jun 2023

Cited by 4 | Viewed by 1937

Abstract

In this paper, an anaerobic digestion (AD) study was conducted on confectionery waste with granular polylactide (PLA) as a cell carrier. Digested sewage sludge (SS) served as the inoculum and buffering agent of systems. This article shows the results of the analyses of [...] Read more.

In this paper, an anaerobic digestion (AD) study was conducted on confectionery waste with granular polylactide (PLA) as a cell carrier. Digested sewage sludge (SS) served as the inoculum and buffering agent of systems. This article shows the results of the analyses of the key experimental properties of PLA, i.e., morphological characteristics of the microstructure, chemical composition and thermal stability of the biopolymer. The evaluation of quantitative and qualitative changes in the genetic diversity of bacterial communities, performed using the state-of-the-art next generation sequencing (NGS) technique, revealed that the material significantly enhanced bacterial proliferation; however, it does not change microbiome biodiversity, as also confirmed via statistical analysis. More intense microbial proliferation (compared to the control sample, without PLA and not digested, CW–control, CW–confectionery waste) may be indicative of the dual role of the biopolymer—support and medium. Actinobacteria (34.87%) were the most abundant cluster in the CW–control, while the most dominant cluster in digested samples was firmicutes: in the sample without the addition of the carrier (CW–dig.) it was 68.27%, and in the sample with the addition of the carrier (CW + PLA) it was only 26.45%, comparable to the control sample (CW–control)—19.45%. Interestingly, the number of proteobacteria decreased in the CW–dig. sample (17.47%), but increased in the CW + PLA sample (39.82%) compared to the CW–control sample (32.70%). The analysis of biofilm formation dynamics using the BioFlux microfluidic system shows a significantly faster growth of the biofilm surface area for the CW + PLA sample. This information was complemented by observations of the morphological characteristics of the microorganisms using fluorescence microscopy. The images of the CW + PLA sample showed carrier sections covered with microbial consortia. Full article

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12 pages, 828 KiB

Open AccessArticle

Genomic Analysis of Corynebacterium diphtheriae Strains Isolated in the Years 2007–2022 with a Report on the Identification of the First Non-Toxigenic tox Gene-Bearing Strain in Poland

by Tomasz Wołkowicz, Katarzyna Zacharczuk and Aleksandra Anna Zasada

Int. J. Mol. Sci. 2023, 24(5), 4612; https://doi.org/10.3390/ijms24054612 - 27 Feb 2023

Cited by 1 | Viewed by 2310

Abstract

Infections caused by non-toxigenic Corynebacterium diphtheriae have been reported every year in Poland since 2004, with the ST8 biovar gravis strains being most commonly isolated. This study analyzed thirty strains isolated between 2017 and 2022 and six previously isolated strains. All the strains [...] Read more.

Infections caused by non-toxigenic Corynebacterium diphtheriae have been reported every year in Poland since 2004, with the ST8 biovar gravis strains being most commonly isolated. This study analyzed thirty strains isolated between 2017 and 2022 and six previously isolated strains. All the strains were characterized using classic methods in terms of species, biovar level, and diphtheria toxin production, as well as by means of whole genome sequencing. The phylogenetic relationship based on SNP analysis was determined. The number of C. diphtheriae infections has been rising in Poland every year with a maximum of 22 cases in the year 2019. Since 2022, only the non-toxigenic gravis ST8 (most common) and mitis ST439 (less common) strains have been isolated. An analysis of the genomes of the ST8 strains showed that they had many potential virulence factors, such as adhesins and iron-uptake systems. The situation rapidly changed in 2022 and strains from different STs were isolated (ST32, 40, and 819). The ST40 biovar mitis strain was found to be non-toxigenic tox gene-bearing (NTTB), with the tox gene inactivated due to a single nucleotide deletion. Such strains were previously isolated in Belarus. The sudden appearance of new C. diphtheriae strains with different STs and the isolation of the first NTTB strain in Poland indicate that C. diphtheriae should be classified as a pathogen of special public health concern. Full article

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25 pages, 6748 KiB

Open AccessArticle

Exopolysaccharide Biosynthesis in Rhizobium leguminosarum bv. trifolii Requires a Complementary Function of Two Homologous Glycosyltransferases PssG and PssI

by Kamil Żebracki, Aleksandra Horbowicz, Małgorzata Marczak, Anna Turska-Szewczuk, Piotr Koper, Klaudia Wójcik, Marceli Romańczuk, Magdalena Wójcik and Andrzej Mazur

Int. J. Mol. Sci. 2023, 24(4), 4248; https://doi.org/10.3390/ijms24044248 - 20 Feb 2023

Cited by 2 | Viewed by 2233

Abstract

The Pss-I region of Rhizobium leguminosarum bv. trifolii TA1 comprises more than 20 genes coding for glycosyltransferases, modifying enzymes, and polymerization/export proteins, altogether determining the biosynthesis of symbiotically relevant exopolysaccharides. In this study, the role of homologous PssG and PssI glycosyltransferases in exopolysaccharide [...] Read more.

