Characterization of Virulence-Associated Traits in Mycoplasma penetrans Strains Acting as Likely Etiological Agents of Idiopathic Nongonococcal Urethritis.

Mycoplasma penetrans is an emerging pathogen with a reduced genome. This bacterium has only previously been cultured from individuals with chronic immunodeficiencies. Here we report the characteristics of 4 M. penetrans isolates from the urine of immunocompetent males with nongonococcal urethritis, in comparison with strain HF-2 from an immunocompromised patient. Several features exhibited distinct differences between these isolates and HF-2. Unlike HF-2, all 4 were resistant to azithromycin. They exhibited greater sialic acid-dependent binding to erythrocytes, gliding motility speed, and H2O2 production than HF-2. All new isolates produced thinner capsules than HF-2. Invasiveness varied, with some isolates being more invasive than HF-2 and some less invasive. Cytotoxicity to HeLa cells was similar to HF-2, and all strains could clear extracellular traps produced by innate immune cells. We conclude that subtle differences among M. penetrans strains may be critical for this organism to establish an infection in an otherwise healthy individual.

Instructional Models for Course-Based Research Experience (CRE) Teaching.

The course-based research experience (CRE) with its documented educational benefits is increasingly being implemented in science, technology, engineering, and mathematics education. This article reports on a study that was done over a period of 3 years to explicate the instructional processes involved in teaching an undergraduate CRE. One hundred and two instructors from the established and large multi-institutional SEA-PHAGES program were surveyed for their understanding of the aims and practices of CRE teaching. This was followed by large-scale feedback sessions with the cohort of instructors at the annual SEA Faculty Meeting and subsequently with a small focus group of expert CRE instructors. Using a qualitative content analysis approach, the survey data were analyzed for the aims of inquiry instruction and pedagogical practices used to achieve these goals. The results characterize CRE inquiry teaching as involving three instructional models: 1) being a scientist and generating data; 2) teaching procedural knowledge; and 3) fostering project ownership. Each of these models is explicated and visualized in terms of the specific pedagogical practices and their relationships. The models present a complex picture of the ways in which CRE instruction is conducted on a daily basis and can inform instructors and institutions new to CRE teaching.

Evaluation of Genome Sequences of the Bacteriophages JeTaime and Luna22.

The mycobacteriophages JeTaime (E cluster) and Luna22 (Q cluster) were isolated from soil in Providence, Rhode Island, and Charleston, South Carolina, respectively, using a Mycobacterium smegmatis mc2 155 host. The genome of JeTaime is 75,099 bp (142 predicted genes), and that of Luna22 is 53,730 bp (87 predicted genes). Both phages exhibit Siphoviridae morphology.

Modelling persistent Mycoplasma pneumoniae biofilm infections in a submerged BEAS-2B bronchial epithelial tissue culture model.

Introduction. Infections with the respiratory pathogen Mycoplasma pneumoniae are often chronic, recurrent and resistant, persisting after antibiotic treatment. M. pneumoniae grown on glass forms protective biofilms, consistent with a role for biofilms in persistence. These biofilms consist of towers of bacteria interspersed with individual adherent cells.Hypothesis/Gap Statement. A tissue culture model for M. pneumoniae biofilms has not been described or evaluated to address whether growth, development and resistance properties are consistent with persistence in the host. Moreover, it is unclear whether the M. pneumoniae cells in the biofilm towers and individual bacterial cells have distinct roles in disease.Aim. We evaluated the properties of biofilms of M. pneumoniae grown on the immortalized human bronchial epithelial cell line BEAS-2B in relation to persistence in the host. We observed nucleation of biofilm towers and the disposition of individual cells in culture, leading to a model of how tower and individual cells contribute to infection and disease.Methodology. With submerged BEAS-2B cells as a substrate, we evaluated growth and development of M. pneumoniae biofilms using scanning electron microscopy and confocal laser scanning microscopy. We characterized resistance to erythromycin and complement using minimum inhibitory concentration assays and quantification of colony forming units. We monitored biofilm tower formation using time-lapse microscopic analysis of host-cell-free M. pneumoniae cultures.Results. Bacteria grown on host cells underwent similar development to those grown without host cells, including tower formation, rounding and incidence of individual cells outside towers. Erythromycin and complement significantly reduced growth of M. pneumoniae. Towers formed exclusively from pre-existing aggregates of bacteria. We discuss a model of the M. pneumoniae biofilm life cycle in which protective towers derive from pre-existing aggregates, and generate individual cytotoxic cells.Conclusion . M. pneumoniae can form protective biofilms in a tissue culture model, implicating biofilms in chronic infections, with aggregates of M. pneumoniae cells being important for establishing infections.

