-
Frontiers in Immunology 2021and are two significant mycoplasmas that infect the urogenital and respiratory tracts of humans. Despite distinct tissue tropisms, they both have similar pathogenic... (Comparative Study)
Comparative Study
and are two significant mycoplasmas that infect the urogenital and respiratory tracts of humans. Despite distinct tissue tropisms, they both have similar pathogenic mechanisms and infect/invade epithelial cells in the respective regions and persist within these cells. However, the pathogenic mechanisms of these species in terms of bacterium-host interactions are poorly understood. To gain insights on this, we infected HeLa cells independently with and and assessed gene expression by whole transcriptome sequencing (RNA-seq) approach. The results revealed that HeLa cells respond to and differently by regulating various protein-coding genes. Though there is a significant overlap between the genes regulated by these species, many of the differentially expressed genes were specific to each species. KEGG pathway and signaling network analyses revealed that the genes specific to are more related to cellular processes. In contrast, the genes specific to infection are correlated with immune response and inflammation, possibly suggesting that has some inherent ability to modulate host immune pathways.
Topics: Epithelial Cells; Gene Expression Profiling; Gene Regulatory Networks; HeLa Cells; Host-Pathogen Interactions; Humans; Mycoplasma genitalium; Mycoplasma pneumoniae; Protein Interaction Maps; RNA-Seq; Signal Transduction; Transcriptome; Exome Sequencing
PubMed: 34707609
DOI: 10.3389/fimmu.2021.738431 -
Scientific Reports Mar 2021Mycoplasmas are fastidious microorganisms, typically characterised by their restricted metabolism and minimalist genome. Although there is reported evidence that some...
Mycoplasmas are fastidious microorganisms, typically characterised by their restricted metabolism and minimalist genome. Although there is reported evidence that some mycoplasmas can develop biofilms little is known about any differences in metabolism in these organisms between the growth states. A systematic metabolomics approach may help clarify differences associated between planktonic and biofilm associated mycoplasmas. In the current study, the metabolomics of two different mycoplasmas of clinical importance (Mycoplasma pneumoniae and Mycoplasma fermentans) were examined using a novel approach involving nuclear magnetic resonance spectroscopy and principle component analysis. Characterisation of metabolic changes was facilitated through the generation of high-density metabolite data and diffusion-ordered spectroscopy that provided the size and structural information of the molecules under examination. This enabled the discrimination between biofilms and planktonic states for the metabolomic profiles of both organisms. This work identified clear biofilm/planktonic differences in metabolite composition for both clinical mycoplasmas and the outcomes serve to establish a baseline understanding of the changes in metabolism observed in these pathogens in their different growth states. This may offer insight into how these organisms are capable of exploiting and persisting in different niches and so facilitate their survival in the clinical setting.
Topics: Biofilms; Diffusion; Magnetic Resonance Spectroscopy; Mycoplasma fermentans; Mycoplasma pneumoniae; Plankton; Principal Component Analysis; Serum
PubMed: 33707544
DOI: 10.1038/s41598-021-84326-2 -
Frontiers in Cellular and Infection... 2017Mycoplasmas are the smallest self-replicating organisms and obligate parasites of a specific vertebrate host. An in-depth analysis of the functional capabilities of...
