Mycoplasma Cells Research Articles

Abstract A009: A new version of NGS-QC-PANEL enables better authentication and characterization of human and mouse samples

Abstract Introduction: We previously reported a deep NGS-based QC panel for the authentication, characterization, and contamination of human and mouse samples [1]. It has visibly higher sensitivity and accuracy than traditional STR and SNP assays in authenticating human and mouse cancer cell lines, patient-derived xenograft tumors, and organoids, and is able to detect both inter- and intra-species contamination. Using NGS data from a single run, it also provides other functionalities, including the detection of mycoplasma contamination and viral infection of HBV/EBV/HPV16/HPV18/HIV viruses; the identification of gender and genetic background; the estimation of genetic drift and the construction of phylogenetic trees for samples with a common ancestor; and the quantification of percentages for individual cell lines in their mixtures. A new version of the NGS-QC-PANEL was developed to further enhance the application of the current version. Methods: The new version has over 2000 pairs of primers, compared to 600 pairs in the previous version. We designed characteristic SNPs for more mouse models and selected the characteristic segments for specific mycoplasma species, more microorganism species, and some vertebrate species. Also, we selected more segments for gender characterization. The primers were designed for these newly added SNPs and segments and incorporated into the previous primer pools. Results: The number of mouse models that can be unambiguously authenticated increases from 32 to 92, in particular, MC38 of 5 different origins can be distinguished. Besides quantifying mycoplasma contamination by calculating a ratio of mycoplasma cells to human/mouse cells, the panel is able to distinguish 85 mycoplasma species. More than 100 virus and bacteria species can be detected. Three more segments, in addition to the existing three, are added to infer the gender of human models. Finally, the panel can detect cross-species contamination from cat, cattle, chicken, dog, duck, guineapig, hamster, monkey, rabbit, rat, sheep based on species-specific segments. Conclusions: The new NGS-QC-PANEL makes the authentication, characterization, and contamination detection for human and mouse samples more accurate, more comprehensive and more in-depth, which will further contribute to quality control and assurance of the preclinical tumor models used in drug discovery.

Cryo-electron tomography reveals the binding and release states of the major adhesion complex from Mycoplasma genitalium.

The nap particle is an immunogenic surface adhesion complex from Mycoplasma genitalium. It is essential for motility and responsible for binding sialylated oligosaccharides on the surface of the host cell. The nap particle is composed of two P140-P110 heterodimers, the structure of which was recently solved. However, the interpretation of the mechanism by which the mycoplasma cells orchestrate adhesion remained challenging. Here, we provide cryo-electron tomography structures at ~11 Å resolution, which allow for the distinction between the bound and released state of the nap particle, displaying the in vivo conformational states. Fitting of the atomically resolved structures reveals that bound sialylated oligosaccharides are stabilized by both P110 and P140. Movement of the stalk domains allows for the transfer of conformational changes from the interior of the cell to the binding pocket, thus having the capability of an active release process. It is likely that the same mechanism can be transferred to other Mycoplasma species that belong to the pneumoniae cluster.

Mechanisms of Long-Term Persistence of Mycoplasmas in Children with Asthma.

Improving the management of children with asthma associated with mycoplasma infection is important. Aim: To study the duration of the persistence of antigens, and DNA in a free state, in the structures of circulating immune complexes (CICs) and living cells of Mycoplasma pneumoniae (Mpn) and Mycoplasma hominis (Mh) in children with asthma. In total, 205 children with asthma from 1 to 14 years were observed. The reaction of aggregate-hemagglutination (AHAA), the direct immunofluorescence reaction (DIF), the reaction of the polymerase chain reaction (PCR), and the culture method were used. In addition, 47 children were re-examined 1.5 months after the treatment of mycoplasma infection with azithromycin. The number of samples positive for antigens and DNA in the free state and in the structures of CICs significantly decreased. Then, 50 blood serum samples containing Mh antigens, and 50 samples containing Mpn antigens were analyzed by culture method. Mh was isolated in 21 (65.5%) of 32 samples containing DNA. Mpn was isolated from antigen-positive samples in nine cases. The presented data indicate the long-term persistence of antigens, and DNA of mycoplasma cells in the free state, in the structure of CICs, as well as in the form of "microcolonies". A high level of CICs can be used to predict the course of the disease and the response to therapy.