The Pss-I region of Rhizobium leguminosarum bv. trifolii TA1 comprises more than 20 genes coding for glycosyltransferases, modifying enzymes, and polymerization/export proteins, altogether determining the biosynthesis of symbiotically relevant exopolysaccharides. In this study, the role of homologous PssG and PssI glycosyltransferases in exopolysaccharide subunit synthesis were analyzed. It was shown that the glycosyltransferase-encoding genes of the Pss-I region were part of a single large transcriptional unit with potential downstream promoters activated in specific conditions. The ΔpssG and ΔpssI mutants produced significantly lower amounts of the exopolysaccharide, while the double deletion mutant ΔpssIΔpssG produced no exopolysaccharide. Complementation of double mutation with individual genes restored exopolysaccharide synthesis, but only to the level similar to that observed for the single ΔpssI or ΔpssG mutants, indicating that PssG and PssI serve complementary functions in the process. PssG and PssI interacted with each other in vivo and in vitro. Moreover, PssI displayed an expanded in vivo interaction network comprising other GTs involved in subunit assembly and polymerization/export proteins. PssG and PssI proteins were shown to interact with the inner membrane through amphipathic helices at their C-termini, and PssG also required other proteins involved in exopolysaccharide synthesis to localize in the membrane protein fraction. Full article

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28 pages, 4383 KiB

Open AccessArticle

The Prolonged Treatment of Salmonella enterica Strains with Human Serum Effects in Phenotype Related to Virulence

by Bożena Futoma-Kołoch, Michał Małaszczuk, Kamila Korzekwa, Małgorzata Steczkiewicz, Andrzej Gamian and Gabriela Bugla-Płoskońska

Int. J. Mol. Sci. 2023, 24(1), 883; https://doi.org/10.3390/ijms24010883 - 3 Jan 2023

Viewed by 5668

Abstract

Salmonella enterica as common pathogens of humans and animals are good model organisms to conduct research on bacterial biology. Because these bacteria can multiply in both the external environments and in the living hosts, they prove their wide adaptability. It has been previously [...] Read more.

Salmonella enterica as common pathogens of humans and animals are good model organisms to conduct research on bacterial biology. Because these bacteria can multiply in both the external environments and in the living hosts, they prove their wide adaptability. It has been previously demonstrated that prolonged exposition of Salmonella serotype O48 cells to normal human serum led to an increase in resistance to sera in connection with the synthesis of very long O-antigen. In this work, we have studied the phenotype connected to virulence of Salmonella enterica strains that were subjected to consecutive passages in 50% human serum from platelet-poor plasma (SPPP). We found that eight passages in SPPP may not be enough for the bacteria to become serum-resistant (S. Typhimurium ATCC 14028, S. Senftenberg). Moreover, C1q and C3c complement components bound to Salmonellae (S. Typhimurium ATCC 14028, S. Hammonia) membrane proteins, which composition has been changed after passaging in sera. Interestingly, passages in SPPP generated genetic changes within gene fljB, which translated to cells’ motility (S. Typhimurium ATCC 14028, S. Erlangen). One strain, S. Hammonia exposed to a serum developed a multi-drug resistance (MDR) phenotype and two S. Isaszeg and S. Erlangen tolerance to disinfectants containing quaternary ammonium salts (QAS). Furthermore, colonial morphotypes of the serum adaptants were similar to those produced by starter cultures. These observations suggest that overcoming stressful conditions is manifested on many levels. Despite great phenotypic diversity occurring after prolonged exposition to SPPP, morphotypes of colonies remained unchanged in basic media. This work is an example in which stable morphotypes distinguished by altered virulence can be confusing during laboratory work with life-threatening strains. Full article

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2022

Jump to: 2023, 2021

13 pages, 293 KiB

Open AccessCommunication

Usefulness of Molecular Methods for Helicobacter pylori Detection in Pediatric Patients and Their Correlation with Histopathological Sydney Classification

by Tomasz Bogiel, Agnieszka Mikucka, Anna Szaflarska-Popławska and Dariusz Grzanka

Int. J. Mol. Sci. 2023, 24(1), 179; https://doi.org/10.3390/ijms24010179 - 22 Dec 2022

Cited by 5 | Viewed by 2518

Abstract

Helicobacter pylori infections, as one of the most prevalent among humans, are generally acquired during childhood, and are one of the main causes of chronic gastritis and peptic ulcer disease. A bacterial culture from a gastric biopsy is the gold standard and is [...] Read more.