Mycoplasma genitalium Biofilms Contain Poly-GlcNAc and Contribute to Antibiotic Resistance.

Mycoplasma genitalium is an important etiologic agent of non-gonococcal urethritis (NGU), known for chronicity and multidrug resistance, in which biofilms may play an integral role. In some bacterial species capable of forming biofilms, extracellular polymeric substances (EPS) composed of poly-N-acetylglucosamine (PNAG) are a crucial component of the matrix. Monosaccharide analysis of M. genitalium strains revealed high abundance of GlcNAc, suggesting a biofilm-specific EPS. Chromatograms also showed high concentrations of galactose and glucose as observed in other mycoplasma species. Fluorescence microscopy of M. genitalium biofilms utilizing fluor-coupled lectins revealed differential staining of biofilm structures. Scanning electron microscopy (SEM) showed increasing maturation over time of bacterial "towers" seen in biofilm development. As seen with Mycoplasma pneumoniae, organisms within fully mature M. genitalium biofilms exhibited loss of cell polarization. Bacteria associated with disrupted biofilms exhibited decreased dose-dependent viability after treatment with antibiotics compared to bacteria with intact biofilms. In addition, growth index analysis demonstrated decreases in metabolism in cultures with disrupted biofilms with antibiotic treatment. Taken together, these data suggest that M. genitalium biofilms are a contributing factor in antibiotic resistance.

Mycoplasma pneumoniae biofilms grown in vitro: traits associated with persistence and cytotoxicity.

The atypical bacterial pathogen Mycoplasma pneumoniae is a leading etiological agent of community-acquired pneumonia in humans; infections are often recalcitrant, recurrent and resistant to antibiotic treatment. These characteristics suggest a mechanism that facilitates long-term colonization in hosts. In an in vitro setting, M. pneumoniae forms biofilms that are unusual in that motility plays no more than a very limited role in their formation and development. Given the unusual nature of M. pneumoniae biofilms, open questions remain concerning phenotypes associated with persistence, such as what properties might favour the bacteria while minimizing host damage. M. pneumoniae also produces several cytotoxic molecules including community-acquired respiratory distress syndrome (CARDS) toxin, H2S and H2O2, but how it deploys these agents during growth is unknown. Whereas several biochemical techniques for biofilm disruption were ineffective, sonication was required for disruption of M. pneumoniae biofilms to generate individual cells for comparative studies, suggesting unusual physical properties likely related to the atypical cell envelope. Nonetheless, like for other bacteria, biofilms were less susceptible to antibiotic inhibition and complement killing than dispersed cells, with resistance increasing as the biofilms matured. CARDS toxin levels and enzymatic activities associated with H2S and H2O2 production were highest during early biofilm formation and decreased over time, suggesting attenuation of virulence in connection with chronic infection. Collectively, these findings result in a model of how M. pneumoniae biofilms contribute to both the establishment and propagation of M. pneumoniae infections, and how both biofilm towers and individual cells participate in persistence and chronic disease.

Development of Mycoplasma pneumoniae biofilms in vitro and the limited role of motility.