Direct Measurement of Kinetic Force Generated by Mycoplasma.

Optical tweezers enable us to measure the force generated by bacterial motility and motor proteins. Here, we describe a method, using optical tweezers and related techniques, to measure the force generated during Mycoplasma gliding. An avidin-conjugated polystyrene bead trapped by a focused laser beam is bound to the surface-biotinylated Mycoplasma cell, which pulls the bead from the trap center of the laser. The force generated by Mycoplasma is calculated from a displacement measured and a spring constant of the laser trap.

Cytoskeletal components can turn wall-less spherical bacteria into kinking helices

Bacterial cell shape is generally determined through an interplay between the peptidoglycan cell wall and cytoplasmic filaments made of polymerized MreB. Indeed, some bacteria (e.g., Mycoplasma) that lack both a cell wall and mreB genes consist of non-motile cells that are spherical or pleomorphic. However, other members of the same class Mollicutes (e.g., Spiroplasma, also lacking a cell wall) display a helical cell shape and kink-based motility, which is thought to rely on the presence of five MreB isoforms and a specific fibril protein. Here, we show that heterologous expression of Spiroplasma fibril and MreB proteins confers helical shape and kinking ability to Mycoplasma capricolum cells. Isoform MreB5 is sufficient to confer helicity and kink propagation to mycoplasma cells. Cryoelectron microscopy confirms the association of cytoplasmic MreB filaments with the plasma membrane, suggesting a direct effect on membrane curvature. However, in our experiments, the heterologous expression of MreBs and fibril did not result in efficient motility in culture broth, indicating that additional, unknown Spiroplasma components are required for swimming.

Traditional protocols and optimization methods lead to absent expression in a mycoplasma cell-free gene expression platform.

Cell-free expression (CFE) systems are one of the main platforms for building synthetic cells. A major drawback is the orthogonality of cell-free systems across species. To generate a CFE system compatible with recently established minimal cell constructs, we attempted to optimize a Mycoplasma bacterium-based CFE system using lysates of the genome-minimized cell JCVI-syn3A (Syn3A) and its close phylogenetic relative Mycoplasma capricolum (Mcap). To produce mycoplasma-derived crude lysates, we systematically tested methods commonly used for bacteria, based on the S30 protocol of Escherichia coli. Unexpectedly, after numerous attempts to optimize lysate production methods or composition of feeding buffer, none of the Mcap or Syn3A lysates supported cell-free gene expression. Only modest levels of in vitro transcription of RNA aptamers were observed. While our experimental systems were intended to perform transcription and translation, our assays focused on RNA. Further investigations identified persistently high ribonuclease (RNase) activity in all lysates, despite removal of recognizable nucleases from the respective genomes and attempts to inhibit nuclease activities in assorted CFE preparations. An alternative method using digitonin to permeabilize the mycoplasma cell membrane produced a lysate with diminished RNase activity yet still was unable to support cell-free gene expression. We found that intact mycoplasma cells poisoned E. coli cell-free extracts by degrading ribosomal RNAs, indicating that the mycoplasma cells, even the minimal cell, have a surface-associated RNase activity. However, it is not clear which gene encodes the RNase. This work summarizes attempts to produce mycoplasma-based CFE and serves as a cautionary tale for researchers entering this field. Graphical

Cell-in-Cell Phenomena in Wall-Less Bacteria: Is It Possible?

This work describes curious structures formed by the mainly phytopathogenic mycoplasma Acholeplasma laidlawii, as well as the human pathogen Ureaplasma parvum cells which resemble cell-in-cell structures of higher eukaryotes and protists. The probable significance of such structures for the mycoplasma cell is discussed. The possibility of their formation in nature and their potential role in the transformation of genetic material, for example, by maintaining (on the one hand) the stability of the genome in the line of generations during asexual reproduction or (on the other hand) the genome plasticity, are substantiated. It should be especially noted that all the arguments presented are based only on morphological data. However, closer attention to unusual structures, the existence of which was shown by electron microscopy images in this case, may prompt researchers to analyze their data more carefully and find something rare and non-trivial among seemingly trivial things. If it is proven by additional methods that cell-in-cell structures can indeed be formed by prokaryotes without a cell wall, this phenomenon may acquire general biological significance.