Helicobacter pylori infections, as one of the most prevalent among humans, are generally acquired during childhood, and are one of the main causes of chronic gastritis and peptic ulcer disease. A bacterial culture from a gastric biopsy is the gold standard and is the only method that has 100% specificity. However, its sensitivity varies, depending on experience of the laboratory staff, applied culture media, specimen transport conditions, biopsy site, and quality of the sample. The same factors compromise all invasive methods and a culture-based H. pylori infection diagnostic, as well as a recent intake of antibiotics, bismuth-containing compounds, and proton pump inhibitors. Molecular methods have been used for clinical microbiology investigation since the beginning of the 21st century. However, their usefulness for H. pylori infections diagnosis remains unclear, especially in pediatric patients. The aim of the study was to assess the incidence of H. pylori infections in a group of 104 pediatric patients and to compare the results of the PCR test with the corresponding histopathological investigation effects. Among the biopsy samples collected from 104 children, 44 (42.3%) were positive in PCR, while 43 (41.3%) and 39 (37.5%) presented histologically-confirmed signs of inflammation and H. pylori colonization, respectively. Moreover, the mean grades of the parameters of the histopathological examination were higher in the group of PCR-positive samples. The compatibility of both research methods was confirmed, emphasizing the usefulness of molecular methods for detecting H. pylori infections in pediatric patients. Considering that the PCR-based method gives reliable results and is less time-consuming and costly, it is worth discussing this method as a new standard in the diagnosis of H. pylori infections, at least among pediatric patients, for which culture-based diagnostics is not sufficient or histopathological examination is negative, while inflammation signs are observed macroscopically. Full article

25 pages, 2652 KiB

Open AccessArticle

Functional Characterization of TetR-like Transcriptional Regulator PA3973 from Pseudomonas aeruginosa

by Karolina Kotecka, Adam Kawalek, Magdalena Modrzejewska-Balcerek, Jan Gawor, Karolina Zuchniewicz, Robert Gromadka and Aneta Agnieszka Bartosik

Int. J. Mol. Sci. 2022, 23(23), 14584; https://doi.org/10.3390/ijms232314584 - 23 Nov 2022

Cited by 3 | Viewed by 3163

Abstract

Pseudomonas aeruginosa, a human opportunistic pathogen, is a common cause of nosocomial infections. Its ability to survive under different conditions relies on a complex regulatory network engaging transcriptional regulators controlling metabolic pathways and capabilities to efficiently use the available resources. P. aeruginosa PA3973 [...] Read more.

Pseudomonas aeruginosa, a human opportunistic pathogen, is a common cause of nosocomial infections. Its ability to survive under different conditions relies on a complex regulatory network engaging transcriptional regulators controlling metabolic pathways and capabilities to efficiently use the available resources. P. aeruginosa PA3973 encodes an uncharacterized TetR family transcriptional regulator. In this study, we applied a transcriptome profiling (RNA-seq), genome-wide identification of binding sites using ChIP-seq, as well as the phenotype analyses to unravel the biological role of PA3973. Transcriptional profiling of P. aeruginosa PAO1161 overexpressing PA3973 showed changes in the mRNA level of 648 genes. Concomitantly, ChIP-seq analysis identified more than 300 PA3973 binding sites in the P. aeruginosa genome. A 13 bp sequence motif was indicated as the binding site of PA3973. The PA3973 regulon encompasses the PA3972-PA3971 genes encoding a probable acyl-CoA dehydrogenase and a thioesterase. In vitro analysis showed PA3973 binding to PA3973p. Accordingly, the lack of PA3973 triggered increased expression of PA3972 and PA3971. The ∆PA3972-71 PAO1161 strain demonstrated impaired growth in the presence of stress-inducing agents hydroxylamine or hydroxyurea, thus suggesting the role of PA3972-71 in pathogen survival upon stress. Overall our results showed that TetR-type transcriptional regulator PA3973 has multiple binding sites in the P. aeruginosa genome and influences the expression of diverse genes, including PA3972-PA3971, encoding proteins with a proposed role in stress response. Full article

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26 pages, 2168 KiB

Open AccessArticle

A New Expression System Based on Psychrotolerant Debaryomyces macquariensis Yeast and Its Application to the Production of Cold-Active β-d-Galactosidase from Paracoccus sp. 32d

by Marta Wanarska, Ewelina Krajewska-Przybyszewska, Monika Wicka-Grochocka, Hubert Cieśliński, Anna Pawlak-Szukalska, Aneta M. Białkowska, Marianna Turkiewicz, Tomasz Florczak, Ewa Gromek, Joanna Krysiak and Natalia Filipowicz

Int. J. Mol. Sci. 2022, 23(19), 11691; https://doi.org/10.3390/ijms231911691 - 2 Oct 2022

Cited by 2 | Viewed by 2547

Abstract

Yeasts provide attractive host/vector systems for heterologous gene expression. The currently used yeast-based expression platforms include mesophilic and thermotolerant species. A eukaryotic expression system working at low temperatures could be particularly useful for the production of thermolabile proteins and proteins that tend to [...] Read more.