Mycoplasma pneumoniae is a bacterial pathogen of humans that is a major causative agent of chronic respiratory disease. M. pneumoniae infections often recur even after successful treatment of symptoms with antibiotics, and resistance to antibiotics is increasing worldwide, with nearly complete resistance in some places. Although biofilms often contribute to chronicity and resistance, M. pneumoniae biofilms remain poorly characterized. Scanning electron microscopy revealed that cells of wild-type (WT) M. pneumoniae strain M129 biofilms, as well as mutants II-3 and II-3R, in vitro became increasingly rounded as the biofilm towers matured over 5 days. The role of gliding motility in biofilm formation was addressed by analyzing differences in biofilm architecture in non-motile mutant II-3R and hypermotile mutant prpC-and by using time-lapse microcinematography to measure flux of cells around biofilm towers. There were no major differences in biofilm architecture between WT and motility mutants, with perhaps a slight tendency for the prpC- cells to spread outside towers during early stages of biofilm formation. Consistent with an insignificant role of motility in biofilm development, flux of cells near towers, which was low, was dominated by exit of cells. Immunofluorescence microscopy revealed that motility-associated attachment organelle (AO) proteins exhibited no discernable changes in localization to foci over time, but immunoblotting identified a decrease in steady-state levels of protein P200, which is required for normal gliding speed, as the WT culture aged. Non-adherent strain II-3 and non-motile strain II-3R also exhibited a steady decrease in P200 steady-state levels, suggesting that the decrease in P200 levels was not a response to changes in gliding behavior during maturation. We conclude that M. pneumoniae cells undergo morphological changes as biofilms mature, motility plays no major role in biofilm development, and P200 loss might be related to maturation of cells. This study helps to characterize potential therapeutic targets for M. pneumoniae infections.

Mycoplasma pneumoniae from the Respiratory Tract and Beyond.

Mycoplasma pneumoniae is an important cause of respiratory tract infections in children as well as adults that can range in severity from mild to life-threatening. Over the past several years there has been much new information published concerning infections caused by this organism. New molecular-based tests for M. pneumoniae detection are now commercially available in the United States, and advances in molecular typing systems have enhanced understanding of the epidemiology of infections. More strains have had their entire genome sequences published, providing additional insights into pathogenic mechanisms. Clinically significant acquired macrolide resistance has emerged worldwide and is now complicating treatment. In vitro susceptibility testing methods have been standardized, and several new drugs that may be effective against this organism are undergoing development. This review focuses on the many new developments that have occurred over the past several years that enhance our understanding of this microbe, which is among the smallest bacterial pathogens but one of great clinical importance.

The Variable Internal Structure of the Mycoplasma penetrans Attachment Organelle Revealed by Biochemical and Microscopic Analyses: Implications for Attachment Organelle Mechanism and Evolution.

Although mycoplasmas have small genomes, many of them, including the HIV-associated opportunist Mycoplasma penetrans, construct a polar attachment organelle (AO) that is used for both adherence to host cells and gliding motility. However, the irregular phylogenetic distribution of similar structures within the mycoplasmas, as well as compositional and ultrastructural differences among these AOs, suggests that AOs have arisen several times through convergent evolution. We investigated the ultrastructure and protein composition of the cytoskeleton-like material of the M. penetrans AO with several forms of microscopy and biochemical analysis, to determine whether the M. penetrans AO was constructed at the molecular level on principles similar to those of other mycoplasmas, such as Mycoplasma pneumoniae and Mycoplasma mobile We found that the M. penetrans AO interior was generally dissimilar from that of other mycoplasmas, in that it exhibited considerable heterogeneity in size and shape, suggesting a gel-like nature. In contrast, several of the 12 potential protein components identified by mass spectrometry of M. penetrans detergent-insoluble proteins shared certain distinctive biochemical characteristics with M. pneumoniae AO proteins, although not with M. mobile proteins. We conclude that convergence between M. penetrans and M. pneumoniae AOs extends to the molecular level, leading to the possibility that the less organized material in both M. pneumoniae and M. penetrans is the substance principally responsible for the organization and function of the AO.IMPORTANCEMycoplasma penetrans is a bacterium that infects HIV-positive patients and may contribute to the progression of AIDS. It attaches to host cells through a structure called an AO, but it is not clear how it builds this structure. Our research is significant not only because it identifies the novel protein components that make up the material within the AO that give it its structure but also because we find that the M. penetrans AO is organized unlike AOs from other mycoplasmas, suggesting that similar structures have evolved multiple times. From this work, we derive some basic principles by which mycoplasmas, and potentially all organisms, build structures at the subcellular level.

Cellular Microbiology of Mycoplasma canis.