Mycoplasmas are the smallest self-replicating organisms and obligate parasites of a specific vertebrate host. An in-depth analysis of the functional capabilities of mycoplasma species is fundamental to understand how some of simplest forms of life on Earth succeeded in subverting complex hosts with highly sophisticated immune systems. In this study we present a genome-scale comparison, focused on identification of functional protein domains, of 80 publically available mycoplasma genomes which were consistently re-annotated using a standardized annotation pipeline embedded in a semantic framework to keep track of the data provenance. We examined the pan- and core-domainome and studied predicted functional capability in relation to host specificity and phylogenetic distance. We show that the pan- and core-domainome of mycoplasma species is closed. A comparison with the proteome of the "minimal" synthetic bacterium JCVI-Syn3.0 allowed us to classify domains and proteins essential for minimal life. Many of those essential protein domains, essential Domains of Unknown Function (DUFs) and essential hypothetical proteins are not persistent across mycoplasma genomes suggesting that mycoplasma species support alternative domain configurations that bypass their essentiality. Based on the protein domain composition, we could separate mycoplasma species infecting blood and tissue. For selected genomes of tissue infecting mycoplasmas, we could also predict whether the host is ruminant, pig or human. Functionally closely related mycoplasma species, which have a highly similar protein domain repertoire, but different hosts could not be separated. This study provides a concise overview of the functional capabilities of mycoplasma species, which can be used as a basis to further understand host-pathogen interaction or to design synthetic minimal life.
Topics: Animals; Bacterial Proteins; Genes, Bacterial; Genome Size; Genome, Bacterial; Host Specificity; Host-Pathogen Interactions; Humans; Mycoplasma; Mycoplasma Infections; Phylogeny; Protein Domains; Proteome; RNA, Ribosomal, 16S; Species Specificity; Spiroplasma; Swine
PubMed: 28224116
DOI: 10.3389/fcimb.2017.00031 -
Veterinary Research Jul 2019Mycoplasma hyopneumoniae and Mycoplasma hyorhinis are two phylogenetically related species colonizing the respiratory tract of pigs but differing in pathogenicity, the...
Mycoplasma hyopneumoniae and Mycoplasma hyorhinis are two phylogenetically related species colonizing the respiratory tract of pigs but differing in pathogenicity, the basis of which is not well resolved. We hypothesize that genes belonging to the species-specific portion of the genome and being non-essential during ideal laboratory growth conditions encode possible virulent determinants and are the driver of interspecies differences. To investigate this, transposon mutant libraries were generated for both species and a transposon sequencing (Tn-seq) method for mycoplasmas was established to identify non-essential genes. Tn-seq datasets combined with bidirectional Blastp analysis revealed that 101 out of a total 678 coding sequences (CDS) are species-specific and non-essential CDS of M. hyopneumoniae strain F7.2C, while 96 out of a total 751 CDS are species-specific and non-essential CDS in the M. hyorhinis strain JF5820. Among these species-specific and non-essential CDS were genes involved in metabolic pathways. In particular, the myo-inositol and the sialic acid pathways were found to be non-essential and therefore could be considered important to the specific pathogenicity of M. hyopneumoniae and M. hyorhinis, respectively. Such pathways could enable the use of an alternative energy source providing an advantage in their specific niche and might be interesting targets to knock out in order to generate attenuated live vaccines.
Topics: Animals; DNA Transposable Elements; Gene Library; Mycoplasma Infections; Mycoplasma hyopneumoniae; Mycoplasma hyorhinis; Pneumonia of Swine, Mycoplasmal; Sequence Analysis, DNA; Swine; Virulence
PubMed: 31324222
DOI: 10.1186/s13567-019-0674-7 -
Poultry Science Jan 2014The survival times of Mycoplasma gallisepticum (Mg) and Mycoplasma synoviae (Ms) on washed and unwashed natural and synthetic kanekalon hair samples over a 5-d period...
The survival times of Mycoplasma gallisepticum (Mg) and Mycoplasma synoviae (Ms) on washed and unwashed natural and synthetic kanekalon hair samples over a 5-d period were evaluated using the color changing unit method for comparison with results of previous studies conducted on natural hair. Regardless of whether synthetic or natural hair samples prewashed with a disinfectant shampoo were spiked with Mg or Ms, all viable organisms rapidly dropped below a count of 1 × 10(1)/mL of culture. Unwashed natural hair seeded with a titer of approximately 1 × 10(6)/mL of viable Mg or Ms decreased to 6 × 10(5)/mL and 6 × 10(3)/mL, respectively, by 4 h postseeding, but no viable Mg or Ms were detected on natural hair from 8 h onwards. By contrast, the titers of Mg and Ms on synthetic hair did not decline from the initial 1 × 10(6)/mL seed dose up to 96 h postseeding, and, in fact, viable Mg and Ms was still detectable at 9 d postinfection. Application of a real-time quantitative single-tube duplex PCR assay confirmed that no proliferation of Mg or Ms had occurred on the synthetic hair samples, the cells simply remained viable. The unexpected finding that Mg and Ms survive for extended periods on synthetic kanekalon hair fibers raises the question of whether attachment to a surface is a prerequisite for the survival and persistence of Mg and Ms in the extra-host environment. Future studies should be aimed at determining whether other synthetic hair types or indeed other types of plastics commonly found in the poultry house offer similar survival advantages to mycoplasmas.