Yeasts provide attractive host/vector systems for heterologous gene expression. The currently used yeast-based expression platforms include mesophilic and thermotolerant species. A eukaryotic expression system working at low temperatures could be particularly useful for the production of thermolabile proteins and proteins that tend to form insoluble aggregates. For this purpose, an expression system based on an Antarctic psychrotolerant yeast Debaryomyces macquariensis strain D50 that is capable of growing at temperatures ranging from 0 to 30 °C has been developed. The optimal physical culture conditions for D. macquariensis D50 in a fermenter are as follows: temperature 20 °C, pH 5.5, aeration rate of 1.5 vvm, and a stirring speed of 300 rpm. Four integrative plasmid vectors equipped with an expression cassette containing the constitutive GAP promoter and CYC1 transcriptional terminator from D. macquariensis D50 were constructed and used to clone and express a gene-encoding cold-active β-d-galactosidase of Paracoccus sp. 32d. The yield was 1150 U/L of recombinant yeast culture. Recombinant D. macquariensis D50 strains were mitotically stable under both selective and non-selective conditions. The D. macquariensis D50 host/vector system has been successfully utilized for the synthesis of heterologous thermolabile protein, and it can be an alternative to other microbial expression systems. Full article

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25 pages, 1156 KiB

Open AccessReview

Molecular Mechanisms of Drug Resistance and Epidemiology of Multidrug-Resistant Variants of Neisseria gonorrhoeae

by Beata Mlynarczyk-Bonikowska, Cezary Kowalewski, Aneta Krolak-Ulinska and Wojciech Marusza

Int. J. Mol. Sci. 2022, 23(18), 10499; https://doi.org/10.3390/ijms231810499 - 10 Sep 2022

Cited by 27 | Viewed by 7927

Abstract

The paper presents various issues related to the increasing drug resistance of Neisseria gonorrhoeae and the occurrence and spread of multidrug-resistant clones. One of the most important is the incidence and evolution of resistance mechanisms of N. gonorrhoeae to beta-lactam antibiotics. Chromosomal resistance [...] Read more.

The paper presents various issues related to the increasing drug resistance of Neisseria gonorrhoeae and the occurrence and spread of multidrug-resistant clones. One of the most important is the incidence and evolution of resistance mechanisms of N. gonorrhoeae to beta-lactam antibiotics. Chromosomal resistance to penicillins and oxyimino-cephalosporins and plasmid resistance to penicillins are discussed. Chromosomal resistance is associated with the presence of mutations in the PBP2 protein, containing mosaic variants and nonmosaic amino acid substitutions in the transpeptidase domain, and their correlation with mutations in the mtrR gene and its promoter regions (the MtrCDE membrane pump repressor) and in several other genes, which together determine reduced sensitivity or resistance to ceftriaxone and cefixime. Plasmid resistance to penicillins results from the production of beta-lactamases. There are different types of beta-lactamases as well as penicillinase plasmids. In addition to resistance to beta-lactam antibiotics, the paper covers the mechanisms and occurrence of resistance to macrolides (azithromycin), fluoroquinolones and some other antibiotics. Moreover, the most important epidemiological types of multidrug-resistant N. gonorrhoeae, prevalent in specific years and regions, are discussed. Epidemiological types are defined as sequence types, clonal complexes and genogroups obtained by various typing systems such as NG-STAR, NG-MAST and MLST. New perspectives on the treatment of N. gonorrhoeae infections are also presented, including new drugs active against multidrug-resistant strains. Full article

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15 pages, 2529 KiB

Open AccessArticle

Genetic Diversity of Tomato Black Ring Virus Satellite RNAs and Their Impact on Virus Replication

by Julia Minicka, Agnieszka Taberska, Aleksandra Zarzyńska-Nowak, Katarzyna Kubska, Daria Budzyńska, Santiago F. Elena and Beata Hasiów-Jaroszewska

Int. J. Mol. Sci. 2022, 23(16), 9393; https://doi.org/10.3390/ijms23169393 - 20 Aug 2022

Cited by 5 | Viewed by 2049

Abstract

Viral satellite RNAs (satRNAs) are small subviral particles that are associated with the genomic RNA of a helper virus (HV). Their replication, encapsidation, and movement depend on the HV. In this paper, we performed a global analysis of the satRNAs associated with different [...] Read more.

Viral satellite RNAs (satRNAs) are small subviral particles that are associated with the genomic RNA of a helper virus (HV). Their replication, encapsidation, and movement depend on the HV. In this paper, we performed a global analysis of the satRNAs associated with different isolates of tomato black ring virus (TBRV). We checked the presence of satRNAs in 42 samples infected with TBRV, performed recombination and genetic diversity analyses, and examined the selective pressure affecting the satRNAs population. We identified 18 satRNAs in total that differed in length and the presence of point mutations. Moreover, we observed a strong effect of selection operating upon the satRNA population. We also constructed infectious cDNA clones of satRNA and examined the viral load of different TBRV isolates in the presence and absence of satRNAs, as well as the accumulation of satRNA molecules on infected plants. Our data provide evidence that the presence of satRNAs significantly affects viral load; however, the magnitude of this effect differs among viral isolates and plant hosts. We also showed a positive correlation between the number of viral genomic RNAs (gRNAs) and satRNAs for two analysed TBRV isolates. Full article

