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Adhesive-invasive E. coli has been suggested to be associated with the development of Crohn's disease (CD). It is assumed that they can provoke the onset of the inflammatory process as a result of the invasion of intestinal epithelial cells and then, due to survival inside macrophages and dendritic cells, stimulate chronic inflammation. In previous reports, we have shown that passage of the CD isolate ZvL2 on minimal medium M9 supplemented with sodium propionate (PA) as a carbon source stimulates and inhibits the adherent-invasive properties and the ability to survive in macrophages. This effect was reversible and not observed for the laboratory strain K12 MG1655. We were able to compare the isogenic strain AIEC in two phenotypes-virulent (ZvL2-PA) and non-virulent (ZvL2-GLU). Unlike ZvL2-GLU, ZvL2-PA activates the production of ROS and cytokines when interacting with neutrophils. The laboratory strain does not cause a similar effect. To activate neutrophils, bacterial opsonization is necessary. Differences in neutrophil NADH oxidase activation and ζ-potential for ZvL2-GLU and ZvL2-PA are associated with changes in membrane protein abundance, as demonstrated by differential 2D electrophoresis and LC-MS. The increase in ROS and cytokine production during the interaction of ZvL2-PA with neutrophils is associated with a rearrangement of the abundance of membrane proteins, which leads to the activation of Rcs and PhoP/Q signaling pathways and changes in the composition and/or modification of LPS. Certain isoforms of OmpA may play a role in the formation of the virulent phenotype of ZvL2-PA and participate in the activation of NADPH oxidase in neutrophils.

Interaction of microbiota with hybrid vaterite-pectin microparticles as an attractive multifunctional vehicle for mucosal delivery should not provoke inflammation. Our purpose was to study the reaction of bacteria E. coli strain Mg1655 and isolate SharL from a patient with Crohn disease on the cultivation with hybrid microparticles and vaterite, and the subsequent activation of neutrophils. Vaterite-pectin microparticles enhanced leakage of ATP from bacteria. For E. coli Mg1655, the concentration of DNA decreased, while intracellular ATP increased. For E. coli SharL, the intracellular ATP decreased with simultaneous growth of DNA. Bacteria and microparticles together did not enhance activation of neutrophils in comparison with the particles per se in the medium without serum and in comparison with bacteria in the medium supplemented with serum; microparticles did not reduce functional activity of neutrophils., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Protease S (PrtS) from Photorhabdus laumondii belongs to the group of protealysin-like proteases (PLPs), which are understudied factors thought to play a role in the interaction of bacteria with other organisms. Since P. laumondii is an insect pathogen and a nematode symbiont, the analysis of the biological functions of PLPs using the PrtS model provides novel data on diverse types of interactions between bacteria and hosts., Methods and Results: Recombinant PrtS was produced in Escherichia coli. Efficient inhibition of PrtS activity by photorin, a recently discovered emfourin-like protein inhibitor from P. laumondii, was demonstrated. The Galleria mellonella was utilized to examine the insect toxicity of PrtS and the impact of PrtS on hemolymph proteins in vitro. The insect toxicity of PrtS is reduced compared to protease homologues from non-pathogenic bacteria and is likely not essential for the infection process. However, using proteomic analysis, potential PrtS targets have been identified in the hemolymph., Conclusions: The spectrum of identified proteins indicates that the function of PrtS is to modulate the insect immune response. Further studies of PLPs' biological role in the PrtS and P. laumondii model must clarify the details of PrtS interaction with the insect immune system during bacterial infection., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

A new Mycoplasma hominis phenotype forming mini-colonies (MC) on agar and distinct from the phenotype forming typical colonies (TC) not only in size, but also in morphology, growth rate, and resistance to adverse factors, has been previously identified. In this study, the phenotype of colonies was determined and a comparative analysis of the amino acid sequence of the main variable antigen Vaa of the laboratory strain N-34 and seven clinical isolates of M. hominis was performed. It is demonstrated that the amino acid sequence of Vaa in clinical isolates forming TC (similar to the laboratory strain N-34) is entirely analogous to that of laboratory strain. Clinical isolates forming MC carry amino acid substitutions in the variable C-terminal region of Vaa, which can contribute to adhesion to eukaryotic cells and immune evasion. The connection between colony phenotype and amino acid sequence of Vaa is established., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