Topics: Acrylonitrile; Detergents; Hair; Mycoplasma gallisepticum; Mycoplasma synoviae; Time Factors; Vinyl Chloride
PubMed: 24570416
DOI: 10.3382/ps.2013-03457 -
MBio Jun 2022Trichomonas vaginalis can host the endosymbiont Mycoplasma hominis, an opportunistic pathogenic bacterium capable of modulating T. vaginalis pathobiology. Recently, a...
Trichomonas vaginalis can host the endosymbiont Mycoplasma hominis, an opportunistic pathogenic bacterium capable of modulating T. vaginalis pathobiology. Recently, a new noncultivable mycoplasma, " Mycoplasma girerdii," has been shown to be closely associated with women affected by trichomoniasis, suggesting a biological association. Although several features of " M. girerdii" have been investigated through genomic analysis, the nature of the potential T. vaginalis-" M. girerdii" consortium and its impact on the biology and pathogenesis of both microorganisms have not yet been explored. Here, we investigate the association between " M. girerdii" and T. vaginalis isolated from patients affected by trichomoniasis, demonstrating their intracellular localization. By using an model system based on single- and double- infection of -free isogenic T. vaginalis, we investigated the ability of the protist to establish a relationship with the bacteria and impact T. vaginalis growth. Our data indicate likely competition between M. hominis and " M. girerdii" while infecting trichomonad cells. Comparative dual-transcriptomics data showed major shifts in parasite gene expression in response to the presence of , including genes associated with energy metabolism and pathogenesis. Consistent with the transcriptomics data, both parasite-mediated hemolysis and binding to host epithelial cells were significantly upregulated in the presence of either species. Taken together, these results support a model in which this microbial association could modulate the virulence of T. vaginalis. T. vaginalis and form a unique case of endosymbiosis that modulates the parasite's pathobiology. Recently, a new nonculturable mycoplasma species (" Mycoplasma girerdii") has been described as closely associated with the protozoon. Here, we report the characterization of this endosymbiotic relationship. Clinical isolates of the parasite demonstrate that mycoplasmas are common among trichomoniasis patients. The relationships are studied by devising an system of single and/or double infections in isogenic protozoan recipients. Comparative growth experiments and transcriptomics data demonstrate that the composition of different microbial consortia influences the growth of the parasite and significantly modulates its transcriptomic profile, including metabolic enzymes and virulence genes such as adhesins and pore-forming proteins. The data on modulation from RNA sequencing (RNA-Seq) correlated closely with those of the cytopathic effect and adhesion to human target cells. We propose the hypothesis that the presence and the quantitative ratios of endosymbionts may contribute to modulating protozoan virulence. Our data highlight the importance of considering pathogenic entities as microbial ecosystems, reinforcing the importance of the development of integrated diagnostic and therapeutic strategies.
Topics: Ecosystem; Female; Humans; Mycoplasma; Mycoplasma hominis; Trichomonas Infections; Trichomonas vaginalis
PubMed: 35608298
DOI: 10.1128/mbio.00918-22 -
International Journal of Molecular... Nov 2021Contamination of cells/tissues by infectious pathogens (e.g., fungi, viruses, or bacteria, including mycoplasma) is a major problem in cell-based transplantation. In...