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14 pages, 1244 KiB

Open AccessArticle

Genotypes and Variants of BKPyV in Organ Donors after Brain Death

by Jacek Furmaga, Marek Kowalczyk, Olga Furmaga-Rokou, Christos A. Rokos, Tomasz Zapolski, Leszek Krakowski, Andrzej Jakubczak and Sławomir Rudzki

Int. J. Mol. Sci. 2022, 23(16), 9173; https://doi.org/10.3390/ijms23169173 - 15 Aug 2022

Cited by 1 | Viewed by 2342

Abstract

Kidney transplantation from a donor with latent BKPyV might be the cause of serious complications, such as BK virus-associated nephropathy. The aim of the study was to determine the prevalence of BKPyV infection in donors after brain death (DBDs), to analyse the molecular [...] Read more.

Kidney transplantation from a donor with latent BKPyV might be the cause of serious complications, such as BK virus-associated nephropathy. The aim of the study was to determine the prevalence of BKPyV infection in donors after brain death (DBDs), to analyse the molecular variation of BKPyV and to compare clinical and inflammation parameters of DBDs infected with various genotypes of BKPyV. BKPyV was investigated in blood and urine samples of 103 DBDs using PCR followed by sequencing and bioinformatic analysis, and the viral load was assessed by qPCR. Clinical parameters, including cellular markers of inflammation were assessed. The results confirm high prevalence of BKPyV (48%),and genotype IV (49%) over genotype I (43%) and the co-infection with genotypes I and IV in 8.2%. Viral load ranged from 10² to 10⁷ copies/mL, with an average of 1.92 × 10⁶ copies/mL. No specific markers for BKPyV infection were detected among the parameters tested. Infection with genotype I may be associated with the adverse impact on thekidney function, while infection with genotype IV was associated with the anemia Not only the viral load but also the genotype of BKPyV may have an impact on the course of infection. Full article

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18 pages, 298 KiB

Open AccessArticle

Prevalence of Plasmid-Mediated Quinolone Resistance (PMQRs) Determinants and Whole Genome Sequence Screening of PMQR-Producing E. coli Isolated from Men Undergoing a Transrectal Prostate Biopsy

by Katarzyna Piekarska, Katarzyna Zacharczuk, Tomasz Wołkowicz and Rafał Gierczyński

Int. J. Mol. Sci. 2022, 23(16), 8907; https://doi.org/10.3390/ijms23168907 - 10 Aug 2022

Cited by 2 | Viewed by 2101

Abstract

Fluoroquinolones (FQs) are recommended as prophylaxis for men undergoing transrectal prostate biopsy (TRUS-Bx). Recent studies suggest a significant share of FQ-resistant rectal flora in post-TRUST-Bx infections. Methods: 435 Enterobacterales isolates from 621 patients attending 12 urological departments in Poland were screened by PCR [...] Read more.

Fluoroquinolones (FQs) are recommended as prophylaxis for men undergoing transrectal prostate biopsy (TRUS-Bx). Recent studies suggest a significant share of FQ-resistant rectal flora in post-TRUST-Bx infections. Methods: 435 Enterobacterales isolates from 621 patients attending 12 urological departments in Poland were screened by PCR for PMQR genes. PMQR-positive isolates were tested for quinolone susceptibility and investigated by whole genome sequencing (WGS) methods. Results: In total, 32 (7.35%) E. coli strains with ciprofloxacin MIC in the range 0.125–32 mg/L harbored at least one PMQR gene. qnrS and qnrB were the most frequent genes detected in 16 and 12 isolates, respectively. WGS was performed for 28 of 32 PMQR-producing strains. A variety of serotypes and sequence types (STs) of E. coli was noticed. All strains carried at least one virulence gene. AMR genes that encoded resistance against different classes of antibiotics were identified. Additionally, five of 13 ciprofloxacin-susceptible E. coli had alterations in codon 83 of the GyrA subunits. Conclusion: This study provides information on the common presence of PMQRs among E. coli, which may explain the cause for development of post-TRUS-Bx infections. High numbers of virulence and antimicrobial resistance genes detected show a potential for analysed strains to develop infections. Full article

20 pages, 2230 KiB

Open AccessArticle

Genomic Insights of First ermB-Positive ST338-SCCmecV_T/CC59 Taiwan Clone of Community-Associated Methicillin-Resistant Staphylococcus aureus in Poland

by Ksenia Szymanek-Majchrzak and Grażyna Młynarczyk

Int. J. Mol. Sci. 2022, 23(15), 8755; https://doi.org/10.3390/ijms23158755 - 6 Aug 2022

Cited by 1 | Viewed by 2934

Abstract

We report the first Polish representative of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA), lukS/F-PV-positive, encoding the ermB gene, as a genetic determinant of constitutive resistance to macrolides, lincosamides, and streptogramin B antibiotics, cMLS-B. This is the first detection of the CA-MRSA strain [...] Read more.

