Innokentii E. Vishnyakov

Extracellular membrane vesicles secreted by mycoplasma Acholeplasma laidlawii PG8 are enriched in virulence proteins

Mycoplasmas (class Mollicutes), the smallest prokaryotes capable of self-replication, as well as Archaea, Gram-positive and Gram-negative bacteria constitutively produce extracellular vesicles (EVs). However, little is known regarding the content and functions of mycoplasma vesicles. Here, we present for the first time a proteomics-based characterisation of extracellular membrane vesicles from Acholeplasma laidlawii PG8. The ubiquitous mycoplasma is widespread in nature, found in humans, animals and plants, and is the causative agent of phytomycoplasmoses and the predominant contaminant of cell cultures. Taking a proteomics approach using LC-ESI-MS/MS, we identified 97 proteins. Analysis of the identified proteins indicated that A. laidlawii-derived EVs are enriched in virulence proteins that may play critical roles in mycoplasma-induced pathogenesis. Our data will help to elucidate the functions of mycoplasma-derived EVs and to develop effective methods to control infections and contaminations of cell cultures by mycoplasmas. In the present study, we have documented for the first time the proteins in EVs secreted by mycoplasma vesicular proteins identified in this study are likely involved in the adaptation of bacteria to stressors, survival in microbial communities and pathogen-host interactions. These findings suggest that the secretion of EVs is an evolutionally conserved and universal process that occurs in organisms from the simplest wall-less bacteria to complex organisms and indicate the necessity of developing new approaches to control infects.

New insights into FtsZ rearrangements during the cell division of Escherichia coli from single‐molecule localization microscopy of fixed cells

FtsZ – a prokaryotic tubulin homolog – is one of the central components of bacterial division machinery. At the early stage of cytokinesis FtsZ forms the so‐called Z‐ring at mid‐cell that guides septum formation. Many approaches were used to resolve the structure of the Z‐ring, however, researchers are still far from consensus on this question. We utilized single‐molecule localization microscopy (SMLM) in combination with immunofluorescence staining to visualize FtsZ in Esherichia coli fixed cells that were grown under slow and fast growth conditions. This approach allowed us to obtain images of FtsZ structures at different stages of cell division and accurately measure Z‐ring dimensions. Analysis of these images demonstrated that Z‐ring thickness increases during constriction, starting at about 70 nm at the beginning of division and increasing by approximately 25% half‐way through constriction.

The identification and characterization of IbpA, a novel α-crystallin-type heat shock protein from mycoplasma.

Abstractα-Crystallin-type small heat shock proteins (sHsps) are expressed in many bacteria, animals, plants, and archaea. Among mycoplasmas (Mollicutes), predicted sHsp homologues so far were found only in the Acholeplasmataceae family. In this report, we describe the cloning and functional characterization of a novel sHsp orthologue, IbpA protein, present in Acholeplasma laidlawii. Importantly, similar to the endogenously expressed sHsp proteins, the recombinant IbpA protein was able to spontaneously generate oligomers in vitro and to rescue chemically denatured bovine insulin from irreversible denaturation and aggregation. Collectively, these data suggest that IbpA is a bona fide member of the sHsps family. The immune-electron microscopy data using specific antibodies against IbpA have revealed different intracellular localization of this protein in A. laidlawii cells upon heat shock, which suggests that IbpA not only may participate in the stabilization of individual polypeptides, but may also play a protective role in the maintenance of various cellular structures upon temperature stress.

FtsZ and bacterial cell division

In this review we describe proteins and supermolecular structures which take part in the division of bacterial cells. FtsZ, a eukaryotic tubulin homolog is a key cell division protein in most prokaryotes. FtsZ, as well as tubulin, is capable of binding and hydrolyzing GTP. The division of a bacterial cell begins with the forming of a so-called divisome. The basis of such a divisome is a contractile ring (Z ring) which encircles the cell about midcell. The Z-ring consists of a bundle of laterally bound protofilaments formed in result of FtsZ polymerization. Z-ring is rigidly bounded to the cytosolic side of the inner membrane with the participation of FtsA, ZipA, FtsW and many other divisome cell division proteins. The ring directs the process of cytokinesis transmitting constriction power to the membrane. The primary structures of the prokaryotic FtsZ family members significantly differ from eukaryotic tubulins except for the sites of GTP binding. There is a high degree of structural homology between these proteins in the region. FtsZ is one of the most conserved proteins in prokaryotes. However, ftsZ genes have not been found in several species of microorganisms with completely sequenced genomes. They include two species of mycoplasmas (Ureaplasma parvum and Mycoplasma mobile), Prostecobacter dejongeii, 10 species of chlamydia and 5 species of archaea. Consequently, these organisms divide without FtsZ participation. The genomes of U. parvum and M. mobile have many open reading frames which encode proteins with unknown functions. A comparison of the primary structures of these hypothetical proteins did not identify any known cell division proteins. We hypothesize that the process of cell division in these organisms should involve proteins similar to FtsZ in function and homologous to FtsZ or other cell division proteins in structure.