High-speed Atomic Force Microscopy Observation of Internal Structure Movements in Living Mycoplasma.

Dozens of Mycoplasma species belonging to the class Mollicutes bind to solid surfaces through the organelle formed at a cell pole and glide in its direction by a unique mechanism. In Mycoplasma mobile, the fastest gliding species in Mycoplasma, the force for gliding is generated by ATP hydrolysis on an internal structure. However, the spatial and temporal behaviors of the internal structures in living cells were unclear. High-speed atomic force microscopy (HS-AFM) is a powerful method to monitor the dynamic behaviors of biomolecules and cells that can be captured while maintaining their active state in aqueous solution. In this protocol, we describe a method to detect their movements using HS-AFM. This protocol should be useful for the studies of many kinds of microorganisms. Graphic abstract: Scanning Mycoplasma cell.

Model of a Mycoplasma Cell

Model of a Mycoplasma Cell

Building Structural Models of a Whole Mycoplasma Cell

Building structural models of entire cells has been a long-standing cross-discipline challenge for the research community, as it requires an unprecedented level of integration between multiple sources of biological data and enhanced methods for computational modeling and visualization. Here, we present the first 3D structural models of an entire Mycoplasma genitalium (MG) cell, built using the CellPACK suite of computational modeling tools. Our model recapitulates the data described in recent whole-cell system biology simulations and provides a structural representation for all MG proteins, DNA and RNA molecules, obtained by combining experimental and homology-modeled structures and lattice-based models of the genome. We establish a framework for gathering, curating and evaluating these structures, exposing current weaknesses of modeling methods and the boundaries of MG structural knowledge, and visualization methods to explore functional characteristics of the genome and proteome. We compare two approaches for data gathering, a manually-curated workflow and an automated workflow that uses homologous structures, both of which are appropriate for the analysis of mesoscale properties such as crowding and volume occupancy. Analysis of model quality provides estimates of the regularization that will be required when these models are used as starting points for atomic molecular dynamics simulations.

Minimalistic mycoplasmas harbor different functional toxin-antitoxin systems.

Mycoplasmas are minute bacteria controlled by very small genomes ranging from 0.6 to 1.4 Mbp. They encompass several important medical and veterinary pathogens that are often associated with a wide range of chronic diseases. The long persistence of mycoplasma cells in their hosts can exacerbate the spread of antimicrobial resistance observed for many species. However, the nature of the virulence factors driving this phenomenon in mycoplasmas is still unclear. Toxin-antitoxin systems (TA systems) are genetic elements widespread in many bacteria that were historically associated with bacterial persistence. Their presence on mycoplasma genomes has never been carefully assessed, especially for pathogenic species. Here we investigated three candidate TA systems in M. mycoides subsp. capri encoding a (i) novel AAA-ATPase/subtilisin-like serine protease module, (ii) a putative AbiEii/AbiEi pair and (iii) a putative Fic/RelB pair. We sequence analyzed fourteen genomes of M. mycoides subsp. capri and confirmed the presence of at least one TA module in each of them. Interestingly, horizontal gene transfer signatures were also found in several genomic loci containing TA systems for several mycoplasma species. Transcriptomic and proteomic data confirmed differential expression profiles of these TA systems during mycoplasma growth in vitro. While the use of heterologous expression systems based on E. coli and B. subtilis showed clear limitations, the functionality and neutralization capacities of all three candidate TA systems were successfully confirmed using M. capricolum subsp. capricolum as a host. Additionally, M. capricolum subsp. capricolum was used to confirm the presence of functional TA system homologs in mycoplasmas of the Hominis and Pneumoniae phylogenetic groups. Finally, we showed that several of these M. mycoides subsp. capri toxins tested in this study, and particularly the subtilisin-like serine protease, could be used to establish a kill switch in mycoplasmas for industrial applications.

Traceability Methods for Cell Line Authentication and Mycoplasma Detection.