We report the first Polish representative of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA), lukS/F-PV-positive, encoding the ermB gene, as a genetic determinant of constitutive resistance to macrolides, lincosamides, and streptogramin B antibiotics, cMLS-B. This is the first detection of the CA-MRSA strain responsible for nosocomial infection in the Warsaw Clinical Hospital. Resistance to β-lactams associates with a composite genetic element, SCCmec cassette type V_T (5C2&5). We assigned the strain to sequence type ST338 (single-locus variant of ST59), clonal complex CC59, spa-type t437, and agr-type I. Genomic-based comparison was designated SO574/12 as an international Taiwan clone, which has been so far described mainly in the Asia-Pacific region. The ermB gene locates on the chromosome within the 14,690 bp mobile element structure, i.e., the MES_PM1-like structure, which also encodes aminoglycoside- and streptothricin-resistance genes. The MES_PM1-like structure is a composite transposon containing Tn551, flanked by direct repeats of IS1216V insertion sequences, which probably originates from Enterococcus. The ermB is preceded by the 273 bp regulatory region that contains the regulatory 84 bp ermBL ORF, encoding the 27 amino acid leader peptides. The latest research suggests that a new leader peptide, ermBL2, also exists in the ermB regulatory region. Therefore, the detailed function of ermBL2 requires further investigations. Full article

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33 pages, 2584 KiB

Open AccessReview

Molecular Mechanisms of Drug Resistance in Staphylococcus aureus

by Beata Mlynarczyk-Bonikowska, Cezary Kowalewski, Aneta Krolak-Ulinska and Wojciech Marusza

Int. J. Mol. Sci. 2022, 23(15), 8088; https://doi.org/10.3390/ijms23158088 - 22 Jul 2022

Cited by 121 | Viewed by 20540

Abstract

This paper discusses the mechanisms of S. aureus drug resistance including: (1) introduction. (2) resistance to beta-lactam antibiotics, with particular emphasis on the mec genes found in the Staphylococcaceae family, the structure and occurrence of SCCmec cassettes, as well as differences in [...] Read more.

This paper discusses the mechanisms of S. aureus drug resistance including: (1) introduction. (2) resistance to beta-lactam antibiotics, with particular emphasis on the mec genes found in the Staphylococcaceae family, the structure and occurrence of SCCmec cassettes, as well as differences in the presence of some virulence genes and its expression in major epidemiological types and clones of HA-MRSA, CA-MRSA, and LA-MRSA strains. Other mechanisms of resistance to beta-lactam antibiotics will also be discussed, such as mutations in the gdpP gene, BORSA or MODSA phenotypes, as well as resistance to ceftobiprole and ceftaroline. (3) Resistance to glycopeptides (VRSA, VISA, hVISA strains, vancomycin tolerance). (4) Resistance to oxazolidinones (mutational and enzymatic resistance to linezolid). (5) Resistance to MLS-B (macrolides, lincosamides, ketolides, and streptogramin B). (6) Aminoglycosides and spectinomicin, including resistance genes, their regulation and localization (plasmids, transposons, class I integrons, SCCmec), and types and spectrum of enzymes that inactivate aminoglycosides. (7). Fluoroquinolones (8) Tetracyclines, including the mechanisms of active protection of the drug target site and active efflux of the drug from the bacterial cell. (9) Mupirocin. (10) Fusidic acid. (11) Daptomycin. (12) Resistance to other antibiotics and chemioterapeutics (e.g., streptogramins A, quinupristin/dalfopristin, chloramphenicol, rifampicin, fosfomycin, trimethoprim) (13) Molecular epidemiology of MRSA. Full article

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15 pages, 3339 KiB

Open AccessArticle

Characterization of the Putative Acylated Cellulose Synthase Operon in Komagataeibacter xylinus E25

by Izabela Szymczak, Agnieszka J. Pietrzyk-Brzezińska, Kajetan Duszyński and Małgorzata Ryngajłło

Int. J. Mol. Sci. 2022, 23(14), 7851; https://doi.org/10.3390/ijms23147851 - 16 Jul 2022

Cited by 6 | Viewed by 2757

Abstract

Bacterial cellulose is a natural polymer with an expanding array of applications. Because of this, the main cellulose producers of the Komagataeibacter genus have been extensively studied with the aim to increase its synthesis or to customize its physicochemical features. Up to now, [...] Read more.