Contamination of cells/tissues by infectious pathogens (e.g., fungi, viruses, or bacteria, including mycoplasma) is a major problem in cell-based transplantation. In this study, we tested a polymerase chain reaction (PCR) method to provide rapid, simple, and sensitive detection of mycoplasma contamination in laboratory cultures for clinical use. This mycoplasma PCR system covers the species (spp.) listed for testing in the 17th revision of the Japanese Pharmacopoeia, and we designed it for use in transplantable retinal cells. Here, we analyzed mycoplasma contamination in induced pluripotent stem cell (iPS cell)-derived transplantable retinal pigment epithelium (RPE) cells. In the spike tests to RPE cells with nine species of class bacteria, including seven spp. and one of each spp. and spp., contamination at the concentration of 100 and 10 CFU/mL were detected with 100% probability in all cases, while 1 CFU/mL had a detection rate of 0-75%. DNA prepared from bacteria species other than class species was not detectable, indicating the specificity of this PCR. While iPS cells and iPS-RPE cells established in our laboratory were all negative by this PCR, some of the commercially available cell lines were positive. Cells for transplantation should never have infection, as once pathogens are implanted into the eyes, they can cause severe intraocular inflammation. Thus, it is imperative to monitor for infections in the transplants, although generally, mycoplasma infection is difficult to detect.
Topics: Cell Line; Cell- and Tissue-Based Therapy; DNA, Bacterial; Humans; Induced Pluripotent Stem Cells; Mycoplasma; Polymerase Chain Reaction; RNA, Ribosomal, 16S; Retinal Pigment Epithelium; Transplantation; Ureaplasma
PubMed: 34830437
DOI: 10.3390/ijms222212555 -
Antimicrobial Agents and Chemotherapy Mar 2019Escalating levels of antibiotic resistance in mycoplasmas, particularly macrolide resistance in and , have narrowed our antibiotic arsenal. Further, mycoplasmas lack a...
Turning the Tide against Antibiotic Resistance by Evaluating Novel, Halogenated Phenazine, Quinoline, and NH125 Compounds against Species Clinical Isolates and Type Strains.
Escalating levels of antibiotic resistance in mycoplasmas, particularly macrolide resistance in and , have narrowed our antibiotic arsenal. Further, mycoplasmas lack a cell wall and do not synthesize folic acid, rendering common antibiotics, such as beta-lactams, vancomycin, sulfonamides, and trimethoprim, of no value. To address this shortage, we screened nitroxoline, triclosan, and a library of 20 novel, halogenated phenazine, quinoline, and NH125 analogues against species and clinical isolates from urine. We tested a subset of these compounds ( = 9) against four mycoplasma type strains (, , , and ) using a validated broth microdilution or agar dilution method. Among 72 species clinical isolates, nitroxoline proved most effective (MIC, 6.25 µM), followed by an -arylated NH125 analogue (MIC, 12.5 µM). NH125 and its analogue had significantly higher MICs against isolates than against isolates, whereas nitroxoline did not. Nitroxoline exhibited bactericidal activity against isolates but bacteriostatic activity against the majority of isolates. Among the type strains, the compounds had the greatest activity against and , with 8 (80%) and 5 (71.4%) isolates demonstrating MICs of ≤12.5 µM, respectively. Triclosan also exhibited lower MICs against and Overall, we identified a promising range of quinoline, halogenated phenazine, and NH125 compounds that showed effectiveness against and and found that nitroxoline, approved for use outside the United States for the treatment of urinary tract infections, and an -arylated NH125 analogue demonstrated low MICs against species isolates.
Topics: Anti-Bacterial Agents; Drug Resistance, Multiple, Bacterial; Humans; Imidazoles; Microbial Sensitivity Tests; Mycoplasma; Mycoplasma Infections; Phenazines; Quinolines; Ureaplasma Infections; Ureaplasma urealyticum
PubMed: 30642935
DOI: 10.1128/AAC.02265-18 -
Journal of Visualized Experiments : JoVE Jun 2011The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. Although...