Mycoplasma canis can infect many mammalian hosts but is best known as a commensal or opportunistic pathogen of dogs. The unexpected presence of M. canis in brains of dogs with idiopathic meningoencephalitis prompted new in vitro studies to help fill the void of basic knowledge about the organism's candidate virulence factors, the host responses that it elicits, and its potential roles in pathogenesis. Secretion of reactive oxygen species and sialidase varied quantitatively (P < 0.01) among strains of M. canis isolated from canine brain tissue or mucosal surfaces. All strains colonized the surface of canine MDCK epithelial and DH82 histiocyte cells and murine C8-D1A astrocytes. Transit through MDCK and DH82 cells was demonstrated by gentamicin protection assays and three-dimensional immunofluorescence imaging. Strains further varied (P < 0.01) in the extents to which they influenced the secretion of tumor necrosis factor alpha (TNF-α) and the neuroendocrine regulatory peptide endothelin-1 by DH82 cells. Inoculation with M. canis also decreased major histocompatibility complex class II (MHC-II) antigen expression by DH82 cells (P < 0.01), while secretion of gamma interferon (IFN-γ), interleukin-6 (IL-6), interleukin-10 (IL-10), and complement factor H was unaffected. The basis for differences in the responses elicited by these strains was not obvious in their genome sequences. No acute cytopathic effects on any homogeneous cell line, or consistent patterns of M. canis polyvalent antigen distribution in canine meningoencephalitis case brain tissues, were apparent. Thus, while it is not likely a primary neuropathogen, M. canis has the capacity to influence meningoencephalitis through complex interactions within the multicellular and neurochemical in vivo milieu.

Potential Molecular Targets for Narrow-Spectrum Agents to Combat Mycoplasma pneumoniae Infection and Disease.

As Mycoplasma pneumoniae macrolide resistance grows and spreads worldwide, it is becoming more important to develop new drugs to prevent infection or limit disease. Because other mycoplasma species have acquired resistance to other classes of antibiotics, it is reasonable to presume that M. pneumoniae can do the same, so switching to commonly used antibiotics like fluoroquinolones will not result in forms of therapy with long-term utility. Moreover, broad-spectrum antibiotics can have serious consequences for the patient, as these drugs may have severe impacts on the natural microbiota of the individual, compromising the health of the patient either short-term or long-term. Therefore, developing narrow-spectrum antibiotics that effectively target only M. pneumoniae and no more than a small portion of the microbiota is likely to yield impactful, positive results that can be used perhaps indefinitely to combat M. pneumoniae. Development of these agents requires a deep understanding of the basic biology of M. pneumoniae, in many areas deeper than what is currently known. In this review, we discuss potential targets for new, narrow-spectrum agents and both the positive and negative aspects of selecting these targets, which include toxic molecules, metabolic pathways, and attachment and motility. By gathering this information together, we anticipate that it will be easier for researchers to evaluate topics of priority for study of M. pneumoniae.

Mycoplasma iowae: relationships among oxygen, virulence, and protection from oxidative stress.

The poultry-associated bacterium Mycoplasma iowae colonizes multiple sites in embryos, with disease or death resulting. Although M. iowae accumulates in the intestinal tract, it does not cause disease at that site, but rather only in tissues that are exposed to atmospheric O2. The activity of M. iowae catalase, encoded by katE, is capable of rapid removal of damaging H2O2 from solution, and katE confers a substantial reduction in the amount of H2O2 produced by Mycoplasma gallisepticum katE transformants in the presence of glycerol. As catalase-producing bacteria are often beneficial to hosts with inflammatory bowel disease, we explored whether M. iowae was exclusively protective against H2O2-producing bacteria in a Caenorhabditis elegans model, whether its protectiveness changed in response to O2 levels, and whether expression of genes involved in H2O2 metabolism and virulence changed in response to O2 levels. We observed that M. iowae was in fact protective against H2O2-producing Streptococcus pneumoniae, but not HCN-producing Pseudomonas aeruginosa, and that M. iowae cells grown in 1% O2 promoted survival of C. elegans to a greater extent than M. iowae cells grown in atmospheric O2. Transcript levels of an M. iowae gene encoding a homolog of Mycoplasma pneumoniae CARDS toxin were 5-fold lower in cells grown in low O2. These data suggest that reduced O2, representing the intestinal environment, triggers M. iowae to reduce its virulence capabilities, effecting a change from a pathogenic mode to a potentially beneficial one.

Mycoplasma pneumoniae, an underutilized model for bacterial cell biology.