Bacterial cellulose is a natural polymer with an expanding array of applications. Because of this, the main cellulose producers of the Komagataeibacter genus have been extensively studied with the aim to increase its synthesis or to customize its physicochemical features. Up to now, the genetic studies in Komagataeibacter have focused on the first cellulose synthase operon (bcsI) encoding the main enzyme complex. However, the role of other accessory cellulose operons has been understudied. Here we aimed to fill this gap by performing a detailed analysis of the second cellulose synthase operon (bcsII), which is putatively linked with cellulose acylation. In this study we harnessed the genome sequence, gene expression and protein structure information of K. xylinus E25 and other Komagataeibacter species to discuss the probable features of bcsII and the biochemical function of its main protein products. The results of our study support the previous hypothesis that bcsII is involved in the synthesis of the acylated polymer and expand it by presenting the evidence that it may also function in the regulation of its attachment to the cell surface and to the crystalline cellulose fibers. Full article

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19 pages, 4335 KiB

Open AccessArticle

Antifungal Effect of Penicillamine Due to the Selective Targeting of L-Homoserine O-Acetyltransferase

by Aleksandra Kuplińska, Kamila Rząd, Marek Wojciechowski, Sławomir Milewski and Iwona Gabriel

Int. J. Mol. Sci. 2022, 23(14), 7763; https://doi.org/10.3390/ijms23147763 - 14 Jul 2022

Cited by 2 | Viewed by 2595

Abstract

Due to the apparent similarity of fungal and mammalian metabolic pathways, the number of established antifungal targets is low, and the identification of novel ones is highly desirable. The results of our studies, presented in this work, indicate that the fungal biosynthetic pathway [...] Read more.

Due to the apparent similarity of fungal and mammalian metabolic pathways, the number of established antifungal targets is low, and the identification of novel ones is highly desirable. The results of our studies, presented in this work, indicate that the fungal biosynthetic pathway of L-methionine, an amino acid essential for humans, seems to be an attractive perspective. The MET2 gene from Candida albicans encoding L-homoserine O-acetyltransferase (CaMet2p), an enzyme catalyzing the first step in that pathway, was cloned and expressed as the native or the oligo-His-tagged fusion protein in Escherichia coli. The recombinant enzymes were purified and characterized for their basic molecular properties and substrate specificities. The purified MET2 gene product revealed the appropriate activity, catalyzed the conversion of L-homoserine (L-Hom) to O-acetyl-L-homoserine (OALH), and exhibited differential sensitivity to several L-Hom or OALH analogues, including penicillamine. Surprisingly, both penicillamine enantiomers (L- and D-Pen) displayed comparable inhibitory effects. The results of the docking of L- and D-Pen to the model of CaMet2p confirmed that both enantiomeric forms of the inhibitor are able to bind to the catalytic site of the enzyme with similar affinities and a similar binding mode. The sensitivity of some fungal cells to L-Pen, depending on the presence or absence of L-Met in the medium, clearly indicate Met2p targeting. Moreover, C. glabrata clinical strains that are resistant to fluconazole displayed a similar susceptibility to L-Pen as the wild-type strains. Our results prove the potential usefulness of Met2p as a molecular target for antifungal chemotherapy. Full article

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30 pages, 1820 KiB

Open AccessReview

Obesity and the Brain

by Jacek Karczewski, Aleksandra Zielińska, Rafał Staszewski, Piotr Eder, Agnieszka Dobrowolska and Eliana B. Souto

Int. J. Mol. Sci. 2022, 23(11), 6145; https://doi.org/10.3390/ijms23116145 - 30 May 2022

Cited by 9 | Viewed by 5719

Abstract

Innate and adaptive immunity are essential for neurodevelopment and central nervous system (CNS) homeostasis; however, the fragile equilibrium between immune and brain cells can be disturbed by any immune dysregulation and cause detrimental effects. Accumulating evidence indicates that, despite the blood–brain barrier (BBB), [...] Read more.

Innate and adaptive immunity are essential for neurodevelopment and central nervous system (CNS) homeostasis; however, the fragile equilibrium between immune and brain cells can be disturbed by any immune dysregulation and cause detrimental effects. Accumulating evidence indicates that, despite the blood–brain barrier (BBB), overactivation of the immune system leads to brain vulnerability that increases the risk of neuropsychiatric disorders, particularly upon subsequent exposure later in life. Disruption of microglial function in later life can be triggered by various environmental and psychological factors, including obesity-driven chronic low-grade inflammation and gut dysbiosis. Increased visceral adiposity has been recognized as an important risk factor for multiple neuropsychiatric conditions. The review aims to present our current understanding of the topic. Full article

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23 pages, 6357 KiB

Open AccessArticle

The Regulatory Circuit Underlying Downregulation of a Type III Secretion System in Yersinia enterocolitica by Transcription Factor OmpR

by Marta Nieckarz, Karolina Jaworska, Adrianna Raczkowska and Katarzyna Brzostek

Int. J. Mol. Sci. 2022, 23(9), 4758; https://doi.org/10.3390/ijms23094758 - 26 Apr 2022

Cited by 2 | Viewed by 3080

Abstract

In a previous study, differential proteomic analysis was used to identify membrane proteins of the human enteropathogen Yersinia enterocolitica, whose levels are influenced by OmpR, the transcriptional regulator in the two-component EnvZ/OmpR system. Interestingly, this analysis demonstrated that at 37 °C, OmpR [...] Read more.