Outer membrane vesicles secreted by pathogenic and nonpathogenic Bacteroides fragilis represent different metabolic activities

Numerous studies are devoted to the intestinal microbiota and intercellular communication maintaining homeostasis. In this regard, vesicles secreted by bacteria represent one of the most popular topics for research. For example, the outer membrane vesicles (OMVs) of Bacteroides fragilis play an important nutritional role with respect to other microorganisms and promote anti-inflammatory effects on immune cells. However, toxigenic B. fragilis (ETBF) contributes to bowel disease, even causing colon cancer. If nontoxigenic B. fragilis (NTBF) vesicles exert a beneficial effect on the intestine, it is likely that ETBF vesicles can be utilized for potential pathogenic implementation. To confirm this possibility, we performed comparative proteomic HPLC-MS/MS analysis of vesicles isolated from ETBF and NTBF. Furthermore, we performed, for the first time, HPLC-MS/MS and GS-MS comparative metabolomic analysis for the vesicles isolated from both strains with subsequent reconstruction of the vesicle metabolic pathways. We utilized fluxomic experiments to validate the reconstructed biochemical reaction activities and finally observed considerable difference in the vesicle proteome and metabolome profiles. Compared with NTBF OMVs, metabolic activity of ETBF OMVs provides their similarity to micro reactors that are likely to be used for long-term persistence and implementing pathogenic potential in the host.

Localization microscopy study of FtsZ structures in E. coli cells during SOS-response

Localization microscopy allows visualization of biological structures with resolution well below the diffraction limit. This is achieved by temporal separation of single fluorophore molecules emission and subsequent localization of them with the precision of few tens of nanometers. This method was previously successfully used to obtain images of FtsZ structures in Escherichia coli cells using FtsZ fusion with fluorescent protein mEos2. In this work we obtained superresolution images of FtsZ structures in fixed E. coli cells using immunocytochemical labeling. Comparison of superresolution FtsZ structures in cells undergoing SOS-response and healthy cells shows that FtsZ structures are partially disassembled during SOS-response, but still retain some periodicity.

Mycoplasma heat shock proteins and their genes

Mycoplasmas (class Mollicutes) are the simplest prokaryotic organisms capable of independent reproduction and are therefore considered a natural model of a “minimal” cell. The systems retained by mycoplasmas in the course of their reductive evolution may be fundamental for all cells. This is the first review to summarize and systematize available information concerning the genes encoding the heat shock proteins (HSP) in mycoplasma. An attempt is made to analyze the presence or absence of the mycoplasma analogues of the major bacterial chaperones and proteases, which determine cell resistance to stresses, as well as protein homeostasis under optimal growth conditions. The data on the mechanisms for the regulation of transcription of the HSP genes in mycoplasma are presented. The properties and functions of the best-characterized mycoplasma HSP, namely, DnaK, DnaJ-like proteins, the GroEL/GroES system, ClpB, and small heat shock proteins (sHSP), are discussed.

Localization of division protein FtsZ in Mycoplasma hominis

The localization of FtsZ protein in M. hominis cells was studied by immunoelectron microscopy with polyclonal antibodies to this protein. Cell polymorphism typical for mycoplasmas was seen on electron microscopic pictures. Among the diversity of cell shapes, we distinguished dumbbell-shaped dividing cells and cells connected with each other by membrane tubules (former constrictions). The label was predominantly observed in the constriction area of dividing M. hominis cells and on thin membrane tubules. A septum and the gold labeling of this structure have not been described before in mycoplasma cells. For the first time, in some rounded and oval cells, colloidal gold particles labeled the entire plasma membrane, probably marking a submembranous contractile ring (Z ring). These observations confirm the implication of FtsZ protein in M. hominis cytokinesis. In some cells, the spiral-like distribution of gold particles was observed. Most likely, FtsZ protofilaments in M. hominis cells form spiral structures similar to Z spirals in Bacillus subtilis and Escherichia coli. Their presence in mycoplasma cells may be considered to be an important argument in favor of Z ring assembly through the reorganization of Z spirals. FtsZ as a bacterial cytoskeleton protein binding with membrane directly or through intermediates may be engaged in maintenance of M. hominis cell shape.

α-crystallin-type heat-shock protein from mycoplasma Acholeplasma laidlawii (Mollicutes)

An increased amount of major heat-shock proteins (HSPs) after heat treatment has been revealed in Acholeplasma laidlawii cells grown in liquid culture, with the quantity of small HSPs, known as P17, being enhanced by hundreds times. The P17 protein was isolated and identified as an α-crystallin-type HSP (α-HSP) by sequencing the N-terminal 15 amino acids of the P17 polypeptide chain followed by finding the corresponding open reading frame (ORF) in the completely sequenced genome of A. laidlawii PG 8A. A computer-based search for homologous ORFs in the genomes of all 14 species of the Mycoplasmataceae family (mycoplasmas themselves) that have been completely sequenced to date yields a negative result. However, among the representatives of the Mollicutes (mycoplasma) class, genes encoding α-HSPs were found in two phytoplasma species (Phytoplasmataceae family) and the acholeplasma examined (Acholeplasmataceae family). It is supposed that the presence or absence of α-HSPs in microorganisms might be related to their inhabitancy; representatives of Acholeplasmataceae and Phytoplasmataceae families mostly reside in plant tissues, which is in contrast to the majority of the Mycoplasmataceae family, which lives in animal and human tissues, i.e., use ecological niches with relatively constant temperature.

Recovery of division process in bacterial cells after induction of SulA protein which is responsible for cytokinesis arrest during SOS-response

SOS-response is an important tool of bacteria intended to protect their genome and thereby allow them to survive under adverse conditions. Recently SOS-response was demonstrated to enhance mutagenesis and thus help bacteria to acquire antibiotic resistance. Due to high significance of this phenomena it seems to be important to investigate processes that allow bacteria to survive after SOS-response activation. In current work the recovery of division process of Escherichia coli cells after division arrest due to expression of SOS-response protein SulA was studied. Data indicate that cells are able to rapidly restore normal division; also nucleoid occlusion seems to be the main septum positioning mechanism during the process. In the course of recovery FtsZ forms helix-like structures, which then transform into Z-rings.