In recent decades, bacterial cell biology has seen great advances, and numerous model systems have been developed to study a wide variety of cellular processes, including cell division, motility, assembly of macromolecular structures, and biogenesis of cell polarity. Considerable attention has been given to these model organisms, which include Escherichia coli, Bacillus subtilis, Caulobacter crescentus, and Myxococcus xanthus. Studies of these processes in the pathogenic bacterium Mycoplasma pneumoniae and its close relatives have also been carried out on a smaller scale, but this work is often overlooked, in part due to this organism's reputation as minimalistic and simple. In this minireview, I discuss recent work on the role of the M. pneumoniae attachment organelle (AO), a structure required for adherence to host cells, in these processes. The AO is constructed from proteins that generally lack homology to those found in other organisms, and this construction occurs in coordination with cell cycle events. The proteins of the M. pneumoniae AO share compositional features with proteins with related roles in model organisms. Once constructed, the AO becomes activated for its role in a form of gliding motility whose underlying mechanism appears to be distinct from that of other gliding bacteria, including Mycoplasma mobile. Together with the FtsZ cytoskeletal protein, motility participates in the cell division process. My intention is to bring this deceptively complex organism into alignment with the better-known model systems.

Reduction of hydrogen peroxide accumulation and toxicity by a catalase from Mycoplasma iowae.

Mycoplasma iowae is a well-established avian pathogen that can infect and damage many sites throughout the body. One potential mediator of cellular damage by mycoplasmas is the production of H2O2 via a glycerol catabolic pathway whose genes are widespread amongst many mycoplasma species. Previous sequencing of M. iowae serovar I strain 695 revealed the presence of not only genes for H2O2 production through glycerol catabolism but also the first documented mycoplasma gene for catalase, which degrades H2O2. To test the activity of M. iowae catalase in degrading H2O2, we studied catalase activity and H2O2 accumulation by both M. iowae serovar K strain DK-CPA, whose genome we sequenced, and strains of the H2O2-producing species Mycoplasma gallisepticum engineered to produce M. iowae catalase by transformation with the M. iowae putative catalase gene, katE. H2O2-mediated virulence by M. iowae serovar K and catalase-producing M. gallisepticum transformants were also analyzed using a Caenorhabditis elegans toxicity assay, which has never previously been used in conjunction with mycoplasmas. We found that M. iowae katE encodes an active catalase that, when expressed in M. gallisepticum, reduces both the amount of H2O2 produced and the amount of damage to C. elegans in the presence of glycerol. Therefore, the correlation between the presence of glycerol catabolism genes and the use of H2O2 as a virulence factor by mycoplasmas might not be absolute.

Giant steps toward understanding a mycoplasma gliding motor.

Mycoplasma mobile carries out gliding motility using a novel motor whose proposed mechanism more closely resembles eukaryotic cytoskeletal motors than other bacterial ones. High-resolution microscopy and techniques that take advantage of the special properties of the mycoplasma cell reveal that this motor propels cells in steps of discrete size.

Biological database of images and genomes: tools for community annotations linking image and genomic information.

Genomic data and biomedical imaging data are undergoing exponential growth. However, our understanding of the phenotype-genotype connection linking the two types of data is lagging behind. While there are many types of software that enable the manipulation and analysis of image data and genomic data as separate entities, there is no framework established for linking the two. We present a generic set of software tools, BioDIG, that allows linking of image data to genomic data. BioDIG tools can be applied to a wide range of research problems that require linking images to genomes. BioDIG features the following: rapid construction of web-based workbenches, community-based annotation, user management and web services. By using BioDIG to create websites, researchers and curators can rapidly annotate a large number of images with genomic information. Here we present the BioDIG software tools that include an image module, a genome module and a user management module. We also introduce a BioDIG-based website, MyDIG, which is being used to annotate images of mycoplasmas.

Type 1 and type 2 strains of Mycoplasma pneumoniae form different biofilms.

Several mycoplasma species have been shown to form biofilms that confer resistance to antimicrobials and which may affect the host immune system, thus making treatment and eradication of the pathogens difficult. The present study shows that the biofilms formed by two strains of the human pathogen Mycoplasma pneumoniae differ quantitatively and qualitatively. Compared with strain UAB PO1, strain M129 grows well but forms biofilms that are less robust, with towers that are less smooth at the margins. A polysaccharide containing N-acetylglucosamine is secreted by M129 into the culture medium but found in tight association with the cells of UAB PO1. The polysaccharide may have a role in biofilm formation, contributing to differences in virulence, chronicity and treatment outcome between strains of M. pneumoniae. The UAB PO1 genome was found to be that of a type 2 strain of M. pneumoniae, whereas M129 is type 1. Examination of other M. pneumoniae isolates suggests that the robustness of the biofilm correlates with the strain type.