Many laboratories struggle with mycoplasma contamination and cell line misidentification when growing cells in culture. These well-documented issues affect the scientific research community and have detrimental downstream effects. Research published with suspect cultures can produce misleading results. There is increasing pressure to verify the integrity of experimental and established cell lines before publishing. Therefore, laboratories need to define how and when to perform these critical tests, analyze the results, and determine action plans if disparities exist. Our laboratory is committed to producing cell lines of the highest quality for use in experiments; thus, we created a surveillance strategy for these potential problems. We developed processes for both testing and tracing cell line authentication and mycoplasma detection data. Using these methods, we can protect the integrity of our patient and commercial cell lines, maintaining reliable cultures for our research.

Rapid and sensitive mycoplasma detection system using image-based deep learning

A major concern in the clinical application of cell therapy is the manufacturing cost of cell products, which mainly depends on quality control. The mycoplasma test, an important biological test in cell therapy, takes several weeks to detect a microorganism and is extremely expensive. Furthermore, the manual detection of mycoplasma from images requires high-level expertise. We hypothesized that a mycoplasma identification program using a convolutional neural network could reduce the test time and improve sensitivity. To this end, we developed a program comprising three parts (mycoplasma detection, prediction, and cell counting) that allows users to evaluate the sample and verify infected/non-infected cells identified by the program. In experiments conducted, stained DNA images of positive and negative control using mycoplasma-infected and non-infected Vero cells, respectively, were used as training data, and the program results were compared with those of conventional methods, such as manual counting based on visual observation. The minimum detectable mycoplasma contaminations for manual counting and the proposed program were 10 and 5 CFU (colony-forming unit), respectively, and the test time for manual counting was 20 times that for the proposed program. These results suggest that the proposed system can realize a low-cost and streamlined manufacturing process for cellular products in cell-based research and clinical applications.

Bacterial Conjugation Protocol for Ruminant Mycoplasmas.

In Mycoplasma agalactiae, two simultaneous processes of DNA transfer have been described that require direct cell-to-cell contact and are similar to conjugation. One involves the self-transmission of an integrative conjugative element (ICE) while the second concerns the horizontal transfer of large and small fragments of chromosomal DNA. Here, we describe an optimized conjugation protocol for the horizontal transfer of ICE or chromosomal DNA carrying antibiotic resistance markers (i.e., tetracycline, gentamicin, puromycin) from donor to recipient mycoplasma cells. Calculation of the conjugation frequencies, selection and characterization of transconjugants are detailed. This protocol has been developed with M. agalactiae but has been successfully used for M. bovis and can be adapted to other related mycoplasma species.

First Report of Candidatus Mycoplasma haemohominis Infection in Australia Causing Persistent Fever in an Animal Carer

Hemotropic mycoplasmas (hemoplasmas) infect animals and humans and can lead to clinical syndromes mainly characterized by hemolytic anemia. A novel pathogen, Candidatus Mycoplasma haemohominis, was recently associated with a case of human hemoplasmosis in Europe. Here we report the first detection of this pathogen in an Australian patient exhibiting persistent fever, hemolytic anemia, and pancytopenia over a 10-month period. After exhaustive negative testing for human infectious diseases, whole genome sequencing (WGS) was performed on the patient's bone marrow aspirate, using an Illumina NextSeq500 platform. Conventional polymerase chain reaction (PCR), followed by Sanger sequencing, was then performed on blood samples using novel Mycoplasma-specific primers targeting the 16S ribosomal RNA gene. In addition, a Mycoplasma-specific fluorescence in situ hybridization (FISH) assay was developed to differentiate Mycoplasma cells from other erythrocyte inclusions (eg, Pappenheimer and Howell-Jolly bodies) which are morphologically similar to bacterial cocci by light microscopy. WGS analysis revealed that approximately 0.04% of the total number of unmapped reads to human genome corresponded to Mycoplasma species. A 1-kb Mycoplasma 16S fragment was successfully amplified by conventional PCR, and sequence analyses revealed 100% identity with Candidatus Mycoplasma haemohominis. FISH confirmed that several (approximately 2%) epierythrocytic inclusions initially observed by light microscopy corresponded to Mycoplasma cells. This represents the second report of hemolytic anemia associated with hemoplasma infection in a human, and the first report of human hemoplasmosis in Australia. This study highlights the importance of new and emerging diagnostic approaches and need for further investigations on the epidemiology of Candidatus Mycoplasma haemohominis in Australia.