The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. Although mycoplasma do not usually kill contaminated cells, they are difficult to detect and can cause a variety of effects on cultured cells, including altered metabolism, slowed proliferation and chromosomal aberrations. In short, mycoplasma contamination compromises the value of those cell lines in providing accurate data for life science research. The sources of mycoplasma contamination in the laboratory are very challenging to completely control. As certain mycoplasma species are found on human skin, they can be introduced through poor aseptic technique. Additionally, they can come from contaminated supplements such as fetal bovine serum, and most importantly from other contaminated cell cultures. Once mycoplasma contaminates a culture, it can quickly spread to contaminate other areas of the lab. Strict adherence to good laboratory practices such as good aseptic technique are key, and routine testing for mycoplasma is highly recommended for successful control of mycoplasma contamination. PCR-based detection of mycoplasma has become a very popular method for routine cell line maintenance. PCR-based detection methods are highly sensitive and can provide rapid results, which allows researchers to respond quickly to isolate and eliminate contamination once it is detected in comparison to the time required using microbiological techniques. The LookOut Mycoplasma PCR Detection Kit is highly sensitive, with a detection limit of only 2 genomes per μl. Taking advantage of the highly specific JumpStart Taq DNA Polymerase and a proprietary primer design, false positives are greatly reduced. The convenient 8-tube format, strips pre-coated with dNTPs, and associated primers helps increase the throughput to meet the needs of customers with larger collections of cell lines. Given the extreme sensitivity of the kit, great care must be taken to prevent inadvertent contamination of samples and reagents. The step-by-step protocol we demonstrate highlights the precautions and practices required for reliable mycoplasma detection. We also show and discuss typical results and their interpretation. Our goal is to ensure the success of researchers using the LookOut Mycoplasma PCR Detection Kit.
Topics: Animals; Humans; Mycoplasma; Polymerase Chain Reaction; Reagent Kits, Diagnostic
PubMed: 21712802
DOI: 10.3791/3057 -
Journal of Biotechnology Oct 2016Fluorescence expression tools for stable and innocuous whole mycoplasma cell labelling have been developed. A Tn4001-derivative mini-transposon affording unmarked,...
Fluorescence expression tools for stable and innocuous whole mycoplasma cell labelling have been developed. A Tn4001-derivative mini-transposon affording unmarked, stable mutagenesis in mycoplasmas was modified to allow the constitutive, high-level expression of mCherry, mKO2 and mNeonGreen. These tools were used to introduce the respective fluorescent proteins as chromosomal tags in the phylogenetically distant species Mycoplasma mycoides subsp. mycoides and Mycoplasma bovis. The production, selection and characterisation of fluorescent clones were straightforward and resulted in the unprecedented observation of red and green fluorescent mycoplasma colonies in the two species, with no apparent cytotoxicity. Equivalent fluorescence expression levels were quantified by flow cytometry in both species, suggesting that these tools can be broadly applied in mycoplasmas. A macrophage infection assay was performed to assess the usefulness of mNeonGreen-expressing strains for monitoring mycoplasma infections, and notably cell invasion. The presence of fluorescent mycoplasmas inside live phagocytic cells was detected and quantified by flow cytometry and corroborated by confocal microscopy, which allowed the identification of individual mycoplasmas in the cytoplasm of infected cells. The fluorescence expression tools developed in this study are suitable for host-pathogen interaction studies and offer innumerable perspectives for the functional analysis of mycoplasmas both in vitro and in vivo.
Topics: Animals; Cattle; Cells, Cultured; Flow Cytometry; Green Fluorescent Proteins; Host-Pathogen Interactions; Molecular Imaging; Mycoplasma; Mycoplasma Infections; Phagocytes; Recombinant Proteins; Reproducibility of Results; Spectrometry, Fluorescence
PubMed: 27497759
DOI: 10.1016/j.jbiotec.2016.08.006