Analysis of energy sources for Mycoplasma penetrans gliding motility.

Mycoplasma penetrans, a potential human pathogen found mainly in HIV-infected individuals, uses a tip structure for both adherence and gliding motility. To improve our understanding of the molecular mechanism of M. penetrans gliding motility, we used chemical inhibitors of energy sources associated with motility of other organisms to determine which of these is used by M. penetrans and also tested whether gliding speed responded to temperature and pH. Mycoplasma penetrans gliding motility was not eliminated in the presence of a proton motive force inhibitor, a sodium motive force inhibitor, or an agent that depletes cellular ATP. At near-neutral pH, gliding speed increased as temperature increased. The absence of a clear chemical energy source for gliding motility and a positive correlation between speed and temperature suggest that energy derived from heat provides the major source of power for the gliding motor of M. penetrans.

Conserved terminal organelle morphology and function in Mycoplasma penetrans and Mycoplasma iowae.

Within the genus Mycoplasma are species whose cells have terminal organelles, polarized structures associated with cytadherence and gliding motility. Mycoplasma penetrans, found mostly in HIV-infected patients, and Mycoplasma iowae, an economically significant poultry pathogen, are members of the Mycoplasma muris phylogenetic cluster. Both species have terminal organelles that interact with host cells, yet the structures in these species, or any in the M. muris cluster, remain uncharacterized. Time-lapse microcinematography of two strains of M. penetrans, GTU-54-6A1 and HF-2, and two serovars of M. iowae, K and N, show that the terminal organelles of both species play a role in gliding motility, with differences in speed within and between the two species. The strains and serovars also differed in their hemadsorption abilities that positively correlated with differences in motility speeds. No morphological differences were observed between M. penetrans and M. iowae by scanning electron microscopy (SEM). SEM and light microscopy of M. penetrans and M. iowae showed the presence of membranous filaments connecting pairs of dividing cells. Breaking of this filament during cell division was observed for M. penetrans by microcinematography, and this suggests a role for motility during division. The Triton X-100-insoluble fractions of M. penetrans and M. iowae consisted of similar structures that were unique compared to those identified in other mycoplasma species. Like other polarized mycoplasmas, M. penetrans and M. iowae have terminal organelles with cytadherence and gliding functions. The difference in function and morphology of the terminal organelles suggests that mycoplasmas have evolved terminal organelles independently of one another.

Insights into the function of Mycoplasma pneumoniae protein P30 from orthologous gene replacement.

The attachment organelles of bacterial species belonging to the Mycoplasma pneumoniae phylogenetic cluster are required for host cytadherence, gliding motility and virulence. Despite being closely related, these bacteria possess distinct cellular morphologies and gliding characteristics. The molecular mechanisms for most attachment organelle phenotypes, including shape and ability to power motility, are obscure. The attachment organelle-associated P30 protein of M. pneumoniae is implicated in both adherence and motility, with mutations negatively impacting cell morphology, adherence, gliding and virulence. To test whether the P30 alleles of different mycoplasma species confer species-specific attachment organelle properties, we created an M. pneumoniae strain in which the Mycoplasma genitalium P30 orthologue, P32, was substituted for the native P30. Selected clones were visualized by scanning electron microscopy to assess morphology and by indirect immunofluorescence microscopy to localize P32. Cytadherence ability and gliding motility were assessed by haemadsorption assay and phase-contrast microcinematography, respectively. Cell and attachment organelle morphologies were indistinguishable from wild-type M. pneumoniae as well as M. pneumoniae II-3 expressing a C-terminally 6×His-tagged P30 construct. P32 was localized to the tip of the attachment organelle of transformant cells. Although a specific role for P30 in species-specific phenotypes was not identified, this first test of orthologous gene replacement in different mycoplasma species demonstrates that the differences in the M. pneumoniae and M. genitalium proteins contribute little if anything to the different attachment organelle phenotypes between these species.