Mycoplasma hominis is an opportunistic human pathogen that causes acute and chronic infections of the urogenital tract. A new form of M. hominis colonies (microcolonies) was isolated, that differed from typical colonies by morphology, size, growth rate, and resistance to unfavorable factors, in particular, to antibiotics. The formation of microcolonies is associated with a switch in energy metabolism towards nucleoside utilization, which leads to a decrease in energy production and a transition to a persistor-like state. Typical and microcolony cultures of M. hominis H-34 were obtained and a comparative analysis of their adhesive-invasive potential, morphology, and size was carried out. It was shown that both typical and microcolonies can effectively attach and penetrate into HeLa cells. Unlike microcolonies, the morphology and size of cells in typical colonies change significantly after HeLa infection. This indicates functional changes in cells of typical colonies during infection., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Mycoplasma hominis is an opportunistic urogenital pathogen in vertebrates. It is a non-glycolytic species that produces energy via arginine degradation. Among genital mycoplasmas, M. hominis is the most commonly reported to play a role in systemic infections and can persist in the host for a long time. However, it is unclear how M. hominis proceeds under arginine limitation. The recent metabolic reconstruction of M. hominis has demonstrated its ability to catabolize deoxyribose phosphate to produce ATP. In this study, we cultivated M. hominis on two different energy sources (arginine and thymidine) and demonstrated the differences in growth rate, antibiotic sensitivity, and biofilm formation. Using label-free quantitative proteomics, we compared the proteome of M. hominis under these conditions. A total of 466 proteins were identified from M. hominis , representing approximately 85% of the predicted proteome, while the levels of 94 proteins changed significantly. As expected, we observed changes in the levels of metabolic enzymes. The energy source strongly affects the synthesis of enzymes related to RNA modifications and ribosome assembly. The translocation of lipoproteins and other membrane-associated proteins was also impaired. Our study, the first global characterization of the proteomic switching of M. hominis in arginine-deficiency media, illustrates energy source-dependent control of pathogenicity factors and can help to determine the mechanisms underlying the interaction between the growth rate and fitness of genome-reduced bacteria., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Evsyutina, Semashko, Galyamina, Kovalchuk, Ziganshin, Ladygina, Fisunov and Pobeguts.)

Introduction. Mycoplasma hominis is a bacterium belonging to the class Mollicutes . It causes acute and chronic infections of the urogenital tract. The main features of this bacterium are an absence of cell wall and a reduced genome size (517-622 protein-encoding genes). Previously, we have isolated morphologically unknown M. hominis colonies called micro-colonies (MCs) from the serum of patients with inflammatory urogenital tract infection. Hypothesis. MCs are functionally different from the typical colonies (TCs) in terms of metabolism and cell division. Aim. To determine the physiological differences between MCs and TCs of M. hominis and elucidate the pathways of formation and growth of MCs by a comparative proteomic analysis of these two morphological forms. Methodology. LC-MS proteomic analysis of TCs and MCs using an Ultimate 3000 RSLC nanoHPLC system connected to a QExactive Plus mass spectrometer. Results. The study of the proteomic profiles of M. hominis colonies allowed us to reconstruct their energy metabolism pathways. In addition to the already known pentose phosphate and arginine deamination pathways, M. hominis can utilise ribose phosphate and deoxyribose phosphate formed by nucleoside catabolism as energy sources. Comparative proteomic HPLC-MS analysis revealed that the proteomic profiles of TCs and MCs were different. We assume that MC cells preferably utilised deoxyribonucleosides, particularly thymidine, as an energy source rather than arginine or ribonucleosides. Utilisation of deoxyribonucleosides is less efficient as compared with that of ribonucleosides and arginine in terms of energy production. Thymidine phosphorylase DeoA is one of the key enzymes of deoxyribonucleosides utilisation. We obtained a DeoA overexpressing mutant that exhibited a phenotype similar to that of MCs, which confirmed our hypothesis. Conclusion. In addition to the two known pathways for energy production (arginine deamination and the pentose phosphate pathway) M. hominis can use deoxyribonucleosides and ribonucleosides. MC cells demonstrate a reorganisation of energy metabolism: unlike TC cells, they preferably utilise deoxyribonucleosides, particularly thymidine, as an energy source rather than arginine or ribonucleosides. Thus MC cells enter a state of energy starvation, which helps them to survive under stress, and in particular, to be resistant to antibiotics.

Mycoplasma gallisepticum belongs to the class Mollicutes and induces severe chronic respiratory disease in chickens. It lacks the cell wall and contains a very small genome and, accordingly, a reduced set of regulatory proteins. It is assumed that one of the regulatory mechanisms in mycoplasmas may be the dynamics of the spatial organization of the chromosome. M. gallisepticum has only two known nucleoid-associated (NAP) histone-like proteins (Hup_1 and Hup_2). To search for new potential NAP that may play a role in the infection process, we isolated nucleoid fractions from M. gallisepticum cells before and after infection of HD3 chicken erythroblast cell line and performed a comparative proteomic analysis of these fractions. We identified several potential NAP that included the components of the terminal organelle and adhesion, VlhA antigen, NADH oxidase, and PykF pyruvate kinase., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