Mbov_0503 Encodes a Novel Cytoadhesin that Facilitates Mycoplasma bovis Interaction with Tight Junctions.

Molecules contributing to microbial cytoadhesion are important virulence factors. In Mycoplasma bovis, a minimal bacterium but an important cattle pathogen, binding to host cells is emerging as a complex process involving a broad range of surface-exposed structures. Here, a new cytoadhesin of M. bovis was identified by producing a collection of individual knock-out mutants and evaluating their binding to embryonic bovine lung cells. The cytoadhesive-properties of this surface-exposed protein, which is encoded by Mbov_0503 in strain HB0801, were demonstrated at both the mycoplasma cell and protein levels using confocal microscopy and ELISA. Although Mbov_0503 disruption was only associated in M. bovis with a partial reduction of its binding capacity, this moderate effect was sufficient to affect M. bovis interaction with the host-cell tight junctions, and to reduce the translocation of this mycoplasma across epithelial cell monolayers. Besides demonstrating the capacity of M. bovis to disrupt tight junctions, these results identified novel properties associated with cytoadhesin that might contribute to virulence and host colonization. These findings provide new insights into the complex interplay taking place between wall-less mycoplasmas and the host-cell surface.

Factors determining microbial colonization of liquid nitrogen storage tanks used for archiving biological samples

The availability of bioresources is a precondition for life science research, medical applications, and diagnostics, but requires a dedicated quality management to guarantee reliable and safe storage. Anecdotal reports of bacterial isolates and sample contamination indicate that organisms may persist in liquid nitrogen (LN) storage tanks. To evaluate the safety status of cryocollections, we systematically screened organisms in the LN phase and in ice layers covering inner surfaces of storage tanks maintained in different biobanking facilities. We applied a culture-independent approach combining cell detection by epifluorescence microscopy with the amplification of group-specific marker genes and high-throughput sequencing of bacterial ribosomal genes. In the LN phase, neither cells nor bacterial 16S rRNA gene copy numbers were detectable (detection limit, 102 cells per ml, 103 gene copies per ml). In several cases, small numbers of bacteria of up to 104 cells per ml and up to 106 gene copies per ml, as well as Mycoplasma, or fungi were detected in the ice phase formed underneath the lids or accumulated at the bottom. The bacteria most likely originated from the stored materials themselves (Elizabethingia, Janthibacterium), the technical environment (Pseudomonas, Acinetobacter, Methylobacterium), or the human microbiome (Bacteroides, Streptococcus, Staphylococcus). In single cases, bacteria, Mycoplasma, fungi, and human cells were detected in the debris at the bottom of the storage tanks. In conclusion, the limited microbial load of the ice phase and in the debris of storage tanks can be effectively avoided by minimizing ice formation and by employing hermetically sealed sample containers.

Proteases as Secreted Exoproteins in Mycoplasmas from Ruminant Lungs and Their Impact on Surface-Exposed Proteins.

Many mycoplasma species are isolated from the ruminant lungs as either saprophytes or true pathogens. These wall-less bacteria possess a minimal genome and reduced metabolic capabilities. Accordingly, they rely heavily on their hosts for the supply of essential metabolites and, notably, peptides. Seven of 13 ruminant lung-associated Mycoplasma (sub)species were shown to possess caseinolytic activity when grown in rich media and assessed with a quantitative fluorescence test. For some species, this activity was detected in spent medium, an indication that proteases were secreted outside the mycoplasma cells. To identify these proteases, we incubated concentrated washed cell pellets in a defined medium and analyzed the supernatants by tandem mass spectrometry. Secreted-protease activity was detected mostly in the species belonging to the Mycoplasma mycoides cluster (MMC) and, to a lesser extent, in Mycoplasma bovirhinis Analyzing a Mycoplasma mycoides subsp. capri strain, chosen as a model, we identified 35 expressed proteases among 55 predicted coding genes, of which 5 were preferentially found in the supernatant. Serine protease S41, acquired by horizontal gene transfer, was responsible for the caseinolytic activity, as demonstrated by zymography and mutant analysis. In an M. capricolum mutant, inactivation of the S41 protease resulted in marked modification of the expression or secretion of 17 predicted surface-exposed proteins. This is an indication that the S41 protease could have a role in posttranslational cleavage of surface-exposed proteins and ectodomain shedding, whose physiological impacts still need to be explored.IMPORTANCE Few studies pertaining to proteases in ruminant mycoplasmas have been reported. Here, we focus on proteases that are secreted outside the mycoplasma cell using a mass spectrometry approach. The most striking result is the identification, within the Mycoplasma mycoides cluster, of a serine protease that is exclusively detected outside the mycoplasma cells and is responsible for casein digestion. This protease may also be involved in the posttranslational processing of surface proteins, as suggested by analysis of mutants showing a marked reduction in the secretion of extracellular proteins. By analogy, this finding may help increase understanding of the mechanisms underlying this ectodomain shedding in other mycoplasma species. The gene encoding this protease is likely to have been acquired via horizontal gene transfer from Gram-positive bacteria and sortase-associated surface proteases. Whether this protease and the associated ectodomain shedding are related to virulence has yet to be ascertained.