In a previous study, differential proteomic analysis was used to identify membrane proteins of the human enteropathogen Yersinia enterocolitica, whose levels are influenced by OmpR, the transcriptional regulator in the two-component EnvZ/OmpR system. Interestingly, this analysis demonstrated that at 37 °C, OmpR negatively affects the level of over a dozen Ysc-Yop proteins, which constitute a type III secretion system (T3SS) that is essential for the pathogenicity of Y. enterocolitica. Here, we focused our analysis on the role of OmpR in the expression and secretion of Yops (translocators and effectors). Western blotting with anti-Yops antiserum and specific anti-YopD, -YopE and -YopH antibodies, confirmed that the production of Yops is down-regulated by OmpR with the greatest negative effect on YopD. The RT-qPCR analysis demonstrated that, while OmpR had a negligible effect on the activity of regulatory genes virF and yscM1, it highly repressed the expression of yopD. OmpR was found to bind to the promoter of the lcrGVsycD-yopBD operon, suggesting a direct regulatory effect. In addition, we demonstrated that the negative regulatory influence of OmpR on the Ysc-Yop T3SS correlated with its positive role in the expression of flhDC, the master regulator of the flagellar-associated T3SS. Full article

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15 pages, 5656 KiB

Open AccessArticle

Lanthanide-Dependent Methanol Metabolism of a Proteobacteria-Dominated Community in a Light Lanthanide-Rich Deep Environment

by Agnieszka Daszczyńska, Tomasz Krucoń, Robert Stasiuk, Marta Koblowska and Renata Matlakowska

Int. J. Mol. Sci. 2022, 23(7), 3947; https://doi.org/10.3390/ijms23073947 - 1 Apr 2022

Cited by 1 | Viewed by 2873

Abstract

This study investigated the occurrence and diversity of proteobacterial XoxF-type methanol dehydrogenases (MDHs) in the microbial community that inhabits a fossil organic matter- and sedimentary lanthanide (Ln³⁺)-rich underground mine environment using a metagenomic and metaproteomic approach. A total of 8 XoxF-encoding [...] Read more.

This study investigated the occurrence and diversity of proteobacterial XoxF-type methanol dehydrogenases (MDHs) in the microbial community that inhabits a fossil organic matter- and sedimentary lanthanide (Ln³⁺)-rich underground mine environment using a metagenomic and metaproteomic approach. A total of 8 XoxF-encoding genes (XoxF-EGs) and 14 protein sequences matching XoxF were identified. XoxF-type MDHs were produced by Alpha-, Beta-, and Gammaproteobacteria represented by the four orders Methylococcales, Nitrosomonadales, Rhizobiales, and Xanthomonadales. The highest number of XoxF-EG- and XoxF-matching protein sequences were affiliated with Nitrosomonadales and Rhizobiales, respectively. Among the identified XoxF-EGs, two belonged to the XoxF1 clade, five to the XoxF4 clade, and one to the XoxF5 clade, while seven of the identified XoxF proteins belonged to the XoxF1 clade, four to the XoxF4 clade, and three to the XoxF5 clade. Moreover, the accumulation of light lanthanides and the presence of methanol in the microbial mat were confirmed. This study is the first to show the occurrence of XoxF in the metagenome and metaproteome of a deep microbial community colonizing a fossil organic matter- and light lanthanide-rich sedimentary environment. The presented results broaden our knowledge of the ecology of XoxF-producing bacteria as well as of the distribution and diversity of these enzymes in the natural environment. Full article

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2021

Jump to: 2023, 2022

16 pages, 3025 KiB

Open AccessArticle

Minigene as a Novel Regulatory Element in Toxin-Antitoxin Systems

by Barbara Kędzierska and Katarzyna Potrykus

Int. J. Mol. Sci. 2021, 22(24), 13389; https://doi.org/10.3390/ijms222413389 - 13 Dec 2021

Cited by 1 | Viewed by 2829

Abstract

The axe-txe type II toxin-antitoxin (TA) system is characterized by a complex and multilayered mode of gene expression regulation. Precise and tight control of this process is crucial to keep the toxin in an appropriate balance with the cognate antitoxin until its activation [...] Read more.

The axe-txe type II toxin-antitoxin (TA) system is characterized by a complex and multilayered mode of gene expression regulation. Precise and tight control of this process is crucial to keep the toxin in an appropriate balance with the cognate antitoxin until its activation is needed for the cell. In this report, we provide evidence that a minigene encoded within the axe-txe operon influences translation of the Txe toxin. This is the first example to date of such a regulatory mechanism identified in the TA modules. Here, in a series of genetic studies, we employed translational reporter gene fusions to establish the molecular basis of this phenomenon. Our results show that translation of the two-codon mini-ORF displays an in cis mode of action, and positively affects the expression of txe, possibly by increasing its mRNA stability through protection from an endonuclease attack. Moreover, we established that the reading frame in which the two cistrons are encoded, as well as the distance between them, are critical parameters that affect the level of such regulation. In addition, by searching for two-codon ORFs we found sequences of several potential minigenes in the leader sequences of several other toxins belonging to the type II TA family. These findings suggest that this type of gene regulation may not only apply for the axe-txe cassette, but could be more widespread among other TA systems. Full article

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