The structure and dynamics of bacterial nucleoids play important roles in regulating gene expression. Bacteria of class Mollicutes and, in particular, mycoplasmas feature extremely reduced genomes. They lack multiple structural proteins of the nucleoid, as well as regulators of gene expression. We studied the organization of Mycoplasma gallisepticum nucleoids in the stationary and exponential growth phases at the structural and protein levels. The growth phase transition results in the structural reorganization of M. gallisepticum nucleoid. In particular, it undergoes condensation and changes in the protein content. The observed changes corroborate with the previously identified global rearrangement of the transcriptional landscape in this bacterium during the growth phase transition. In addition, we identified that the glycolytic enzyme enolase functions as a nucleoid structural protein in this bacterium. It is capable of non-specific DNA binding and can form fibril-like complexes with DNA., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Fisunov, Zubov, Pobeguts, Varizhuk, Galyamina, Evsyutina, Semashko, Manuvera, Kovalchuk, Ziganshin, Barinov, Klinov and Govorun.)

It is assumed that unknown mechanisms can be involved in adaptation Mycoplasma gallisepticum to unfavorable factors, one of these can be local rearrangements of the structure and spatial organization of the chromosome. To study these mechanisms, we obtained a culture of M. gallisepticum with synchronized division and isolated the nucleoid fraction from this culture by the method of mild cell lysis and centrifugation in a sucrose gradient. Liquid chromatography-mass spectrometry analysis of the proteome showed that in comparison with the cell lysate, the nucleoid fraction was enriched with DNA-binding proteins. This analysis will help to find new nucleoid-associated proteins and to study their dynamics, distribution, and their role during infection and under stress conditions., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Mycoplasma gallisepticum (M. gallisepticum) belongs to the class of Mollicutes. It causes chronic respiratory disease in avian species. It is characterized by lack of cell wall and reduced genome size. As a result of genome reduction, M. gallisepticum has a limited variety of DNA-binding proteins (DBP) and transcription factors. Consequently, the diversity of DNA-binding proteins and transcription factors (TF) in M. gallisepticum is limited in comparison with related bacteria such as Bacillus subtilis . Studies have shown, however, that mycoplasmas demonstrate a wide range of differential expression of genes in response to various stress factors, which promotes effective adaptation to unfavorable conditions. We assume that in the case of mycoplasmas, which are characterized by a combination of the reduction of known gene expression regulation systems and a high adaptive potential, the coordination of gene expression can be provided due to local changes in the structure and spatial organization of the chromosome. The study of the dynamic changes of the proteomic profile of M. gallisepticum nucleoid may assist in revealing its mechanisms of functioning, regulation of chromosome organization and stress adaptation including its changes upon invasion of the host cells., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that might affect the work described in this article., (© 2021 Published by Elsevier Inc.)

Culturing of Mycoplasma hominis in the presence of arginine and thymidine and subsequent comparative proteomic analysis of cells showed that, in addition to the already known arginine dihydrolase pathway of energy metabolism, M. hominis can utilize deoxyribose phosphates formed as a result of catabolism of pyrimidine nucleosides. In this case, a sharp deceleration of cell growth was observed. This allows M. hominis to occupy new niches in the host organism and survive under competitive conditions when the main sources of energy are unavailable., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Ribosomes contain a number of modifications in rRNA, the function of which is unclear. Here we show--using proteomic analysis and dual fluorescence reporter in vivo assays--that m(2)G966 and m(5)C967 in 16S rRNA of Escherichia coli ribosomes are necessary for correct attenuation of tryptophan (trp) operon. Expression of trp operon is upregulated in the strain where RsmD and RsmB methyltransferases were deleted, which results in the lack of m(2)G966 and m(5)C967 modifications. The upregulation requires the trpL attenuator, but is independent of the promotor of trp operon, ribosome binding site of the trpE gene, which follows trp attenuator and even Trp codons in the trpL sequence. Suboptimal translation initiation efficiency in the rsmB/rsmD knockout strain is likely to cause a delay in translation relative to transcription which causes misregulation of attenuation control of trp operon.