Mycoplasmas under experimental antimicrobial selection: The unpredicted contribution of horizontal chromosomal transfer.

Horizontal Gene Transfer was long thought to be marginal in Mycoplasma a large group of wall-less bacteria often portrayed as minimal cells because of their reduced genomes (ca. 0.5 to 2.0 Mb) and their limited metabolic pathways. This view was recently challenged by the discovery of conjugative exchanges of large chromosomal fragments that equally affected all parts of the chromosome via an unconventional mechanism, so that the whole mycoplasma genome is potentially mobile. By combining next generation sequencing to classical mating and evolutionary experiments, the current study further explored the contribution and impact of this phenomenon on mycoplasma evolution and adaptation using the fluoroquinolone enrofloxacin (Enro), for selective pressure and the ruminant pathogen Mycoplasma agalactiae, as a model organism. For this purpose, we generated isogenic lineages that displayed different combination of spontaneous mutations in Enro target genes (gyrA, gyrB, parC and parE) in association to gradual level of resistance to Enro. We then tested whether these mutations can be acquired by a susceptible population via conjugative chromosomal transfer knowing that, in our model organism, the 4 target genes are scattered in three distinct and distant loci. Our data show that under antibiotic selective pressure, the time scale of the mutational pathway leading to high-level of Enro resistance can be readily compressed into a single conjugative step, in which several EnroR alleles were transferred from resistant to susceptible mycoplasma cells. In addition to acting as an accelerator for antimicrobial dissemination, mycoplasma chromosomal transfer reshuffled genomes beyond expectations and created a mosaic of resistant sub-populations with unpredicted and unrelated features. Our findings provide insights into the process that may drive evolution and adaptability of several pathogenic Mycoplasma spp. via an unconventional conjugative mechanism.

Mycoplasmas are no exception to extracellular vesicles release: Revisiting old concepts.

Release of extracellular vesicles (EV) by Gram-negative and positive bacteria is being frequently reported. EV are nano-sized, membrane-derived, non-self-replicating, spherical structures shed into the extracellular environment that could play a role in bacteria-host interactions. Evidence of EV production in bacteria belonging to the class Mollicutes, which are wall-less, is mainly restricted to the genus Acholeplasma and is scanty for the Mycoplasma genus that comprises major human and animal pathogens. Here EV release by six Mycoplasma (sub)species of clinical importance was investigated. EV were obtained under nutritional stress conditions, purified by ultracentrifugation and observed by electron microscopy. The membrane proteins of EV from three different species were further identified by mass spectrometry as a preliminary approach to determining their potential role in host-pathogen interactions. EV were shown to be released by all six (sub)species although their quantities and sizes (30–220 nm) were very variable. EV purification was complicated by the minute size of viable mycoplasmal cells. The proteins of EV-membranes from three (sub)species included major components of host-pathogen interactions, suggesting that EV could contribute to make the host-pathogen interplay more complex. The process behind EV release has yet to be deciphered, although several observations demonstrated their active release from the plasma membrane of living cells. This work shed new light on old concepts of “elementary bodies” and “not-cell bound antigens”.