To investigate the dynamic cellular response to a condition change, selective labeling of the nascent proteome is necessary. Here, we report a method combining click chemistry protein labeling with 2D DIGE. To test the relevance of the method, we compared nascent proteomes of actively growing bacterial cells with that of cells exposed to protein synthesis inhibitor, erythromycin. Cells were incubated with methionine analog, homopropargyl glycin, and their nascent proteome was selectively labeled with monosulfonated neutral Cy3 and Cy5 azides specially synthesized for this purpose. Following fluorescent labeling, the protein samples were mixed and subjected to standard 2D DIGE separation. The method allowed us to reveal a dramatic reduction of newly synthesized proteins upon erythromycin treatment, while the total proteome was not significantly affected. Additionally, several proteins, whose synthesis was resistant to erythromycin, were identified., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We present the complete genome sequence and proteogenomic map for Acholeplasma laidlawii PG-8A (class Mollicutes, order Acholeplasmatales, family Acholeplasmataceae). The genome of A. laidlawii is represented by a single 1,496,992-bp circular chromosome with an average G+C content of 31 mol%. This is the longest genome among the Mollicutes with a known nucleotide sequence. It contains genes of polymerase type I, SOS response, and signal transduction systems, as well as RNA regulatory elements, riboswitches, and T boxes. This demonstrates a significant capability for the regulation of gene expression and mutagenic response to stress. Acholeplasma laidlawii and phytoplasmas are the only Mollicutes known to use the universal genetic code, in which UGA is a stop codon. Within the Mollicutes group, only the sterol-nonrequiring Acholeplasma has the capacity to synthesize saturated fatty acids de novo. Proteomic data were used in the primary annotation of the genome, validating expression of many predicted proteins. We also detected posttranslational modifications of A. laidlawii proteins: phosphorylation and acylation. Seventy-four candidate phosphorylated proteins were found: 16 candidates are proteins unique to A. laidlawii, and 11 of them are surface-anchored or integral membrane proteins, which implies the presence of active signaling pathways. Among 20 acylated proteins, 14 contained palmitic chains, and six contained stearic chains. No residue of linoleic or oleic acid was observed. Acylated proteins were components of mainly sugar and inorganic ion transport systems and were surface-anchored proteins with unknown functions., (Copyright © 2011, American Society for Microbiology. All Rights Reserved.)

Acylation of the N-terminal Cys residue is an essential, ubiquitous, and uniquely bacterial posttranslational modification that allows anchoring of proteins to the lipid membrane. In gram-negative bacteria, acylation proceeds through three sequential steps requiring lipoprotein diacylglyceryltransferase, lipoprotein signal peptidase, and finally lipoprotein N-acyltransferase. The apparent lack of genes coding for recognizable homologs of lipoprotein N-acyltransferase in gram-positive bacteria and Mollicutes suggests that the final step of the protein acylation process may be absent in these organisms. In this work, we monitored the acylation state of eight major lipoproteins of the mollicute Acholeplasma laidlawii using a combination of standard two-dimensional gel electrophoresis protein separation, blotting to nitrocellulose membranes, and MALDI-MS identification of modified N-terminal tryptic peptides. We show that for each A. laidlawii lipoprotein studied a third fatty acid in an amide linkage on the N-terminal Cys residue is present, whereas diacylated species were not detected. The result thus proves that A. laidlawii encodes a lipoprotein N-acyltransferase activity. We hypothesize that N-acyltransferases encoded by genes non-homologous to N-acyltransferases of gram-negative bacteria are also present in other mollicutes and gram-positive bacteria.

Mollicutes (mycoplasmas) have been recognized as highly evolved prokaryotes with an extremely small genome size and very limited coding capacity. Thus, they may serve as a model of a 'minimal cell': a cell with the lowest possible number of genes yet capable of autonomous self-replication. We present the results of a comparative analysis of proteomes of three mycoplasma species: A. laidlawii, M. gallisepticum, and M. mobile. The core proteome components found in the three mycoplasma species are involved in fundamental cellular processes which are necessary for the free living of cells. They include replication, transcription, translation, and minimal metabolism. The members of the proteome core seem to be tightly interconnected with a number of interactions forming core interactome whether or not additional species-specific proteins are located on the periphery. We also obtained a genome core of the respective organisms and compared it with the proteome core. It was found that the genome core encodes 73 more proteins than the proteome core. Apart of proteins which may not be identified due to technical limitations, there are 24 proteins that seem to not be expressed under the optimal conditions.

The productive internalization in the host cell of Chlamydia trachomatis elementary bodies and their infectivity depends on the degree of reduction of disulfide bonds in the outer envelope of the elementary body. We have hypothesized that the reducing agent may be intracellular glutathione (GSH). Three approaches were used to modulate the intracellular GSH concentration: (1) treatment of cells with buthionine sulfoximine, which causes irreversible inhibition of GSH biosynthesis; (2) hydrogen peroxide-induced oxidation of GSH by intracellular glutathione peroxidases; and (3) treatment of cells with N-acetyl-l-cysteine (NAC), a precursor of glutathione. In the first two cases, we observed a four- to sixfold inhibition of C. trachomatis infection, whereas in NAC-treated cells we detected an increase in the size of chlamydial inclusions. Using a proteomics approach, we showed that the inhibition of chlamydial infection does not combine with alterations in protein expression patterns after cell treatment. These results suggest that GSH plays a key role in the reduction of disulfide bonds in the C. trachomatis outer envelope at an initial stage of the infection., (Copyright © 2010 Elsevier Inc. All rights reserved.)